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Sample records for host cell mitochondria

  1. Beet yellow stunt virus in cells of Sonchus oleraceus L. and its relation to host mitochondria.

    PubMed

    Esau, K

    1979-10-15

    In Sonchus oleraceus L. (Asteraceae) infected with the beet yellow stunt virus (BYSV) the virions are found in phloem cells, including the sieve elements. In parenchymatous phloem cells, the virus is present mainly in the cytoplasm. In some parenchymatous cells, containing massive accumulations of virus, the flexuous rodlike virus particles are found partly inserted into mitochondrial cristae. The mitochondrial envelope is absent where virus is present in the cristae. A similar relation between virus and host mitochondria apparently has not been recorded for any other plant virus.

  2. Induction of cell death after localization to the host cell mitochondria by the Mycobacterium tuberculosis PE_PGRS33 protein.

    PubMed

    Cadieux, Nathalie; Parra, Marcela; Cohen, Hannah; Maric, Dragan; Morris, Sheldon L; Brennan, Michael J

    2011-03-01

    PE_PGRS33 is the most studied member of the unique PE family of mycobacterial proteins. These proteins are composed of a PE domain (Pro-Glu motif), a linker region and a PGRS domain (polymorphic GC-rich-repetitive sequence). Previous studies have shown that PE_PGRS33 is surface-exposed, constitutively expressed during growth and infection, involved in creating antigenic diversity, and able to induce death in transfected or infected eukaryotic cells. In this study, we showed that PE_PGRS33 co-localizes to the mitochondria of transfected cells, a phenomenon dependent on the linker region and the PGRS domain, but not the PE domain. Using different genetic fusions and chimeras, we also demonstrated a direct correlation between localization to the host mitochondria and the induction of cell death. Finally, although all constructs localizing to the mitochondria did induce apoptosis, only the wild-type PE_PGRS33 with its own PE domain also induced primary necrosis, indicating a potentially important role for the PE domain. Considering the importance of primary necrosis in Mycobacterium tuberculosis dissemination during natural infection, the PE_PGRS33 protein may play a crucial role in the pathogenesis of tuberculosis.

  3. Vibrio cholerae Porin OmpU Induces Caspase-independent Programmed Cell Death upon Translocation to the Host Cell Mitochondria.

    PubMed

    Gupta, Shelly; Prasad, G V R Krishna; Mukhopadhaya, Arunika

    2015-12-25

    Porins, a major class of outer membrane proteins in Gram-negative bacteria, primarily act as transport channels. OmpU is one of the major porins of human pathogen, Vibrio cholerae. In the present study, we show that V. cholerae OmpU has the ability to induce target cell death. Although OmpU-mediated cell death shows some characteristics of apoptosis, such as flipping of phosphatidylserine in the membrane as well as cell size shrinkage and increased cell granularity, it does not show the caspase-3 activation and DNA laddering pattern typical of apoptotic cells. Increased release of lactate dehydrogenase in OmpU-treated cells indicates that the OmpU-mediated cell death also has characteristics of necrosis. Further, we show that the mechanism of OmpU-mediated cell death involves major mitochondrial changes in the target cells. We observe that OmpU treatment leads to the disruption of mitochondrial membrane potential, resulting in the release of cytochrome c and apoptosis-inducing factor (AIF). AIF translocates to the host cell nucleus, implying that it has a crucial role in OmpU-mediated cell death. Finally, we observe that OmpU translocates to the target cell mitochondria, where it directly initiates mitochondrial changes leading to mitochondrial membrane permeability transition and AIF release. Partial blocking of AIF release by cyclosporine A in OmpU-treated cells further suggests that OmpU may be inducing the opening of the mitochondrial permeability transition pore. All of these results lead us to the conclusion that OmpU induces cell death in target cells in a programmed manner in which mitochondria play a central role.

  4. Vibrio cholerae Porin OmpU Induces Caspase-independent Programmed Cell Death upon Translocation to the Host Cell Mitochondria*

    PubMed Central

    Gupta, Shelly; Prasad, G. V. R. Krishna; Mukhopadhaya, Arunika

    2015-01-01

    Porins, a major class of outer membrane proteins in Gram-negative bacteria, primarily act as transport channels. OmpU is one of the major porins of human pathogen, Vibrio cholerae. In the present study, we show that V. cholerae OmpU has the ability to induce target cell death. Although OmpU-mediated cell death shows some characteristics of apoptosis, such as flipping of phosphatidylserine in the membrane as well as cell size shrinkage and increased cell granularity, it does not show the caspase-3 activation and DNA laddering pattern typical of apoptotic cells. Increased release of lactate dehydrogenase in OmpU-treated cells indicates that the OmpU-mediated cell death also has characteristics of necrosis. Further, we show that the mechanism of OmpU-mediated cell death involves major mitochondrial changes in the target cells. We observe that OmpU treatment leads to the disruption of mitochondrial membrane potential, resulting in the release of cytochrome c and apoptosis-inducing factor (AIF). AIF translocates to the host cell nucleus, implying that it has a crucial role in OmpU-mediated cell death. Finally, we observe that OmpU translocates to the target cell mitochondria, where it directly initiates mitochondrial changes leading to mitochondrial membrane permeability transition and AIF release. Partial blocking of AIF release by cyclosporine A in OmpU-treated cells further suggests that OmpU may be inducing the opening of the mitochondrial permeability transition pore. All of these results lead us to the conclusion that OmpU induces cell death in target cells in a programmed manner in which mitochondria play a central role. PMID:26559970

  5. A new hypothesis of pathogenesis based on the divorce between mitochondria and their host cells: possible relevance for Alzheimer's disease.

    PubMed

    Agnati, L F; Guidolin, D; Baluska, F; Leo, G; Barlow, P W; Carone, C; Genedani, S

    2010-06-01

    On the basis of not only the endosymbiotic theory of eukaryotic cell organization and evolution but also of observations of transcellular communication via Tunneling NanoTubes (TNTs), the hypothesis is put forward that when mitochondria, which were once independently living prokaryote-like organisms, are subjected to detrimental genetic, toxic, or environmental conditions, including age-related endogenous factors, they can regress towards their original independent state. At that point, they can become potentially pathogenic intruders within their eukaryotic host cell. Because of the protoplasmic disequilibrium caused by an altered, or mutated, mitochondral population, certain host cells with a minimal capacity for self-renewal, such as dopaminergic neurons, risk a loss of function and degenerate. It is also proposed that altered mitochondria, as well as their mutated mtDNA, can migrate, via TNTs, into adjacent cells. In this way, neurodegenerative states are propagated between cells (glia and/or neurons) of the Central Nervous System (CNS) and that this leads to conditions such as Alzheimer's and Parkinson's disease. This proposal finds indirect support from observations on rotenone-poisoned glioblastoma cells which have been co-cultured with non-poisoned cells. Immunocytochemical techniques revealed that mitochondria, moving along the TNTs, migrated from the poisoned cells towards the healthy cells. It has also been demonstrated by means of immunocytochemistry that, in glioblastoma cell cultures, Amyloid Precursor Protein (APP) is present in TNTs, hence it may migrate from one cell to neighbouring cells. This datum may be of high relevance for a better understanding of Alzheimer's Disease (AD) since molecular, cellular, and animal model studies have revealed that the formation of amyloid beta (Abeta) and other derivatives of the APP are key pathogenic factors in AD, causing mitochondrial dysfunction, free radical generation, oxidative damage, and inflammation

  6. Stem cell mitochondria during aging.

    PubMed

    Min-Wen, Jason Chua; Jun-Hao, Elwin Tan; Shyh-Chang, Ng

    2016-04-01

    Mitochondria are the central hubs of cellular metabolism, equipped with their own mitochondrial DNA (mtDNA) blueprints to direct part of the programming of mitochondrial oxidative metabolism and thus reactive oxygen species (ROS) levels. In stem cells, many stem cell factors governing the intricate balance between self-renewal and differentiation have been found to directly regulate mitochondrial processes to control stem cell behaviors during tissue regeneration and aging. Moreover, numerous nutrient-sensitive signaling pathways controlling organismal longevity in an evolutionarily conserved fashion also influence stem cell-mediated tissue homeostasis during aging via regulation of stem cell mitochondria. At the genomic level, it has been demonstrated that heritable mtDNA mutations and variants affect mammalian stem cell homeostasis and influence the risk for human degenerative diseases during aging. Because such a multitude of stem cell factors and signaling pathways ultimately converge on the mitochondria as the primary mechanism to modulate cellular and organismal longevity, it would be most efficacious to develop technologies to therapeutically target and direct mitochondrial repair in stem cells, as a unified strategy to combat aging-related degenerative diseases in the future.

  7. [Dialogues between cell nuclei and mitochondria].

    PubMed

    Szewczyk, Maciej; Stępień, Piotr P

    2016-01-01

    Mitochondria are not only ATP producing organelles, but they play pivotal roles in apoptosis, neurodegeneration, cancer and aging. Mammalian mitochondrial genome is a small DNA molecule of about 16.5 kb, encoding less than 20 polypeptides and a set of ribosomal RNAs and tRNAs. In order to ensure proper cell functioning a continous communication between cell nucleus and mitochondria must be maintained. This review presents novel developments in the field of nucleo-mitochondrial communications. We discuss the import of regulatory cytosolic miRNAs into mitochondria, export of RNA from mitochondria, the existence of novel 3 polypeptides encoded by the mitochondrial genome and the transfer of mitochondrial DNA to nuclear genomes. Mechanisms of these processes and their significance for cellular homeostasis are poorly known and present an important challenge for molecular biology.

  8. Microbiota-mitochondria inter-talk: consequence for microbiota-host interaction.

    PubMed

    Saint-Georges-Chaumet, Yann; Edeas, Marvin

    2016-02-01

    New discoveries in metagenomics and clinical research have highlighted the importance of the gut microbiota for human health through the regulation of the host immune response and energetic metabolism. The microbiota interacts with host cells in particular by intermingling with the mitochondrial activities. This mitochondria-microbiota cross-talk is intriguing because mitochondria share many common structural and functional features with the prokaryotic world. Several studies reported a correlation between microbiota quality and diversity and mitochondrial function. The mitochondrial production of reactive oxygen species (ROS) plays an important role during the innate immune response and inflammation, and is often targeted by pathogenic bacteria. Data suggest that excessive mitochondrial ROS production may affect ROS signaling induced by the microbiota to regulate the gut epithelial barrier. Finally, the microbiota releases metabolites that can directly interfere with the mitochondrial respiratory chain and ATP production. Short chain fatty acids have beneficial effects on mitochondrial activity. All these data suggest that the microbiota targets mitochondria to regulate its interaction with the host. Imbalance of this targeting may result in a pathogenic state as observed in numerous studies. The challenge to find new treatments will be to find strategies to modulate the quality and diversity of the microbiota rather than acting on microbiota metabolites and microbiota-related factors.

  9. Malaria parasite liver stages render host hepatocytes susceptible to mitochondria-initiated apoptosis

    PubMed Central

    Kaushansky, A; Metzger, P G; Douglass, A N; Mikolajczak, S A; Lakshmanan, V; Kain, H S; Kappe, S HI

    2013-01-01

    Intracellular eukaryotic parasites and their host cells constitute complex, coevolved cellular interaction systems that frequently cause disease. Among them, Plasmodium parasites cause a significant health burden in humans, killing up to one million people annually. To succeed in the mammalian host after transmission by mosquitoes, Plasmodium parasites must complete intracellular replication within hepatocytes and then release new infectious forms into the blood. Using Plasmodium yoelii rodent malaria parasites, we show that some liver stage (LS)-infected hepatocytes undergo apoptosis without external triggers, but the majority of infected cells do not, and can also resist Fas-mediated apoptosis. In contrast, apoptosis is dramatically increased in hepatocytes infected with attenuated parasites. Furthermore, we find that blocking total or mitochondria-initiated host cell apoptosis increases LS parasite burden in mice, suggesting that an anti-apoptotic host environment fosters parasite survival. Strikingly, although LS infection confers strong resistance to extrinsic host hepatocyte apoptosis, infected hepatocytes lose their ability to resist apoptosis when anti-apoptotic mitochondrial proteins are inhibited. This is demonstrated by our finding that B-cell lymphoma 2 family inhibitors preferentially induce apoptosis in LS-infected hepatocytes and significantly reduce LS parasite burden in mice. Thus, targeting critical points of susceptibility in the LS-infected host cell might provide new avenues for malaria prophylaxis. PMID:23928701

  10. Analysis of mitochondria isolated from single cells.

    PubMed

    Johnson, Ryan D; Navratil, Marian; Poe, Bobby G; Xiong, Guohua; Olson, Karen J; Ahmadzadeh, Hossein; Andreyev, Dmitry; Duffy, Ciarán F; Arriaga, Edgar A

    2007-01-01

    Bulk studies are not suitable to describe and study cell-to-cell variation, which is of high importance in biological processes such as embryogenesis, tissue differentiation, and disease. Previously, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to measure the properties of organelles isolated from millions of cells. As such, these bulk measurements reported average properties for the organelles of cell populations. Similar measurements for organelles released from single cells would be highly relevant to describe the subcellular variations among cells. Toward this goal, here we introduce an approach to analyze the mitochondria released from single mammalian cells. Osteosarcoma 143B cells are labeled with either the fluorescent mitochondrion-specific 10-N-nonyl acridine orange (NAO) or via expression of the fluorescent protein DsRed2. Subsequently, a single cell is introduced into the CE-LIF capillary where the organelles are released by a combined treatment of digitonin and trypsin. After this treatment, an electric field is applied and the released organelles electromigrate toward the LIF detector. From an electropherogram, the number of detected events per cell, their individual electrophoretic mobilities, and their individual fluorescence intensities are calculated. The results obtained from DsRed2 labeling, which is retained in intact mitochondria, and NAO labeling, which labels all mitochondria, are the basis for discussion of the strengths and limitations of this single-cell approach.

  11. Stem cells, mitochondria and aging.

    PubMed

    Ahlqvist, Kati J; Suomalainen, Anu; Hämäläinen, Riikka H

    2015-11-01

    Decline in metabolism and regenerative potential of tissues are common characteristics of aging. Regeneration is maintained by somatic stem cells (SSCs), which require tightly controlled energy metabolism and genomic integrity for their homeostasis. Recent data indicate that mitochondrial dysfunction may compromise this homeostasis, and thereby contribute to tissue degeneration and aging. Progeroid Mutator mouse, accumulating random mtDNA point mutations in their SSCs, showed disturbed SSC homeostasis, emphasizing the importance of mtDNA integrity for stem cells. The mechanism involved changes in cellular redox-environment, including subtle increase in reactive oxygen species (H₂O₂and superoxide anion), which did not cause oxidative damage, but disrupted SSC function. Mitochondrial metabolism appears therefore to be an important regulator of SSC fate determination, and defects in it in SSCs may underlie premature aging. Here we review the current knowledge of mitochondrial contribution to SSC dysfunction and aging. This article is part of a Special Issue entitled: Mitochondrial Dysfunction in Aging.

  12. Mitochondria and Energetic Depression in Cell Pathophysiology

    PubMed Central

    Seppet, Enn; Gruno, Marju; Peetsalu, Ants; Gizatullina, Zemfira; Nguyen, Huu Phuc; Vielhaber, Stefan; Wussling, Manfred H.P.; Trumbeckaite, Sonata; Arandarcikaite, Odeta; Jerzembeck, Doreen; Sonnabend, Maria; Jegorov, Katharina; Zierz, Stephan; Striggow, Frank; Gellerich, Frank N.

    2009-01-01

    Mitochondrial dysfunction is a hallmark of almost all diseases. Acquired or inherited mutations of the mitochondrial genome DNA may give rise to mitochondrial diseases. Another class of disorders, in which mitochondrial impairments are initiated by extramitochondrial factors, includes neurodegenerative diseases and syndromes resulting from typical pathological processes, such as hypoxia/ischemia, inflammation, intoxications, and carcinogenesis. Both classes of diseases lead to cellular energetic depression (CED), which is characterized by decreased cytosolic phosphorylation potential that suppresses the cell’s ability to do work and control the intracellular Ca2+ homeostasis and its redox state. If progressing, CED leads to cell death, whose type is linked to the functional status of the mitochondria. In the case of limited deterioration, when some amounts of ATP can still be generated due to oxidative phosphorylation (OXPHOS), mitochondria launch the apoptotic cell death program by release of cytochrome c. Following pronounced CED, cytoplasmic ATP levels fall below the thresholds required for processing the ATP-dependent apoptotic cascade and the cell dies from necrosis. Both types of death can be grouped together as a mitochondrial cell death (MCD). However, there exist multiple adaptive reactions aimed at protecting cells against CED. In this context, a metabolic shift characterized by suppression of OXPHOS combined with activation of aerobic glycolysis as the main pathway for ATP synthesis (Warburg effect) is of central importance. Whereas this type of adaptation is sufficiently effective to avoid CED and to control the cellular redox state, thereby ensuring the cell survival, it also favors the avoidance of apoptotic cell death. This scenario may underlie uncontrolled cellular proliferation and growth, eventually resulting in carcinogenesis. PMID:19564950

  13. Evaluation of respiration of mitochondria in cancer cells exposed to mitochondria-targeted agents.

    PubMed

    Kluckova, Katarina; Dong, Lan-Feng; Bajzikova, Martina; Rohlena, Jakub; Neuzil, Jiri

    2015-01-01

    Respiration is one of the major functions of mitochondria, whereby these vital organelles use oxygen to produce energy. Many agents that may be of potential clinical relevance act by targeting mitochondria, where they may suppress mitochondrial respiration. It is therefore important to evaluate this process and understand how this is modulated by small molecules. Here, we describe the general methodology to assess respiration in cultured cells, followed by the evaluation of the effect of one anticancer agent targeted to mitochondria on this process, and also how to assess this in tumor tissue.

  14. Mitochondria-specific accumulation of amyloid β induces mitochondrial dysfunction leading to apoptotic cell death.

    PubMed

    Cha, Moon-Yong; Han, Sun-Ho; Son, Sung Min; Hong, Hyun-Seok; Choi, Young-Ju; Byun, Jayoung; Mook-Jung, Inhee

    2012-01-01

    Mitochondria are best known as the essential intracellular organelles that host the homeostasis required for cellular survival, but they also have relevance in diverse disease-related conditions, including Alzheimer's disease (AD). Amyloid β (Aβ) peptide is the key molecule in AD pathogenesis, and has been highlighted in the implication of mitochondrial abnormality during the disease progress. Neuronal exposure to Aβ impairs mitochondrial dynamics and function. Furthermore, mitochondrial Aβ accumulation has been detected in the AD brain. However, the underlying mechanism of how Aβ affects mitochondrial function remains uncertain, and it is questionable whether mitochondrial Aβ accumulation followed by mitochondrial dysfunction leads directly to neuronal toxicity. This study demonstrated that an exogenous Aβ(1-42) treatment, when applied to the hippocampal cell line of mice (specifically HT22 cells), caused a deleterious alteration in mitochondria in both morphology and function. A clathrin-mediated endocytosis blocker rescued the exogenous Aβ(1-42)-mediated mitochondrial dysfunction. Furthermore, the mitochondria-targeted accumulation of Aβ(1-42) in HT22 cells using Aβ(1-42) with a mitochondria-targeting sequence induced the identical morphological alteration of mitochondria as that observed in the APP/PS AD mouse model and exogenous Aβ(1-42)-treated HT22 cells. In addition, subsequent mitochondrial dysfunctions were demonstrated in the mitochondria-specific Aβ(1-42) accumulation model, which proved indistinguishable from the mitochondrial impairment induced by exogenous Aβ(1-42)-treated HT22 cells. Finally, cellular toxicity was directly induced by mitochondria-targeted Aβ(1-42) accumulation, which mimics the apoptosis process in exogenous Aβ(1-42)-treated HT22 cells. Taken together, these results indicate that mitochondria-targeted Aβ(1-42) accumulation is the necessary and sufficient condition for Aβ-mediated mitochondria impairments, and leads

  15. Redox proteins in mammalian cell death: an evolutionarily conserved function in mitochondria and prokaryotes.

    PubMed

    Punj, Vasu; Chakrabarty, A M

    2003-04-01

    Mammalian cell mitochondria are believed to have prokaryotic ancestry. Mitochondria are not only the powerhouse of energy generation within the eukaryotic cell but they also play a major role in inducing apoptotic cell death through release of redox proteins such as cytochrome c and the apoptosis-inducing factor (AIF), a flavoprotein with NADH oxidase activity. Recent evidence indicates that some present day prokaryotes release redox proteins that induce apoptosis in mammalian cells through stabilization of the tumour suppressor protein p53. p53 interacts with mitochondria either directly or through activation of the genes for pro-apoptotic proteins such as Bax or NOXA or genes that encode redox enzymes responsible for the production of reactive oxygen species (ROS). The analogy between the ancient ancestors of present day bacteria, the mitochondria, and the present day bacteria with regard to their ability to release redox proteins for triggering mammalian cell death is an interesting example of functional conservation during the hundreds of millions of years of evolution. It is possible that the ancestors of the present day prokaryotes released redox proteins to kill the ancestors of the eukaryotes. During evolution of the mitochondria from prokaryotes as obligate endosymbionts, the mitochondria maintained the same functions to programme their own host cell death.

  16. Mitochondria: An intriguing target for killing tumour-initiating cells.

    PubMed

    Yan, Bing; Dong, Lanfeng; Neuzil, Jiri

    2016-01-01

    Tumour-initiating cells (TICs) play a pivotal role in cancer initiation, metastasis and recurrence, as well as in resistance to therapy. Therefore, development of drugs targeting TICs has become a focus of contemporary research. Mitochondria have emerged as a promising target of anti-cancer therapies due to their specific role in cancer metabolism and modulation of apoptotic pathways. Mitochondria of TICs possess special characteristics, some of which can be utilised to design drugs specifically targeting these cells. In this paper, we will review recent research on TICs and their mitochondria, and introduce drugs that kill these cells by way of mitochondrial targeting.

  17. The role of mitochondria in metabolism and cell death.

    PubMed

    Vakifahmetoglu-Norberg, Helin; Ouchida, Amanda Tomie; Norberg, Erik

    2017-01-15

    Mitochondria are complex organelles that play a central role in energy metabolism, control of stress responses and are a hub for biosynthetic processes. Beyond its well-established role in cellular energetics, mitochondria are critical mediators of signals to propagate various cellular outcomes. In addition mitochondria are the primary source of intracellular reactive oxygen species (ROS) generation and are involved in cellular Ca(2+) homeostasis, they contain a self-destructive arsenal of apoptogenic factors that can be unleashed to promote cell death, thus displaying a shared platform for metabolism and apoptosis. In the present review, we will give a brief account on the integration of mitochondrial metabolism and apoptotic cell death.

  18. Late acquisition of mitochondria by a host with chimeric prokaryotic ancestry

    PubMed Central

    Pittis, Alexandros A.; Gabaldón, Toni

    2016-01-01

    The origin of eukaryotes stands as a major conundrum in biology1. Current evidence indicates that the Last Eukaryotic Common Ancestor (LECA) already possessed many eukaryotic hallmarks, including a complex subcellular organization1–3. In addition, the lack of evolutionary intermediates challenges the elucidation of the relative order of emergence of eukaryotic traits. Mitochondria are ubiquitous organelles derived from an alpha-proteobacterial endosymbiont4. Different hypotheses disagree on whether mitochondria were acquired early or late during eukaryogenesis5. Similarly, the nature and complexity of the receiving host are debated, with models ranging from a simple prokaryotic host to an already complex proto-eukaryote1,3,6,7. Most competing scenarios can be roughly grouped into either mito-early, which consider the driving force of eukaryogenesis to be mitochondrial endosymbiosis into a simple host, or mito-late, which postulate that a significant complexity predated mitochondrial endosymbiosis3. Here we provide evidence for late mitochondrial endosymbiosis. We used phylogenomics to directly test whether proto-mitochondrial proteins were acquired earlier or later than other LECA proteins. We found that LECA protein families of alpha-proteobacterial ancestry and of mitochondrial localization show the shortest phylogenetic distances to their closest prokaryotic relatives, when compared to proteins of different prokaryotic origin or cellular localization. Altogether, our results shed new light on a long-standing question and provide compelling support for the late acquisition of mitochondria into a host that already had a proteome of chimeric phylogenetic origin. We argue that mitochondrial endosymbiosis was one of the ultimate steps in eukaryogenesis and that it provided the definitive selective advantage to mitochondria-bearing eukaryotes over less complex forms. PMID:26840490

  19. Late acquisition of mitochondria by a host with chimaeric prokaryotic ancestry.

    PubMed

    Pittis, Alexandros A; Gabaldón, Toni

    2016-03-03

    The origin of eukaryotes stands as a major conundrum in biology. Current evidence indicates that the last eukaryotic common ancestor already possessed many eukaryotic hallmarks, including a complex subcellular organization. In addition, the lack of evolutionary intermediates challenges the elucidation of the relative order of emergence of eukaryotic traits. Mitochondria are ubiquitous organelles derived from an alphaproteobacterial endosymbiont. Different hypotheses disagree on whether mitochondria were acquired early or late during eukaryogenesis. Similarly, the nature and complexity of the receiving host are debated, with models ranging from a simple prokaryotic host to an already complex proto-eukaryote. Most competing scenarios can be roughly grouped into either mito-early, which consider the driving force of eukaryogenesis to be mitochondrial endosymbiosis into a simple host, or mito-late, which postulate that a significant complexity predated mitochondrial endosymbiosis. Here we provide evidence for late mitochondrial endosymbiosis. We use phylogenomics to directly test whether proto-mitochondrial proteins were acquired earlier or later than other proteins of the last eukaryotic common ancestor. We find that last eukaryotic common ancestor protein families of alphaproteobacterial ancestry and of mitochondrial localization show the shortest phylogenetic distances to their closest prokaryotic relatives, compared with proteins of different prokaryotic origin or cellular localization. Altogether, our results shed new light on a long-standing question and provide compelling support for the late acquisition of mitochondria into a host that already had a proteome of chimaeric phylogenetic origin. We argue that mitochondrial endosymbiosis was one of the ultimate steps in eukaryogenesis and that it provided the definitive selective advantage to mitochondria-bearing eukaryotes over less complex forms.

  20. miRNAs in mtDNA-less cell mitochondria

    PubMed Central

    Dasgupta, N; Peng, Y; Tan, Z; Ciraolo, G; Wang, D; Li, R

    2015-01-01

    The novel regulation mechanism in mtDNA-less cells was investigated. Very low mtDNA copy in mtDNA-less 206 ρ° cells was identified. But no 13 mitochondria-specific proteins were translated in 206 ρ° cells. Their mitochondrial respiration complexes V, III and II were 86.5, 29.4 and 49.6% of 143B cells, respectively. Complexes I and IV completely lack in 206 ρ° cells. Non-mitochondrial respiration to generate ATP in 206 ρ° cells was discovered. The expression levels of some mitochondrial RNAs including 12S rRNA, COX1, COX2, COX3, ND4 and ND5 were low. However, ND1, ND3 and Cyto b were not expressed in 206 ρ° cells. Unequal transcription of mitochondrial RNAs indicated the post-transcriptional cleavage and processing mechanisms in the regulation of mitochondrial gene expression in 206 ρ° cells. MicroRNAs (miRNAs) may modulate these mitochondrial RNA expression in these cells. RNA-induced silencing complex indeed within 206 ρ° cell mitochondria indicated miRNAs in 206 ρ° cell mitochondria. miRNA profile in mtDNA-less 206 ρ° cells was studied by next-generation sequencing of small RNAs. Several mitochondria-enriched miRNAs such as miR-181c-5p and miR-146a-5p were identified in 206 ρ° cell mitochondria. miR-181c-5p and miR-146a-5p had 23 and 19 potential targets on mitochondrial RNAs respectively, and these two miRNAs had multiple targets on mitochondria-associated messenger RNAs encoded by nuclear genes. These data provided the first direct evidence that miRNAs were imported into mitochondria and regulated mitochondrial RNA expressions. PMID:27551440

  1. Horizontal transfer of whole mitochondria restores tumorigenic potential in mitochondrial DNA-deficient cancer cells

    PubMed Central

    Dong, Lan-Feng; Kovarova, Jaromira; Bajzikova, Martina; Bezawork-Geleta, Ayenachew; Svec, David; Endaya, Berwini; Sachaphibulkij, Karishma; Coelho, Ana R; Sebkova, Natasa; Ruzickova, Anna; Tan, An S; Kluckova, Katarina; Judasova, Kristyna; Zamecnikova, Katerina; Rychtarcikova, Zuzana; Gopalan, Vinod; Andera, Ladislav; Sobol, Margarita; Yan, Bing; Pattnaik, Bijay; Bhatraju, Naveen; Truksa, Jaroslav; Stopka, Pavel; Hozak, Pavel; Lam, Alfred K; Sedlacek, Radislav; Oliveira, Paulo J; Kubista, Mikael; Agrawal, Anurag; Dvorakova-Hortova, Katerina; Rohlena, Jakub; Berridge, Michael V; Neuzil, Jiri

    2017-01-01

    Recently, we showed that generation of tumours in syngeneic mice by cells devoid of mitochondrial (mt) DNA (ρ0 cells) is linked to the acquisition of the host mtDNA. However, the mechanism of mtDNA movement between cells remains unresolved. To determine whether the transfer of mtDNA involves whole mitochondria, we injected B16ρ0 mouse melanoma cells into syngeneic C57BL/6Nsu9-DsRed2 mice that express red fluorescent protein in their mitochondria. We document that mtDNA is acquired by transfer of whole mitochondria from the host animal, leading to normalisation of mitochondrial respiration. Additionally, knockdown of key mitochondrial complex I (NDUFV1) and complex II (SDHC) subunits by shRNA in B16ρ0 cells abolished or significantly retarded their ability to form tumours. Collectively, these results show that intact mitochondria with their mtDNA payload are transferred in the developing tumour, and provide functional evidence for an essential role of oxidative phosphorylation in cancer. DOI: http://dx.doi.org/10.7554/eLife.22187.001 PMID:28195532

  2. Mitochondria. Cell cycle-dependent regulation of mitochondrial preprotein translocase.

    PubMed

    Harbauer, Angelika B; Opalińska, Magdalena; Gerbeth, Carolin; Herman, Josip S; Rao, Sanjana; Schönfisch, Birgit; Guiard, Bernard; Schmidt, Oliver; Pfanner, Nikolaus; Meisinger, Chris

    2014-11-28

    Mitochondria play central roles in cellular energy conversion, metabolism, and apoptosis. Mitochondria import more than 1000 different proteins from the cytosol. It is unknown if the mitochondrial protein import machinery is connected to the cell division cycle. We found that the cyclin-dependent kinase Cdk1 stimulated assembly of the main mitochondrial entry gate, the translocase of the outer membrane (TOM), in mitosis. The molecular mechanism involved phosphorylation of the cytosolic precursor of Tom6 by cyclin Clb3-activated Cdk1, leading to enhanced import of Tom6 into mitochondria. Tom6 phosphorylation promoted assembly of the protein import channel Tom40 and import of fusion proteins, thus stimulating the respiratory activity of mitochondria in mitosis. Tom6 phosphorylation provides a direct means for regulating mitochondrial biogenesis and activity in a cell cycle-specific manner.

  3. The cellular energy crisis: mitochondria and cell death.

    PubMed

    Waterhouse, Nigel J

    2003-01-01

    Exploding nuclear reactors, environmental destruction, and global warming; the danger of energy production is clear. It is quite remarkable that in this modern age, where power usage is at a premium, we find that even on a cellular level, generation of large quantities of power comes at a cost. Mitochondria, which produce the majority of cellular energy in the form of ATP, have recently been shown to play an essential role in the death of a cell by a process known as apoptosis. During apoptosis, the integrity of mitochondria is compromised and various pro-apoptotic proteins are released into the cytoplasm. This results in activation of caspases, proteases that orchestrate the death of the cell. Cells in which apoptosis is inhibited upstream of mitochondria generally maintain the potential to proliferate, whereas inhibition of caspases downstream of mitochondria generally only delays cell death. Although breaches of the mitochondrial outer membrane result in the release of proteins that are important for respiration, mitochondria appear capable of maintaining at least some of their functions, including ATP production, even after this event. This has important implications both for the mechanism of outer-membrane permeabilization and the mechanism by which the cells eventually die in the absence of caspase activity. The events surrounding the breach of the mitochondrial outer membrane during apoptosis have therefore received much interest over the past few years.

  4. Construction of photoenergetic mitochondria in cultured mammalian cells.

    PubMed

    Hara, Kiyotaka Y; Wada, Takeyoshi; Kino, Kuniki; Asahi, Toru; Sawamura, Naoya

    2013-01-01

    The proton motive force (PMF) is bio-energetically important for various cellular reactions to occur. We developed PMF-photogenerating mitochondria in cultured mammalian cells. An archaebacterial rhodopsin, delta-rhodopsin, which is a light-driven proton pump derived from Haloterrigena turkmenica, was expressed in the mitochondria of CHO-K1 cells. The constructed stable CHO-K1 cell lines showed suppression of cell death induced by rotenone, a pesticide that inhibits mitochondrial complex I activity involved in PMF generation through the electron transport chain. Delta-rhodopsin was also introduced into the mitochondria of human neuroblastoma SH-SY5Y cells. The constructed stable SH-SY5Y cell lines showed suppression of dopaminergic neuronal cell death induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an inducer of Parkinson's disease models, which acts through inhibition of complex I activity. These results suggest that the light-activated proton pump functioned as a PMF generator in the mitochondria of mammalian cells, and suppressed cell death induced by inhibition of respiratory PMF generation.

  5. Power surge: supporting cells "fuel" cancer cell mitochondria.

    PubMed

    Martinez-Outschoorn, Ubaldo E; Sotgia, Federica; Lisanti, Michael P

    2012-01-04

    An emerging paradigm in tumor metabolism is that catabolism in host cells "fuels" the anabolic growth of cancer cells via energy transfer. A study in Nature Medicine (Nieman et al., 2011) supports this; they show that triglyceride catabolism in adipocytes drives ovarian cancer metastasis by providing fatty acids as mitochondrial fuels.

  6. Light-dependent intracellular positioning of mitochondria in Arabidopsis thaliana mesophyll cells.

    PubMed

    Islam, Md Sayeedul; Niwa, Yasuo; Takagi, Shingo

    2009-06-01

    Mitochondria, the power house of the cell, are one of the most dynamic cell organelles. Although there are several reports on actin- or microtubule-dependent movement of mitochondria in plant cells, intracellular positioning and motility of mitochondria under different light conditions remain open questions. Mitochondria were visualized in living Arabidopsis thaliana leaf cells using green fluorescent protein fused to a mitochondrion-targeting signal. In darkness, mitochondria were distributed randomly in palisade cells. In contrast, mitochondria accumulated along the periclinal walls, similar to the accumulation response of chloroplasts, when treated with weak blue light (470 nm, 4 micromol m(-2) s(-1)). Under strong blue light (100 micromol m(-2) s(-1)), mitochondria occupied the anticlinal positions similar to the avoidance response of chloroplasts and nuclei. While strong red light (660 nm, 100 micromol m(-2) s(-1)) induced the accumulation of mitochondria along the inner periclinal walls, green light exhibited little effect on the distribution of mitochondria. In addition, the mode of movement of individual mitochondria along the outer periclinal walls under different light conditions was precisely analyzed by time-lapse fluorescence microscopy. A gradual increase in the number of static mitochondria located in the vicinity of chloroplasts with a time period of blue light illumination clearly demonstrated the accumulation response of mitochondria. Light-induced co-localization of mitochondria with chloroplasts strongly suggested their mutual metabolic interactions. This is the first characterization of the light-dependent redistribution of mitochondria in plant cells.

  7. Mitochondria in the midgut epithelial cells of sugarcane borer parasitized by Cotesia flavipes (Cameron, 1891).

    PubMed

    Pinheiro, D O; Silva, M D; Gregório, E A

    2010-02-01

    The sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae) has been controlled by Cotesia flavipes (Hymenoptera: Braconidae); however, very little is known about the effect of the parasitism in the host organs, including the midgut. This work aims to verify mitochondrial alteration in the different midgut epithelial cells of D. saccharalis parasitized by C. flavipes. Midgut fragments (anterior and posterior region) of both non-parasitized and parasitized larvae were processed for transmission electron microscopy. The mitochondria of midgut epithelial cell in the parasitized larvae exhibit morphological alteration, represented by matrix rarefaction and vacuolisation. These mitochondrial alterations are more pronounced in the anterior midgut region during the parasitism process, mainly in the columnar cell.

  8. Transition metal catalysis in the mitochondria of living cells

    NASA Astrophysics Data System (ADS)

    Tomás-Gamasa, María; Martínez-Calvo, Miguel; Couceiro, José R.; Mascareñas, José L.

    2016-09-01

    The development of transition metal catalysts capable of promoting non-natural transformations within living cells can open significant new avenues in chemical and cell biology. Unfortunately, the complexity of the cell makes it extremely difficult to translate standard organometallic chemistry to living environments. Therefore, progress in this field has been very slow, and many challenges, including the possibility of localizing active metal catalysts into specific subcellular sites or organelles, remain to be addressed. Herein, we report a designed ruthenium complex that accumulates preferentially inside the mitochondria of mammalian cells, while keeping its ability to react with exogenous substrates in a bioorthogonal way. Importantly, we show that the subcellular catalytic activity can be used for the confined release of fluorophores, and even allows selective functional alterations in the mitochondria by the localized transformation of inert precursors into uncouplers of the membrane potential.

  9. Transition metal catalysis in the mitochondria of living cells

    PubMed Central

    Tomás-Gamasa, María; Martínez-Calvo, Miguel; Couceiro, José R.; Mascareñas, José L.

    2016-01-01

    The development of transition metal catalysts capable of promoting non-natural transformations within living cells can open significant new avenues in chemical and cell biology. Unfortunately, the complexity of the cell makes it extremely difficult to translate standard organometallic chemistry to living environments. Therefore, progress in this field has been very slow, and many challenges, including the possibility of localizing active metal catalysts into specific subcellular sites or organelles, remain to be addressed. Herein, we report a designed ruthenium complex that accumulates preferentially inside the mitochondria of mammalian cells, while keeping its ability to react with exogenous substrates in a bioorthogonal way. Importantly, we show that the subcellular catalytic activity can be used for the confined release of fluorophores, and even allows selective functional alterations in the mitochondria by the localized transformation of inert precursors into uncouplers of the membrane potential. PMID:27600651

  10. Energy transfer in "parasitic" cancer metabolism: mitochondria are the powerhouse and Achilles' heel of tumor cells.

    PubMed

    Martinez-Outschoorn, Ubaldo E; Pestell, Richard G; Howell, Anthony; Tykocinski, Mark L; Nagajyothi, Fnu; Machado, Fabiana S; Tanowitz, Herbert B; Sotgia, Federica; Lisanti, Michael P

    2011-12-15

    It is now widely recognized that the tumor microenvironment promotes cancer cell growth and metastasis via changes in cytokine secretion and extracellular matrix remodeling. However, the role of tumor stromal cells in providing energy for epithelial cancer cell growth is a newly emerging paradigm. For example, we and others have recently proposed that tumor growth and metastasis is related to an energy imbalance. Host cells produce energy-rich nutrients via catabolism (through autophagy, mitophagy, and aerobic glycolysis), which are then transferred to cancer cells to fuel anabolic tumor growth. Stromal cell-derived L-lactate is taken up by cancer cells and is used for mitochondrial oxidative phosphorylation (OXPHOS) to produce ATP efficiently. However, "parasitic" energy transfer may be a more generalized mechanism in cancer biology than previously appreciated. Two recent papers in Science and Nature Medicine now show that lipolysis in host tissues also fuels tumor growth. These studies demonstrate that free fatty acids produced by host cell lipolysis are re-used via beta-oxidation (beta-OX) in cancer cell mitochondria. Thus, stromal catabolites (such as lactate, ketones, glutamine and free fatty acids) promote tumor growth by acting as high-energy onco-metabolites. As such, host catabolism, via autophagy, mitophagy and lipolysis, may explain the pathogenesis of cancer-associated cachexia and provides exciting new druggable targets for novel therapeutic interventions. Taken together, these findings also suggest that tumor cells promote their own growth and survival by behaving as a "parasitic organism." Hence, we propose the term "Parasitic Cancer Metabolism" to describe this type of metabolic coupling in tumors. Targeting tumor cell mitochondria (OXPHOS and beta-OX) would effectively uncouple tumor cells from their hosts, leading to their acute starvation. In this context, we discuss new evidence that high-energy onco-metabolites (produced by the stroma) can

  11. Isolation of mitochondria by gentle cell membrane disruption, and their subsequent characterization.

    PubMed

    Shibata, Takahiro; Yamashita, Saki; Hirusaki, Kotoe; Katoh, Kaoru; Ohta, Yoshihiro

    2015-08-07

    Mitochondria play a key role in several physiological processes as in integrating signals in the cell. However, understanding of the mechanism by which mitochondria sense and respond to signals has been limited due to the lack of an appropriate model system. In this study, we developed a method to isolate and characterize mitochondria without cell homogenization. By gently pipetting cells treated with streptolysin-O, a pore-forming membrane protein, we disrupted the cell membrane and were able to isolate both elongated and spherical mitochondria. Fluorescence imaging combined with super resolution microscopy showed that both the outer and inner membranes of the elongated mitochondria isolated using the newly developed method were intact. In addition, a FRET-based ATP sensor expressed in the mitochondrial matrix demonstrated that ATP generation by FoF1-ATPase in the isolated elongated mitochondria was as high as that in intracellular mitochondria. On the other hand, some of the spherical mitochondria isolated with this method had the outer membrane that no longer encapsulated the inner membrane. In addition, all mitochondria isolated using conventional procedures involving homogenization were spherical, many of them had damaged membranes, and low levels of ATP generation. Our results suggest that elongated mitochondria isolated from cells through gentle cell membrane disruption using a pore-forming protein tend to be more similar to intracellular mitochondria, having an intact membrane system and higher activity than spherical mitochondria.

  12. Mitochondria, the Cell Cycle, and the Origin of Sex via a Syncytial Eukaryote Common Ancestor.

    PubMed

    Garg, Sriram G; Martin, William F

    2016-07-02

    Theories for the origin of sex traditionally start with an asexual mitosing cell and add recombination, thereby deriving meiosis from mitosis. Though sex was clearly present in the eukaryote common ancestor, the order of events linking the origin of sex and the origin of mitosis is unknown. Here, we present an evolutionary inference for the origin of sex starting with a bacterial ancestor of mitochondria in the cytosol of its archaeal host. We posit that symbiotic association led to the origin of mitochondria and gene transfer to host's genome, generating a nucleus and a dedicated translational compartment, the eukaryotic cytosol, in which-by virtue of mitochondria-metabolic energy was not limiting. Spontaneous protein aggregation (monomer polymerization) and Adenosine Tri-phosphate (ATP)-dependent macromolecular movement in the cytosol thereby became selectable, giving rise to continuous microtubule-dependent chromosome separation (reduction division). We propose that eukaryotic chromosome division arose in a filamentous, syncytial, multinucleated ancestor, in which nuclei with insufficient chromosome numbers could complement each other through mRNA in the cytosol and generate new chromosome combinations through karyogamy. A syncytial (or coenocytic, a synonym) eukaryote ancestor, or Coeca, would account for the observation that the process of eukaryotic chromosome separation is more conserved than the process of eukaryotic cell division. The first progeny of such a syncytial ancestor were likely equivalent to meiospores, released into the environment by the host's vesicle secretion machinery. The natural ability of archaea (the host) to fuse and recombine brought forth reciprocal recombination among fusing (syngamy and karyogamy) progeny-sex-in an ancestrally meiotic cell cycle, from which the simpler haploid and diploid mitotic cell cycles arose. The origin of eukaryotes was the origin of vertical lineage inheritance, and sex was required to keep vertically

  13. Mitochondria single nucleotide variation across six blood cell types.

    PubMed

    Zhang, Pan; Samuels, David C; Wang, Jing; Zhao, Shilin; Shyr, Yu; Guo, Yan

    2016-05-01

    It has been shown that heteroplasmic mitochondrial DNA variants can be tissue specific. However, whether mitochondrial DNA variants are specific by blood cell types has not been investigated. Motivated by this question and using mitochondria sequences extracted from RNAseq data from six distinct blood cell types (neutrophil, monocyte, myeloid dendritic, natural killer, T and B), we thoroughly compared SNPs and heteroplasmies among these cell types. Each cell type from each subject was sequenced at four time points used as biological replicates. We found that mitochondria content is low in neutrophil compared to the other five blood cell types. Subsequent analysis on the other five blood cell types showed that at the SNP level, there was no discrepancy. At the heteroplasmy level, we observed good concordances among all blood cell types. However, the allele frequencies of the heteroplasmy differed between blood cell types for certain heteroplasmic sites. Furthermore, we identified five tri-allelic sites (1610, 2617, 8303, 12146, 13710) that are likely caused by RNA editing. Three out of these five sites are located at the ninth position of tRNA genes, and are likely resulting from post-transcriptional methylation.

  14. Oxidation of H2S in mammalian cells and mitochondria.

    PubMed

    Abou-Hamdan, Abbas; Guedouari-Bounihi, Hala; Lenoir, Véronique; Andriamihaja, Mireille; Blachier, François; Bouillaud, Frédéric

    2015-01-01

    Hydrogen sulfide (H2S) is the third gasotransmitter described in mammals. These gasotransmitters (H2S, CO, and NO) are small molecules able to diffuse freely across membranes and thus susceptible to reach easily intracellular targets, one of which is the respiratory enzyme cytochrome oxidase subject to complete inhibition by low micromolar concentrations of these gases. However in contrast to NO or CO, H2S can be metabolized by a sulfide quinone reductase feeding the mitochondrial respiratory chain with the hydrogen atoms of sulfide. Sulfide is thus a two-sided molecule: substrate or poison according to the concentration. The aim of this chapter is to present a mean to monitor sulfide oxidation by isolated mitochondria or cells and to summarize how the properties of this amazing couple (mitochondria and sulfide) translate into practical and conceptual consequences.

  15. Axin expression reduces staurosporine-induced mitochondria-mediated cell death in HeLa cells.

    PubMed

    Shin, Jee-Hye; Kim, Hyun-wook; Rhyu, Im Joo; Song, Ki-Joon; Kee, Sun-Ho

    2012-10-01

    Cytoplasmic axin expression frequently produces punctuate structures in cells, but the nature of axin puncta has not been fully elucidated. In an effort to analyze cytoplasmic axin puncta, we established HeLa cells expressing axin in a doxycycline-inducible manner (HeLa-Axin). We observed that axin accumulated in an aggregate-like pattern in perinuclear areas and appeared to be associated with mitochondria, Golgi apparatus, and endoplasmic reticulum (ER), but not lysosomes. Further biochemical analysis suggested that some part of the cytoplasmic axin pool was associated with mitochondria. In addition, mitochondrial proteins [i.e., cytochrome oxidase IV (CoxIV) and cytochrome c] were slightly higher in HeLa-Axin cells than in HeLa-EV cells, suggesting altered mitochondrial degradation. HeLa-Axin cells were then treated with staurosporine (STS) to determine if the mitochondria-induced apoptosis pathway was altered. Compared to STS-treated control cells (HeLa-EV), HeLa-Axin cells had less STS-induced cytotoxicity and reduced caspase-3 activation and PARP cleavage. Given that mitochondria outer membrane potential was unchanged, HeLa-Axin cells might be relatively resistant to STS-mediated mitochondrial damage. Mitochondria associated with axin aggregates were resistant to detergent-mediated permeabilization. These results suggest that axin forms aggregate-like structures in association with mitochondria, which render mitochondria resistant to STS-induced membrane damage and cytotoxicity.

  16. Computational properties of mitochondria in T cell activation and fate

    PubMed Central

    Dupont, Geneviève

    2016-01-01

    In this article, we review how mitochondrial Ca2+ transport (mitochondrial Ca2+ uptake and Na+/Ca2+ exchange) is involved in T cell biology, including activation and differentiation through shaping cellular Ca2+ signals. Based on recent observations, we propose that the Ca2+ crosstalk between mitochondria, endoplasmic reticulum and cytoplasm may form a proportional–integral–derivative (PID) controller. This PID mechanism (which is well known in engineering) could be responsible for computing cellular decisions. In addition, we point out the importance of analogue and digital signal processing in T cell life and implication of mitochondrial Ca2+ transport in this process. PMID:27852805

  17. Xenogenic transfer of isolated murine mitochondria into human rho0 cells can improve respiratory function.

    PubMed

    Katrangi, Eyad; D'Souza, Gerard; Boddapati, Sarathi V; Kulawiec, Mariola; Singh, Keshav K; Bigger, Brian; Weissig, Volkmar

    2007-12-01

    Mitochondrial DNA mutations are the direct cause of several physiological disorders and are also associated with the aging process. The modest progress made over the past two decades towards manipulating the mitochondrial genome and understanding its function within living mammalian cells means that cures for mitochondrial DNA mutations are still elusive. Here, we report that transformed mammalian cells internalize exogenous isolated mitochondria upon simple co-incubation. We first demonstrate the physical presence of internalized mitochondria within recipient cells using fluorescence microscopy. Second, we show that xenogenic transfer of murine mitochondria into human cells lacking functional mitochondria can functionally restore respiration in cells lacking mtDNA. Third, utilizing the natural competence of isolated mitochondria to take up linear DNA molecules, we demonstrate the feasibility of using cellular internalization of isolated exogenous mitochondria as a potential tool for studying mitochondrial genetics in living mammalian cells.

  18. The oncolytic peptide LTX-315 induces cell death and DAMP release by mitochondria distortion in human melanoma cells

    PubMed Central

    Eike, Liv-Marie; Yang, Nannan; Rekdal, Øystein; Sveinbjørnsson, Baldur

    2015-01-01

    Host defense peptides (HDPs) are naturally occurring molecules found in most species, in which they play a significant role in the first line defense against intruding pathogens, and several HDPs have been shown to possess anticancer activity. Structure-activity relationship studies on the HDP bovine lactoferricin revealed a de novo design of a nonamer peptide LTX-315, with oncolytic properties. In the present study, we investigated the oncolytic activity of LTX-315 in human melanoma cells (A375). LTX-315 induced a rapid plasma membrane disruption and cell death within 2 hours. At a low concentration, fluorescence-labeled LTX-315 was internalized and accumulated in cytoplasmic vacuoles in close proximity to the mitochondria. The mitochondrial membrane potential was shown to depolarize as a consequence of LTX-315 treatment and at ultrastructural level, the mitochondria morphology was significantly altered. Release of danger signals (DAMPs) such as ATP, Cytochrome C and HMGB1 into the cell supernatant of cultured cells was evident minutes after peptide treatment. The oncolytic effect of LTX-315 involving perturbation of both the cell membrane and the mitochondria with subsequent release of DAMPs may highlight the ability of LTX-315 to induce complete regression and long-term protective immune responses as previously reported in experimental animal models. PMID:26472184

  19. Mitochondria, Chloroplasts in Animal and Plant Cells: Significance of Conformational Matching.

    PubMed

    Stefano, George B; Snyder, Christopher; Kream, Richard M

    2015-07-17

    Many commonalities between chloroplasts and mitochondria exist, thereby suggesting a common origin via a bacterial ancestor capable of enhanced ATP-dependent energy production functionally linked to cellular respiration and photosynthesis. Accordingly, the molecular evolution/retention of the catalytic Qo quinol oxidation site of cytochrome b complexes as the tetrapeptide PEWY sequence functionally underlies the common retention of a chemiosmotic proton gradient mechanism for ATP synthesis in cellular respiration and photosynthesis. Furthermore, the dual regulatory targeting of mitochondrial and chloroplast gene expression by mitochondrial transcription termination factor (MTERF) proteins to promote optimal energy production and oxygen consumption further advances these evolutionary contentions. As a functional consequence of enhanced oxygen utilization and production, significant levels of reactive oxygen species (ROS) may be generated within mitochondria and chloroplasts, which may effectively compromise cellular energy production following prolonged stress/inflammationary conditions. Interestingly, both types of organelles have been identified in selected animal cells, most notably specialized digestive cells lining the gut of several species of Sacoglossan sea slugs. Termed kleptoplasty or kleptoplastic endosymbiosis, functional chloroplasts from algal food sources are internalized and stored within digestive cells to provide the host with dual energy sources derived from mitochondrial and photosynthetic processes. Recently, the observation of internalized algae within embryonic tissues of the spotted salamander strongly suggest that developmental processes within a vertebrate organism may require photosynthetic endosymbiosis as an internal regulator. The dual presence of mitochondria and functional chloroplasts within specialized animal cells indicates a high degree of biochemical identity, stereoselectivity, and conformational matching that are the likely

  20. Dysfunction of mitochondria Ca2+ uptake in cystic fibrosis airway epithelial cells.

    PubMed

    Antigny, Fabrice; Girardin, Nathalie; Raveau, Dorothée; Frieden, Maud; Becq, Frédéric; Vandebrouck, Clarisse

    2009-07-01

    In the genetic disease cystic fibrosis (CF), the most common mutation F508del promotes the endoplasmic reticulum (ER) retention of misfolded CF proteins. Furthermore, in homozygous F508del-CFTR airway epithelial cells, the histamine Ca(2+) mobilization is abnormally increased. Because the uptake of Ca(2+) by mitochondria during Ca(2+) influx or Ca(2+) release from ER stores may be crucial for maintaining a normal Ca(2+) homeostasis, we compared the mitochondria morphology and distribution by transmission electron microscopy technique and the mitochondria membrane potential variation (DeltaPsi(mit)) using a fluorescent probe (TMRE) on human CF (CF-KM4) and non-CF (MM39) tracheal serous gland cell lines. Confocal imaging of Rhod-2-AM-loaded or of the mitochondrial targeted cameleon 4mtD3cpv-transfected human CF and non-CF cells, were used to examine the ability of mitochondria to sequester intracellular Ca(2+). The present study reveals that (i) the mitochondria network is fragmented in F508del-CFTR cells, (ii) the DeltaPsi(mit) of CF mitochondria is depolarized compared non-CF mitochondria, and (iii) the CF mitochondria Ca(2+) uptake is reduced compared non-CF cells. We propose that these defects in airway epithelial F508del-CFTR cells are the consequence of mitochondrial membrane depolarization leading to a deficient mitochondrial Ca(2+) uptake.

  1. Involvement of S6K1 in mitochondria function and structure in HeLa cells.

    PubMed

    Park, Jisoo; Tran, Quangdon; Mun, Kisun; Masuda, Kouhei; Kwon, So Hee; Kim, Seon-Hwan; Kim, Dong-Hoon; Thomas, George; Park, Jongsun

    2016-12-01

    The major biological function of mitochondria is to generate cellular energy through oxidative phosphorylation. Apart from cellular respiration, mitochondria also play a key role in signaling processes, including aging and cancer metabolism. It has been shown that S6K1-knockout mice are resistant to obesity due to enhanced beta-oxidation, with an increased number of large mitochondria. Therefore, in this report, the possible involvement of S6K1 in regulating mitochondria dynamics and function has been investigated in stable lenti-shS6K1-HeLa cells. Interestingly, S6K1-stably depleted HeLa cells showed phenotypical changes in mitochondria morphology. This observation was further confirmed by detailed image analysis of mitochondria shape. Corresponding molecular changes were also observed in these cells, such as the induction of mitochondrial fission proteins (Drp1 and Fis1). Oxygen consumption is elevated in S6K1-depeleted HeLa cells and FL5.12 cells. In addition, S6K1 depletion leads to enhancement of ATP production in cytoplasm and mitochondria. However, the relative ratio of mitochondrial ATP to cytoplasmic ATP is actually decreased in lenti-shS6K1-HeLa cells compared to control cells. Lastly, induction of mitophagy was found in lenti-shS6K1-HeLa cells with corresponding changes of mitochondria shape on electron microscope analysis. Taken together, our results indicate that S6K1 is involved in the regulation of mitochondria morphology and function in HeLa cells. This study will provide novel insights into S6K1 function in mitochondria-mediated cellular signaling.

  2. A Highly Photostable Hyperbranched Polyglycerol-Based NIR Fluorescence Nanoplatform for Mitochondria-Specific Cell Imaging.

    PubMed

    Dong, Chunhong; Liu, Zhongyun; Liu, Junqing; Wu, Changzhu; Neumann, Falko; Wang, Hanjie; Schäfer-Korting, Monika; Kleuser, Burkhard; Chang, Jin; Li, Wenzhong; Ma, Nan; Haag, Rainer

    2016-09-01

    Considering the critical role of mitochondria in the life and death of cells, non-invasive long-term tracking of mitochondria has attracted considerable interest. However, a high-performance mitochondria-specific labeling probe with high photostability is still lacking. Herein a highly photostable hyperbranched polyglycerol (hPG)-based near-infrared (NIR) quantum dots (QDs) nanoplatform is reported for mitochondria-specific cell imaging. Comprising NIR Zn-Cu-In-S/ZnS QDs as extremely photostable fluorescent labels and alkyl chain (C12 )/triphenylphosphonium (TPP)-functionalized hPG derivatives as protective shell, the tailored QDs@hPG-C12 /TPP nanoprobe with a hydrodynamic diameter of about 65 nm exhibits NIR fluorescence, excellent biocompatibility, good stability, and mitochondria-targeted ability. Cell uptake experiments demonstrate that QDs@hPG-C12 /TPP displays a significantly enhanced uptake in HeLa cells compared to nontargeted QDs@hPG-C12 . Further co-localization study indicates that the probe selectively targets mitochondria. Importantly, compared with commercial deep-red mitochondria dyes, QDs@hPG-C12 /TPP possesses superior photostability under continuous laser irradiation, indicating great potential for long-term mitochondria labeling and tracking. Moreover, drug-loaded QDs@hPG-C12 /TPP display an enhanced tumor cell killing efficacy compared to nontargeted drugs. This work could open the door to the construction of organelle-targeted multifunctional nanoplatforms for precise diagnosis and high-efficient tumor therapy.

  3. Non-invasive radiofrequency treatment effect on mitochondria in pancreatic cancer cells

    PubMed Central

    Curley, Steven A.; Palalon, Flavio; Lu, Xiaolin; Koshkina, Nadezhda V.

    2015-01-01

    Background Development of novel therapeutic approaches for cancer therapy is important, especially for tumors that have poor response or develop resistance to standard chemotherapy and radiation. We discovered that non-invasive radiofrequency (RF) fields can affect cancer, but not normal cells, inhibit progression of tumors in mice, and enhance anticancer effect of chemotherapy. However, it remains unclear what physiological and molecular mechanisms this treatment induces inside cells. Here, we studied the effect of RF treatment on mitochondria in human pancreatic cancer cells. Methods Morphology of mitochondria in cells was studied by electron microscopy. Alteration of mitochondrial membrane potential (Δψ) was accessed with Mitotracker probe. Respiratory activity of mitochondria was evaluated by changes in oxygen consumption rates (OCR) determined with MitoStress kit. Production of intracellular reactive oxygen species (ROS) was performed using flow cytometry. Colocalization of mitochondria and autophagosome markers in cells was done by fluorescence immunostaining and confocal microscopy analysis. Results RF changed morphology of mitochondria in cancer cells, altered polarization of the mitochondrial membrane, substantially impaired mitochondrial respiration, and increased ROS production, which indicate on the RF-induced stress on mitochondria. We also observed frequent colocalization of the autophagosome marker LC3B with the mitochondrial marker Tom20 inside cancer cells after RF exposure indicating on the presence of mitochondria in the autophagosomes. This suggests that RF-induced stress can damage mitochondria and induce elimination of damaged organelle via autophagy. Conclusion RF treatment impaired the function of mitochondria in cancer cells. Therefore, mitochondria can represent one of the targets of the RF treatment. PMID:24986120

  4. Spaceflight and clinorotation cause cytoskeleton and mitochondria changes and increases in apoptosis in cultured cells

    NASA Astrophysics Data System (ADS)

    Schatten, Heide; Lewis, Marian L.; Chakrabarti, Amitabha

    2001-08-01

    The cytoskeleton is a complex network of fibers that is sensitive to environmental factors including microgravity and altered gravitational forces. Cellular functions such as transport of cell organelles depend on cytoskeletal integrity; regulation of cytoskeletal activity plays a role in cell maintenance, cell division, and apoptosis. Here we report cytoskeletal and mitochondria alterations in cultured human lymphocyte (Jurkat) cells after exposure to spaceflight and in insect cells of Drosophila melanogaster (Schneider S-1) after exposure to conditions created by clinostat rotation. Jurkat cells were flown on the space shuttle in Biorack cassettes while Schneider S-1 cells were exposed to altered gravity forces as produced by clinostat rotation. The effects of both treatments were similar in the different cell types. Fifty percent of cells displayed effects on the microtubule network in both cell lines. Under these experimental conditions mitochondria clustering and morphological alterations of mitochondrial cristae was observed to various degrees after 4 and 48 hours of culture. Jurkat cells underwent cell divisions during exposure to spaceflight but a large number of apoptotic cells was also observed. Similar results were obtained in Schneider S-1 cells cultured under clinostat rotation. Both cell lines displayed mitochondria abnormalities and mitochondria clustering toward one side of the cells which is interpreted to be the result of microtubule disruption and failure of mitochondria transport along microtubules. The number of mitochondria was increased in cells exposed to altered gravity while cristae morphology was severely affected indicating altered mitochondria function. These results show that spaceflight as well as altered gravity produced by clinostat rotation affects microtubule and mitochondria organization and results in increases in apoptosis.

  5. Spaceflight and clinorotation cause cytoskeleton and mitochondria changes and increases in apoptosis in cultured cells

    NASA Technical Reports Server (NTRS)

    Schatten, H.; Lewis, M. L.; Chakrabarti, A.

    2001-01-01

    The cytoskeleton is a complex network of fibers that is sensitive to environmental factors including microgravity and altered gravitational forces. Cellular functions such as transport of cell organelles depend on cytoskeletal integrity; regulation of cytoskeletal activity plays a role in cell maintenance, cell division, and apoptosis. Here we report cytoskeletal and mitochondria alterations in cultured human lymphocyte (Jurkat) cells after exposure to spaceflight and in insect cells of Drosophila melanogaster (Schneider S-1) after exposure to conditions created by clinostat rotation. Jurkat cells were flown on the space shuttle in Biorack cassettes while Schneider S-1 cells were exposed to altered gravity forces as produced by clinostat rotation. The effects of both treatments were similar in the different cell types. Fifty percent of cells displayed effects on the microtubule network in both cell lines. Under these experimental conditions mitochondria clustering and morphological alterations of mitochondrial cristae was observed to various degrees after 4 and 48 hours of culture. Jurkat cells underwent cell divisions during exposure to spaceflight but a large number of apoptotic cells was also observed. Similar results were obtained in Schneider S-1 cells cultured under clinostat rotation. Both cell lines displayed mitochondria abnormalities and mitochondria clustering toward one side of the cells which is interpreted to be the result of microtubule disruption and failure of mitochondria transport along microtubules. The number of mitochondria was increased in cells exposed to altered gravity while cristae morphology was severely affected indicating altered mitochondria function. These results show that spaceflight as well as altered gravity produced by clinostat rotation affects microtubule and mitochondria organization and results in increases in apoptosis. Grant numbers: NAG 10-0224, NAG2-985. c 2001. Elsevier Science Ltd. All rights reserved.

  6. Role of mitochondria-associated hexokinase II in cancer cell death induced by 3-bromopyruvate.

    PubMed

    Chen, Zhao; Zhang, Hui; Lu, Weiqin; Huang, Peng

    2009-05-01

    It has long been observed that cancer cells rely more on glycolysis to generate ATP and actively use certain glycolytic metabolic intermediates for biosynthesis. Hexokinase II (HKII) is a key glycolytic enzyme that plays a role in the regulation of the mitochondria-initiated apoptotic cell death. As a potent inhibitor of hexokinase, 3-bromopyruvate (3-BrPA) is known to inhibit cancer cell energy metabolism and trigger cell death, supposedly through depletion of cellular ATP. The current study showed that 3-BrPA caused a covalent modification of HKII protein and directly triggered its dissociation from mitochondria, leading to a specific release of apoptosis-inducing factor (AIF) from the mitochondria to cytosol and eventual cell death. Co-immunoprecipitation revealed a physical interaction between HKII and AIF. Using a competitive peptide of HKII, we showed that the dissociation of hexokinase II from mitochondria alone could cause apoptotic cell death, especially in the mitochondria-deficient rho(0) cells that highly express HKII. Interestingly, the dissociation of HKII itself did not directly affect the mitochondrial membrane potential, ROS generation, and oxidative phosphorylation. Our study suggests that the physical association between HKII and AIF is important for the normal localization of AIF in the mitochondria, and disruption of this protein complex by 3-BrPA leads to their release from the mitochondria and eventual cell death.

  7. Hexokinase II–derived cell-penetrating peptide targets mitochondria and triggers apoptosis in cancer cells

    PubMed Central

    Woldetsadik, Abiy D.; Vogel, Maria C.; Rabeh, Wael M.; Magzoub, Mazin

    2017-01-01

    Overexpression of mitochondria-bound hexokinase II (HKII) in cancer cells plays an important role in their metabolic reprogramming and protects them against apoptosis, thereby facilitating their growth and proliferation. Here, we show that covalently coupling a peptide corresponding to the mitochondrial membrane–binding N-terminal 15 aa of HKII (pHK) to a short, penetration-accelerating sequence (PAS) enhances the cellular uptake, mitochondrial localization, and cytotoxicity of the peptide in HeLa cells. Further analysis revealed that pHK-PAS depolarized mitochondrial membrane potential, inhibited mitochondrial respiration and glycolysis, and depleted intracellular ATP levels. The effects of pHK-PAS were correlated with dissociation of endogenous full-length HKII from mitochondria and release of cytochrome c. Of significance, pHK-PAS treatment of noncancerous HEK293 cells resulted in substantially lower cytotoxicity. Thus, pHK-PAS effectively disrupted the mitochondria-HKII association in cancer cells, which led to mitochondrial dysfunction and, finally, apoptosis. Our results demonstrate the potential of the pHK-PAS cell-penetrating peptide as a novel therapeutic strategy in cancer.—Woldetsadik, A. D., Vogel, M. C., Rabeh, W. M., Magzoub, M. Hexokinase II–derived cell-penetrating peptide targets mitochondria and triggers apoptosis in cancer cells.

  8. Aquatic viruses induce host cell death pathways and its application.

    PubMed

    Reshi, Latif; Wu, Jen-Leih; Wang, Hao-Ven; Hong, Jiann-Ruey

    2016-01-04

    Virus infections of mammalian and animal cells consist of a series of events. As intracellular parasites, viruses rely on the use of host cellular machinery. Through the use of cell culture and molecular approaches over the past decade, our knowledge of the biology of aquatic viruses has grown exponentially. The increase in aquaculture operations worldwide has provided new approaches for the transmission of aquatic viruses that include RNA and DNA viruses. Therefore, the struggle between the virus and the host for control of the cell's death machinery is crucial for survival. Viruses are obligatory intracellular parasites and, as such, must modulate apoptotic pathways to control the lifespan of their host to complete their replication cycle. This paper updates the discussion on the detailed mechanisms of action that various aquatic viruses use to induce cell death pathways in the host, such as Bad-mediated, mitochondria-mediated, ROS-mediated and Fas-mediated cell death circuits. Understanding how viruses exploit the apoptotic pathways of their hosts may provide great opportunities for the development of future potential therapeutic strategies and pathogenic insights into different aquatic viral diseases.

  9. [Evolution of mitochondria].

    PubMed

    Litoshenko, A Ia

    2002-01-01

    Until recently, the origin and evolution of mitochondria was explained by the serial endosymbiosis hypothesis. This hypothesis posits that contemporary mitochondria are the direct descendants of a bacterial endosymbiont, which was settled in a nucleus-containing amitochondriate host cell. Results of the mitochondrial gene sequences support a monophyletic origin of the mitochondria from a single eubacterial ancestor shared with a subdivision of the alpha-proteobacteria. In recent years, the complete sequences of the vast variety of mitochondrial and eubacterial genomes were determined. These data indicate that the mitochondrial genome evolved from a common ancestor of all extant eukaryotes and assume a possibility that the mitochondrial and nuclear constituents of the eukaryotic cell originated simultaneously.

  10. Redox homeostasis protects mitochondria through accelerating ROS conversion to enhance hypoxia resistance in cancer cells.

    PubMed

    Li, Pengying; Zhang, Dongyang; Shen, Lingxiao; Dong, Kelei; Wu, Meiling; Ou, Zhouluo; Shi, Dongyun

    2016-03-09

    Mitochondria are the powerhouses of eukaryotic cells and the main source of reactive oxygen species (ROS) in hypoxic cells, participating in regulating redox homeostasis. The mechanism of tumor hypoxia tolerance, especially the role of mitochondria in tumor hypoxia resistance remains largely unknown. This study aimed to explore the role of mitochondria in tumor hypoxia resistance. We observed that glycolysis in hypoxic cancer cells was up-regulated more rapidly, with far lesser attenuation in aerobic oxidation, thus contributing to a more stable ATP/ADP ratio. In hypoxia, cancer cells rapidly convert hypoxia-induced O(2˙)(-) into H2O2. H2O2 is further decomposed by a relatively stronger antioxidant system, causing ROS levels to increase lesser compared to normal cells. The moderate ROS leads to an appropriate degree of autophagy, eliminating the damaged mitochondria and offering nutrients to promote mitochondria fusion, thus protects mitochondria and improves hypoxia tolerance in cancer. The functional mitochondria could enable tumor cells to flexibly switch between glycolysis and oxidative phosphorylation to meet the different physiological requirements during the hypoxia/re-oxygenation cycling of tumor growth.

  11. Redox homeostasis protects mitochondria through accelerating ROS conversion to enhance hypoxia resistance in cancer cells

    PubMed Central

    Li, Pengying; Zhang, Dongyang; Shen, Lingxiao; Dong, Kelei; Wu, Meiling; Ou, Zhouluo; Shi, Dongyun

    2016-01-01

    Mitochondria are the powerhouses of eukaryotic cells and the main source of reactive oxygen species (ROS) in hypoxic cells, participating in regulating redox homeostasis. The mechanism of tumor hypoxia tolerance, especially the role of mitochondria in tumor hypoxia resistance remains largely unknown. This study aimed to explore the role of mitochondria in tumor hypoxia resistance. We observed that glycolysis in hypoxic cancer cells was up-regulated more rapidly, with far lesser attenuation in aerobic oxidation, thus contributing to a more stable ATP/ADP ratio. In hypoxia, cancer cells rapidly convert hypoxia-induced O2·− into H2O2. H2O2 is further decomposed by a relatively stronger antioxidant system, causing ROS levels to increase lesser compared to normal cells. The moderate ROS leads to an appropriate degree of autophagy, eliminating the damaged mitochondria and offering nutrients to promote mitochondria fusion, thus protects mitochondria and improves hypoxia tolerance in cancer. The functional mitochondria could enable tumor cells to flexibly switch between glycolysis and oxidative phosphorylation to meet the different physiological requirements during the hypoxia/re-oxygenation cycling of tumor growth. PMID:26956544

  12. Modified host cells with efflux pumps

    DOEpatents

    Dunlop, Mary J.; Keasling, Jay D.; Mukhopadhyay, Aindrila

    2016-08-30

    The present invention provides for a modified host cell comprising a heterologous expression of an efflux pump capable of transporting an organic molecule out of the host cell wherein the organic molecule at a sufficiently high concentration reduces the growth rate of or is lethal to the host cell.

  13. A novel bifunctional mitochondria-targeted anticancer agent with high selectivity for cancer cells

    PubMed Central

    He, Huan; Li, Dong-Wei; Yang, Li-Yun; Fu, Li; Zhu, Xun-Jin; Wong, Wai-Kwok; Jiang, Feng-Lei; Liu, Yi

    2015-01-01

    Mitochondria have recently emerged as novel targets for cancer therapy due to its important roles in fundamental cellular function. Discovery of new chemotherapeutic agents that allow for simultaneous treatment and visualization of cancer is urgent. Herein, we demonstrate a novel bifunctional mitochondria-targeted anticancer agent (FPB), exhibiting both imaging capability and anticancer activity. It can selectively accumulate in mitochondria and induce cell apoptosis. Notably, it results in much higher toxicity toward cancer cells owing to much higher uptake by cancer cells. These features make it highly attractive in cancer imaging and treatment. PMID:26337336

  14. Morphological and biochemical characterization of mitochondria in Torpedo red blood cells.

    PubMed

    Pica, A; Scacco, S; Papa, F; De Nitto, E; Papa, S

    2001-02-01

    A study is presented on the morphology and respiratory functions of mitochondria from Torpedo marmorata red blood cells. In vivo staining of red blood cells and transmission electron microscopy showed the existence of a considerable number of vital and orthodox mitochondria which decreased from young erythroblasts to mature erythrocytes from 60-50 to 30-20 per cell. In erythrocytes mitochondria exhibited a canonical, functional respiratory chain. The content and activity of cytochromes in erythrocytes were, however, significantly lower as compared to mammalian tissues.

  15. Mitochondrial transit peptide exhibits cell penetration ability and efficiently delivers macromolecules to mitochondria.

    PubMed

    Jain, Aastha; Chugh, Archana

    2016-09-01

    Mitochondrial malfunction under various circumstances can lead to a variety of disorders. Effective targeting of macromolecules (drugs) is important for restoration of mitochondrial function and treatment of related disorders. We have designed a novel cell-penetrating mitochondrial transit peptide (CpMTP) for delivery of macromolecules to mitochondria. Comparison between properties of cell-penetrating peptides (CPPs) and mitochondrial signal sequences enabled prediction of peptides with dual ability for cellular translocation and mitochondrial localization. Among the predicted peptides, CpMTP translocates across HeLa cells and shows successful delivery of noncovalently conjugated cargo molecules to mitochondria. CpMTP may have applications in transduction and transfection of mitochondria for therapeutics.

  16. Mitochondria Are Gate-keepers of T Cell Function by Producing the ATP That Drives Purinergic Signaling*

    PubMed Central

    Ledderose, Carola; Bao, Yi; Lidicky, Markus; Zipperle, Johannes; Li, Linglin; Strasser, Katharina; Shapiro, Nathan I.; Junger, Wolfgang G.

    2014-01-01

    T cells play a central role in host defense. ATP release and autocrine feedback via purinergic receptors has been shown to regulate T cell function. However, the sources of the ATP that drives this process are not known. We found that stimulation of T cells triggers a spike in cellular ATP production that doubles intracellular ATP levels in <30 s and causes prolonged ATP release into the extracellular space. Cell stimulation triggered rapid mitochondrial Ca2+ uptake, increased oxidative phosphorylation, a drop in mitochondrial membrane potential (Δψm), and the accumulation of active mitochondria at the immune synapse of stimulated T cells. Inhibition of mitochondria with CCCP, KCN, or rotenone blocked intracellular ATP production, ATP release, intracellular Ca2+ signaling, induction of the early activation marker CD69, and IL-2 transcription in response to cell stimulation. These findings demonstrate that rapid activation of mitochondrial ATP production fuels the purinergic signaling mechanisms that regulate T cells and define their role in host defense. PMID:25070895

  17. Branchial mitochondria-rich cells in the dogfish Squalus acanthias.

    PubMed

    Wilson, Jonathan M; Morgan, John D; Vogl, A Wayne; Randall, David J

    2002-06-01

    In marine teleost fishes, the gill mitochondria-rich cells (MRCs) are responsible for NaCl elimination; however, in elasmobranch fishes, the specialized rectal gland is considered to be the most important site for salt secretion. The role of the gills in elasmobranch ion regulation, although clearly shown to be secondary, is not well characterized. In the present study, we investigated some morphological properties of the branchial MRCs and the localization, and activity of the important ionoregulatory enzyme Na(+)/K(+)-ATPase, under control conditions and following rectal gland removal (1 month) in the spiny dogfish, Squalus acanthias. A clear correlation can be made between MRC numbers and the levels of Na(+)/K(+)-ATPase activity in crude gill homogenates (r(2)=-0.69). Strong Na(+)/K(+)-ATPase immunoreactivity is also clearly associated with the basolateral membrane of these MRCs. In addition, the dogfish were able to maintain ionic balance after rectal gland removal. These results all suggest a possible role of the dogfish gill in salt secretion. MRCs were, however, unresponsive to rectal gland removal in terms of changes in number, fine structure and Na(+)/K(+)-ATPase activity, as might be expected if they were compensating for the loss of salt secretion by the rectal gland. Thus, the specific role that these MRCs play in ion regulation in the dogfish remains to be determined

  18. Porcine parvovirus infection induces apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated pathway

    SciTech Connect

    Zhang, Hongling; Huang, Yong; Du, Qian; Luo, Xiaomao; Zhang, Liang; Zhao, Xiaomin; Tong, Dewen

    2015-01-09

    Highlights: • PPV reduces PK-15 cells viability by inducing apoptosis. • PPV infection induces apoptosis through mitochondria-mediated pathway. • PPV infection activates p53 to regulate the mitochondria apoptotic signaling. - Abstract: Porcine parvovirus (PPV) infection has been reported to induce the cytopathic effects (CPE) in some special host cells and contribute the occurrence of porcine parvovirus disease, but the molecular mechanisms underlying PPV-induced CPE are not clear. In this study, we investigated the morphological and molecular changes of porcine kidney cell line (PK-15 cells) infected with PPV. The results showed that PPV infection inhibited the viability of PK-15 cells in a time and concentration dependent manner. PPV infection induced typical apoptotic features including chromatin condensation, apoptotic body formation, nuclear fragmentation, and Annexin V-binding activity. Further studies showed that Bax was increased and translocated to mitochondria, whereas Bcl-2 was decreased in PPV-infected cells, which caused mitochondrial outer-membrane permeabilization, resulting in the release of mitochondrial cytochrome c, followed by caspase-9 and caspase-3 activation. However, the expression of Fas and Fas ligand (FasL) did not appear significant changes in the process of PPV-induced apoptosis. Moreover, PPV infection activated p53 signaling, which was involved in the activation of apoptotic signaling induced by PPV infection via regulation of Bax and Bcl-2. Taken together, our results demonstrated that PPV infection induced apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated apoptosis pathway. This study may contribute to shed light on the molecular pathogenesis of PPV infection.

  19. Mitochondrial peroxiredoxin-5 as potential modulator of mitochondria-ER crosstalk in MPP+-induced cell death.

    PubMed

    De Simoni, Stéphanie; Linard, Dominique; Hermans, Emmanuel; Knoops, Bernard; Goemaere, Julie

    2013-05-01

    Peroxiredoxin-5 (PRDX5) is an antioxidant enzyme which differs from the other peroxiredoxins with regards to its enzymatic mechanism, its high affinity for organic peroxides and peroxynitrite and its wide subcellular distribution. In particular, the mitochondrial isoform of PRDX5 confers a remarkable cytoprotection toward oxidative stress to mammalian cells. Mitochondrial dysfunction and disruption of Ca²⁺ homeostasis are implicated in neurodegeneration. Growing evidence supports that endoplasmic reticulum (ER) could operate in tandem with mitochondria to regulate intracellular Ca²⁺ fluxes in neurodegenerative processes. Here, we overexpressed mitochondrial PRDX5 in SH-SY5Y cells to dissect the role of this enzyme in 1-methyl-4-phenylpyridinium (MPP)⁺-induced cell death. Our data show that mitochondria-dependent apoptosis triggered by MPP⁺, assessed by the measurement of caspase-9 activation and mitochondrial DNA damage, is prevented by mitochondrial PRDX5 overexpression. Moreover, PRDX5 overexpression blocks the increase in intracellular Ca²⁺, Ca²⁺-dependent activation of calpains and Bax cleavage. Finally, using Ca²⁺ channel inhibitors (Nimodipine, Dantrolene and 2-APB), we show that Ca²⁺ release arises essentially from ER stores through 1,4,5-inositol-trisphosphate receptors (IP3 R). Altogether, our results suggest that the MPP⁺ mitochondrial pathway of apoptosis is regulated by mitochondrial PRDX5 in a process that could involve redox modulation of Ca²⁺ transporters via a crosstalk between mitochondria and ER.

  20. Mitochondria: a sulfhydryl oxidase and fission GTPase connect mitochondrial dynamics with pluripotency in embryonic stem cells.

    PubMed

    Wilkerson, Donald C; Sankar, Uma

    2011-09-01

    Mitochondria have long been recognized as cellular energy power houses that also regulate cellular redox signaling to arbitrate cell survival. Recent studies of mitochondria in stem cells (SCs) demonstrate that they have critical roles beyond this traditional view. Embryonic (E) SCs, termed pluripotent for their ability to differentiate into all cell types within an organism, maintain a limited number of morphologically undifferentiated (electron translucent and poorly formed cristae) mitochondria. As these cells differentiate, their mitochondria undergo a tightly choreographed gain of number, mass and morphological complexity. Therefore, mechanisms that regulate mitochondrial growth, localization, division and partition must play active roles in the maintenance of pluripotency and execution of differentiation. Aberrant mitochondrial dynamics are associated with a plethora of human disorders, for which SCs hold curative potential. Hence, a comprehensive understanding of the mechanisms that regulate mitochondrial dynamics and function in SCs and their overall relationship to the maintenance of pluripotency is pivotal for the progression of therapeutic regenerative medicine.

  1. Biophysical Investigation of the Ironome of Human Jurkat Cells and Mitochondria

    PubMed Central

    Jhurry, Nema D.; Chakrabarti, Mrinmoy; McCormick, Sean P.; Holmes-Hampton, Gregory P.; Lindahl, Paul A.

    2012-01-01

    The speciation of iron in intact human Jurkat leukemic cells and their isolated mitochondria was assessed using biophysical methods. Large-scale cultures were grown in medium enriched with 57Fe citrate. Mitochondria were isolated anaerobically to prevent oxidation of iron centers. 5 K Mössbauer spectra of cells were dominated by a sextet due to ferritin. They also exhibited an intense central quadrupole doublet due to S = 0 [Fe4S4]2+ clusters and low-spin (LS) FeII heme centers. Spectra of isolated mitochondria were largely devoid of ferritin but contained the central doublet and features arising from what appear to be FeIII oxyhydroxide (phosphate) nanoparticles. Spectra from both cells and mitochondria contained a low-intensity doublet from non-heme high-spin (NHHS) FeII species. A portion of these species may constitute the ‘labile iron pool’ (LIP) proposed in cellular Fe trafficking. Such species might engage in Fenton chemistry to generate reactive oxygen species. EPR spectra of cells and mitochondria exhibited signals from reduced Fe/S clusters, and HS FeIII heme and non-heme species. The basal redox state of mitochondria within cells is reduced as monitored by heme redox states; this redox poise is unaltered during the anaerobic isolation of the organelle. Contributions from heme a, b and c centers were quantified using electronic absorption spectroscopy. Metal concentrations in cells and mitochondria were measured using ICP-MS. Results were collectively assessed to estimate the concentrations of various Fe-containing species in mitochondria and whole cells – the first “ironome” profile of a human cell. PMID:22726227

  2. Whole cell cryo-electron tomography suggests mitochondria divide by budding.

    PubMed

    Hu, Guo-Bin

    2014-08-01

    Eukaryotes rely on mitochondrial division to guarantee that each new generation of cells acquires an adequate number of mitochondria. Mitochondrial division has long been thought to occur by binary fission and, more recently, evidence has supported the idea that binary fission is mediated by dynamin-related protein (Drp1) and the endoplasmic reticulum. However, studies to date have depended on fluorescence microscopy and conventional electron microscopy. Here, we utilize whole cell cryo-electron tomography to visualize mitochondrial division in frozen hydrated intact HeLa cells. We observe a large number of relatively small mitochondria protruding from and connected to large mitochondria or mitochondrial networks. Therefore, this study provides evidence that mitochondria divide by budding.

  3. Effects of granulosa cell mitochondria transfer on the early development of bovine embryos in vitro.

    PubMed

    Hua, Song; Zhang, Yong; Li, Xiang-Chen; Ma, Li-Bing; Cao, Jun-Wei; Dai, Jin-Po; Li, Rong

    2007-01-01

    The objective of this study was to determine the effect of exogenous mitochondria obtained from granulosa cells on the development of bovine embryos in vitro. We classified cumulus oocyte complexes (COCs) as good (G)- and poor (P)-quality oocytes based on cytoplasmic appearance and cumulus characteristics, and assessed mtDNA copy numbers in the G and P oocytes with real-time polymerase chain reaction (PCR). The mitochondria were isolated by fractionation and suspended in mitochondria injection buffer (MIB). Part one of the experiment consisted of the following treatments: (1) G-oocytes + sperm, (2) P-oocytes + mitochondria + MIB + sperm, (3) P-oocytes + MIB + sperm, and (4) P-oocytes + sperm. In part 2, oocytes were parthenogenetically activated. The treatments were: (1) G-oocytes, (2) P-oocytes + mitochondria + MIB, (3) P-oocytes + MIB, and (4) P-oocytes alone. The results indicated a significant difference in mtDNA copy number between G (361 113 +/- 147 114) and P (198 293 +/- 174 178) oocytes (p < 0.01). The rates of morula, blastocyst, and hatched blastocysts derived from P-oocytes + mitochondria were similar to those of G-oocytes, but significantly higher than P-oocytes without exogenous mitochondria in both the ICSI and parthenogenetic activation experiments. We found no difference in blastomere numbers between G-oocytes and P-oocytes + mitochondria in either experiment, but blastomere numbers in these two groups were significantly higher than in P-oocyte groups without exogenous mitochondria. These data suggest that mtDNA content is very important for early embryo development. Furthermore, the transfer of mitochondria from the same breed may improve embryo quality during preimplantation development.

  4. RNA metabolism in plant mitochondria.

    PubMed

    Hammani, Kamel; Giegé, Philippe

    2014-06-01

    Mitochondria are essential for the eukaryotic cell and are derived from the endosymbiosis of an α-proteobacterial ancestor. Compared to other eukaryotes, RNA metabolism in plant mitochondria is complex and combines bacterial-like traits with novel features that evolved in the host cell. These complex RNA processes are regulated by families of nucleus-encoded RNA-binding proteins. Transcription is particularly relaxed and is initiated from multiple promoters covering the entire genome. The variety of RNA precursors accumulating in mitochondria highlights the importance of post-transcriptional processes to determine the size and abundance of transcripts. Here we review RNA metabolism in plant mitochondria, from RNA transcription to translation, with a special focus on their unique features that are controlled by trans-factors.

  5. Symbiotic bacteria in oocyte and ovarian cell mitochondria of the tick Ixodes ricinus: biology and phylogenetic position.

    PubMed

    Rymaszewska, Anna

    2007-04-01

    Under natural conditions, eukaryote cells may contain bacteria. Arthropods such as ticks, insects or mites are a group particularly favoured by the obligate intracellular bacteria. While arthropods are vectors for some of them, other bacteria inhabit invertebrate host cells having entered mutualistic interactions. Such endosymbionts dwell usually in the host cell vacuoles or cytoplasm but have been also reported from tick oocyte mitochondria. The microorganisms contribute to mitochondria degradation, but their colonies are not eliminated from the tick cells affected. So far, such bacteria have been detected in three research centres. The Italian centre has reported on results of microscope and molecular analyses, while the Polish centre published molecular data. The Danish centre registered a 16S rRNA gene fragment in GenBank. Independent comparisons of the 16S rRNA gene sequences, carried out in the Italian and Polish centres, confirmed that the nucleotide sequences of the Ixodes ricinus endosymbionts formed a single clade with certain non-identified tick bacterium species isolated from the tick Haemaphysalis wellingtoni. On the other hand, pathogenic species of the genera Anaplasma, Ehrlichia and Rickettsia detected in I. ricinus as well as symbionts of the genus Wolbachia present in Culex pipiens and Drosophila simulans have been placed at a different site on the phylogenetic tree.

  6. MIRO1 influences the morphology and intracellular distribution of mitochondria during embryonic cell division in Arabidopsis.

    PubMed

    Yamaoka, Shohei; Nakajima, Masaki; Fujimoto, Masaru; Tsutsumi, Nobuhiro

    2011-02-01

    Regulating the morphology and intracellular distribution of mitochondria is essential for embryo development in animals. However, the importance of such regulation is not clearly defined in plants. The evolutionarily conserved Miro proteins are known to be involved in the regulation of mitochondrial morphology and motility. We previously demonstrated that MIRO1, an Arabidopsis thaliana orthologue of the Miro protein, is required for embryogenesis. An insertional mutation in the MIRO1 gene causes arrest of embryonic cell division, leading to abortion of the embryo at an early stage. Here we investigated the role of MIRO1 in the regulation of mitochondrial behaviour in egg cells and early-stage embryos using GFP-labeled mitochondria. Two-photon laser scanning microscopy revealed that, in miro1 mutant egg cells, mitochondria are abnormally enlarged, although egg cell formation is nearly unaffected. After fertilization and subsequent zygotic cell division, the homozygous miro1 mutant two-celled embryo contained a significantly reduced number of mitochondria in its apical cell compared with the wild type, suggesting that the miro1 mutation inhibits proper intracellular distribution of mitochondria, leading to an arrest of embryonic cell division. Our findings suggest that proper mitochondrial morphology and intracellular distribution are maintained by MIRO1 and are vital for embryonic cell division.

  7. Mitochondria-rich cells in the astigmatic mites, Diplaegidia columbae (Buchholz) (Analgidae) and Falculifer rostratus (Buchholz) (Falculiferidae) (Acari: Astigmata).

    PubMed

    Witaliński, Wojciech; Liana, Marcin

    2010-09-01

    Mitochondria are well-characterized intracellular organelles usually concentrated in locations of high energy consumption. Light microscopic and transmission electron microscopic observations of the internal anatomy of the feather mites Diplaegidia columbae and Falculifer rostratus were conducted. In the anterior half of the bodies of the mites, we found several dozen of distinctive mitochondria-rich (MR) cells filled with abundant, large mitochondria. Mitochondria are placed individually or enclosed in small groups within an elaborated lamellar system forming a mitochondria-lamellae complex (MLC). The role of the MLC as well as the MR cells is not clear at present, but their involvement in heat generation is hypothesized and briefly discussed.

  8. DNA damage and mitochondria dysfunction in cell apoptosis induced by nonthermal air plasma

    SciTech Connect

    Kim, G. J.; Lee, J. K.; Kim, W.; Kim, K. T.

    2010-01-11

    Nonthermal plasma is known to induce animal cell death but the mechanism is not yet clear. Here, cellular and biochemical regulation of cell apoptosis is demonstrated for plasma treated cells. Surface type nonthermal air plasma triggered apoptosis of B16F10 mouse melanoma cancer cells causing DNA damage and mitochondria dysfunction. Plasma treatment activated caspase-3, apoptosis executioner. The plasma treated cells also accumulated gamma-H2A.X, marker for DNA double strand breaks, and p53 tumor suppressor gene as a response to DNA damage. Interestingly, cytochrome C was released from mitochondria and its membrane potential was changed significantly.

  9. Toward genetic transformation of mitochondria in mammalian cells using a recoded drug-resistant selection marker.

    PubMed

    Yoon, Young Geol; Koob, Michael Duane

    2011-04-20

    Due to technical difficulties, the genetic transformation of mitochondria in mammalian cells is still a challenge. In this report, we described our attempts to transform mammalian mitochondria with an engineered mitochondrial genome based on selection using a drug resistance gene. Because the standard drug-resistant neomycin phosphotransferase confers resistance to high concentrations of G418 when targeted to the mitochondria, we generated a recoded neomycin resistance gene that uses the mammalian mitochondrial genetic code to direct the synthesis of this protein in the mitochondria, but not in the nucleus (mitochondrial version). We also generated a universal version of the recoded neomycin resistance gene that allows synthesis of the drug-resistant proteins both in the mitochondria and nucleus. When we transfected these recoded neomycin resistance genes that were incorporated into the mouse mitochondrial genome clones into mouse tissue culture cells by electroporation, no DNA constructs were delivered into the mitochondria. We found that the universal version of the recoded neomycin resistance gene was expressed in the nucleus and thus conferred drug resistance to G418 selection, while the synthetic mitochondrial version of the gene produced no background drug-resistant cells from nuclear transformation. These recoded synthetic drug-resistant genes could be a useful tool for selecting mitochondrial genetic transformants as a precise technology for mitochondrial transformation is developed.

  10. Mitochondria are required for ATM activation by extranuclear oxidative stress in cultured human hepatoblastoma cell line Hep G2 cells

    SciTech Connect

    Morita, Akinori; Tanimoto, Keiji; Murakami, Tomoki; Morinaga, Takeshi; Hosoi, Yoshio

    2014-01-24

    Highlights: • Oxidative ATM activation can occur in the absence of nuclear DNA damage response. • The oxidized Hep G2 cells were subjected to subcellular fractionation. • The obtained results suggest that the ATM activation occurs in mitochondria. • ATM failed to respond to oxidative stress in mitochondria-depleted Hep G2 cells. • Mitochondria are required for the oxidative activation of ATM. - Abstract: Ataxia–telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in DNA damage response (DDR). A recent study reported that oxidized ATM can be active in the absence of DDR. However, the issue of where ATM is activated by oxidative stress remains unclear. Regarding the localization of ATM, two possible locations, namely, mitochondria and peroxisomes are possible. We report herein that ATM can be activated when exposed to hydrogen peroxide without inducing nuclear DDR in Hep G2 cells, and the oxidized cells could be subjected to subcellular fractionation. The first detergent-based fractionation experiment revealed that active, phosphorylated ATM was located in the second fraction, which also contained both mitochondria and peroxisomes. An alternative fractionation method involving homogenization and differential centrifugation, which permits the light membrane fraction containing peroxisomes to be produced, but not mitochondria, revealed that the light membrane fraction contained only traces of ATM. In contrast, the heavy membrane fraction, which mainly contained mitochondrial components, was enriched in ATM and active ATM, suggesting that the oxidative activation of ATM occurs in mitochondria and not in peroxisomes. In Rho 0-Hep G2 cells, which lack mitochondrial DNA and functional mitochondria, ATM failed to respond to hydrogen peroxide, indicating that mitochondria are required for the oxidative activation of ATM. These findings strongly suggest that ATM can be activated in response to oxidative stress in mitochondria

  11. Salmonella - at home in the host cell.

    PubMed

    Malik-Kale, Preeti; Jolly, Carrie E; Lathrop, Stephanie; Winfree, Seth; Luterbach, Courtney; Steele-Mortimer, Olivia

    2011-01-01

    The Gram-negative bacterium Salmonella enterica has developed an array of sophisticated tools to manipulate the host cell and establish an intracellular niche, for successful propagation as a facultative intracellular pathogen. While Salmonella exerts diverse effects on its host cell, only the cell biology of the classic "trigger"-mediated invasion process and the subsequent development of the Salmonella-containing vacuole have been investigated extensively. These processes are dependent on cohorts of effector proteins translocated into host cells by two type III secretion systems (T3SS), although T3SS-independent mechanisms of entry may be important for invasion of certain host cell types. Recent studies into the intracellular lifestyle of Salmonella have provided new insights into the mechanisms used by this pathogen to modulate its intracellular environment. Here we discuss current knowledge of Salmonella-host interactions including invasion and establishment of an intracellular niche within the host.

  12. Mitochondria released by cells undergoing TNF-α-induced necroptosis act as danger signals.

    PubMed

    Maeda, A; Fadeel, B

    2014-07-03

    Necrosis leads to the release of so-called damage-associated molecular patterns (DAMPs), which may provoke inflammatory responses. However, the release of organelles from dying cells, and the consequences thereof have not been documented before. We demonstrate here that mitochondria are released from cells undergoing tumor necrosis factor-α (TNF-α)-induced, receptor-interacting protein (RIP)1-dependent necroptosis, a form of programmed necrosis. The released, purified mitochondria were determined to be intact as they did not emit appreciable amounts of mitochondrial DNA (mtDNA). Pharmacological inhibition of dynamin-related protein 1 (Drp1) prevented mitochondrial fission in TNF-α-triggered cells, but this did not block necroptosis nor the concomitant release of mitochondria. Importantly, primary human macrophages and dendritic cells engulfed mitochondria from necroptotic cells leading to modulation of macrophage secretion of cytokines and induction of dendritic cell maturation. Our results show that intact mitochondria are released from necroptotic cells and suggest that these organelles act as bona fide danger signals.

  13. Transformations of the macromolecular landscape at mitochondria during DNA-damage-induced apoptotic cell death.

    PubMed

    Yadav, N; Pliss, A; Kuzmin, A; Rapali, P; Sun, L; Prasad, P; Chandra, D

    2014-10-09

    Apoptosis is a dynamic process regulated by mitochondrion critical for cellular respiration and survival. Execution of apoptosis is mediated by multiple protein signaling events at mitochondria. Initiation and progression of apoptosis require numerous apoptogenic factors that are either released from or sequestered in mitochondria, which may transform the biomolecular makeup of the organelle. In this communication, using Raman microspectroscopy, we demonstrate that transformation in biomolecular composition of mitochondrion may be used as apoptosis marker in an individual cell. For the first time, we show that significant changes occur in the concentrations of RNA, DNA, protein, and lipid constituents of mitochondria during apoptosis. The structural analysis of proteins on mitochondria demonstrated a decrease in α-helix secondary structure content, and an increase in the levels of random coils and β-sheets on mitochondria. This may represent an additional hallmark of apoptosis. Strikingly, we observed nearly identical changes in macromolecular content of mitochondria both in the presence and absence of a key proapoptotic protein, Bax (Bcl-2-associated X protein). Increased DNA level in mitochondria corresponded with higher mitochondrial DNA (mtDNA), cellular reactive oxygen species (ROS), and mitochondrial ROS production. Upregulation of polymerase-γ (POLG), mitochondrial helicase Twinkle, and mitochondrial transcription factor A (Tfam) in response to DNA damage correlated with increased mtDNA and RNA synthesis. Elevated activity of oxidative phosphorylation complexes supports functional mitochondrial respiration during apoptosis. Thus, we define previously unknown dynamic correlation of macromolecular structure of mitochondria and apoptosis progression in the presence and absence of Bax protein. These findings open up a new approach for monitoring physiological status of cells by non invasive single-cell method.

  14. Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts

    PubMed Central

    Williamson, Chad D.; Wong, Daniel S.; Bozidis, Petros; Zhang, Aiping; Colberg-Poley, Anamaris M.

    2015-01-01

    Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub-domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria-associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated. Because of its documented role in lipid and ceramide transfer from the ER to mitochondria, a method to purify MAM from infected cells was also developed. Two robust procedures were developed to efficiently isolate mitochondria, ER, and MAM fractions while providing substantial protein yields from HCMV-infected primary fibroblasts and from transfected HeLa cells. Furthermore, this unit includes protocols for isolation of detergent resistant membranes from subcellular fractions as well as techniques that allow visualization of the mitochondria network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients. PMID:26331984

  15. Binding of the host-specific toxins from Helminthosporium maydis race T and Phyllosticta maydis to mitochondria isolated from Zea mays

    SciTech Connect

    Frantzen, K.A.

    1985-01-01

    Helminthosphorium maydis race I and Phyllosticta maydis, the causal agents of southern and yellow corn leaf blights, respectively, produce host-specific toxins. The toxic specificity of these natural products is identical to the host-specificity of the pathogens for certain varieties of corn. Susceptible genotypes carry the Texas type of cytoplasmic male sterility. Isolated mitochondria from susceptible plant species are highly sensitive to these toxins, whereas other plant species, including resistant corn varieties, and their mitochondria are not. The mitochondrion may be the primary cellular site of action for these toxins. The toxins from H. maydis and P. maydis were tritiated by reduction with borotritide salts. The labeled products had a high specific activity (3.8 to 8 Ci/mmole), high biological activity, and specificity identical to that of the native toxins. A filtration binding assay was developed to investigate the binding characteristics of these labeled toxins to isolated mitochondria. Mitochondria isolated from both cytoplasmic male sterile (Texas) and normal corn demonstrated similar binding characteristics including ligand displaceable binding with both labeled toxins. Ligand displaceable binding was also detectable in mitochondria from soybeans, a nonhost plant for these fungi. The ability to displace the bound labeled toxins was generally correlated with the biological activity of the competing toxin. The results of this study suggest that a receptor site hypothesis for the mode of action of these toxins may not be valid.

  16. Mitochondria in mesenchymal stem cell biology and cell therapy: From cellular differentiation to mitochondrial transfer.

    PubMed

    Hsu, Yi-Chao; Wu, Yu-Ting; Yu, Ting-Hsien; Wei, Yau-Huei

    2016-04-01

    Mesenchymal stem cells (MSCs) are characterized to have the capacity of self-renewal and the potential to differentiate into mesoderm, ectoderm-like and endoderm-like cells. MSCs hold great promise for cell therapies due to their multipotency in vitro and therapeutic advantage of hypo-immunogenicity and lower tumorigenicity. Moreover, it has been shown that MSCs can serve as a vehicle to transfer mitochondria into cells after cell transplantation. Mitochondria produce most of the energy through oxidative phosphorylation in differentiated cells. It has been increasingly clear that the switch of energy supply from glycolysis to aerobic metabolism is essential for successful differentiation of MSCs. Post-translational modifications of proteins have been established to regulate mitochondrial function and metabolic shift during MSCs differentiation. In this article, we review and provide an integrated view on the roles of different protein kinases and sirtuins in the maintenance and differentiation of MSCs. Importantly, we provide evidence to suggest that alteration in the expression of Sirt3 and Sirt5 and relative changes in the acylation levels of mitochondrial proteins might be involved in the activation of mitochondrial function and adipogenic differentiation of adipose-derived MSCs. We summarize their roles in the regulation of mitochondrial biogenesis and metabolism, oxidative responses and differentiation of MSCs. On the other hand, we discuss recent advances in the study of mitochondrial dynamics and mitochondrial transfer as well as their roles in the differentiation and therapeutic application of MSCs to improve cell function in vitro and in animal models. Accumulating evidence has substantiated that the therapeutic potential of MSCs is conferred not only by cell replacement and paracrine effects but also by transferring mitochondria into injured tissues or cells to modulate the cellular metabolism in situ. Therefore, elucidation of the underlying mechanisms

  17. Biocavity laser spectroscopy of genetically altered yeast cells and isolated yeast mitochondria

    NASA Astrophysics Data System (ADS)

    Gourley, Paul L.; Hendricks, Judy K.; McDonald, Anthony E.; Copeland, R. Guild; Naviaux, Robert K.; Yaffe, Michael P.

    2006-02-01

    We report an analysis of 2 yeast cell mutants using biocavity laser spectroscopy. The two yeast strains differed only by the presence or absence of mitochondrial DNA. Strain 104 is a wild-type (ρ +) strain of the baker's yeast, Saccharomyces cerevisiae. Strain 110 was derived from strain 104 by removal of its mitochondrial DNA (mtDNA). Removal of mtDNA causes strain 110 to grow as a "petite" (ρ -), named because it forms small colonies (of fewer cells because it grows more slowly) on agar plates supplemented with a variety of different carbon sources. The absence of mitochondrial DNA results in the complete loss of all the mtDNA-encoded proteins and RNAs, and loss of the pigmented, heme-containing cytochromes a and b. These cells have mitochondria, but the mitochondria lack the normal respiratory chain complexes I, III, IV, and V. Complex II is preserved because its subunits are encoded by genes located in nuclear DNA. The frequency distributions of the peak shifts produced by wild-type and petite cells and mitochondria show striking differences in the symmetry and patterns of the distributions. Wild-type ρ + cells (104) and mitochondria produced nearly symmetric, Gaussian distributions. The ρ - cells (110) and mitochondria showed striking asymmetry and skew that appeared to follow a Poisson distribution.

  18. A fluorescent carbon-dots-based mitochondria-targetable nanoprobe for peroxynitrite sensing in living cells.

    PubMed

    Wu, Xiaoxue; Sun, Shan; Wang, Yuhui; Zhu, Jiali; Jiang, Kai; Leng, Yumin; Shu, Qinghai; Lin, Hengwei

    2017-04-15

    Mitochondria, the power generators in cell, are a primary organelle of oxygen consumption and a main source of reactive oxygen/nitrogen species (ROS/RNS). Peroxynitrite (ONOO(-)), known as a kind of RNS, has been considered to be a significant factor in many cell-related biological processes, and there is great desire to develop fluorescent probes that can sensitively and selectively detect peroxynitrite in living cells. Herein, we developed a fluorescent carbon-dots (C-dots) based mitochondria-targetable nanoprobe with high sensitivity and selectivity for peroxynitrite sensing in living cells. The C-dots with its surface rich in amino groups was synthesized using o-phenylenediamine as carbon precursor, and it could be covalently conjugated with a mitochondria-targeting moiety, i.e. triphenylphosphonium (TPP). In the presence of peroxynitrite, the fluorescence of the constructed nanoprobe (C-dots-TPP) was efficiently quenched via a mechanism of photoinduced electron transfer (PET). The nanoprobe exhibited relatively high sensitivity (limit of detection: 13.5nM) and selectivity towards peroxynitrite in aqueous buffer. The performance of the nanoprobe for fluorescence imaging of peroxynitrite in mitochondria was investigated. The results demonstrated that the nanoprobe showed fine mitochondria-targeting ability and imaging contrast towards peroxynitrite in living cells. We anticipate that the proposed nanoprobe will provide a facile tool to explore the role played by peroxynitrite in cytobiology.

  19. Myo19 Ensures Symmetric Partitioning of Mitochondria and Coupling of Mitochondrial Segregation to Cell Division

    PubMed Central

    Rohn, Jennifer L.; Patel, Jigna V.; Neumann, Beate; Bulkescher, Jutta; Mchedlishvili, Nunu; McMullan, Rachel C.; Quintero, Omar A.; Ellenberg, Jan; Baum, Buzz

    2014-01-01

    Summary During animal cell division, an actin-based ring cleaves the cell into two. Problems with this process can cause chromosome missegregation and defects in cytoplasmic inheritance and the partitioning of organelles, which in turn are associated with human diseases [1–3]. Although much is known about how chromosome segregation is coupled to cell division, the way organelles coordinate their inheritance during partitioning to daughter cells is less well understood. Here, using a high-content live-imaging small interfering RNA screen, we identify Myosin-XIX (Myo19) as a novel regulator of cell division. Previously, this actin-based motor was shown to control the interphase movement of mitochondria [4]. Our analysis shows that Myo19 is indeed localized to mitochondria and that its silencing leads to defects in the distribution of mitochondria within cells and in mitochondrial partitioning at division. Furthermore, many Myo19 RNAi cells undergo stochastic division failure—a phenotype that can be mimicked using a treatment that blocks mitochondrial fission and rescued by decreasing mitochondrial fusion, implying that mitochondria can physically interfere with cytokinesis. Strikingly, using live imaging we also observe the inappropriate movement of mitochondria to the poles of spindles in cells depleted for Myo19 as they enter anaphase. Since this phenocopies the results of an acute loss of actin filaments in anaphase, these data support a model whereby the Myo19 actin-based motor helps to control mitochondrial movement to ensure their faithful segregation during division. The presence of DNA within mitochondria makes their inheritance an especially important aspect of symmetrical cell division. PMID:25447992

  20. Myo19 ensures symmetric partitioning of mitochondria and coupling of mitochondrial segregation to cell division.

    PubMed

    Rohn, Jennifer L; Patel, Jigna V; Neumann, Beate; Bulkescher, Jutta; Mchedlishvili, Nunu; McMullan, Rachel C; Quintero, Omar A; Ellenberg, Jan; Baum, Buzz

    2014-11-03

    During animal cell division, an actin-based ring cleaves the cell into two. Problems with this process can cause chromosome missegregation and defects in cytoplasmic inheritance and the partitioning of organelles, which in turn are associated with human diseases. Although much is known about how chromosome segregation is coupled to cell division, the way organelles coordinate their inheritance during partitioning to daughter cells is less well understood. Here, using a high-content live-imaging small interfering RNA screen, we identify Myosin-XIX (Myo19) as a novel regulator of cell division. Previously, this actin-based motor was shown to control the interphase movement of mitochondria. Our analysis shows that Myo19 is indeed localized to mitochondria and that its silencing leads to defects in the distribution of mitochondria within cells and in mitochondrial partitioning at division. Furthermore, many Myo19 RNAi cells undergo stochastic division failure--a phenotype that can be mimicked using a treatment that blocks mitochondrial fission and rescued by decreasing mitochondrial fusion, implying that mitochondria can physically interfere with cytokinesis. Strikingly, using live imaging we also observe the inappropriate movement of mitochondria to the poles of spindles in cells depleted for Myo19 as they enter anaphase. Since this phenocopies the results of an acute loss of actin filaments in anaphase, these data support a model whereby the Myo19 actin-based motor helps to control mitochondrial movement to ensure their faithful segregation during division. The presence of DNA within mitochondria makes their inheritance an especially important aspect of symmetrical cell division.

  1. AMPK activity regulates trafficking of mitochondria to the leading edge during cell migration and matrix invasion

    PubMed Central

    Cunniff, Brian; McKenzie, Andrew J.; Heintz, Nicholas H.; Howe, Alan K.

    2016-01-01

    Cell migration is a complex behavior involving many energy-expensive biochemical events that iteratively alter cell shape and location. Mitochondria, the principal producers of cellular ATP, are dynamic organelles that fuse, divide, and relocate to respond to cellular metabolic demands. Using ovarian cancer cells as a model, we show that mitochondria actively infiltrate leading edge lamellipodia, thereby increasing local mitochondrial mass and relative ATP concentration and supporting a localized reversal of the Warburg shift toward aerobic glycolysis. This correlates with increased pseudopodial activity of the AMP-activated protein kinase (AMPK), a critically important cellular energy sensor and metabolic regulator. Furthermore, localized pharmacological activation of AMPK increases leading edge mitochondrial flux, ATP content, and cytoskeletal dynamics, whereas optogenetic inhibition of AMPK halts mitochondrial trafficking during both migration and the invasion of three-dimensional extracellular matrix. These observations indicate that AMPK couples local energy demands to subcellular targeting of mitochondria during cell migration and invasion. PMID:27385336

  2. Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

    PubMed Central

    Lampl, Thomas; Crum, Jo A.; Davis, Taylor A.; Milligan, Carol; Del Gaizo Moore, Victoria

    2015-01-01

    Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput. PMID:25866954

  3. Interrelations between the Parasitophorous Vacuole of Toxoplasma gondii and Host Cell Organelles

    NASA Astrophysics Data System (ADS)

    Cardoso Magno, Rodrigo; Cobra Straker, Lorian; de Souza, Wanderley; Attias, Marcia

    2005-04-01

    Toxoplasma gondii, the causative agent of toxoplasmosis, is capable of actively penetrating and multiplying in any nucleated cell of warm-blooded animals. Its survival strategies include escape from fusion of the parasitophorous vacuole with host cell lysosomes and rearrangement of host cell organelles in relation to the parasitophorous vacuole. In this article we report the rearrangement of host cell organelles and elements of the cytoskeleton of LLCMK2 cells, a lineage derived from green monkey kidney epithelial cells, in response to infection by T. gondii tachyzoites. Transmission electron microscopy made on flat embedded monolayers cut horizontally to the apical side of the cells or field emission scanning electron microscopy of monolayers scraped with scotch tape before sputtering showed that association of mitochondria to the vacuole is much less frequent than previously described. On the other hand, all parasitophorous vacuoles were surrounded by elements of the endoplasmic reticulum. These data were complemented by observations by laser scanning microscopy using fluorescent probes from mitochondria and endoplasmic reticulum and reinforced by three-dimensional reconstruction from serial sections observed by transmission electron microscopy and labeling of mitochondria and endoplasmic reticulum by fluorescent probes.

  4. Crosstalk from non-cancerous mitochondria can inhibit tumor properties of metastatic cells by suppressing oncogenic pathways.

    PubMed

    Kaipparettu, Benny Abraham; Ma, Yewei; Park, Jun Hyoung; Lee, Tin-Lap; Zhang, Yiqun; Yotnda, Patricia; Creighton, Chad J; Chan, Wai-Yee; Wong, Lee-Jun C

    2013-01-01

    Mitochondrial-nucleus cross talks and mitochondrial retrograde regulation can play a significant role in cellular properties. Transmitochondrial cybrid systems (cybrids) are an excellent tool to study specific effects of altered mitochondria under a defined nuclear background. The majority of the studies using the cybrid model focused on the significance of specific mitochondrial DNA variations in mitochondrial function or tumor properties. However, most of these variants are benign polymorphisms without known functional significance. From an objective of rectifying mitochondrial defects in cancer cells and to establish mitochondria as a potential anticancer drug target, understanding the role of functional mitochondria in reversing oncogenic properties under a cancer nuclear background is very important. Here we analyzed the potential reversal of oncogenic properties of a highly metastatic cell line with the introduction of non-cancerous mitochondria. Cybrids were established by fusing the mitochondria DNA depleted 143B TK- ρ0 cells from an aggressive osteosarcoma cell line with mitochondria from benign breast epithelial cell line MCF10A, moderately metastatic breast cancer cell line MDA-MB-468 and 143B cells. In spite of the uniform cancerous nuclear background, as observed with the mitochondria donor cells, cybrids with benign mitochondria showed high mitochondrial functional properties including increased ATP synthesis, oxygen consumption and respiratory chain activities compared to cybrids with cancerous mitochondria. Interestingly, benign mitochondria could reverse different oncogenic characteristics of 143B TK(-) cell including cell proliferation, viability under hypoxic condition, anti-apoptotic properties, resistance to anti-cancer drug, invasion, and colony formation in soft agar, and in vivo tumor growth in nude mice. Microarray analysis suggested that several oncogenic pathways observed in cybrids with cancer mitochondria are inhibited in cybrids with

  5. Parkin elimination of mitochondria is important for maintenance of lens epithelial cell ROS levels and survival upon oxidative stress exposure.

    PubMed

    Brennan, Lisa; Khoury, Josef; Kantorow, Marc

    2017-01-01

    Age-related cataract is associated with oxidative stress and death of lens epithelial cells (LECs) whose survival is dependent on functional mitochondrial populations. Oxidative stress-induced depolarization/damage of LEC mitochondria results in increased reactive oxygen species (ROS) levels and cell death suggesting the need for a LEC mechanism to remove mitochondria depolarized/damaged upon oxidative stress exposure to prevent ROS release and LEC death. To date, a mechanism(s) for removal of depolarized/damaged LEC mitochondria has yet to be identified and the importance of eliminating oxidative stress-damaged mitochondria to prevent LEC ROS release and death has not been established. Here, we demonstrate that Parkin levels increase in LECs exposed to H2O2-oxidative stress. We establish that Parkin translocates to LEC mitochondria depolarized upon oxidative stress exposure and that Parkin recruits p62/SQSTM1 to depolarized LEC mitochondria. We demonstrate that translocation of Parkin results in the elimination of depolarized/damaged mitochondria and that Parkin clearance of LEC mitochondria is dependent on its ubiquitin ligase activity. Importantly, we demonstrate that Parkin elimination of damaged LEC mitochondria results in reduced ROS levels and increased survival upon oxidative stress exposure. These results establish that Parkin functions to eliminate LEC mitochondria depolarized/damaged upon oxidative stress exposure and that elimination of damaged mitochondria by Parkin is important for LEC homeostasis and survival. The data also suggest that mitochondrial quality control by Parkin could play a role in lens transparency.

  6. Edaravone Decreases Paraquat Toxicity in A549 Cells and Lung Isolated Mitochondria

    PubMed Central

    Shokrzadeh, Mohammad; Shaki, Fatemeh; Mohammadi, Ebrahim; Rezagholizadeh, Neda; Ebrahimi, Fatemeh

    2014-01-01

    Edaravone, an antioxidant and radical scavenger, showed protective effects against oxidative stress-like condition. Paraquat (PQ) is toxic herbicide considerable evidence suggests that oxidative stress and mitochondrial dysfunction contribute to PQ toxicity. In this study, protective effect of edaravone against PQ induced toxicity and reactive oxygen species (ROS) generation in A549 cells and lung isolated mitochondria were evaluated. A549 cells and lung isolated mitochondria were divided into control group, PQ group, edaravone group and PQ plus edaravone-pretreated group. Cellular and mitochondrial viability assayed using MTT test and ROS generations in both cellular and mitochondrial fraction were determined by fluorometry using DCFH-DA as indicator. Our results showed that edaravone (5–100 µM) prevented PQ (500 µM) induced cytotoxicity in A549 cells that the best protective effect was observed at concentration of 50 µM of edaravone. In addition, PQ-induced ROS generation in A549 cells significantly inhibited by edaravone. Moreover, PQ decreased mitochondria viability and also increased ROS generation in lung isolated mitochondria that edaravone (25–400 µM) markedly inhibited these toxic effects. In overall, the results of this study suggest that lung mitochondria maintenance is essential for maintaining PQt cytotoxicity and Edaravone is a protective drug against PQ toxicity in-vitro. PMID:25237364

  7. Altered intestinal microbiota–host mitochondria crosstalk in new onset Crohn's disease

    PubMed Central

    Mottawea, Walid; Chiang, Cheng-Kang; Mühlbauer, Marcus; Starr, Amanda E.; Butcher, James; Abujamel, Turki; Deeke, Shelley A.; Brandel, Annette; Zhou, Hu; Shokralla, Shadi; Hajibabaei, Mehrdad; Singleton, Ruth; Benchimol, Eric I.; Jobin, Christian; Mack, David R.; Figeys, Daniel; Stintzi, Alain

    2016-01-01

    Intestinal microbial dysbiosis is associated with Crohn's disease (CD). However, the mechanisms leading to the chronic mucosal inflammation that characterizes this disease remain unclear. In this report, we use systems-level approaches to study the interactions between the gut microbiota and host in new-onset paediatric patients to evaluate causality and mechanisms of disease. We report an altered host proteome in CD patients indicative of impaired mitochondrial functions. In particular, mitochondrial proteins implicated in H2S detoxification are downregulated, while the relative abundance of H2S microbial producers is increased. Network correlation analysis reveals that Atopobium parvulum controls the central hub of H2S producers. A. parvulum induces pancolitis in colitis-susceptible interleukin-10-deficient mice and this phenotype requires the presence of the intestinal microbiota. Administrating the H2S scavenger bismuth mitigates A. parvulum-induced colitis in vivo. This study reveals that host–microbiota interactions are disturbed in CD and thus provides mechanistic insights into CD pathogenesis. PMID:27876802

  8. Induction of apoptosis in human cancer cells by targeting mitochondria with gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Mkandawire, M. M.; Lakatos, M.; Springer, A.; Clemens, A.; Appelhans, D.; Krause-Buchholz, U.; Pompe, W.; Rödel, G.; Mkandawire, M.

    2015-06-01

    A major challenge in designing cancer therapies is the induction of cancer cell apoptosis, although activation of intrinsic apoptotic pathways by targeting gold nanoparticles to mitochondria is promising. We report an in vitro procedure targeting mitochondria with conjugated gold nanoparticles and investigating effects on apoptosis induction in the human breast cancer cell line Jimt-1. Gold nanoparticles were conjugated to a variant of turbo green fluorescent protein (mitoTGFP) harbouring an amino-terminal mitochondrial localization signal. Au nanoparticle conjugates were further complexed with cationic maltotriose-modified poly(propylene imine) third generation dendrimers. Fluorescence and transmission electron microscopy revealed that Au nanoparticle conjugates were directed to mitochondria upon transfection, causing partial rupture of the outer mitochondrial membrane, triggering cell death. The ability to target Au nanoparticles into mitochondria of breast cancer cells and induce apoptosis reveals an alternative application of Au nanoparticles in photothermal therapy of cancer.A major challenge in designing cancer therapies is the induction of cancer cell apoptosis, although activation of intrinsic apoptotic pathways by targeting gold nanoparticles to mitochondria is promising. We report an in vitro procedure targeting mitochondria with conjugated gold nanoparticles and investigating effects on apoptosis induction in the human breast cancer cell line Jimt-1. Gold nanoparticles were conjugated to a variant of turbo green fluorescent protein (mitoTGFP) harbouring an amino-terminal mitochondrial localization signal. Au nanoparticle conjugates were further complexed with cationic maltotriose-modified poly(propylene imine) third generation dendrimers. Fluorescence and transmission electron microscopy revealed that Au nanoparticle conjugates were directed to mitochondria upon transfection, causing partial rupture of the outer mitochondrial membrane, triggering cell

  9. IL-1β induces apoptosis and autophagy via mitochondria pathway in human degenerative nucleus pulposus cells

    PubMed Central

    Shen, Jieliang; Xu, Shengxi; Zhou, Hao; Liu, Huzhe; Jiang, Wei; Hao, Jie; Hu, Zhenming

    2017-01-01

    IL-1β has been reported highly expressed in degenerative intervertebral disc, and our previous study indicated IL-1β facilitates apoptosis of human degenerative nucleus pulposus (NP) cell. However, the underlying molecular mechanism remains unclear. We here demonstrate that IL-1β played a significantly pro-apoptotic effect under serum deprivation. IL-1β decreased Bcl-2/Bax ratio and enhanced cytochrome C released from mitochondria to cytosol, which proved mitochondria-meidated apoptosis was induced. Subsequently, mitochondria damage was detected under IL-1β stimualtion. In addition, IL-1β-mediated injuried mitochondria contributes to activate autophagy. However, pretreatment with the autophagy inhibitor 3-methyladenine showed the potential in further elevating the apoptosis rate induced by IL-1β in NP cells. Our results indicated that the mitochondrial pathway was involved in IL-1β-induced apoptosis of NP cells. Meanwhile, the damaged mitochondria-induced autophagy played a protective role against apoptosis, suggesting a postive feedback mechanism under inflammatory stress. PMID:28120948

  10. SNARE-Mediated Cholesterol Movement to Mitochondria Supports Steroidogenesis in Rodent Cells

    PubMed Central

    Lin, Ye; Hou, Xiaoming; Shen, Wen-Jun; Hanssen, Ruth; Khor, Victor K.; Cortez, Yuan; Roseman, Ann N.; Azhar, Salman

    2016-01-01

    Vesicular transport involving soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins is known to be responsible for many major cellular activities. In steroidogenic tissues, chronic hormone stimulation results in increased expression of proteins involved in the steroidogenic pathway, whereas acute hormone stimulation prompts the rapid transfer of cholesterol to the inner mitochondrial membrane to be utilized as substrate for steroid hormone production. Several different pathways are involved in supplying cholesterol to mitochondria, but mobilization of stored cholesteryl esters appears to initially constitute the preferred source; however, the mechanisms mediating this cholesterol transfer are not fully understood. To study the potential contribution of SNARE proteins in steroidogenesis, we examined the expression levels of various SNARE proteins in response to hormone stimulation in steroidogenic tissues and cells and established an in vitro mitochondria reconstitution assay system to assess the contribution of various SNARE proteins on cholesterol delivery for steroidogenesis. Our results from reconstitution experiments along with knockdown studies in rat primary granulosa cells and in a Leydig cell line show that soluble N-ethylmaleimide sensitive factor attachment protein-α, synaptosomal-associated protein of 25 kDa, syntaxin-5, and syntaxin-17 facilitate the transport of cholesterol to mitochondria. Thus, although StAR is required for efficient cholesterol movement into mitochondria for steroidogenesis, specific SNAREs participate and are necessary to mediate cholesterol movement to mitochondria. PMID:26771535

  11. The ER-mitochondria interface: the social network of cell death.

    PubMed

    Grimm, Stefan

    2012-02-01

    When cellular organelles communicate bad things can happen. Recent findings uncovered that the junction between the endoplasmic reticulum (ER) and the mitochondria holds a crucial role for cell death regulation. Not only does this locale connect the two best-known organelles in apoptosis, numerous regulators of cell death are concentrated at this spot, providing a terrain for intense signal transfers. Ca2+ is the most prominent signalling factor that is released from the ER and, at high concentration, mediates the transfer of an apoptosis signal to mitochondria as the executioner organelle for cell death. An elaborate array of checks and balances is fine-tuning this process including Bcl-2 family members. Moreover, MAMs, "mitochondria-associated membranes", are distinct membrane sections at the ER that are in close contact with mitochondria and have been found to exchange lipids and lipid-derived molecules such as ceramide for apoptosis induction. Recent work has also described a reverse transfer of apoptosis signals, from mitochondria to the ER, via cytochrome c release and prolonged IP3R opening or through the mitochondrial fission factor Fis1 and Bap31 at the ER, which form the ARCosome, a novel caspase-activation complex.

  12. Ca2+ transport by mitochondria from L1210 mouse ascites tumor cells.

    PubMed

    Reynafarje, B; Lehninger, A L

    1973-06-01

    Mitochondria isolated from the ascites form of L1210 mouse leukemia cells readily accumulate Ca(2+) from the suspending medium and eject H(+) during oxidation of succinate in the presence of phosphate and Mg(2+), with normal stoichiometry between Ca(2+) uptake and electron transport. Ca(2+) loads up to 1600 ng-atoms per mg of protein are attained. As is the case in mitochondria from normal tissues, Ca(2+) uptake takes precedence over oxidative phosphorylation. However, Ca(2+) transport by the L-1210 mitochondria is unusual in other respects, which may possibly have general significance in tumor cells. The apparent affinity of the L1210 mitochondria for Ca(2+) in stimulation of oxygen uptake is about 3-fold greater than in normal liver mitochondria; moreover, the maximal rate of Ca(2+) transport is also considerably higher. Furthermore, when Ca(2+) pulses are added to L1210 mitochondria in the absence of phosphate or other permeant anions, much larger amounts of Ca(2+) are bound and H(+) ejected per atom of oxygen consumed than in the presence of phosphate; up to 7 Ca(2+) ions are bound per pair of electrons passing each energy-conserving site of the electron-transport chain. Such "superstoichiometry" of Ca(2+) uptake can be accounted for by two distinct types of respiration-dependent interaction of Ca(2+) with the L1210 mitochondria. One is the stimulation of oxygen consumption, which is achieved by relatively low concentrations of Ca(2+) (K(m) congruent with 8 muM) and is accompanied by binding of Ca(2+) up to 40 ng-atoms per mg of protein. The second process, also dependent on electron transport, is the binding of further Ca(2+) from the medium in exchange with previously stored membrane-bound protons, in which the affinity for Ca(2+) is much lower (K(m) congruent with 120 muM).

  13. Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts.

    PubMed

    Williamson, Chad D; Wong, Daniel S; Bozidis, Petros; Zhang, Aiping; Colberg-Poley, Anamaris M

    2015-09-01

    Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub-domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria-associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated. Because of its documented role in lipid and ceramide transfer from the ER to mitochondria, a method to purify MAM from infected cells was also developed. Two robust procedures were developed to efficiently isolate mitochondria, ER, and MAM fractions while providing substantial protein yields from HCMV-infected primary fibroblasts and from transfected HeLa cells. Furthermore, this unit includes protocols for isolation of detergent resistant membranes from subcellular fractions as well as techniques that allow visualization of the mitochondrial network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients.

  14. Pathogenesis of ganglion "cell death" in glaucoma and neuroprotection: focus on ganglion cell axonal mitochondria.

    PubMed

    Osborne, Neville N

    2008-01-01

    Retinal ganglion cell axons within the globe are functionally specialized being richly provided with many mitochondria. The mitochondria produce the high energy requirement for nerve conduction in the unmyelinated part of the ganglion cell axons. We have proposed that in the initiation of glaucoma, an alteration in the quality of blood flow dynamics in the optic nerve head causes a compromise in the retinal ganglion cell axon energy requirement, rendering the ganglion cells susceptible to additional insults. One secondary insult might be light entering the eye to further affect ganglion cell axon mitochondrial function. Other insults to the ganglion cells might be substances (e.g., glutamate, nitric oxide, TNF-alpha) released from astrocytes. These effects ultimately cause ganglion cell death because of the inability of mitochondria to maintain normal function. We therefore suggest that ganglion cell apoptosis in glaucoma is both receptor and mitochondrial mediated. Agents targeted specifically at enhancing ganglion cell mitochondrial energy production should therefore be beneficial in a disease like glaucoma. Ganglion cell death in glaucoma might therefore, in principle, not be unlike the pathophysiology of numerous neurological disorders involving energy dysregulation and oxidative stress. The trigger(s) for ganglion cell apoptosis in glaucoma is/are likely to be multifactorial, and the rationale for targeting impaired energy production as a possibility of improving a patient's quality of life is based on logic derived from laboratory studies where neuronal apoptosis is shown to occur via different mechanisms. Light-induced neuronal apoptosis is likely to be more relevant to ganglion cell death in glaucoma than, for example, neuronal apoptosis associated with Parkinson's disease. Logic suggests that enhancing mitochondrial function generally will slow down ganglion cell apoptosis and therefore benefit glaucoma patients. On the basis of our laboratory studies, we

  15. ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.

    PubMed

    Lewis, Samantha C; Uchiyama, Lauren F; Nunnari, Jodi

    2016-07-15

    Mitochondrial DNA (mtDNA) encodes RNAs and proteins critical for cell function. In human cells, hundreds to thousands of mtDNA copies are replicated asynchronously, packaged into protein-DNA nucleoids, and distributed within a dynamic mitochondrial network. The mechanisms that govern how nucleoids are chosen for replication and distribution are not understood. Mitochondrial distribution depends on division, which occurs at endoplasmic reticulum (ER)-mitochondria contact sites. These sites were spatially linked to a subset of nucleoids selectively marked by mtDNA polymerase and engaged in mtDNA synthesis--events that occurred upstream of mitochondrial constriction and division machine assembly. Our data suggest that ER tubules proximal to nucleoids are necessary but not sufficient for mtDNA synthesis. Thus, ER-mitochondria contacts coordinate licensing of mtDNA synthesis with division to distribute newly replicated nucleoids to daughter mitochondria.

  16. Biochemistry, proteomics, and phosphoproteomics of plant mitochondria from non-photosynthetic cells

    PubMed Central

    Havelund, Jesper F.; Thelen, Jay J.; Møller, Ian M.

    2013-01-01

    Mitochondria fulfill some basic roles in all plant cells. They supply the cell with energy in the form of ATP and reducing equivalents [NAD(P)H] and they provide the cell with intermediates for a range of biosynthetic pathways. In addition to this, mitochondria contribute to a number of specialized functions depending on the tissue and cell type, as well as environmental conditions. We will here review the biochemistry and proteomics of mitochondria from non-green cells and organs, which differ from those of photosynthetic organs in a number of respects. We will briefly cover purification of mitochondria and general biochemical properties such as oxidative phosphorylation. We will then mention a few adaptive properties in response to water stress, seed maturation and germination, and the ability to function under hypoxic conditions. The discussion will mainly focus on Arabidopsis cell cultures, etiolated germinating rice seedlings and potato tubers as model plants. It will cover the general proteome as well as the posttranslational modification protein phosphorylation. To date 64 phosphorylated mitochondrial proteins with a total of 103 phosphorylation sites have been identified. PMID:23494127

  17. Mitochondria Biogenesis and Bioenergetics Gene Profiles in Isogenic Prostate Cells with Different Malignant Phenotypes

    PubMed Central

    Burch, Tanya C.; Rhim, Johng S.

    2016-01-01

    Background. The most significant hallmarks of cancer are directly or indirectly linked to deregulated mitochondria. In this study, we sought to profile mitochondria associated genes in isogenic prostate cell lines with different tumorigenic phenotypes from the same patient. Results. Two isogenic human prostate cell lines RC77N/E (nonmalignant cells) and RC77T/E (malignant cells) were profiled for expression of mitochondrial biogenesis and energy metabolism genes by qRT-PCR using the Human Mitochondria and the Mitochondrial Energy Metabolism RT2 PCR arrays. Forty-seven genes were differentially regulated between the two cell lines. The interaction and regulatory networks of these genes were generated by Ingenuity Pathway Analysis. UCP2 was the most significantly upregulated gene in primary adenocarcinoma cells in the current study. The overexpression of UCP2 upon malignant transformation was further validated using human prostatectomy clinical specimens. Conclusions. This study demonstrates the overexpression of multiple genes that are involved in mitochondria biogenesis, bioenergetics, and modulation of apoptosis. These genes may play a role in malignant transformation and disease progression. The upregulation of some of these genes in clinical samples indicates that some of the differentially transcribed genes could be the potential targets for therapeutic interventions. PMID:27478826

  18. Biased inheritance of mitochondria during asymmetric cell division in the mouse oocyte.

    PubMed

    Dalton, Caroline M; Carroll, John

    2013-07-01

    A fundamental rule of cell division is that daughter cells inherit half the DNA complement and an appropriate proportion of cellular organelles. The highly asymmetric cell divisions of female meiosis present a different challenge because one of the daughters, the polar body, is destined to degenerate, putting at risk essential maternally inherited organelles such as mitochondria. We have therefore investigated mitochondrial inheritance during the meiotic divisions of the mouse oocyte. We find that mitochondria are aggregated around the spindle by a dynein-mediated mechanism during meiosis I, and migrate together with the spindle towards the oocyte cortex. However, at cell division they are not equally segregated and move instead towards the oocyte-directed spindle pole and are excluded from the polar body. We show that this asymmetrical inheritance in favour of the oocyte is not caused by bias in the spindle itself but is dependent on an intact actin cytoskeleton, spindle-cortex proximity, and cell cycle progression. Thus, oocyte-biased inheritance of mitochondria is a variation on rules that normally govern organelle segregation at cell division, and ensures that essential maternally inherited mitochondria are retained to provide ATP for early mammalian development.

  19. Campylobacter jejuni cell lysates differently target mitochondria and lysosomes on HeLa cells.

    PubMed

    Canonico, B; Campana, R; Luchetti, F; Arcangeletti, M; Betti, M; Cesarini, E; Ciacci, C; Vittoria, E; Galli, L; Papa, S; Baffone, W

    2014-08-01

    Campylobacter jejuni is the most common cause of bacterial gastroenteritis in humans. The synthesis of cytolethal distending toxin appears essential in the infection process. In this work we evaluated the sequence of lethal events in HeLa cells exposed to cell lysates of two distinct strains, C. jejuni ATCC 33291 and C. jejuni ISS3. C. jejuni cell lysates (CCLys) were added to HeLa cell monolayers which were analysed to detect DNA content, death features, bcl-2 and p53 status, mitochondria/lysosomes network and finally, CD54 and CD59 alterations, compared to cell lysates of C. jejuni 11168H cdtA mutant. We found mitochondria and lysosomes differently targeted by these bacterial lysates. Death, consistent with apoptosis for C. jejuni ATCC 33291 lysate, occurred in a slow way (>48 h); concomitantly HeLa cells increase their endolysosomal compartment, as a consequence of toxin internalization besides a simultaneous and partial lysosomal destabilization. C. jejuni CCLys induces death in HeLa cells mainly via a caspase-dependent mechanism although a p53 lysosomal pathway (also caspase-independent) seems to appear in addition. In C. jejuni ISS3-treated cells, the p53-mediated oxidative degradation of mitochondrial components seems to be lost, inducing the deepest lysosomal alterations. Furthermore, CD59 considerably decreases, suggesting both a degradation or internalisation pathway. CCLys-treated HeLa cells increase CD54 expression on their surface, because of the action of lysate as its double feature of toxin and bacterial peptide. In conclusion, we revealed that C. jejuni CCLys-treated HeLa cells displayed different features, depending on the particular strain.

  20. A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation

    SciTech Connect

    Park, So Jung; Park, Young Jun; Shin, Ji Hyun; Kim, Eun Sung; Hwang, Jung Jin; Jin, Dong-Hoon; Kim, Jin Cheon; Cho, Dong-Hyung

    2011-05-13

    Highlights: {yields} We screened and identified Tyrphostin A9, a receptor tyrosine kinase inhibitor as a strong mitochondria fission inducer. {yields} Tyrphostin A9 treatment promotes mitochondria dysfunction and contributes to cytotoxicity in cancer cells. {yields} Tyrphostin A9 induces apoptotic cell death through a Drp1-mediated pathway. {yields} Our studies suggest that Tyrphostin A9 induces mitochondria fragmentation and apoptotic cell death via Drp1 dependently. -- Abstract: Mitochondria dynamics controls not only their morphology but also functions of mitochondria. Therefore, an imbalance of the dynamics eventually leads to mitochondria disruption and cell death. To identify specific regulators of mitochondria dynamics, we screened a bioactive chemical compound library and selected Tyrphostin A9, a tyrosine kinase inhibitor, as a potent inducer of mitochondrial fission. Tyrphostin A9 treatment resulted in the formation of fragmented mitochondria filament. In addition, cellular ATP level was decreased and the mitochondrial membrane potential was collapsed in Tyr A9-treated cells. Suppression of Drp1 activity by siRNA or over-expression of a dominant negative mutant of Drp1 inhibited both mitochondrial fragmentation and cell death induced by Tyrpohotin A9. Moreover, treatment of Tyrphostin A9 also evoked mitochondrial fragmentation in other cells including the neuroblastomas. Taken together, these results suggest that Tyrphostin A9 induces Drp1-mediated mitochondrial fission and apoptotic cell death.

  1. Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells

    PubMed Central

    Wang, X; Gerdes, H-H

    2015-01-01

    Tunneling nanotubes (TNTs) are F-actin-based membrane tubes that form between cells in culture and in tissues. They mediate intercellular communication ranging from electrical signalling to the transfer of organelles. Here, we studied the role of TNTs in the interaction between apoptotic and healthy cells. We found that pheochromocytoma (PC) 12 cells treated with ultraviolet light (UV) were rescued when cocultured with untreated PC12 cells. UV-treated cells formed a different type of TNT with untreated PC12 cells, which was characterized by continuous microtubule localized inside these TNTs. The dynamic behaviour of mCherry-tagged end-binding protein 3 and the accumulation of detyrosinated tubulin in these TNTs indicate that they are regulated structures. In addition, these TNTs show different biophysical properties, for example, increased diameter allowing dye entry, prolonged lifetime and decreased membrane fluidity. Further studies demonstrated that microtubule-containing TNTs were formed by stressed cells, which had lost cytochrome c but did not enter into the execution phase of apoptosis characterized by caspase-3 activation. Moreover, mitochondria colocalized with microtubules in TNTs and transited along these structures from healthy to stressed cells. Importantly, impaired formation of TNTs and untreated cells carrying defective mitochondria were unable to rescue UV-treated cells in the coculture. We conclude that TNT-mediated transfer of functional mitochondria reverse stressed cells in the early stages of apoptosis. This provides new insights into the survival mechanisms of damaged cells in a multicellular context. PMID:25571977

  2. Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells.

    PubMed

    VanLinden, Magali R; Dölle, Christian; Pettersen, Ina K N; Kulikova, Veronika A; Niere, Marc; Agrimi, Gennaro; Dyrstad, Sissel E; Palmieri, Ferdinando; Nikiforov, Andrey A; Tronstad, Karl Johan; Ziegler, Mathias

    2015-11-13

    The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD(+) biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD(+) in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD(+) content, we have expressed plant and yeast mitochondrial NAD(+) carriers in human cells and observed a profound increase in mitochondrial NAD(+). None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD(+) content. Surprisingly, constitutive redistribution of NAD(+) from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD(+) transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD(+) levels. These results suggest that a mitochondrial NAD(+) transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD(+) synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells.

  3. Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells

    SciTech Connect

    Kwon, ChangHyuk; Tak, Hyosun; Rho, Mina; Chang, Hae Ryung; Kim, Yon Hui; Kim, Kyung Tae; Balch, Curt; Lee, Eun Kyung; Nam, Seungyoon

    2014-03-28

    Highlights: • piRNA sequences were mapped to human mitochondrial (mt) genome. • We inspected small RNA-Seq datasets from somatic cell mt subcellular fractions. • Piwi and piRNA transcripts are present in mammalian somatic cancer cell mt fractions. - Abstract: Piwi-interacting RNAs (piRNAs) are 26–31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWI and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies.

  4. Heptachlor induced mitochondria-mediated cell death via impairing electron transport chain complex III

    SciTech Connect

    Hong, Seokheon; Kim, Joo Yeon; Hwang, Joohyun; Shin, Ki Soon; Kang, Shin Jung

    2013-08-09

    Highlights: •Heptachlor inhibited mitochondrial electron transport chain complex III activity. •Heptachlor promoted generation of reactive oxygen species. •Heptachlor induced Bax activation. •Heptachlor induced mitochondria-mediated and caspase-dependent apoptosis. -- Abstract: Environmental toxins like pesticides have been implicated in the pathogenesis of Parkinson’s disease (PD). Epidemiological studies suggested that exposures to organochlorine pesticides have an association with an increased PD risk. In the present study, we examined the mechanism of toxicity induced by an organochlorine pesticide heptachlor. In a human dopaminergic neuroblastoma SH-SY5Y cells, heptachlor induced both morphological and functional damages in mitochondria. Interestingly, the compound inhibited mitochondrial electron transport chain complex III activity. Rapid generation of reactive oxygen species and the activation of Bax were then detected. Subsequently, mitochondria-mediated, caspase-dependent apoptosis followed. Our results raise a possibility that an organochlorine pesticide heptachlor can act as a neurotoxicant associated with PD.

  5. Contrasting Lifestyles Within the Host Cell

    PubMed Central

    Case, Elizabeth Di Russo; Samuel, James E.

    2015-01-01

    CHAPTER SUMMARY Intracellular bacterial pathogens have evolved to exploit the protected niche provided within the boundaries of a eukaryotic host cell. Upon entering a host cell, some bacteria can evade the adaptive immune response of its host, and replicate in a relatively nutrient-rich environment devoid of competition from other host flora. Growth within a host cell is not without its hazards, however. Many pathogens enter their hosts through receptor-mediated endocytosis or phagocytosis, two intracellular trafficking pathways that terminate in a highly degradative organelle, the phagolysosome. This usually deadly compartment is maintained at a low pH, and contains degradative enzymes, and reactive oxygen species resulting in an environment to which few bacterial species are adapted. Some intracellular pathogens, like Shigella, Listeria, Francisella, and Rickettsia escape the phagosome to replicate within the cytosol of the host cell. Bacteria that remain within a vacuole either alter the trafficking of their initial phagosomal compartment or adapt to survive within the harsh environment it will soon become. In this chapter, we focus on the mechanisms by which different vacuolar pathogens either evade lysosomal fusion, as in the case of Mycobacterium and Chlamydia, or allow interaction with lysosomes to varying degrees, such as Brucella and Coxiella, and their specific adaptations to inhabit a replicative niche. PMID:26999394

  6. TMX1 determines cancer cell metabolism as a thiol-based modulator of ER–mitochondria Ca2+ flux

    PubMed Central

    Herrera-Cruz, Maria Sol; Gesson, Kevin; Lou, Phing-How; Lucchinetti, Eliana; Zaugg, Michael; Baksh, Shairaz

    2016-01-01

    The flux of Ca2+ from the endoplasmic reticulum (ER) to mitochondria regulates mitochondria metabolism. Within tumor tissue, mitochondria metabolism is frequently repressed, leading to chemotherapy resistance and increased growth of the tumor mass. Therefore, altered ER–mitochondria Ca2+ flux could be a cancer hallmark, but only a few regulatory proteins of this mechanism are currently known. One candidate is the redox-sensitive oxidoreductase TMX1 that is enriched on the mitochondria-associated membrane (MAM), the site of ER–mitochondria Ca2+ flux. Our findings demonstrate that cancer cells with low TMX1 exhibit increased ER Ca2+, accelerated cytosolic Ca2+ clearance, and reduced Ca2+ transfer to mitochondria. Thus, low levels of TMX1 reduce ER–mitochondria contacts, shift bioenergetics away from mitochondria, and accelerate tumor growth. For its role in intracellular ER–mitochondria Ca2+ flux, TMX1 requires its thioredoxin motif and palmitoylation to target to the MAM. As a thiol-based tumor suppressor, TMX1 increases mitochondrial ATP production and apoptosis progression. PMID:27502484

  7. tRNA Biology in Mitochondria

    PubMed Central

    Salinas-Giegé, Thalia; Giegé, Richard; Giegé, Philippe

    2015-01-01

    Mitochondria are the powerhouses of eukaryotic cells. They are considered as semi-autonomous because they have retained genomes inherited from their prokaryotic ancestor and host fully functional gene expression machineries. These organelles have attracted considerable attention because they combine bacterial-like traits with novel features that evolved in the host cell. Among them, mitochondria use many specific pathways to obtain complete and functional sets of tRNAs as required for translation. In some instances, tRNA genes have been partially or entirely transferred to the nucleus and mitochondria require precise import systems to attain their pool of tRNAs. Still, tRNA genes have also often been maintained in mitochondria. Their genetic arrangement is more diverse than previously envisaged. The expression and maturation of mitochondrial tRNAs often use specific enzymes that evolved during eukaryote history. For instance many mitochondria use a eukaryote-specific RNase P enzyme devoid of RNA. The structure itself of mitochondrial encoded tRNAs is also very diverse, as e.g., in Metazoan, where tRNAs often show non canonical or truncated structures. As a result, the translational machinery in mitochondria evolved adapted strategies to accommodate the peculiarities of these tRNAs, in particular simplified identity rules for their aminoacylation. Here, we review the specific features of tRNA biology in mitochondria from model species representing the major eukaryotic groups, with an emphasis on recent research on tRNA import, maturation and aminoacylation. PMID:25734984

  8. Distribution of cytoskeletal structures and organelles of the host cell during evolution of the intracellular parasitism by Trypanosoma cruzi.

    PubMed

    Carvalho, T M; Ferreira, A G; Coimbra, E S; Rosestolato, C T; De Souza, W

    1999-07-01

    The distribution of microtubules, microfilaments, mitochondria, Golgi complex and endosomes/lysosomes was analyzed in Vero cells allowed to interact for different periods of time with the pathogenic protozoan Trypanosoma cruzi and observed by confocal laser scanning microscopy. Microtubules were revealed using a mouse monoclonal anti-alpha-tubulin antibody. Actin filaments were revealed using phalloidin-rhodamine. To identify mitochondria, endosomes/lysosomes and the Golgi complex the cells were labelled with Rhodamine 123, Lucifer yellow and C6-NBD-ceramide, respectively. During cell invasion actin filaments concentrate at the site of parasite penetration in some, but not in all cells, probably depending upon the mechanism used by the trypomastigote form to penetrate into the host cells. Following internalization the trypomastigote form gradually changes into the amastigote form, disruption of the parasitophorous vacuole membrane takes place and the amastigote form enters in direct contact with host cell structures and organelles, and starts to divide. The presence of the parasite in the cytoplasm of the host cell did not induce significant changes in the distribution of actin filaments, microtubules, the Golgi complex, mitochondria and endosomes/lysosomes during the first 48 h of infection. Amastigote forms were seen close to the microtubules. After 72 h of interaction, the number of microtubules and microfilaments around the parasites was reduced and lysosomes and mitochondria were seen in between the parasites.

  9. Metabolic integration during the evolutionary origin of mitochondria.

    PubMed

    Searcy, Dennis G

    2003-08-01

    Although mitochondria provide eukaryotic cells with certain metabolic advantages, in other ways they may be disadvantageous. For example, mitochondria produce reactive oxygen species that damage both nucleocytoplasm and mitochondria, resulting in mutations, diseases, and aging. The relationship of mitochondria to the cytoplasm is best understood in the context of evolutionary history. Although it is clear that mitochondria evolved from symbiotic bacteria, the exact nature of the initial symbiosis is a matter of continuing debate. The exchange of nutrients between host and symbiont may have differed from that between the cytoplasm and mitochondria in modern cells. Speculations about the initial relationships include the following. (1) The pre-mitochondrion may have been an invasive, parasitic bacterium. The host did not benefit. (2) The relationship was a nutritional syntrophy based upon transfer of organic acids from host to symbiont. (3) The relationship was a syntrophy based upon H2 transfer from symbiont to host, where the host was a methanogen. (4) There was a syntrophy based upon reciprocal exchange of sulfur compounds. The last conjecture receives support from our detection in eukaryotic cells of substantial H2S-oxidizing activity in mitochondria, and sulfur-reducing activity in the cytoplasm.

  10. Bax translocation into mitochondria during dihydroartemisinin(DHA)-induced apoptosis in human lung adenocarcinoma cells

    NASA Astrophysics Data System (ADS)

    Lu, Ying-ying; Chen, Tong-sheng; Qu, Jun-Le

    2009-02-01

    Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, isolated from the traditional Chinese herb Artemisia annua, has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathways. However, the molecular mechanisms are not well understood. This study was investigated in human lung adenocarconoma ASTC-a-1 cell line and aimed to determine whether the apoptotic process was mediated by Bax activation and translocation during DHA-induced apoptosis. In this study, DHA induced a time-dependent apoptotic cell death, which was assayed by Cell Counting Kit (CCK-8) and Hoechst 33258 staining. Detection of Bax aggregation and translocation to mitochondria was observed in living cells which were co-transfected with GFP-Bax and Dsred-mito plasmid using confocal fluorescence microscope technique. Overall, these results demonstrated that Bax activation and translocation to mitochondria occurred during DHA-induced apoptosis.

  11. [MORPHO-FUNCTIONAL CHARACTERISTICS OF BLOOD CELL MITOCHONDRIA IN BRONCHIAL ASTHMA].

    PubMed

    Denisenko, Yu k; Novgorodtseva, Tp; Kondrat'eva, E V; Zhukova, N; Antonyuk, M W; Knyshova, V V; Mineeva, E e

    2015-01-01

    We undertook a structural and functional study of blood cell mitochondria in 25 patients with controlled mild bronchial asthma (BA) including evaluation of blood saturation with oxygen, carboxyhemoglobin level in blood and carbon monoxide content in the exhaled air. Membrane potential of leukocyte mitochondria was determined based on the results of flow cytofluorimetry and fatty acid (FA) composition in platelet mitochondrial membranes measured by GLC. It was shown that the absence of clinical symptoms of BA during remission was associated with a reduction of membrane potential and a change of FA composition resulting in the depletion of the basal pool of saturated (12:0, 14:0, 18:0) and polyunsaturated (20:4n-6, 20:5n-3, 22:5n-3, 22:4n-6) FA. These changes in the structural and functional state of blood cell mitochondria in patients with BA are signs of disordered energy-producing activity, membrane permeability and transmembrane transport suggesting the development of mitochondrial dysfunction and cellular hypoxia. A deeper insight into the role of the structural and functional state of blood cell mitochondria in the formation of respiratory disorders will facilitate early detection of the risk and complications of bronchial obstruction.

  12. Bartonella entry mechanisms into mammalian host cells.

    PubMed

    Eicher, Simone C; Dehio, Christoph

    2012-08-01

    The Gram-negative genus Bartonella comprises arthropod-borne pathogens that typically infect mammals in a host-specific manner. Bartonella bacilliformis and Bartonella quintana are human-specific pathogens, while several zoonotic bartonellae specific for diverse animal hosts infect humans as an incidental host. Clinical manifestations of Bartonella infections range from mild symptoms to life-threatening disease. Following transmission by blood-sucking arthropods or traumatic contact with infected animals, bartonellae display sequential tropisms towards endothelial and possibly other nucleated cells and erythrocytes, the latter in a host-specific manner. Attachment to the extracellular matrix (ECM) and to nucleated cells is mediated by surface-exposed bacterial adhesins, in particular trimeric autotransporter adhesins (TAAs). The subsequent engulfment of the pathogen into a vacuolar structure follows a unique series of events whereby the pathogen avoids the endolysosomal compartments. For Bartonella henselae and assumingly most other species, the infection process is aided at different steps by Bartonella effector proteins (Beps). They are injected into host cells through the type IV secretion system (T4SS) VirB/D4 and subvert host cellular functions to favour pathogen uptake. Bacterial binding to erythrocytes is mediated by Trw, another T4SS, in a strictly host-specific manner, followed by pathogen-forced uptake involving the IalB invasin and subsequent replication and persistence within a membrane-bound intra-erythrocytic compartment.

  13. Helicobacter pylori vacuolating cytotoxin A (VacA) engages the mitochondrial fission machinery to induce host cell death

    PubMed Central

    Jain, Prashant; Luo, Zhao-Qing; Blanke, Steven R.

    2011-01-01

    A number of pathogenic bacteria target mitochondria to modulate the host's apoptotic machinery. Studies here revealed that infection with the human gastric pathogen Helicobacter pylori disrupts the morphological dynamics of mitochondria as a mechanism to induce host cell death. The vacuolating cytotoxin A (VacA) is both essential and sufficient for inducing mitochondrial network fragmentation through the mitochondrial recruitment and activation of dynamin-related protein 1 (Drp1), which is a critical regulator of mitochondrial fission within cells. Inhibition of Drp1-induced mitochondrial fission within VacA-intoxicated cells inhibited the activation of the proapoptotic Bcl-2–associated X (Bax) protein, permeabilization of the mitochondrial outer membrane, and cell death. Our data reveal a heretofore unrecognized strategy by which a pathogenic microbe engages the host's apoptotic machinery. PMID:21903925

  14. Iron alters cell survival in a mitochondria-dependent pathway in ovarian cancer cells.

    PubMed

    Bauckman, Kyle; Haller, Edward; Taran, Nicholas; Rockfield, Stephanie; Ruiz-Rivera, Abigail; Nanjundan, Meera

    2015-03-01

    The role of iron in the development of cancer remains unclear. We previously reported that iron reduces cell survival in a Ras/mitogen-activated protein kinase (MAPK)-dependent manner in ovarian cells; however, the underlying downstream pathway leading to reduced survival was unclear. Although levels of intracellular iron, ferritin/CD71 protein and reactive oxygen species did not correlate with iron-induced cell survival changes, we identified mitochondrial damage (via TEM) and reduced expression of outer mitochondrial membrane proteins (translocase of outer membrane: TOM20 and TOM70) in cell lines sensitive to iron. Interestingly, Ru360 (an inhibitor of the mitochondrial calcium uniporter) reversed mitochondrial changes and restored cell survival in HEY ovarian carcinoma cells treated with iron. Further, cells treated with Ru360 and iron also had reduced autophagic punctae with increased lysosomal numbers, implying cross-talk between these compartments. Mitochondrial changes were dependent on activation of the Ras/MAPK pathway since treatment with a MAPK inhibitor restored expression of TOM20/TOM70 proteins. Although glutathione antioxidant levels were reduced in HEY treated with iron, extracellular glutamate levels were unaltered. Strikingly, oxalomalate (inhibitor of aconitase, involved in glutamate production) reversed iron-induced responses in a similar manner to Ru360. Collectively, our results implicate iron in modulating cell survival in a mitochondria-dependent manner in ovarian cancer cells.

  15. Steroidogenesis in MA-10 mouse Leydig cells is altered via fatty acid import into the mitochondria.

    PubMed

    Rone, Malena B; Midzak, Andrew S; Martinez-Arguelles, Daniel B; Fan, Jinjiang; Ye, Xiaoying; Blonder, Josip; Papadopoulos, Vassilios

    2014-10-01

    Mitochondria are home to many cellular processes, including oxidative phosphorylation and fatty acid metabolism, and in steroid-synthesizing cells, they are involved in cholesterol import and metabolism, which is the initiating step in steroidogenesis. The formation of macromolecular protein complexes aids in the regulation and efficiency of these mitochondrial functions, though because of their dynamic nature, they are hard to identify. To overcome this problem, we used Blue-Native PAGE with whole-gel mass spectrometry on isolated mitochondria from control and hormone-treated MA-10 mouse tumor Leydig cells. The presence of multiple mitochondrial protein complexes was shown. Although these were qualitatively similar under control and human chorionic gonadotropin (hCG)-stimulated conditions, quantitative differences in the components of the complexes emerged after hCG treatment. A prominent decrease was observed with proteins involved in fatty acid import into the mitochondria, implying that mitochondrial beta-oxidation is not essential for steroidogenesis. To confirm this observation, we inhibited fatty acid import utilizing the CPT1a inhibitor etomoxir, resulting in increased steroid production. Conversely, stimulation of mitochondrial beta-oxidation with metformin resulted in a dose-dependent reduction in steroidogenesis. These changes were accompanied by changes in mitochondrial respiration and in the lactic acid formed during glycolysis. Taken together, these results suggest that upon hormonal stimulation, mitochondria efficiently import cholesterol for steroid production at the expense of other lipids necessary for energy production, specifically fatty acids required for beta-oxidation.

  16. Endoplasmic reticulum and mitochondria interplay mediates apoptotic cell death: relevance to Parkinson's disease.

    PubMed

    Arduíno, Daniela Moniz; Esteves, A Raquel; Cardoso, Sandra M; Oliveira, Catarina R

    2009-09-01

    Sporadic Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra pars compacta. Many cellular mechanisms are thought to be involved in the death of these specific neurons in PD, including oxidative stress, changes of intracellular calcium homeostasis, and mitochondrial dysfunction. Since recent studies have revealed that also endoplasmic reticulum (ER) stress in conjunction with abnormal protein degradation can contribute to the PD pathophysiology, we investigated here the molecular mechanisms underlying the interplay between ER and mitochondria and its relevance in the control of neuronal cell death in PD. We observed that MPP+ induced changes in the mitochondrial function, affecting mitochondrial membrane potential and electron transport chain function. Likewise, it was also evident the unfolded protein response activation by an overexpression of GRP78 protein. Moreover, stress stimuli caused the release of Ca2+ from the ER that consistently induced mitochondrial Ca2+ uptake, with a rise of mitochondrial matrix free Ca2+. Besides, Ca2+ release inhibition prevented MPP+ mediated mitochondria-dependent caspases activation. Our findings show that ER and mitochondria are in a close communication, establishing a dynamic ER-Ca2+-mitochondria interconnection that can play a prominent role in the neuronal cell death induction under particular stressful circumstances of PD pathology.

  17. The role of mitochondria in stem cell biology.

    PubMed

    Nesti, Claudia; Pasquali, Livia; Vaglini, Francesca; Siciliano, Gabriele; Murri, Luigi

    2007-06-01

    This mini-review summarizes the current literature on the role of mitochondrial DNA mutations and mitochondrial metabolism in stem cell biology. The possible uses of stem cells as a therapeutic tool in mitochondrial disorders are also reported.

  18. Comparison of three methods for mitochondria isolation from the human liver cell line (HepG2)

    PubMed Central

    Azimzadeh, Pedram; Asadzadeh Aghdaei, Hamid; Tarban, Peyman; Akhondi, Mohammad Mahdi; Shirazi, Abolfazl; Khorram Khorshid, Hamid Reza

    2016-01-01

    Aim: The aim of this study was to evaluate and compare three available methods for mitochondrial isolation from a human cell line to predict the best method for each probable application. Background: Organelle isolation is gaining importance in experimental laboratory settings. Mitochondrial dysfunction may affect tumorgenesis process. There are some evidences that transplantation of healthy, intact and active mitochondria into cells containing defective mitochondria may reduce the proliferation. Therefore, isolated mitochondria could be considered as an effective tool for assessment and management of mitochondrial related disorders. Patients and methods: Mitochondrial isolation from the human liver cell line (HepG2) was performed using two commercially available kits, including Qproteome (Qiagen) and MITOISO2 (Sigma-Aldrich), as well as a manual method. Integrity of inner membrane of mitochondria was assessed by JC-1 staining. Activity of isolated mitochondria was evaluated by DCFH-DA staining, and total yield of isolated mitochondria determined by micro-Lowry method. Finally, relative quantification using Real-time PCR was conducted to compare the mtDNA copy number of mitochondria isolated by three different methods. Results: Compared to other methods, manual kit resulted in higher yields of total amount of mitochondrial protein and mtDNA copy numbers. Isolated mitochondria by Qproteome kit, showed a higher activity. Finally, the integrity of inner-membrane of isolated mitochondria was significantly higher in Qproteome when compared with the other two methods. Conclusion: Due to differences in quality, quantity and activity of isolated mitochondria using three techniques discussed here, the method in which best-suited to each research project should be selected according to the distinct features of isolated mitochondria. PMID:27099670

  19. TARGETING THE MITOCHONDRIA ACTIVATES TWO INDEPENDENT CELL DEATH PATHWAYS IN THE OVARIAN CANCER STEM CELLS

    PubMed Central

    Alvero, Ayesha B.; Montagna, Michele K.; Holmberg, Jennie C.; Craveiro, Vinicius; Brown, David; Mor, Gil

    2013-01-01

    Cancer stem cells are responsible for tumor initiation and chemo-resistance. In ovarian cancer, the CD44+/MyD88+ ovarian cancer stem cells (OCSCs) are also able to repair the tumor and serve as tumor vascular progenitors. Targeting these cells is therefore necessary to improve treatment outcome and patient survival. The previous demonstration that the OCSCs are resistant to apoptotic cell death induced by conventional chemotherapy agents suggests that other forms of targeted therapy should be explored. We show in this study that targeting mitochondrial bioenergetics is a potent stimulus to induce caspase-independent cell death in a panel of OCSCs. Treatment of these cells with the novel isoflavone derivative, NV-128, significantly depressed mitochondrial function exhibited by decrease in ATP, Cox-I, and Cox-IV levels, and increase in mitochondrial superoxide and hydrogen peroxide. This promotes a state of “cellular starvation” that activates two independent pathways: 1) AMPKα1 pathway leading to mTOR inhibition; and 2) mitochondrial MEK/ERK pathway leading to loss of mitochondrial membrane potential. The demonstration that a compound can specifically target the mitochondria to induce cell death in this otherwise chemo-resistant cell population opens a new venue for treating ovarian cancer patients. PMID:21677151

  20. The Mitochondria-Mediate Apoptosis of Lepidopteran Cells Induced by Azadirachtin

    PubMed Central

    Huang, Jingfei; Lv, Chaojun; Hu, Meiying; Zhong, Guohua

    2013-01-01

    Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, this seems not to be the case in Drosophila, an insect model organism; thus more in-depth studies of insect cell apoptosis are necessary. In the present study, mitochondrial involvement during azadirachtin- and camptothecin-induced apoptosis in Spodoptera frugiperda Sf9 cells (isolated from Spodoptera frugiperda pupal ovarian tissue) was investigated. The results showed that both azadirachtin and camptothecin could induce apoptosis in Sf9 cells. Reactive oxygen species (ROS) generation, activation of mitochondrial permeability transition pores (MPTPs) and loss of mitochondrial membrane potential (MMP) were observed very early during apoptosis and were followed subsequently by the release of cytochrome-c from the mitochondria. Furthermore, the results also revealed that the opening of MPTPs and the loss of MMP induced by azadirachtin could be significantly inhibited by the permeability transition pore (PTP) inhibitor cyclosporin A (CsA), which was used to identify the key role of mitochondria in the apoptosis of Sf9 cells. However, in camptothecin-treated Sf9 cells, CsA could not suppress the opening of MPTPs and the loss of MMP when apoptosis was induced. The data from caspase-3 and caspase-9 activity assays and detection of apoptosis by morphological observation and flow cytometry also uncovered the different effect of CsA on the two botanical apoptosis inducers. Although different mechanisms of apoptosis induction exist, our study revealed that mitochondria play a crucial role in insect cell line apoptosis. PMID:23516491

  1. Purity matters: A workflow for the valid high-resolution lipid profiling of mitochondria from cell culture samples.

    PubMed

    Kappler, Lisa; Li, Jia; Häring, Hans-Ulrich; Weigert, Cora; Lehmann, Rainer; Xu, Guowang; Hoene, Miriam

    2016-02-19

    Subcellular lipidomics is a novel field of research that requires the careful combination of several pre-analytical and analytical steps. To define a reliable strategy for mitochondrial lipid profiling, we performed a systematic comparison of different mitochondria isolation procedures by western blot analyses and comprehensive high-resolution lipidomics. Using liver-derived HepG2 cells, we compared three common mitochondria isolation methods, differential centrifugation (DC), ultracentrifugation (UC) and a magnetic bead-assisted method (MACS). In total, 397 lipid species, including 32 cardiolipins, could be quantified in only 100 μg (by protein) of purified mitochondria. Mitochondria isolated by UC showed the highest enrichment in the mitochondria-specific cardiolipins as well as their precursors, phosphatidylglycerols. Mitochondrial fractions obtained by the commonly used DC and the more recent MACS method contained substantial contaminations by other organelles. Employing these isolation methods when performing lipidomics analyses from cell culture mitochondria may lead to inaccurate results. To conclude, we present a protocol how to obtain reliable mitochondria-specific lipid profiles from cell culture samples and show that quality controls are indispensable when performing mitochondria lipidomics.

  2. Lysosome-associated membrane proteins (LAMPs) regulate intracellular positioning of mitochondria in MC3T3-E1 cells.

    PubMed

    Rajapakshe, Anupama R; Podyma-Inoue, Katarzyna A; Terasawa, Kazue; Hasegawa, Katsuya; Namba, Toshimitsu; Kumei, Yasuhiro; Yanagishita, Masaki; Hara-Yokoyama, Miki

    2015-02-01

    The intracellular positioning of both lysosomes and mitochondria meets the requirements of degradation and energy supply, which are respectively the two major functions for cellular maintenance. The positioning of both lysosomes and mitochondria is apparently affected by the nutrient status of the cells. However, the mechanism coordinating the positioning of the organelles has not been sufficiently elucidated. Lysosome-associated membrane proteins-1 and -2 (LAMP-1 and LAMP-2) are highly glycosylated proteins that are abundant in lysosomal membranes. In the present study, we demonstrated that the siRNA-mediated downregulation of LAMP-1, LAMP-2 or their combination enhanced the perinuclear localization of mitochondria, in the pre-osteoblastic cell line MC3T3-E1. On the other hand, in the osteocytic cell line MLO-Y4, in which both the lysosomes and mitochondria originally accumulate in the perinuclear region and mitochondria also fill dendrites, the effect of siRNA of LAMP-1 or LAMP-2 was barely observed. LAMPs are not directly associated with mitochondria, and there do not seem to be any accessory molecules commonly required to recruit the motor proteins to lysosomes and mitochondria. Our results suggest that LAMPs may regulate the positioning of lysosomes and mitochondria. A possible mechanism involving the indirect and context-dependent action of LAMPs is discussed.

  3. Purity matters: A workflow for the valid high-resolution lipid profiling of mitochondria from cell culture samples

    PubMed Central

    Kappler, Lisa; Li, Jia; Häring, Hans-Ulrich; Weigert, Cora; Lehmann, Rainer; Xu, Guowang; Hoene, Miriam

    2016-01-01

    Subcellular lipidomics is a novel field of research that requires the careful combination of several pre-analytical and analytical steps. To define a reliable strategy for mitochondrial lipid profiling, we performed a systematic comparison of different mitochondria isolation procedures by western blot analyses and comprehensive high-resolution lipidomics. Using liver-derived HepG2 cells, we compared three common mitochondria isolation methods, differential centrifugation (DC), ultracentrifugation (UC) and a magnetic bead-assisted method (MACS). In total, 397 lipid species, including 32 cardiolipins, could be quantified in only 100 μg (by protein) of purified mitochondria. Mitochondria isolated by UC showed the highest enrichment in the mitochondria-specific cardiolipins as well as their precursors, phosphatidylglycerols. Mitochondrial fractions obtained by the commonly used DC and the more recent MACS method contained substantial contaminations by other organelles. Employing these isolation methods when performing lipidomics analyses from cell culture mitochondria may lead to inaccurate results. To conclude, we present a protocol how to obtain reliable mitochondria-specific lipid profiles from cell culture samples and show that quality controls are indispensable when performing mitochondria lipidomics. PMID:26892142

  4. Hyperglycemia induces apoptosis and p53 mobilization to mitochondria in RINm5F cells.

    PubMed

    Ortega-Camarillo, C; Guzmán-Grenfell, A M; García-Macedo, R; Rosales-Torres, A M; Avalos-Rodríguez, A; Durán-Reyes, G; Medina-Navarro, R; Cruz, M; Díaz-Flores, M; Kumate, J

    2006-01-01

    The mechanisms related to hyperglycemia-induced pancreatic beta-cell apoptosis are poorly defined. Rat insulin-producing cells (RINm5F) cultured in high glucose concentrations (30 mM) showed increased apoptosis and protein p53 translocation to mitochondria. In addition, hyperglycemia induced both the disruption of mitochondrial membrane potential (Delta psi (m)), and an increase in reactive oxygen species (ROS), as shown by fluorescence changes of JC-1 and dichlorodihydrofluorescein-diacetate (DCDHF-DA), respectively. The increased intracellular ROS by high glucose exposure was blunted by mitochondrial-function and NADPH-oxidase inhibitors. We postulate that the concomitant mobilization of p53 protein to the mitochondria and the subsequent changes on the Delta psi (m), lead to an important pancreatic beta-cell apoptosis mechanism induced by oxidative stress caused by hyperglycemia.

  5. P53 mediates amosite asbestos-induced alveolar epithelial cell mitochondria-regulated apoptosis.

    PubMed

    Panduri, Vijayalakshmi; Surapureddi, Sailesh; Soberanes, Saul; Weitzman, Sigmund A; Chandel, Navdeep; Kamp, David W

    2006-04-01

    Asbestos causes pulmonary toxicity in part by generating reactive oxygen species that cause DNA damage. We previously showed that the mitochondria-regulated (intrinsic) death pathway mediates alveolar epithelial cell (AEC) DNA damage and apoptosis. Because p53 regulates the DNA damage response in part by inducing intrinsic cell death, we determined whether p53-dependent transcriptional activity mediates asbestos-induced AEC mitochondrial dysfunction and apoptosis. We show that inhibitors of p53-dependent transcriptional activation (pifithrin and type 16-E6 protein) block asbestos-induced AEC mitochondrial membrane potential change (DeltaPsim), caspase 9 activation, and apoptosis. We demonstrate that asbestos activates p53 promoter activity, mRNA levels, protein expression, and Bax and p53 mitochondrial translocation. Further, pifithrin, E6, phytic acid, or rho(0)-A549 cells (cells incapable of mitochondrial reactive oxygen species production) block asbestos-induced p53 activation. Finally, we show that asbestos augments p53 expression in cells at the bronchoalveolar duct junctions of rat lungs and that phytic acid prevents this. These data suggest that p53-dependent transcription pathways mediate asbestos-induced AEC mitochondria-regulated apoptosis. This suggests an important interactive effect between p53 and the mitochondria in the pathogenesis of asbestos-induced pulmonary toxicity that may have broader implications for our understanding of pulmonary fibrosis and lung cancer.

  6. Neural Cell Apoptosis Induced by Microwave Exposure Through Mitochondria-dependent Caspase-3 Pathway

    PubMed Central

    Zuo, Hongyan; Lin, Tao; Wang, Dewen; Peng, Ruiyun; Wang, Shuiming; Gao, Yabing; Xu, Xinping; Li, Yang; Wang, Shaoxia; Zhao, Li; Wang, Lifeng; Zhou, Hongmei

    2014-01-01

    To determine whether microwave (MW) radiation induces neural cell apoptosis, differentiated PC12 cells and Wistar rats were exposed to 2.856GHz for 5min and 15min, respectively, at an average power density of 30 mW/cm2. JC-1 and TUNEL staining detected significant apoptotic events, such as the loss of mitochondria membrane potential and DNA fragmentation, respectively. Transmission electron microscopy and Hoechst staining were used to observe chromatin ultrastructure and apoptotic body formation. Annexin V-FITC/PI double staining was used to quantify the level of apoptosis. The expressions of Bax, Bcl-2, cytochrome c, cleaved caspase-3 and PARP were examined by immunoblotting or immunocytochemistry. Caspase-3 activity was measured using an enzyme-linked immunosorbent assay. The results showed chromatin condensation and apoptotic body formation in neural cells 6h after microwave exposure. Moreover, the mitochondria membrane potential decreased, DNA fragmentation increased, leading to an increase in the apoptotic cell percentage. Furthermore, the ratio of Bax/Bcl-2, expression of cytochrome c, cleaved caspase-3 and PARP all increased. In conclusion, microwave radiation induced neural cell apoptosis via the classical mitochondria-dependent caspase-3 pathway. This study may provide the experimental basis for further investigation of the mechanism of the neurological effects induced by microwave radiation. PMID:24688304

  7. Th17 cells and Mucosal Host Defense

    PubMed Central

    Aujla, Shean J.; Dubin, Patricia J.; Kolls, Jay K.

    2008-01-01

    Th17 cells are a new lineage of T-cells that are controlled by the transcription factor RORγt and develop independent of GATA-3, T-bet, Stat 4 and Stat 6. Novel effector molecules produced by these cells include IL-17A, IL-17F, IL-22, and IL-26. IL-17RA binds IL-17A and IL-17F and is critical for host defense against extracellular planktonic bacteria by regulating chemokine gradients for neutrophil emigration into infected tissue sites as well as host granulopoiesis. Moreover IL-17 and IL-22 regulate the production of antimicrobial proteins in mucosal epithelium. Although TGF-β1 and IL-6 have been shown to be critical for development of Th17 cells from naïve precursors, IL-23 is also important in regulating IL-17 release in mucosal tissues in response to infectious stimuli. Compared to Th1 cells, IL-23 and IL-17 show limited roles in controlling host defense against primary infections with intracellular bacteria such as Mycobacterium tuberculosis suggesting a predominate role of the Th17 lineage in host defense against extracellular pathogens. However in the setting of chronic biofilm infections, as that occurs with Cystic Fibrosis or bronchetctasis, Th17 cells may be key contributors of tissue injury. PMID:18054248

  8. Novel localisation and possible function of LIN7 and IRSp53 in mitochondria of HeLa cells.

    PubMed

    Ferrari, Ilaria; Crespi, Arianna; Fornasari, Diego; Pietrini, Grazia

    2016-08-01

    By means of immunofluorescence and subcellular fractionation experiments, we here demonstrate mitochondrial distribution of LIN7 and IRSp53 in HeLa cells. These peripheral proteins displayed a tight association with mitochondria and coimmunoprecipitated from mitochondrial fractions. In line with a role for LIN7 in the regulation of IRSp53 activity on actin dynamics, the morphology of mitochondria was similarly altered by changing the expression levels of either each protein or both, whereas mitochondrial morphology was preserved in cells overexpressing IRSp53 deleted of its binding domains for LIN7 (IRSp53Δ5) or for actin polymerisation modulators (IRSp53ΔSH3). In particular, the overexpression of full length LIN7 and/or IRSp53 increased the percentage of cells with short mitochondria, while downregulation of the endogenous proteins by shRNAs increased the amount of cells with elongated and perinuclear clustered mitochondria. These mitochondria were only partially resistant to fragmentation induced by dissipation of the mitochondrial membrane potential (i.e. treatment with sodium azide), whereas mitochondria were fully protected by the fission defective mutant Drp1 K38A. Overexpression of LIN7 or IRSp53 did not prevent the formation of hyperfused mitochondria in cells coexpressing the Drp1 K38A mutant, thus suggesting that LIN7-IRSp53 complex requires functional Drp1 to regulate mitochondrial morphology.

  9. Mitochondria play an important role in the cell proliferation suppressing activity of berberine

    PubMed Central

    Yan, Xiao-Jin; Yu, Xuan; Wang, Xin-Pei; Jiang, Jing-Fei; Yuan, Zhi-Yi; Lu, Xi; Lei, Fan; Xing, Dong-Ming

    2017-01-01

    After being studied for approximately a century, berberine (BBR) has been found to act on various targets and pathways. A great challenge in the pharmacological analysis of BBR at present is to identify which target(s) plays a decisive role. In the study described herein, a rescue experiment was designed to show the important role of mitochondria in BBR activity. A toxic dose of BBR was applied to inhibit cell proliferation and mitochondrial activity, then α-ketobutyrate (AKB), an analogue of pyruvate that serves only as an electron receptor of NADH, was proven to partially restore cell proliferation. However, mitochondrial morphology damage and TCA cycle suppression were not recovered by AKB. As the AKB just help to regenerate NAD+, which is make up for part function of mitochondrial, the recovered cell proliferation stands for the contribution of mitochondria to the activity of BBR. Our results also indicate that BBR suppresses tumour growth and reduces energy charge and mitochondrial DNA (mtDNA) copy number in a HepG2 xenograft model. In summary, our study suggests that mitochondria play an important role in BBR activity regarding tumour cell proliferation and metabolism. PMID:28181523

  10. Mitochondria and cell death: outer membrane permeabilization and beyond.

    PubMed

    Tait, Stephen W G; Green, Douglas R

    2010-09-01

    Mitochondrial outer membrane permeabilization (MOMP) is often required for activation of the caspase proteases that cause apoptotic cell death. Various intermembrane space (IMS) proteins, such as cytochrome c, promote caspase activation following their mitochondrial release. As a consequence, mitochondrial outer membrane integrity is highly controlled, primarily through interactions between pro- and anti-apoptotic members of the B cell lymphoma 2 (BCL-2) protein family. Following MOMP by pro-apoptotic BCL-2-associated X protein (BAX) or BCL-2 antagonist or killer (BAK), additional regulatory mechanisms govern the mitochondrial release of IMS proteins and caspase activity. MOMP typically leads to cell death irrespective of caspase activity by causing a progressive decline in mitochondrial function, although cells can survive this under certain circumstances, which may have pathophysiological consequences.

  11. Mitochondria modify exercise-induced development of stem cell-derived neurons in the adult brain.

    PubMed

    Steib, Kathrin; Schäffner, Iris; Jagasia, Ravi; Ebert, Birgit; Lie, D Chichung

    2014-05-07

    Neural stem cells in the adult mammalian hippocampus continuously generate new functional neurons, which modify the hippocampal network and significantly contribute to cognitive processes and mood regulation. Here, we show that the development of new neurons from stem cells in adult mice is paralleled by extensive changes to mitochondrial mass, distribution, and shape. Moreover, exercise-a strong modifier of adult hippocampal neurogenesis-accelerates neuronal maturation and induces a profound increase in mitochondrial content and the presence of mitochondria in dendritic segments. Genetic inhibition of the activity of the mitochondrial fission factor dynamin-related protein 1 (Drp1) inhibits neurogenesis under basal and exercise conditions. Conversely, enhanced Drp1 activity furthers exercise-induced acceleration of neuronal maturation. Collectively, these results indicate that adult hippocampal neurogenesis requires adaptation of the mitochondrial compartment and suggest that mitochondria are targets for enhancing neurogenesis-dependent hippocampal plasticity.

  12. GLP-1 promotes mitochondrial metabolism in vascular smooth muscle cells by enhancing endoplasmic reticulum-mitochondria coupling.

    PubMed

    Morales, Pablo E; Torres, Gloria; Sotomayor-Flores, Cristian; Peña-Oyarzún, Daniel; Rivera-Mejías, Pablo; Paredes, Felipe; Chiong, Mario

    2014-03-28

    Incretin GLP-1 has important metabolic effects on several tissues, mainly through the regulation of glucose uptake and usage. One mechanism for increasing cell metabolism is modulating endoplasmic reticulum (ER)-mitochondria communication, as it allows for a more efficient transfer of Ca(2+) into the mitochondria, thereby increasing activity. Control of glucose metabolism is essential for proper vascular smooth muscle cell (VSMC) function. GLP-1 has been shown to produce varied metabolic actions, but whether it regulates glucose metabolism in VSMC remains unknown. In this report, we show that GLP-1 increases mitochondrial activity in the aortic cell line A7r5 by increasing ER-mitochondria coupling. GLP-1 increases intracellular glucose and diminishes glucose uptake without altering glycogen content. ATP, mitochondrial potential and oxygen consumption increase at 3h of GLP-1 treatment, paralleled by increased Ca(2+) transfer from the ER to the mitochondria. Furthermore, GLP-1 increases levels of Mitofusin-2 (Mfn2), an ER-mitochondria tethering protein, via a PKA-dependent mechanism. Accordingly, PKA inhibition and Mfn2 down-regulation prevented mitochondrial Ca(2+) increases in GLP-1 treated cells. Inhibiting both Ca(2+) release from the ER and Ca(2+) entry into mitochondria as well as diminishing Mfn2 levels blunted the increase in mitochondrial activity in response to GLP-1. Altogether, these results strongly suggest that GLP-1 increases ER-mitochondria communication in VSMC, resulting in higher mitochondrial activity.

  13. Ultrasensitive fluorescent ratio imaging probe for the detection of glutathione ultratrace change in mitochondria of cancer cells.

    PubMed

    Zhang, Hua; Wang, Caixia; Wang, Kui; Xuan, Xiaopeng; Lv, Qingzhang; Jiang, Kai

    2016-11-15

    Glutathione (GSH) ultratrace change in mitochondria of cancer cells can mildly and effectively induce cancer cells apoptosis in early stage. Thus, if GSH ultratrace change in mitochondria of cancer cells could be recognized and imaged, it will be beneficial for fundamental research of cancer therapy. There have reported a lot of fluorescent probes for GSH, but the fluorescent probe with ultrasensitivity and high selectivity for the ratio imaging of GSH ultratrace changes in mitochondria of cancer cells is scarce. Herein, based on different reaction mechanism of sulfonamide under different pH, a sulfonamide-based reactive ratiometric fluorescent probe (IQDC-M) was reported for the recognizing and imaging of GSH ultratrace change in mitochondria of cancer cells. The detection limit of IQDC-M for GSH ultratrace change is low to 2.02nM, which is far less than 1.0‰ of endogenic GSH in living cells. And during the recognition process, IQDC-M can emit different fluorescent signals at 520nm and 592nm, which results in it recognizing GSH ultratrace change on ratio mode. More importantly, IQDC-M recognizing GSH ultratrace change specifically occurs in mitochondria of cancer cells because of appropriate water/oil amphipathy (log P) of IQDC-M. So, these make IQDC-M possible to image and monitor GSH ultratrace change in mitochondria during cancer cells apoptosis for the first time.

  14. Cancer cell mitochondria confer apoptosis resistance and promote metastasis.

    PubMed

    Kulawiec, Mariola; Owens, Kjerstin M; Singh, Keshav K

    2009-07-01

    Mutations in mtDNA are found in most cancers. In this study, we studied the role of cancer cell mutant mtDNA in tumorigenesis. We sequenced the entire mitochondrial genome of three different breast cancer cell lines and found that all three, MCF7, MDA-MB-231 and MDA-MB-435, contained mutations in mtDNA. MDA-MB-435 cells contained a mutation in the tRNA(Leu(CUN)) gene known to be involved in pathogenesis of mitochondrial diseases. We generated a mutant cybrid (cytoplasmic hybrid) by repopulating the recipient rho(0) (completely devoid of mtDNA) cells with donor mtDNA derived from an enucleated MDA-MB-435 breast cancer cell line. An isogenic wild-type cybrid was produced by transfer of normal mtDNA from a healthy donor. When compared to the wild type, we found that mutant mtDNA increases mitochondrial membrane potential. However, this increase in mitochondrial membrane potential was not associated with increase in reactive oxygen species (ROS) production. MtDNA mutations conferred resistance to apoptosis triggered by etoposide. Our study also revealed that mutations in mtDNA increase metastatic potential. Using a tail-vein model of metastasis in a mouse model, we show that the mutant cybrid metastatizes to the lungs and forms macrometastic foci. Additionally we found that mutations in mtDNA constitutively activate the PI3/Akt pathway that contributes to increased metastatis. Together our study demonstrates that mutant mtDNA promotes apoptotic resistance and metastasis in a mouse model.

  15. Mitochondria mediates caspase-dependent and independent retinal cell death in Staphylococcus aureus endophthalmitis

    PubMed Central

    Singh, P K; Kumar, A

    2016-01-01

    Bacterial endophthalmitis, a vision-threatening complication of ocular surgery or trauma, is characterized by increased intraocular inflammation and retinal tissue damage. Although significant vision loss in endophthalmitis has been linked to retinal cell death, the underlying mechanisms of cell death remain elusive. In this study, using a mouse model of Staphylococcus aureus endophthalmitis and cultured human retinal Müller glia (MIO-M1 cell line), we demonstrate that S. aureus caused significant apoptotic cell death in the mouse retina and Müller glia, as evidenced by increased number of terminal dUTP nick end labeling and Annexin V and propidium iodide-positive cells. Immunohistochemistry and western blot studies revealed the reduction in mitochondrial membrane potential (JC-1 staining), release of cytochrome c into the cytosol, translocation of Bax to the mitochondria and the activation of caspase-9 and -3 in S. aureus-infected retina/retinal cells. In addition, the activation of PARP-1 and the release of apoptosis inducing factor from mitochondria was also observed in S. aureus-infected retinal cells. Inhibition studies using pan-caspase (Q-VD-OPH) and PARP-1 (DPQ) inhibitors showed significant reduction in S. aureus-induced retinal cell death both in vivo and in vitro. Together, our findings demonstrate that in bacterial endophthalmitis, retinal cells undergo apoptosis in the both caspase-dependent and independent manners, and mitochondria have a central role in this process. Hence, targeting the identified signaling pathways may provide the rationale to design therapeutic interventions to prevent bystander retinal tissue damage in bacterial endophthalmitis. PMID:27551524

  16. Targetable fluorescent probe for monitoring exogenous and endogenous NO in mitochondria of living cells.

    PubMed

    Yu, Haibo; Zhang, Xinfu; Xiao, Yi; Zou, Wei; Wang, Liping; Jin, Liji

    2013-08-06

    Nitric oxide (NO) is a ubiquitous cellular messenger molecule in the cardiovascular, nervous, and immune systems. Mitochondrion is the main area where endogenous NO is synthesized by inducible NOS enzymes in mammalian cells. Thus, real-time monitoring NO in mitochondria is very meaningful for NO chemical biology. Although a variety of fluorescent probes for NO have been successfully developed, they are not suited for detecting mitochondrial NO because none of them can specifically localize in mitochondria. Herein, Mito-Rh-NO, the first mitochondria-targetable "turn-on" fluorescent probe for NO, has been developed through attaching a triphenylphosphonium to a rhodamine spirolactam. The characteristics of this probe are as following: (1) Mito-Rh-NO exhibits high sensitivity toward NO. In solution, Mito-Rh-NO responds to NO by significant fluorescence enhancement up to 60-fold, and its NO detection limit is as low as 4.0 nM. (2) The NO sensing of Mito-Rh-NO is highly selective, which will not interfere with the other reactive oxygen and nitrogen species. (3) Mito-Rh-NO has a low cytotoxic effect: after being treated with 10 μM Mito-Rh-NO for 24 h, the survival rate is higher than 90%. (4) Mito-Rh-NO specifically localizing in mitochondria: colocalization experiment of Mito-Rh-NO and Rh 123, a typical mitotracker, shows the merged fluorescent microcopy image with a high Pearson's colocalization coefficient 0.92 and overlap coefficient 0.99. (5) Mito-Rh-NO demonstrates high applicability for real-time monitoring of mitochondrial NO in live cells. Both the exogenous NO released by the donor NOC13 and endogenous NO generated in cells under stimulation have been visualized under confocal microscopy.

  17. A Mitochondria-Targeted Nitroxide/Hemigramicidin S Conjugate Protects Mouse Embryonic Cells Against Gamma Irradiation

    SciTech Connect

    Jiang Jianfei; Belikova, Natalia A.; Hoye, Adam T.; Zhao Qing; Epperly, Michael W.; Greenberger, Joel S.; Wipf, Peter; Kagan, Valerian E.

    2008-03-01

    Purpose: To evaluate the in vitro radioprotective effect of the mitochondria-targeted hemigramicidin S-conjugated 4-amino-2,2,6,6-tetramethyl-piperidine-N-oxyl (hemi-GS-TEMPO) 5-125 in {gamma}-irradiated mouse embryonic cells and adenovirus-12 SV40 hybrid virus transformed human bronchial epithelial cells BEAS-2B and explore the mechanisms involved in its radioprotective effect. Methods and Materials: Cells were incubated with 5-125 before (10 minutes) or after (1 hour) {gamma}-irradiation. Superoxide generation was determined by using dihydroethidium assay, and lipid oxidation was quantitated by using a fluorescence high-performance liquid chromatography-based Amplex Red assay. Apoptosis was characterized by evaluating the accumulation of cytochrome c in the cytosol and externalization of phosphatidylserine on the cell surface. Cell survival was measured by means of a clonogenic assay. Results: Treatment (before and after irradiation) of cells with 5-125 at low concentrations (5, 10, and 20 {mu}M) effectively suppressed {gamma}-irradiation-induced superoxide generation, cardiolipin oxidation, and delayed irradiation-induced apoptosis, evaluated by using cytochrome c release and phosphatidylserine externalization. Importantly, treatment with 5-125 increased the clonogenic survival rate of {gamma}-irradiated cells. In addition, 5-125 enhanced and prolonged {gamma}-irradiation-induced G{sub 2}/M phase arrest. Conclusions: Radioprotection/mitigation by hemi-GS-TEMPO likely is caused by its ability to act as an electron scavenger and prevent superoxide generation, attenuate cardiolipin oxidation in mitochondria, and hence prevent the release of proapoptotic factors from mitochondria. Other mechanisms, including cell-cycle arrest at the G{sub 2}/M phase, may contribute to the protection.

  18. Microsporidia infection impacts the host cell's cycle and reduces host cell apoptosis.

    PubMed

    Martín-Hernández, Raquel; Higes, Mariano; Sagastume, Soledad; Juarranz, Ángeles; Dias-Almeida, Joyce; Budge, Giles E; Meana, Aránzazu; Boonham, Neil

    2017-01-01

    Intracellular parasites can alter the cellular machinery of host cells to create a safe haven for their survival. In this regard, microsporidia are obligate intracellular fungal parasites with extremely reduced genomes and hence, they are strongly dependent on their host for energy and resources. To date, there are few studies into host cell manipulation by microsporidia, most of which have focused on morphological aspects. The microsporidia Nosema apis and Nosema ceranae are worldwide parasites of honey bees, infecting their ventricular epithelial cells. In this work, quantitative gene expression and histology were studied to investigate how these two parasites manipulate their host's cells at the molecular level. Both these microsporidia provoke infection-induced regulation of genes involved in apoptosis and the cell cycle. The up-regulation of buffy (which encodes a pro-survival protein) and BIRC5 (belonging to the Inhibitor Apoptosis protein family) was observed after infection, shedding light on the pathways that these pathogens use to inhibit host cell apoptosis. Curiously, different routes related to cell cycle were modified after infection by each microsporidia. In the case of N. apis, cyclin B1, dacapo and E2F2 were up-regulated, whereas only cyclin E was up-regulated by N. ceranae, in both cases promoting the G1/S phase transition. This is the first report describing molecular pathways related to parasite-host interactions that are probably intended to ensure the parasite's survival within the cell.

  19. Evolution and cell physiology. 2. The evolution of cell signaling: from mitochondria to Metazoa.

    PubMed

    Blackstone, Neil W

    2013-11-01

    The history of life is a history of levels-of-selection transitions. Each transition requires mechanisms that mediate conflict among the lower-level units. In the origins of multicellular eukaryotes, cell signaling is one such mechanism. The roots of cell signaling, however, may extend to the previous major transition, the origin of eukaryotes. Energy-converting protomitochondria within a larger cell allowed eukaryotes to transcend the surface-to-volume constraints inherent in the design of prokaryotes. At the same time, however, protomitochondria can selfishly allocate energy to their own replication. Metabolic signaling may have mediated this principal conflict in several ways. Variation of the protomitochondria was constrained by stoichiometry and strong metabolic demand (state 3) exerted by the protoeukaryote. Variation among protoeukaryotes was increased by the sexual stage of the life cycle, triggered by weak metabolic demand (state 4), resulting in stochastic allocation of protomitochondria to daughter cells. Coupled with selection, many selfish protomitochondria could thus be removed from the population. Hence, regulation of states 3 and 4, as, for instance, provided by the CO2/soluble adenylyl cyclase/cAMP pathway in mitochondria, was critical for conflict mediation. Subsequently, as multicellular eukaryotes evolved, metabolic signaling pathways employed by eukaryotes to mediate conflict within cells could now be co-opted into conflict mediation between cells. For example, in some fungi, the CO2/soluble adenylyl cyclase/cAMP pathway regulates the transition from yeast to forms with hyphae. In animals, this pathway regulates the maturation of sperm. While the particular features (sperm and hyphae) are distinct, both may involve between-cell conflicts that required mediation.

  20. Characterization of mitochondria in cisplatin-resistant human ovarian carcinoma cells.

    PubMed

    Hirama, Masanori; Isonishi, Seiji; Yasuda, Makoto; Ishikawa, Hiroshi

    2006-11-01

    One of the mechanisms of cisplatin cell cytotoxicity is the mitochondria-associated induction of apoptosis. The morphological or functional change of mitochondria in cisplatin-resistant cells has already been reported. Herein we present additional data describing the mitochondrial genomic and functional changes in cisplatin- resistant cells. Cisplatin increased the level of apoptotic cells in cisplatin-sensitive human ovarian carcinoma OV 2008 and C13 cells by 3.90+/-1.01 (SD; N=3) (p<0.01)-fold and 2.03+/-0.20 (SD; N=3) (p<0.01)-fold compared to the basal apoptotic level. This indicates a lower level induction of apoptosis by 50% in cisplatin-resistant OV 2008/C13 *5.25 variant (C13) cells. In both cell types, cisplatin cytotoxicity is mostly inhibited by the caspase-9 inhibitor as well as the caspase-3 inhibitor, Ac-DEVD-CHO, suggesting that the mitochondrial downstream event was functioning well in both the C13 cells and in OV 2008 cells. Mitochondrial transmembrane potential (DeltaPsim) determined by flow cytometry using DiOC6-stained cells revealed a significant depolarization of C13 cells as compared to OV 2008 cells. Treatment of these cells with cisplatin or hydrogen peroxide induces complete mitochondrial DNA damage in OV 2008 cells, while only partial DNA-destruction is observed in C13 cells, strongly suggesting that mitochondria are resistant to cisplatin and oxidative stress response. Continuous oxygen consumption of these cells monitored by a multi-channel dissolved oxygen meter is 1.70-fold higher in OV 2008 cells than C13 cells and the oxygen consumption was decreased by 30% in C13 cells, suggesting mitochondrial respiratory malfunction in these cells. The hypothesis generated here is that mitochondrial DNA resistance to cisplatin and oxidative stress response might be one of the main characteristics concerning the lower level of apoptosis induced by cisplatin. However, the mechanism by which the mitochondrial DNA encoded molecule is involved in

  1. Inhibition of oxidative phosphorylation in ascites tumor mitochondria and cells by intramitochondrial Ca2+.

    PubMed

    Villalobo, A; Lehninger, A L

    1980-03-25

    Accumulation of Ca2+ (+ phosphate) by respiring mitochondria from Ehrlich ascites or AS30-D hepatoma tumor cells inhibits subsequent phosphorylating respiration in response to ADP. The respiratory chain is still functional since a proton-conducting uncoupler produces a normal stimulation of electron transport. The inhibition of phosphorylating respiration is caused by intramitochondrial Ca2+ (+ phosphate). ATP + Mg2+ together, but not singly, prevents the inhibitory action of Ca2+. Neither AMP, GTP, GDP, nor any other nucleoside 5'-triphosphate or 5'-diphosphate could replace ATP in this effect. Phosphorylating respiration on NAD(NADP)-linked substrates was much more susceptible to the inhibitory effect of intramitochondrial Ca2+ than succinate-linked respiration. Significant inhibition of oxidative phosphorylation is given by the endogenous Ca2+ present in freshly isolated tumor mitochondria. The phosphorylating respiration of permeabilized Ehrlich ascites tumor cells is also inhibited by Ca2+ accumulated by the mitochondria in situ. Possible causes of the Ca2+-induced inhibition of oxidative phosphorylation are considered.

  2. A sensitive and specific Raman probe based on bisarylbutadiyne for live cell imaging of mitochondria.

    PubMed

    Yamakoshi, Hiroyuki; Palonpon, Almar; Dodo, Kosuke; Ando, Jun; Kawata, Satoshi; Fujita, Katsumasa; Sodeoka, Mikiko

    2015-02-01

    We previously showed that bisarylbutadiyne (BADY), which has a conjugated diyne structure, exhibits an intense peak in the cellular Raman-silent region. Here, we synthesized a mitochondria-selective Raman probe by linking bisphenylbutadiyne with triphenylphosphonium, a well-known mitochondrial targeting moiety. This probe, named MitoBADY, has a Raman peak 27 times more intense than that of 5-ethynyl-2'-deoxyuridine. Raman microscopy using submicromolar extracellular probe concentrations successfully visualized mitochondria in living cells. A full Raman spectrum is acquired at each pixel of the scanned sample, and we showed that simultaneous Raman imaging of MitoBADY and endogenous cellular biomolecules can be achieved in a single scan. MitoBADY should be useful for the study of mitochondrial dynamics.

  3. Acanthamoeba induces cell-cycle arrest in host cells.

    PubMed

    Sissons, James; Alsam, Selwa; Jayasekera, Samantha; Kim, Kwang Sik; Stins, Monique; Khan, Naveed Ahmed

    2004-08-01

    Acanthamoeba can cause fatal granulomatous amoebic encephalitis (GAE) and eye keratitis. However, the pathogenesis and pathophysiology of these emerging diseases remain unclear. In this study, the effects of Acanthamoeba on the host cell cycle using human brain microvascular endothelial cells (HBMEC) and human corneal epithelial cells (HCEC) were determined. Two isolates of Acanthamoeba belonging to the T1 genotype (GAE isolate) and T4 genotype (keratitis isolate) were used, which showed severe cytotoxicity on HBMEC and HCEC, respectively. No tissue specificity was observed in their ability to exhibit binding to the host cells. To determine the effects of Acanthamoeba on the host cell cycle, a cell-cycle-specific gene array was used. This screened for 96 genes specific for host cell-cycle regulation. It was observed that Acanthamoeba inhibited expression of genes encoding cyclins F and G1 and cyclin-dependent kinase 6, which are proteins important for cell-cycle progression. Moreover, upregulation was observed of the expression of genes such as GADD45A and p130 Rb, associated with cell-cycle arrest, indicating cell-cycle inhibition. Next, the effect of Acanthamoeba on retinoblastoma protein (pRb) phosphorylation was determined. pRb is a potent inhibitor of G1-to-S cell-cycle progression; however, its function is inhibited upon phosphorylation, allowing progression into S phase. Western blotting revealed that Acanthamoeba abolished pRb phosphorylation leading to cell-cycle arrest at the G1-to-S transition. Taken together, these studies demonstrated for the first time that Acanthamoeba inhibits the host cell cycle at the transcriptional level, as well as by modulating pRb phosphorylation using host cell-signalling mechanisms. A complete understanding of Acanthamoeba-host cell interactions may help in developing novel strategies to treat Acanthamoeba infections.

  4. Diversity in host clone performance within a Chinese hamster ovary cell line.

    PubMed

    O'Callaghan, Peter M; Berthelot, Maud E; Young, Robert J; Graham, James W A; Racher, Andrew J; Aldana, Dulce

    2015-01-01

    Much effort has been expended to improve the capabilities of individual Chinese hamster ovary (CHO) host cell lines to synthesize recombinant therapeutic proteins (rPs). However, given the increasing variety in rP molecular types and formats it may be advantageous to employ a toolbox of CHO host cell lines in biomanufacturing. Such a toolbox would contain a panel of hosts with specific capabilities to synthesize certain molecular types at high volumetric concentrations and with the correct product quality (PQ). In this work, we examine a panel of clonally derived host cell lines isolated from CHOK1SV for the ability to manufacture two model proteins, an IgG4 monoclonal antibody (Mab) and an Fc-fusion protein (etanercept). We show that these host cell lines vary in their relative ability to synthesize these proteins in transient and stable pool production format. Furthermore, we examined the PQ attributes of the stable pool-produced Mab and etanercept (by N-glycan ultra performance liquid chromatography (UPLC) and liquid chromatography - tandem mass spectrometry (LC-MS/MS), respectively), and uncovered substantial variation between the host cell lines in Mab N-glycan micro-heterogeneity and etanercept N and O-linked macro-heterogeneity. To further investigate the capabilities of these hosts to act as cell factories, we examined the glycosylation pathway gene expression profiles as well as the levels of endoplasmic reticulum (ER) and mitochondria in the untransfected hosts. We uncovered a moderate correlation between ER mass and the volumetric product concentration in transient and stable pool Mab production. This work demonstrates the utility of leveraging diversity within the CHOK1SV pool to identify new host cell lines with different performance characteristics.

  5. The T cell receptor gamma chain alternate reading frame protein (TARP), a prostate-specific protein localized in mitochondria.

    PubMed

    Maeda, Hiroshi; Nagata, Satoshi; Wolfgang, Curt D; Bratthauer, Gary L; Bera, Tapan K; Pastan, Ira

    2004-06-04

    We previously showed that mRNA encoding TARP (T cell receptor gamma chain alternate reading frame protein) is exclusively expressed in the prostate in males and is up-regulated by androgen in LNCaP cells, an androgen-sensitive prostate cancer cell line. We have now developed an anti-TARP monoclonal antibody named TP1, and show that TARP protein is up-regulated by androgen in both LNCaP and MDA-PCa-2b cells. We used TP1 to determine the subcellular localization of TARP by Western blotting following subcellular fractionation and immunocytochemistry. Both methods showed that TARP is localized in the mitochondria of LNCaP cells, MDA-PCa-2b cells, and PC-3 cells transfected with a TARP-expressing plasmid. We also transfected a plasmid encoding TARP fused to green fluorescent protein into LNCaP, MDA-Pca-2b, and PC-3 cells and confirmed its specific mitochondrial localization in living cells. Fractionation of mitochondria shows that TARP is located in the outer mitochondrial membrane. Immunohistochemistry using a human prostate cancer sample showed that TP1 reacted in a dot-like cytoplasmic pattern consistent with the presence of TARP in mitochondria. These data demonstrate that TARP is the first prostate-specific protein localizing in mitochondria and indicate that TARP, an androgen-regulated protein, may act on mitochondria to carry out its biological functions.

  6. Frataxin-bypassing Isu1: characterization of the bypass activity in cells and mitochondria.

    PubMed

    Yoon, Heeyong; Knight, Simon A B; Pandey, Alok; Pain, Jayashree; Zhang, Yan; Pain, Debkumar; Dancis, Andrew

    2014-04-01

    Frataxin is a conserved mitochondrial protein, and deficiency underlies the neurodegenerative disease Friedreich's ataxia. Frataxin interacts with the core machinery for Fe-S cluster assembly in mitochondria. Recently we reported that in frataxin-deleted yeast strains, a spontaneously occurring mutation in one of two genes encoding redundant Isu scaffold proteins, bypassed the mutant phenotypes. In the present study we created strains expressing a single scaffold protein, either Isu1 or the bypass mutant M107I Isu1. Our results show that in the frataxin-deletion strain expressing the bypass mutant Isu1, cell growth, Fe-S cluster protein activities, haem proteins and iron homoeostasis were restored to normal or close to normal. The bypass effects were not mediated by changes in Isu1 expression level. The persulfide-forming activity of the cysteine desulfurase was diminished in the frataxin deletion (∆yfh1 ISU1) and was improved by expression of the bypass Isu1 (∆yfh1 M107I ISU1). The addition of purified bypass M107I Isu1 protein to a ∆yfh1 lysate conferred similar enhancement of cysteine desulfurase as did frataxin, suggesting that this effect contributed to the bypass mechanism. Fe-S cluster-forming activity in isolated mitochondria was stimulated by the bypass Isu1, albeit at a lower rate. The rescuing effects of the bypass Isu1 point to ways that the core defects in Friedreich's ataxia mitochondria can be restored.

  7. Microsporidia infection impacts the host cell's cycle and reduces host cell apoptosis

    PubMed Central

    Higes, Mariano; Sagastume, Soledad; Juarranz, Ángeles; Dias-Almeida, Joyce; Budge, Giles E.; Meana, Aránzazu; Boonham, Neil

    2017-01-01

    Intracellular parasites can alter the cellular machinery of host cells to create a safe haven for their survival. In this regard, microsporidia are obligate intracellular fungal parasites with extremely reduced genomes and hence, they are strongly dependent on their host for energy and resources. To date, there are few studies into host cell manipulation by microsporidia, most of which have focused on morphological aspects. The microsporidia Nosema apis and Nosema ceranae are worldwide parasites of honey bees, infecting their ventricular epithelial cells. In this work, quantitative gene expression and histology were studied to investigate how these two parasites manipulate their host’s cells at the molecular level. Both these microsporidia provoke infection-induced regulation of genes involved in apoptosis and the cell cycle. The up-regulation of buffy (which encodes a pro-survival protein) and BIRC5 (belonging to the Inhibitor Apoptosis protein family) was observed after infection, shedding light on the pathways that these pathogens use to inhibit host cell apoptosis. Curiously, different routes related to cell cycle were modified after infection by each microsporidia. In the case of N. apis, cyclin B1, dacapo and E2F2 were up-regulated, whereas only cyclin E was up-regulated by N. ceranae, in both cases promoting the G1/S phase transition. This is the first report describing molecular pathways related to parasite-host interactions that are probably intended to ensure the parasite’s survival within the cell. PMID:28152065

  8. Binding of host-selective toxin analogs to mitochondria from normal and Texas male sterile cytoplasm maize

    SciTech Connect

    Frantzen, K.A.; Daly, J.M.; Knoche, H.W.

    1987-04-01

    Tritium-labeled toxin analogs were prepared by reduction with NaB/sup 3/H/sub 4/ of either the toxin from Helminthosporium maydis race T or a toxin component from Phyllosticta maydis. These reduced analogs had high radiochemical specific activities, high biological activities, and plant specificities identical to the native toxins. A filtration assay was developed to test the binding of these labeled analogs to isolated mitochondria. Binding was not energy dependent nor was there measurable matrical uptake. The analogs were shown to be lipophilic, a characteristic which gave rise to considerable nondisplaceable binding. Under conditions limiting nondisplaceable binding, the displaceable binding was shown to be linear with respect to toxin concentration and unsaturable. No significant differences were observed in the binding characteristics between the mitochondria from normal and male-sterile (Texas) cytoplasm maize. The findings suggest that, at physiologically relevant concentrations, these toxin analogs permeate the membranes of susceptible and resistant mitochondria alike. The lack of demonstrable specific binding does not rule out the involvement of a classical receptor site but does indicate that other kinds of molecular interactions may be involved in the mechanisms for toxicity and specificity.

  9. Superoxide produced in the matrix of mitochondria enhances methylmercury toxicity in human neuroblastoma cells.

    PubMed

    Mailloux, Ryan J; Yumvihoze, Emmanuel; Chan, Hing Man

    2015-12-15

    The mechanism of intracellular metabolism of methylmercury (MeHg) is not fully known. It has been shown that superoxide (O2(-)), the proximal reactive oxygen species (ROS) generated by mitochondria, is responsible for MeHg demethylation. Here, we investigated the impact of different mitochondrial respiratory inhibitors, namely rotenone and antimycin A, on the O2(-)mediated degradation of MeHg in human neuroblastoma cells SH-K-SN. We also utilized paraquat (PQ) which generates O2(-) in the mitochondrial matrix. We found that the cleavage of the carbon-metal bond in MeHg was highly dependent on the topology of O2(-) production by mitochondria. Both rotenone and PQ, which increase O2(-) in the mitochondrial matrix at a dose-dependent manner, enhanced the conversion of MeHg to inorganic mercury (iHg). Surprisingly, antimycin A, which prompts emission of O2(-) into the intermembrane space, did not have the same effect even though antimycin A induced a dose dependent increase in O2(-) emission. Rotenone and PQ also enhanced the toxicity of sub-toxic doses (0.1 μM) MeHg which correlated with the accumulation of iHg in mitochondria and depletion of mitochondrial protein thiols. Taken together, our results demonstrate that MeHg degradation is mediated by mitochondrial O2(-), specifically within the matrix of mitochondria when O2(-) is in adequate supply. Our results also show that O2(-) amplifies MeHg toxicity specifically through its conversion to iHg and subsequent interaction with protein cysteine thiols (R-SH). The implications of our findings in mercury neurotoxicity are discussed herein.

  10. An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell.

    PubMed

    Coffey, Michael J; Sleebs, Brad E; Uboldi, Alessandro D; Garnham, Alexandra; Franco, Magdalena; Marino, Nicole D; Panas, Michael W; Ferguson, David Jp; Enciso, Marta; O'Neill, Matthew T; Lopaticki, Sash; Stewart, Rebecca J; Dewson, Grant; Smyth, Gordon K; Smith, Brian J; Masters, Seth L; Boothroyd, John C; Boddey, Justin A; Tonkin, Christopher J

    2015-11-18

    Infection by Toxoplasma gondii leads to massive changes to the host cell. Here, we identify a novel host cell effector export pathway that requires the Golgi-resident aspartyl protease 5 (ASP5). We demonstrate that ASP5 cleaves a highly constrained amino acid motif that has similarity to the PEXEL-motif of Plasmodium parasites. We show that ASP5 matures substrates at both the N- and C-terminal ends of proteins and also controls trafficking of effectors without this motif. Furthermore, ASP5 controls establishment of the nanotubular network and is required for the efficient recruitment of host mitochondria to the vacuole. Assessment of host gene expression reveals that the ASP5-dependent pathway influences thousands of the transcriptional changes that Toxoplasma imparts on its host cell. All these changes result in attenuation of virulence of Δasp5 tachyzoites in vivo. This work characterizes the first identified machinery required for export of Toxoplasma effectors into the infected host cell.

  11. An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell

    PubMed Central

    Coffey, Michael J; Sleebs, Brad E; Uboldi, Alessandro D; Garnham, Alexandra; Franco, Magdalena; Marino, Nicole D; Panas, Michael W; Ferguson, David JP; Enciso, Marta; O'Neill, Matthew T; Lopaticki, Sash; Stewart, Rebecca J; Dewson, Grant; Smyth, Gordon K; Smith, Brian J; Masters, Seth L; Boothroyd, John C; Boddey, Justin A; Tonkin, Christopher J

    2015-01-01

    Infection by Toxoplasma gondii leads to massive changes to the host cell. Here, we identify a novel host cell effector export pathway that requires the Golgi-resident aspartyl protease 5 (ASP5). We demonstrate that ASP5 cleaves a highly constrained amino acid motif that has similarity to the PEXEL-motif of Plasmodium parasites. We show that ASP5 matures substrates at both the N- and C-terminal ends of proteins and also controls trafficking of effectors without this motif. Furthermore, ASP5 controls establishment of the nanotubular network and is required for the efficient recruitment of host mitochondria to the vacuole. Assessment of host gene expression reveals that the ASP5-dependent pathway influences thousands of the transcriptional changes that Toxoplasma imparts on its host cell. All these changes result in attenuation of virulence of Δasp5 tachyzoites in vivo. This work characterizes the first identified machinery required for export of Toxoplasma effectors into the infected host cell. DOI: http://dx.doi.org/10.7554/eLife.10809.001 PMID:26576949

  12. Selective Vulnerability of Cancer Cells by Inhibition of Ca(2+) Transfer from Endoplasmic Reticulum to Mitochondria.

    PubMed

    Cárdenas, César; Müller, Marioly; McNeal, Andrew; Lovy, Alenka; Jaňa, Fabian; Bustos, Galdo; Urra, Felix; Smith, Natalia; Molgó, Jordi; Diehl, J Alan; Ridky, Todd W; Foskett, J Kevin

    2016-03-15

    In the absence of low-level ER-to-mitochondrial Ca(2+) transfer, ATP levels fall, and AMPK-dependent, mTOR-independent autophagy is induced as an essential survival mechanism in many cell types. Here, we demonstrate that tumorigenic cancer cell lines, transformed primary human fibroblasts, and tumors in vivo respond similarly but that autophagy is insufficient for survival, and cancer cells die while their normal counterparts are spared. Cancer cell death is due to compromised bioenergetics that can be rescued with metabolic substrates or nucleotides and caused by necrosis associated with mitotic catastrophe during their proliferation. Our findings reveal an unexpected dependency on constitutive Ca(2+) transfer to mitochondria for viability of tumorigenic cells and suggest that mitochondrial Ca(2+) addiction is a feature of cancer cells.

  13. Exogenous cardiolipin localizes to mitochondria and prevents TAZ knockdown-induced apoptosis in myeloid progenitor cells.

    PubMed

    Ikon, Nikita; Su, Betty; Hsu, Fong-Fu; Forte, Trudy M; Ryan, Robert O

    2015-08-21

    The concentration and composition of cardiolipin (CL) in mitochondria are altered in age-related heart disease, Barth Syndrome, and other rare genetic disorders, resulting in mitochondrial dysfunction. To explore whether exogenous CL can be delivered to cells, CL was combined with apolipoprotein A-I to generate water-soluble, nanoscale complexes termed nanodisks (ND). Mass spectrometry of HL60 myeloid progenitor cell extracts revealed a 30-fold increase in cellular CL content following incubation with CL-ND. When CL-ND containing a fluorescent CL analogue was employed, confocal microscopy revealed CL localization to mitochondria. The ability of CL-ND to elicit a physiological response was examined in an HL60 cell culture model of Barth Syndrome neutropenia. siRNA knockdown of the phospholipid transacylase, tafazzin (TAZ), induced apoptosis in these cells. When TAZ knockdown cells were incubated with CL-ND, the apoptotic response was attenuated. Thus, CL-ND represent a potential intervention strategy for replenishment of CL in Barth Syndrome, age-related heart disease, and other disorders characterized by depletion of this key mitochondrial phospholipid.

  14. Exogenous cardiolipin localizes to mitochondria and prevents TAZ knockdown-induced apoptosis in myeloid progenitor cells

    PubMed Central

    Ikon, Nikita; Su, Betty; Hsu, Fong-Fu; Forteand, Trudy M.; Ryan, Robert O.

    2015-01-01

    The concentration and composition of cardiolipin (CL) in mitochondria are altered in age-related heart disease, Barth Syndrome, and other rare genetic disorders, resulting in mitochondrial dysfunction. To explore whether exogenous CL can be delivered to cells, CL was combined with apolipoprotein A-I to generate water-soluble, nanoscale complexes termed nanodisks (ND). Mass spectrometry HL60 myeloid progenitor cell extracts revealed a 30-fold increase in cellular CL content following incubation with CL-ND. When CL-ND containing a fluorescent CL analogue was employed, confocal microscopy revealed CL localization to mitochondria. The ability of CL-ND to elicit a physiological response was examined in an HL60 cell culture model of Barth Syndrome neutropenia. siRNA knockdown of the phospholipid transacylase, tafazzin (TAZ), induced apoptosis in these cells. When TAZ knockdown cells were incubated with CL-ND, the apoptotic response was attenuated. Thus, CL-ND represent a potential intervention strategy for replenishment of CL in Barth Syndrome, age-related heart disease, and other disorders characterized by depletion of this key mitochondrial phospholipid. PMID:26164234

  15. Involvement of mitochondria and metacaspase elevation in harpin Pss-induced cell death of Saccharomyces cerevisiae.

    PubMed

    Sripriya, Paranthaman; Vedantam, Lakshmi Vasudev; Podile, Appa Rao

    2009-08-15

    Expression of a proteinaceous elicitor harpin(Pss,) encoded by hrpZ of Pseudomonas syringae pv. syringae 61, under GAL1 promoter in Saccharomyces cerevisiae Y187 resulted in galactose-inducible yeast cell death (YCD). Extracellular treatment of harpin did not affect the growth of yeast. The observed YCD was independent of the stage of cell cycle. "Petite" mutant of S. cerevisiae Y187 pYEUT-hrpZ was insensitive to cell death indicating the involvement of mitochondria in this YCD. Loss in mitochondrial potential, but no leakage of Cytochrome c from mitochondria into the cytosol, were notable features in harpin(Pss)-induced YCD. Cyclosporin A had no effect on hrpZ expressing yeast cells, further confirmed that there was no release of Cytochrome c. Elevation of caspase activity has been reported for the first time in this form of cell death induced by harpin expression. Release of reactive oxygen species and clear loss of membrane integrity were evident with the absence of nuclear fragmentation and chromosomal condensation, while annexin V and propidium iodide staining showed features typical of necrosis.

  16. Cryptococcal Cell Morphology Affects Host Cell Interactions and Pathogenicity

    PubMed Central

    Nielsen, Judith N.; Charlier, Caroline; Baltes, Nicholas J.; Chrétien, Fabrice; Heitman, Joseph; Dromer, Françoise; Nielsen, Kirsten

    2010-01-01

    Cryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 µm. Cell enlargement was observed in vivo, producing cells up to 100 µm. These morphological changes in cell size affected pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement was stimulated by coinfection with strains of opposite mating type, and ste3aΔ pheromone receptor mutant strains had reduced cell enlargement. Finally, analysis of DNA content in this novel cell type revealed that these enlarged cells were polyploid, uninucleate, and produced daughter cells in vivo. These results describe a novel mechanism by which C. neoformans evades host phagocytosis to allow survival of a subset of the population at early stages of infection. Thus, morphological changes play unique and specialized roles during infection. PMID:20585559

  17. Imaging of the Actin Cytoskeleton and Mitochondria in Fixed Budding Yeast Cells.

    PubMed

    Higuchi-Sanabria, Ryo; Swayne, Theresa C; Boldogh, Istvan R; Pon, Liza A

    2016-01-01

    The budding yeast Saccharomyces cerevisiae is widely used as a model system to study the organization and function of the cytoskeleton. In the past, its small size, rounded shape, and rigid cell wall created obstacles to explore the cell biology of this model eukaryote. It is now possible to acquire and analyze high-resolution and super-resolution multidimensional images of the yeast cell. As a result, imaging of yeast has emerged as an important tool in eukaryotic cell biology. This chapter describes labeling methods and optical approaches for visualizing the cytoskeleton and interactions of the actin cytoskeleton with mitochondria in fixed yeast cells using wide-field and super-resolution fluorescence microscopy.

  18. Clearance of Damaged Mitochondria Through PINK1 Stabilization by JNK and ERK MAPK Signaling in Chlorpyrifos-Treated Neuroblastoma Cells.

    PubMed

    Park, Jae Hyeon; Ko, Juyeon; Park, Yun Sun; Park, Jungyun; Hwang, Jungwook; Koh, Hyun Chul

    2017-04-01

    Mitochondrial quality control and clearance of damaged mitochondria through mitophagy are important cellular activities. Studies have shown that PTEN-induced putative protein kinase 1 (PINK1) and Parkin play central roles in triggering mitophagy; however, little is known regarding the mechanism by which PINK1 modulates mitophagy in response to reactive oxygen species (ROS)-induced stress. In this study, chlorpyrifos (CPF)-induced ROS caused mitochondrial damage and subsequent engulfing of mitochondria in double-membrane autophagic vesicles, indicating that clearance of damaged mitochondria is due to mitophagy. CPF treatment resulted in PINK1 stabilization on the outer mitochondrial membrane and subsequently increased Parkin recruitment from the cytosol to the abnormal mitochondria. We found that PINK1 physically interacts with Parkin in the mitochondria of CPF-treated cells. Furthermore, a knockdown of PINK1 strongly inhibited the LC3-II protein level by blocking Parkin recruitment. This indicates that CPF-induced mitophagy is due to PINK1 stabilization in mitochondria. We observed that PINK1 stabilization was selectively regulated by ROS-mediated c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling activation but not p38 signaling. In the mitochondria of CPF-exposed cells, pretreatment with specific inhibitors of JNK and ERK1/2 significantly decreased PINK1 stabilization and Parkin recruitment and blocked the LC3-II protein level. Specifically, JNK and ERK1/2 inhibition also dramatically blocked the interaction between PINK1 and Parkin. Our results demonstrated that PINK1 regulation plays a critical role in CPF-induced mitophagy. The simple interpretation of these results is that JNK and ERK1/2 signaling regulates PINK1/Parkin-dependent mitophagy in the mitochondria of CPF-treated cells. Overall, this study proposes a novel molecular regulatory mechanism of PINK1 stabilization under CPF exposure.

  19. Morphofunctional effects of mitotane on mitochondria in human adrenocortical cancer cells.

    PubMed

    Poli, Giada; Guasti, Daniele; Rapizzi, Elena; Fucci, Rossella; Canu, Letizia; Bandini, Alessandra; Cini, Nicoletta; Bani, Daniele; Mannelli, Massimo; Luconi, Michaela

    2013-08-01

    At present, mitotane (MTT) represents the first-line pharmacological approach for the treatment of advanced adrenocortical carcinoma (ACC). Despite clear evidence that the drug can reduce the clinical signs of steroid excess in secreting ACC, the mechanism mediating the possible toxic effect of MTT on tumor cells still remains obscure. This study investigated the intracellular events underlying the toxic effect of MTT by studying qualitative and quantitative alterations in mitochondrial morphology and functions in human adrenocortical cancer cell lines, H295R and SW13. Increasing concentrations of MTT resulted in rapid intracellular accumulation and conversion of the drug. Cytostatic and cytotoxic effects were evident at doses corresponding to the therapeutic window (30-50 μM) through an apoptotic mechanism involving caspase 3/7. Electron microscopic analysis of cell mitochondria displayed MTT-induced dose- and time-dependent alterations in the morphology of the organelle. These alterations were characterized by a marked swelling and a decrease in the number of respiratory cristae, accompanied by a significant depolarization of the mitochondrial membrane potential, finally leading to the disruption of the organelle. A drastic reduction of oxygen consumption was observed due to mitochondrial membrane damage, which was accompanied by a decrease in the levels of VDAC1 integral membrane channel. These findings contribute to better understand the intracellular mechanism of action of MTT in ACC cells, showing that its cytotoxic effect seems to be mainly mediated by an apoptotic process activated by the disruption of mitochondria.

  20. Concepts of papillomavirus entry into host cells.

    PubMed

    Day, Patricia M; Schelhaas, Mario

    2014-02-01

    Papillomaviruses enter basal cells of stratified epithelia. Assembly of new virions occurs in infected cells during terminal differentiation. This unique biology is reflected in the mechanism of entry. Extracellularly, the interaction of nonenveloped capsids with several host cell proteins, after binding, results in discrete conformational changes. Asynchronous internalization occurs over several hours by an endocytic mechanism related to, but distinct from macropinocytosis. Intracellular trafficking leads virions through the endosomal system, and from late endosomes to the trans-Golgi-network, before nuclear delivery. Here, we discuss the existing data with the aim to synthesize an integrated model of the stepwise process of entry, thereby highlighting key open questions. Additionally, we relate data from experiments with cultured cells to in vivo results.

  1. Plant cell proliferation inside an inorganic host.

    PubMed

    Perullini, Mercedes; Rivero, María Mercedes; Jobbágy, Matías; Mentaberry, Alejandro; Bilmes, Sara A

    2007-01-10

    In recent years, much attention has been paid to plant cell culture as a tool for the production of secondary metabolites and the expression of recombinant proteins. Plant cell immobilization offers many advantages for biotechnological processes. However, the most extended matrices employed, such as calcium-alginate, cannot fully protect entrapped cells. Sol-gel chemistry of silicates has emerged as an outstanding strategy to obtain biomaterials in which living cells are truly protected. This field of research is rapidly developing and a large number of bacteria and yeast-entrapping ceramics have already been designed for different applications. But even mild thermal and chemical conditions employed in sol-gel synthesis may result harmful to cells of higher organisms. Here we present a method for the immobilization of plant cells that allows cell growth at cavities created inside a silica matrix. Plant cell proliferation was monitored for a 6-month period, at the end of which plant calli of more than 1 mm in diameter were observed inside the inorganic host. The resulting hybrid device had good mechanical stability and proved to be an effective barrier against biological contamination, suggesting that it could be employed for long-term plant cell entrapment applications.

  2. Pancratistatin selectively targets cancer cell mitochondria and reduces growth of human colon tumor xenografts.

    PubMed

    Griffin, Carly; Karnik, Aditya; McNulty, James; Pandey, Siyaram

    2011-01-01

    The naturally occurring Amaryllidaceae alkaloid pancratistatin exhibits potent apoptotic activity against a large panel of cancer cells lines and has an insignificant effect on noncancerous cell lines, although with an elusive cellular target. Many current chemotherapeutics induce apoptosis via genotoxic mechanisms and thus have low selectivity. The observed selectivity of pancratistatin for cancer cells promoted us to consider the hypothesis that this alkaloid targets cancer cell mitochondria rather than DNA or its replicative machinery. In this study, we report that pancratistatin decreased mitochondrial membrane potential and induced apoptotic nuclear morphology in p53-mutant (HT-29) and wild-type p53 (HCT116) colorectal carcinoma cell lines, but not in noncancerous colon fibroblast (CCD-18Co) cells. Interestingly, pancratistatin was found to be ineffective against mtDNA-depleted (ρ(0)) cancer cells. Moreover, pancratistatin induced cell death in a manner independent of Bax and caspase activation, and did not alter β-tubulin polymerization rate nor cause double-stranded DNA breaks. For the first time we report the efficacy of pancratistatin in vivo against human colorectal adenocarcinoma xenografts. Intratumor administration of pancratistatin (3 mg/kg) caused significant reduction in the growth of subcutaneous HT-29 tumors in Nu/Nu mice (n = 6), with no apparent toxicity to the liver or kidneys as indicated by histopathologic analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Altogether, this work suggests that pancratistatin may be a novel mitochondria-targeting compound that selectively induces apoptosis in cancer cells and significantly reduces tumor growth.

  3. Mitochondrial transfer from Wharton's jelly-derived mesenchymal stem cells to mitochondria-defective cells recaptures impaired mitochondrial function.

    PubMed

    Lin, Hung-Yu; Liou, Chia-Wei; Chen, Shang-Der; Hsu, Te-Yao; Chuang, Jiin-Haur; Wang, Pei-Wen; Huang, Sheng-Teng; Tiao, Mao-Meng; Chen, Jin-Bor; Lin, Tsu-Kung; Chuang, Yao-Chung

    2015-05-01

    Adult mesenchymal stem cell (MSC)-conducted mitochondrial transfer has been recently shown to rescue cellular bioenergetics and prevent cell death caused by mitochondrial dysfunction. Wharton's jelly-derived MSCs (WJMSCs) harvested from postpartum umbilical cords are an accessible and abundant source of stem cells. This study aimed to determine the capability of WJMSCs to transfer their own mitochondria and rescue impaired oxidative phosphorylation (OXPHOS) and bioenergetics caused by mitochondrial DNA defects. To do this, WJMSCs were co-cultured with mitochondrial DNA (mtDNA)-depleted ρ(0) cells and the recapture of mitochondrial function was evaluated. WJMSCs were shown to be capable of transferring their own mitochondria into ρ(0) cells and underwent interorganellar mixture within these cells. Permissive culture media (BrdU-containing and pyruvate- and uridine-free) sieved out a survival cell population from the co-cultured WJMSCs (BrdU-sensitive) and ρ(0) cells (pyruvate/uridine-free). The survival cells had mtDNA identical to that of WJMSCs, whereas they expressed cellular markers identical to that of ρ(0) cells. Importantly, these ρ(0)-plus -WJMSC-mtDNA (ρ(+W)) cells recovered the expression of mtDNA-encoded proteins and exhibited functional oxygen consumption and respiratory control, as well as the activity of electron transport chain (ETC) complexes I, II, III and IV. In addition, ETC complex V-inhibitor-sensitive ATP production and metabolic shifting were also recovered. Furthermore, cellular behaviors including attachment-free proliferation, aerobic viability and OXPHOS-reliant cellular motility were also regained after mitochondrial transfer by WJMSCs. The therapeutic effect of WJMSCs-derived mitochondrial transfer was able to stably sustain for at least 45 passages. In conclusion, this study suggests that WJMSCs may serve as a potential therapeutic strategy for diseases linked to mitochondrial dysfunction through the donation of healthy

  4. Solanine Induces Mitochondria-Mediated Apoptosis in Human Pancreatic Cancer Cells

    PubMed Central

    Sun, Hongwei; Lv, Chongqing; Yang, Longlong; Wang, Yingxiu; Zhang, Qingshun; Yu, Suhui; Kong, Hongru; Wang, Meng; Xie, Jianming; Zhang, Chunwu; Zhou, Mengtao

    2014-01-01

    Steroid alkaloids have been suggested as potential anticancer compounds. However, the underlying mechanisms of how steroid alkaloids inhibit the tumor growth are largely unknown. Here, we reported that solanine, a substance of steroid alkaloids, has a positive effect on the inhibition of pancreatic cancer cell growth in vitro and in vivo. In pancreatic cancer cells and nu/nu nude mice model, we found that solanine inhibited cancer cells growth through caspase-3 dependent mitochondrial apoptosis. Mechanically, solanine promotes the opening of mitochondrial membrane permeability transition pore (MPTP) by downregulating the Bcl-2/Bax ratio; thereafter, Cytochrome c and Smac are released from mitochondria into cytosol to process the caspase-3 zymogen into an activated form. Moreover, we found that the expression of tumor metastasis related proteins, MMP-2 and MMP-9, was also decreased in the cells treated with solanine. Therefore, our results suggested that solanine was an effective compound for the treatment of pancreatic cancer. PMID:24949471

  5. Cancer Cell Mitochondria Targeting by Pancratistatin Analogs is Dependent on Functional Complex II and III.

    PubMed

    Ma, Dennis; Pignanelli, Christopher; Tarade, Daniel; Gilbert, Tyler; Noel, Megan; Mansour, Fadi; Adams, Scott; Dowhayko, Alexander; Stokes, Kyle; Vshyvenko, Sergey; Hudlicky, Tomas; McNulty, James; Pandey, Siyaram

    2017-02-21

    Enhanced mitochondrial stability and decreased dependence on oxidative phosphorylation confer an acquired resistance to apoptosis in cancer cells, but may present opportunities for therapeutic intervention. The compound pancratistatin (PST) has been shown to selectively induce apoptosis in cancer cells. However, its low availability in nature has hindered its clinical advancement. We synthesized PST analogs and a medium-throughput screen was completed. Analogs SVTH-7, -6, and -5 demonstrated potent anti-cancer activity greater than PST and several standard chemotherapeutics. They disrupted mitochondrial function, activated the intrinsic apoptotic pathway, and reduced growth of tumor xenografts in vivo. Interestingly, the pro-apoptotic effects of SVTH-7 on cancer cells and mitochondria were abrogated with the inhibition of mitochondrial complex II and III, suggesting mitochondrial or metabolic vulnerabilities may be exploited by this analog. This work provides a scaffold for characterizing distinct mitochondrial and metabolic features of cancer cells and reveals several lead compounds with high therapeutic potential.

  6. Solanine induces mitochondria-mediated apoptosis in human pancreatic cancer cells.

    PubMed

    Sun, Hongwei; Lv, Chongqing; Yang, Longlong; Wang, Yingxiu; Zhang, Qingshun; Yu, Suhui; Kong, Hongru; Wang, Meng; Xie, Jianming; Zhang, Chunwu; Zhou, Mengtao

    2014-01-01

    Steroid alkaloids have been suggested as potential anticancer compounds. However, the underlying mechanisms of how steroid alkaloids inhibit the tumor growth are largely unknown. Here, we reported that solanine, a substance of steroid alkaloids, has a positive effect on the inhibition of pancreatic cancer cell growth in vitro and in vivo. In pancreatic cancer cells and nu/nu nude mice model, we found that solanine inhibited cancer cells growth through caspase-3 dependent mitochondrial apoptosis. Mechanically, solanine promotes the opening of mitochondrial membrane permeability transition pore (MPTP) by downregulating the Bcl-2/Bax ratio; thereafter, Cytochrome c and Smac are released from mitochondria into cytosol to process the caspase-3 zymogen into an activated form. Moreover, we found that the expression of tumor metastasis related proteins, MMP-2 and MMP-9, was also decreased in the cells treated with solanine. Therefore, our results suggested that solanine was an effective compound for the treatment of pancreatic cancer.

  7. Cancer Cell Mitochondria Targeting by Pancratistatin Analogs is Dependent on Functional Complex II and III

    PubMed Central

    Ma, Dennis; Pignanelli, Christopher; Tarade, Daniel; Gilbert, Tyler; Noel, Megan; Mansour, Fadi; Adams, Scott; Dowhayko, Alexander; Stokes, Kyle; Vshyvenko, Sergey; Hudlicky, Tomas; McNulty, James; Pandey, Siyaram

    2017-01-01

    Enhanced mitochondrial stability and decreased dependence on oxidative phosphorylation confer an acquired resistance to apoptosis in cancer cells, but may present opportunities for therapeutic intervention. The compound pancratistatin (PST) has been shown to selectively induce apoptosis in cancer cells. However, its low availability in nature has hindered its clinical advancement. We synthesized PST analogs and a medium-throughput screen was completed. Analogs SVTH-7, -6, and -5 demonstrated potent anti-cancer activity greater than PST and several standard chemotherapeutics. They disrupted mitochondrial function, activated the intrinsic apoptotic pathway, and reduced growth of tumor xenografts in vivo. Interestingly, the pro-apoptotic effects of SVTH-7 on cancer cells and mitochondria were abrogated with the inhibition of mitochondrial complex II and III, suggesting mitochondrial or metabolic vulnerabilities may be exploited by this analog. This work provides a scaffold for characterizing distinct mitochondrial and metabolic features of cancer cells and reveals several lead compounds with high therapeutic potential. PMID:28220885

  8. Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics.

    PubMed

    Pancrazi, Laura; Di Benedetto, Giulietta; Colombaioni, Laura; Della Sala, Grazia; Testa, Giovanna; Olimpico, Francesco; Reyes, Aurelio; Zeviani, Massimo; Pozzan, Tullio; Costa, Mario

    2015-11-10

    Forkhead box g1 (Foxg1) is a nuclear-cytosolic transcription factor essential for the forebrain development and involved in neurodevelopmental and cancer pathologies. Despite the importance of this protein, little is known about the modalities by which it exerts such a large number of cellular functions. Here we show that a fraction of Foxg1 is localized within the mitochondria in cell lines, primary neuronal or glial cell cultures, and in the mouse cortex. Import of Foxg1 in isolated mitochondria appears to be membrane potential-dependent. Amino acids (aa) 277-302 were identified as critical for mitochondrial localization. Overexpression of full-length Foxg1 enhanced mitochondrial membrane potential (ΔΨm) and promoted mitochondrial fission and mitosis. Conversely, overexpression of the C-term Foxg1 (aa 272-481), which is selectively localized in the mitochondrial matrix, enhanced organelle fusion and promoted the early phase of neuronal differentiation. These findings suggest that the different subcellular localizations of Foxg1 control the machinery that brings about cell differentiation, replication, and bioenergetics, possibly linking mitochondrial functions to embryonic development and pathological conditions.

  9. Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

    PubMed Central

    Pancrazi, Laura; Di Benedetto, Giulietta; Colombaioni, Laura; Della Sala, Grazia; Testa, Giovanna; Olimpico, Francesco; Reyes, Aurelio; Zeviani, Massimo; Pozzan, Tullio; Costa, Mario

    2015-01-01

    Forkhead box g1 (Foxg1) is a nuclear-cytosolic transcription factor essential for the forebrain development and involved in neurodevelopmental and cancer pathologies. Despite the importance of this protein, little is known about the modalities by which it exerts such a large number of cellular functions. Here we show that a fraction of Foxg1 is localized within the mitochondria in cell lines, primary neuronal or glial cell cultures, and in the mouse cortex. Import of Foxg1 in isolated mitochondria appears to be membrane potential-dependent. Amino acids (aa) 277–302 were identified as critical for mitochondrial localization. Overexpression of full-length Foxg1 enhanced mitochondrial membrane potential (ΔΨm) and promoted mitochondrial fission and mitosis. Conversely, overexpression of the C-term Foxg1 (aa 272–481), which is selectively localized in the mitochondrial matrix, enhanced organelle fusion and promoted the early phase of neuronal differentiation. These findings suggest that the different subcellular localizations of Foxg1 control the machinery that brings about cell differentiation, replication, and bioenergetics, possibly linking mitochondrial functions to embryonic development and pathological conditions. PMID:26508630

  10. Targeting drugs to mitochondria.

    PubMed

    Heller, Anne; Brockhoff, Gero; Goepferich, Achim

    2012-09-01

    Mitochondria are of an increasing interest in pharmaceutical and medical research since it has been reported that dysfunction of these organelles contributes to several diseases with a great diversity of clinical appearance. By the fact that mitochondria are located inside the cell and, in turn, origins of mitochondrial diseases or targets of drugs are located inside mitochondria, a drug molecule has to cross several barriers. This is a severe drawback for the selective accumulation of drug molecules in mitochondria. Therefore, targeting strategies such as direct drug modification or encapsulation into nanocarriers have to be applied to achieve an accumulation of drug molecules in these organelles. In this review, it will be demonstrated how properties and dysfunctions of mitochondria are generating a need for the development of mitochondria specific therapies. Furthermore, intracellular targets of mitochondrial diseases, strategies to utilize mitochondrial specificities and targeting approaches will be discussed. Finally, techniques to investigate mitochondrial characteristics and functionality are reviewed.

  11. Counting Legionella cells within single amoeba host cells.

    PubMed

    Buse, Helen Y; Ashbolt, Nicholas J

    2012-03-01

    Here we present the first attempt to quantify Legionella pneumophila cell numbers within individual amoeba hosts that may be released into engineered water systems. The maximum numbers of culturable L. pneumophila cells grown within Acanthamoeba polyphaga and Naegleria fowleri were 1,348 (mean, 329) and 385 (mean, 44) CFU trophozoite(-1), respectively.

  12. Counting Legionella cells within single amoeba host cells

    EPA Science Inventory

    Here we present the first attempt to quantify L. pneumophila cell numbers within individual amoebae hosts that may be released into engineered water systems. The maximum numbers of culturable L. pneumophila cells grown within Acanthamoeba polyphaga and Naegleria fowleri were 134...

  13. Distinct profiles of human embryonic stem cell metabolism and mitochondria identified by oxygen.

    PubMed

    Lees, Jarmon G; Rathjen, Joy; Sheedy, John R; Gardner, David K; Harvey, Alexandra J

    2015-10-01

    Oxygen is a powerful regulator of cell function and embryonic development. It has previously been determined that oxygen regulates human embryonic stem (hES) cell glycolytic and amino acid metabolism, but the effects on mitochondria are as yet unknown. Two hES cell lines (MEL1, MEL2) were analyzed to determine the role of 5% (physiological) and 20% (atmospheric) oxygen in regulating mitochondrial activity. In response to extended physiological oxygen culture, MEL2 hES cells displayed reduced mtDNA content, mitochondrial mass and expression of metabolic genes TFAM, NRF1, PPARa and MT-ND4. Furthermore, MEL2 hES cell glucose consumption, lactate production and amino acid turnover were elevated under physiological oxygen. In stark contrast, MEL1 hES cell amino acid and carbohydrate use and mitochondrial function were relatively unaltered in response to oxygen. Furthermore, differentiation kinetics were delayed in the MEL1 hES cell line following BMP4 treatment. Here we report the first incidence of metabolic dysfunction in a hES cell population, defined as a failure to respond to oxygen concentration through the modulation of metabolism, demonstrating that hES cells can be perturbed during culture despite exhibiting the defining characteristics of pluripotent cells. Collectively, these data reveal a central role for oxygen in the regulation of hES cell metabolism and mitochondrial function, whereby physiological oxygen promotes glucose flux and suppresses mitochondrial biogenesis and gene expression.

  14. Momordica charantia Extract Induces Apoptosis in Human Cancer Cells through Caspase- and Mitochondria-Dependent Pathways

    PubMed Central

    Li, Chia-Jung; Tsang, Shih-Fang; Tsai, Chun-Hao; Tsai, Hsin-Yi; Chyuan, Jong-Ho; Hsu, Hsue-Yin

    2012-01-01

    Plants are an invaluable source of potential new anti-cancer drugs. Momordica charantia is one of these plants with both edible and medical value and reported to exhibit anticancer activity. To explore the potential effectiveness of Momordica charantia, methanol extract of Momordica charantia (MCME) was used to evaluate the cytotoxic activity on four human cancer cell lines, Hone-1 nasopharyngeal carcinoma cells, AGS gastric adenocarcinoma cells, HCT-116 colorectal carcinoma cells, and CL1-0 lung adenocarcinoma cells, in this study. MCME showed cytotoxic activity towards all cancer cells tested, with the approximate IC50 ranging from 0.25 to 0.35 mg/mL at 24 h. MCME induced cell death was found to be time-dependent in these cells. Apoptosis was demonstrated by DAPI staining and DNA fragmentation analysis using agarose gel electrophoresis. MCME activated caspase-3 and enhanced the cleavage of downstream DFF45 and PARP, subsequently leading to DNA fragmentation and nuclear condensation. The apoptogenic protein, Bax, was increased, whereas Bcl-2 was decreased after treating for 24 h in all cancer cells, indicating the involvement of mitochondrial pathway in MCME-induced cell death. These findings indicate that MCME has cytotoxic effects on human cancer cells and exhibits promising anti-cancer activity by triggering apoptosis through the regulation of caspases and mitochondria. PMID:23091557

  15. Development and plasticity of mitochondria and electrical properties of the cell membrane in blowfly photoreceptors.

    PubMed

    Rudolf, Jerneja; Meglič, Andrej; Zupančič, Gregor; Belušič, Gregor

    2014-07-01

    Blowfly photoreceptors are highly energy demanding sensory systems. Their information processing efficiency is enabled by the high temporal resolution of the cell membrane, requiring heavy metabolic support by the mitochondria. We studied the developmental changes of the mitochondrial apparatus and electrical properties of the photoreceptor membrane in the white eyed Calliphora vicina Chalky. Using in vivo microspectrophotometry and Western blot analysis, we found an age-dependent increase in the concentration of mitochondrial pigments. The maximal change occurred during the first week. The age-related changes were smaller in dark-bred than in light-bred flies. The mitochondrial pigment content increased after the switch from dark to light rearing and decreased after the switch from light to dark rearing. The electrical parameters of the photoreceptors were investigated with intracellular recordings. The resting membrane resistance and time constant decreased significantly after eclosion. The decrease was again most significant during the first week of adult life, paralleled with changes in the Na/K pump-dependent hyperpolarizing afterpotential. We conclude that the photoreceptor mitochondria exhibit remarkable ontogenetic and phenotypic plasticity, because the quantity of mitochondrial pigments tightly follows the development of the cell membrane as well as the energy demands of the photoreceptors under different rearing conditions.

  16. Axin is expressed in mitochondria and suppresses mitochondrial ATP synthesis in HeLa cells.

    PubMed

    Shin, Jee-Hye; Kim, Hyun-Wook; Rhyu, Im Joo; Kee, Sun-Ho

    2016-01-01

    Many recent studies have revealed that axin is involved in numerous cellular functions beyond the negative regulation of β-catenin-dependent Wnt signaling. Previously, an association of ectopic axin with mitochondria was observed. In an effort to investigate the relationship between axin and mitochondria, we found that axin expression suppressed cellular ATP production, which was more apparent as axin expression levels increased. Also, mitochondrial expression of axin was observed using two axin-expressing HeLa cell models: doxycycline-inducible ectopic axin expression (HeLa-axin) and axin expression enhanced by long-term treatment with XAV939 (HeLa-XAV). In biochemical analysis, axin is associated with oxidative phosphorylation (OXPHOS) complex IV and is involved in defects in the assembly of complex IV-containing supercomplexes. Functionally, axin expression reduced the activity of OXPHOS complex IV and the oxygen consumption rate (OCR), suggesting axin-mediated mitochondrial dysfunction. Subsequent studies using various inhibitors of Wnt signaling showed that the reduction in cellular ATP levels was weaker in cases of ICAT protein expression and treatment with iCRT3 or NSC668036 compared with XAV939 treatment, suggesting that XAV939 treatment affects ATP synthesis in addition to suppressing Wnt signaling activity. Axin-mediated regulation of mitochondrial function may be an additional mechanism to Wnt signaling for regulation of cell growth.

  17. Neurons Are Host Cells for Mycobacterium tuberculosis

    PubMed Central

    Randall, Philippa J.; Hsu, Nai-Jen; Lang, Dirk; Cooper, Susan; Sebesho, Boipelo; Allie, Nasiema; Keeton, Roanne; Francisco, Ngiambudulu M.; Salie, Sumayah; Labuschagné, Antoinette; Quesniaux, Valerie; Ryffel, Bernhard; Kellaway, Lauriston

    2014-01-01

    Mycobacterium tuberculosis infection of the central nervous system is thought to be initiated once the bacilli have breached the blood brain barrier and are phagocytosed, primarily by microglial cells. In this study, the interactions of M. tuberculosis with neurons in vitro and in vivo were investigated. The data obtained demonstrate that neurons can act as host cells for M. tuberculosis. M. tuberculosis bacilli were internalized by murine neuronal cultured cells in a time-dependent manner after exposure, with superior uptake by HT22 cells compared to Neuro-2a cells (17.7% versus 9.8%). Internalization of M. tuberculosis bacilli by human SK-N-SH cultured neurons suggested the clinical relevance of the findings. Moreover, primary murine hippocampus-derived neuronal cultures could similarly internalize M. tuberculosis. Internalized M. tuberculosis bacilli represented a productive infection with retention of bacterial viability and replicative potential, increasing 2- to 4-fold within 48 h. M. tuberculosis bacillus infection of neurons was confirmed in vivo in the brains of C57BL/6 mice after intracerebral challenge. This study, therefore, demonstrates neurons as potential new target cells for M. tuberculosis within the central nervous system. PMID:24566619

  18. Neurons are host cells for Mycobacterium tuberculosis.

    PubMed

    Randall, Philippa J; Hsu, Nai-Jen; Lang, Dirk; Cooper, Susan; Sebesho, Boipelo; Allie, Nasiema; Keeton, Roanne; Francisco, Ngiambudulu M; Salie, Sumayah; Labuschagné, Antoinette; Quesniaux, Valerie; Ryffel, Bernhard; Kellaway, Lauriston; Jacobs, Muazzam

    2014-05-01

    Mycobacterium tuberculosis infection of the central nervous system is thought to be initiated once the bacilli have breached the blood brain barrier and are phagocytosed, primarily by microglial cells. In this study, the interactions of M. tuberculosis with neurons in vitro and in vivo were investigated. The data obtained demonstrate that neurons can act as host cells for M. tuberculosis. M. tuberculosis bacilli were internalized by murine neuronal cultured cells in a time-dependent manner after exposure, with superior uptake by HT22 cells compared to Neuro-2a cells (17.7% versus 9.8%). Internalization of M. tuberculosis bacilli by human SK-N-SH cultured neurons suggested the clinical relevance of the findings. Moreover, primary murine hippocampus-derived neuronal cultures could similarly internalize M. tuberculosis. Internalized M. tuberculosis bacilli represented a productive infection with retention of bacterial viability and replicative potential, increasing 2- to 4-fold within 48 h. M. tuberculosis bacillus infection of neurons was confirmed in vivo in the brains of C57BL/6 mice after intracerebral challenge. This study, therefore, demonstrates neurons as potential new target cells for M. tuberculosis within the central nervous system.

  19. Caffeic acid phenethyl ester induces mitochondria-mediated apoptosis in human myeloid leukemia U937 cells.

    PubMed

    Jin, Un-Ho; Song, Kwon-Ho; Motomura, Muneo; Suzuki, Ikukatsu; Gu, Yeun-Hwa; Kang, Yun-Jeong; Moon, Tae-Chul; Kim, Cheorl-Ho

    2008-03-01

    Caffeic acid phenyl ester (CAPE), a biologically active ingredient of propolis, has several interesting biological properties including antioxidant, anti-inflammatory, antiviral, immunostimulatory, anti-angiogenic, anti-invasive, anti-metastatic and carcinostatic activities. Recently, several groups have reported that CAPE is cytotoxic to tumor cells but not to normal cells. In this study, we investigated the mechanism of CAPE-induced apoptosis in human myeloid leukemia U937 cells. Treatment of U937 cells with CAPE decreased cell viability in a dose-dependent and time-dependent manner. DNA fragmentation assay revealed the typical ladder profile of oligonucleosomal fragments in CAPE-treated U937 cells. In addition, as evidenced by the nuclear DAPI staining experiment, we observed that the nuclear condensation, a typical phenotype of apoptosis, was found in U937 cells treated with 5 microg/ml of CAPE. Therefore, it was suggested that CAPE is a potent agent inducing apoptosis in U937 cells. Apoptotic action of the CAPE was accompanied by release of cytochrome C, reduction of Bcl-2 expression, increase of Bax expression, activation/cleavage of caspase-3 and activation/cleavage of PARP in U937 cells, but not by Fas protein, an initial mediator in the death signaling, or by phospho-eIF2 alpha and CHOP, crucial mediators in ER-mediated apoptosis. From the results, it was concluded that CAPE induces the mitochondria-mediated apoptosis but not death receptors- or ER-mediated apoptosis in U937 cells.

  20. Curcumin induces p53-independent necrosis in H1299 cells via a mitochondria-associated pathway.

    PubMed

    Li, Feie; Chen, Xi; Xu, Bing; Zhou, Hua

    2015-11-01

    Curcumin has been shown to have various therapeutic and/or adjuvant therapeutic effects on human cancers, as it inhibits cancer cell proliferation and induces apoptosis through p53-dependent molecular pathways. However, numerous cancer cell types bear a mutant p53 gene, and whether curcumin has any therapeutic effects on p53-deficient/mutant cancer cells has remained elusive. The present study sought to determine whether curcumin exerts any anti-proliferative and cytotoxic effects on the p53-deficient H1299 human lung cancer cell line via a p53-independent mechanism. An MTT assay and flow cytometric analysis indicated that curcumin significantly decreased cell proliferation and induced necrotic cell death. Western blot analysis of the cytosolic and mitochondrial fractions of H1299 cells as well as a fluorometric caspase assay indicated that curcumin-induced necrosis was mitochondria- and caspase-dependent, and resulted in cytochrome c release. Of note, this necrotic cell death was reduced following inhibition of B-cell lymphoma‑2 (Bcl-2)‑associated X protein (Bax) or Bcl‑2 homologous antagonist killer (Bak) as well as overexpression of Bcl-2. In conclusion, the present study suggested that curcumin-induced necrotic cell death was mediated via a p53-independent molecular pathway, which was associated with Bax and Bak translocation, caspase activation and cytochrome c release.

  1. 5,8,11,14-Eicosatetraynoic acid-induced destruction of mitochondria in human prostate cells (PC-3).

    SciTech Connect

    Anderson, K. M.; Seed, T. M.; Wilson, D. E.; Harris, J. E.; Biological and Medical Research; Rush Medical Coll.

    1992-01-01

    Culturing human prostate PC-3 cells for 4, 24, or 72 h in the presence of 5,8,11,14-eicosatetraynoic acid (ETYA), an inhibitor of arachidonic acid metabolism and cholesterol biosynthesis, markedly altered the morphology and reduced the number of mitochondria in the treated cells. Using quantitative electron microscopic morphometry, we documented changes in the number, form, area, matrix density, and integrity of the cristae and limiting membranes of mitochondria in cells cultured with ETYA. The inhibition of cholesterol synthesis or the substitution of ETYA for polyunsaturated fatty acids in the inner membrane may participate in the disruption of the mitochondria, which resembles the morphologic sequelae of oxidative stress. If sufficiently extensive, these changes could contribute to the inhibition of cellular proliferation by ETYA.

  2. Preparation and use of MitoPY1 for imaging hydrogen peroxide in mitochondria of live cells

    PubMed Central

    Dickinson, Bryan C; Lin, Vivian S; Chang, Christopher J

    2014-01-01

    Mitochondria peroxy yellow 1 (MitoPY1) is a small-molecule fluorescent probe that selectively tracks to the mitochondria of live biological specimens and responds to local fluxes of hydrogen peroxide (H2O2) by a turn-on fluorescence enhancement. This bifunctional dye uses a triphenylphosphonium targeting group and a boronate-based molecular switch to selectively respond to H2O2 over competing reactive oxygen species (ROS) within the mitochondria. MitoPY1 can be used to measure mitochondrial H2O2 levels in both cell culture and tissue models. In this protocol, we describe the synthesis of MitoPY1 and how to use this chemical tool to visualize mitochondrial H2O2 in live cells. The preparation of MitoPY1 is anticipated to take 7–10 d, and assays involving microscopy of cultured mammalian cells can be performed in 1–2 d. PMID:23722262

  3. Proteome-scale identification and characterization of mitochondria targeting proteins of Mycobacterium avium subspecies paratuberculosis: Potential virulence factors modulating host mitochondrial function.

    PubMed

    Rana, Aarti; Kumar, Devender; Rub, Abdur; Akhter, Yusuf

    2015-07-01

    Mycobacterium avium subsp. paratuberculosis is the etiological agent of Johne's Disease among ruminants. During the course of infection, it expresses a number of proteins for its successful persistence inside the host that cause variety of physiological abnormalities in the host. Mitochondrion is one of the attractive targets for pathogenic bacteria. Employing a proteome-wide sequence and structural signature based approach we have identified 46 M. avium subsp. paratuberculosis proteins as potential targets for the host mitochondrial targeting. These may act as virulence factors modulating mitochondrial physiology for bacterial survival and immune evasion inside the host cells.

  4. Recruitment of BAD by the Chlamydia trachomatis vacuole correlates with host-cell survival.

    PubMed

    Verbeke, Philippe; Welter-Stahl, Lynn; Ying, Songmin; Hansen, Jon; Häcker, Georg; Darville, Toni; Ojcius, David M

    2006-05-01

    Chlamydiae replicate intracellularly in a vacuole called an inclusion. Chlamydial-infected host cells are protected from mitochondrion-dependent apoptosis, partly due to degradation of BH3-only proteins. The host-cell adapter protein 14-3-3beta can interact with host-cell apoptotic signaling pathways in a phosphorylation-dependent manner. In Chlamydia trachomatis-infected cells, 14-3-3beta co-localizes to the inclusion via direct interaction with a C. trachomatis-encoded inclusion membrane protein. We therefore explored the possibility that the phosphatidylinositol-3 kinase (PI3K) pathway may contribute to resistance of infected cells to apoptosis. We found that inhibition of PI3K renders C. trachomatis-infected cells sensitive to staurosporine-induced apoptosis, which is accompanied by mitochondrial cytochrome c release. 14-3-3beta does not associate with the Chlamydia pneumoniae inclusion, and inhibition of PI3K does not affect protection against apoptosis of C. pneumoniae-infected cells. In C. trachomatis-infected cells, the PI3K pathway activates AKT/protein kinase B, which leads to maintenance of the pro-apoptotic protein BAD in a phosphorylated state. Phosphorylated BAD is sequestered via 14-3-3beta to the inclusion, but it is released when PI3K is inhibited. Depletion of AKT through short-interfering RNA reverses the resistance to apoptosis of C. trachomatis-infected cells. BAD phosphorylation is not maintained and it is not recruited to the inclusion of Chlamydia muridarum, which protects poorly against apoptosis. Thus, sequestration of BAD away from mitochondria provides C. trachomatis with a mechanism to protect the host cell from apoptosis via the interaction of a C. trachomatis-encoded inclusion protein with a host-cell phosphoserine-binding protein.

  5. Upregulation of TFAM and mitochondria copy number in human lymphoblastoid cells.

    PubMed

    Chakrabarty, Sanjiban; D'Souza, Reena Reshma; Kabekkodu, Shama Prasada; Gopinath, Puthiya M; Rossignol, Rodrigue; Satyamoorthy, Kapaettu

    2014-03-01

    Mitochondria are central to several physiological and pathological conditions in humans. In the present study, we performed copy number analysis of nuclear encoded mitochondrial genes, in peripheral blood mononuclear cells (PBMCs) and its representative lymphoblastoid cells (LCLs). We have observed hyper diploid copies of mitochondrial transcription factor A (TFAM) gene in the LCLs along with increased mtDNA copy number, mitochondrial mass, intracellular ROS and mitochondrial membrane potential, suggesting elevated mitochondrial biogenesis in LCLs. Gene expression analysis confirmed TFAM over-expression in LCLs when compared to PBMC. Based on our observation, we suggest that increased copy number of TFAM gene upregulates its expression, increases mtDNA copy numbers and protects it from oxidative stress induced damage in the transformed LCLs.

  6. Danger signaling protein HMGB1 induces a distinct form of cell death accompanied by formation of giant mitochondria.

    PubMed

    Gdynia, Georg; Keith, Martina; Kopitz, Jürgen; Bergmann, Marion; Fassl, Anne; Weber, Alexander N R; George, Julie; Kees, Tim; Zentgraf, Hans-Walter; Wiestler, Otmar D; Schirmacher, Peter; Roth, Wilfried

    2010-11-01

    Cells dying by necrosis release the high-mobility group box 1 (HMGB1) protein, which has immunostimulatory effects. However, little is known about the direct actions of extracellular HMGB1 protein on cancer cells. Here, we show that recombinant human HMGB1 (rhHMGB1) exerts strong cytotoxic effects on malignant tumor cells. The rhHMGB1-induced cytotoxicity depends on the presence of mitochondria and leads to fast depletion of mitochondrial DNA, severe damage of the mitochondrial proteome by toxic malondialdehyde adducts, and formation of giant mitochondria. The formation of giant mitochondria is independent of direct nuclear signaling events, because giant mitochondria are also observed in cytoplasts lacking nuclei. Further, the reactive oxygen species scavenger N-acetylcysteine as well as c-Jun NH(2)-terminal kinase blockade inhibited the cytotoxic effect of rhHMGB1. Importantly, glioblastoma cells, but not normal astrocytes, were highly susceptible to rhHMGB1-induced cell death. Systemic treatment with rhHMGB1 results in significant growth inhibition of xenografted tumors in vivo. In summary, rhHMGB1 induces a distinct form of cell death in cancer cells, which differs from the known forms of apoptosis, autophagy, and senescence, possibly representing an important novel mechanism of specialized necrosis. Further, our findings suggest that rhHMGB1 may offer therapeutic applications in treatment of patients with malignant brain tumors.

  7. Ceramide-Induced Apoptosis in Renal Tubular Cells: A Role of Mitochondria and Sphingosine-1-Phoshate

    PubMed Central

    Ueda, Norishi

    2015-01-01

    Ceramide is synthesized upon stimuli, and induces apoptosis in renal tubular cells (RTCs). Sphingosine-1 phosphate (S1P) functions as a survival factor. Thus, the balance of ceramide/S1P determines ceramide-induced apoptosis. Mitochondria play a key role for ceramide-induced apoptosis by altered mitochondrial outer membrane permeability (MOMP). Ceramide enhances oligomerization of pro-apoptotic Bcl-2 family proteins, ceramide channel, and reduces anti-apoptotic Bcl-2 proteins in the MOM. This process alters MOMP, resulting in generation of reactive oxygen species (ROS), cytochrome C release into the cytosol, caspase activation, and apoptosis. Ceramide regulates apoptosis through mitogen-activated protein kinases (MAPKs)-dependent and -independent pathways. Conversely, MAPKs alter ceramide generation by regulating the enzymes involving ceramide metabolism, affecting ceramide-induced apoptosis. Crosstalk between Bcl-2 family proteins, ROS, and many signaling pathways regulates ceramide-induced apoptosis. Growth factors rescue ceramide-induced apoptosis by regulating the enzymes involving ceramide metabolism, S1P, and signaling pathways including MAPKs. This article reviews evidence supporting a role of ceramide for apoptosis and discusses a role of mitochondria, including MOMP, Bcl-2 family proteins, ROS, and signaling pathways, and crosstalk between these factors in the regulation of ceramide-induced apoptosis of RTCs. A balancing role between ceramide and S1P and the strategy for preventing ceramide-induced apoptosis by growth factors are also discussed. PMID:25751724

  8. Mitochondria defects are involved in lead-acetate-induced adult hematopoietic stem cell decline.

    PubMed

    Liu, Jun; Jia, Dao-Yong; Cai, Shi-Zhong; Li, Cheng-Peng; Zhang, Meng-Si; Zhang, Yan-Yan; Yan, Chong-Huai; Wang, Ya-Ping

    2015-05-19

    Occupational high-grade lead exposure has been reduced in recent decades as a result of increased regulation. However, environmental lead exposure remains widespread, and is associated with severe toxicity implicated in human diseases. We performed oral intragastric administration of various dose lead acetate to adult Sprague Dawley rats to define the role of lead exposure in hematopoietic stem cells (HSCs) function, and to clarify its underlying mechanism. Lead acetate-exposed rats exhibited developmental abnormalities in myeloid and lymphoid lineages, and a significant decline in immune functions. It also showed HSCs functional decline associated with senescent phenotype with low grade lead acetate exposure or apoptotic phenotype with relative higher grade dose exposure. Mechanistic exploration showed a significant increase in reactive oxygen species (ROS) in the lead acetate-exposed CD90(+)CD45(-) compartment, which correlated with functional defects in cellular mitochondria. Furthermore, in vivo treatment with the antioxidant vitamin C led to reversion of the CD90(+)CD45(-) compartment functional decline. These results indicate that lead acetate perturbs the hematopoietic balance of adult HSCs, associated with cellular mitochondria defects, increased intracellular ROS generation.

  9. T-cell death following immune activation is mediated by mitochondria-localized SARM.

    PubMed

    Panneerselvam, P; Singh, L P; Selvarajan, V; Chng, W J; Ng, S B; Tan, N S; Ho, B; Chen, J; Ding, J L

    2013-03-01

    Following acute-phase infection, activated T cells are terminated to achieve immune homeostasis, failure of which results in lymphoproliferative and autoimmune diseases. We report that sterile α- and heat armadillo-motif-containing protein (SARM), the most conserved Toll-like receptors adaptor, is proapoptotic during T-cell immune response. SARM expression is significantly reduced in natural killer (NK)/T lymphoma patients compared with healthy individuals, suggesting that decreased SARM supports NK/T-cell proliferation. T cells knocked down of SARM survived and proliferated more significantly compared with wild-type T cells following influenza infection in vivo. During activation of cytotoxic T cells, the SARM level fell before rising, correlating inversely with cell proliferation and subsequent T-cell clearance. SARM knockdown rescued T cells from both activation- and neglect-induced cell deaths. The mitochondria-localized SARM triggers intrinsic apoptosis by generating reactive oxygen species and depolarizing the mitochondrial potential. The proapoptotic function is attributable to the C-terminal sterile alpha motif and Toll/interleukin-1 receptor domains. Mechanistically, SARM mediates intrinsic apoptosis via B cell lymphoma-2 (Bcl-2) family members. SARM suppresses B cell lymphoma-extra large (Bcl-xL) and downregulates extracellular signal-regulated kinase phosphorylation, which are cell survival effectors. Overexpression of Bcl-xL and double knockout of Bcl-2 associated X protein and Bcl-2 homologous antagonist killer substantially reduced SARM-induced apoptosis. Collectively, we have shown how T-cell death following infection is mediated by SARM-induced intrinsic apoptosis, which is crucial for T-cell homeostasis.

  10. Mps1 kinase regulates tumor cell viability via its novel role in mitochondria

    PubMed Central

    Zhang, X; Ling, Y; Guo, Y; Bai, Y; Shi, X; Gong, F; Tan, P; Zhang, Y; Wei, C; He, X; Ramirez, A; Liu, X; Cao, C; Zhong, H; Xu, Q; Ma, R Z

    2016-01-01

    Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells. PMID:27383047

  11. Helium-neon laser irradiation stimulates cell proliferation through photostimulatory effects in mitochondria.

    PubMed

    Hu, Wan-Ping; Wang, Jeh-Jeng; Yu, Chia-Li; Lan, Cheng-Che E; Chen, Gow-Shing; Yu, Hsin-Su

    2007-08-01

    Previous reports have shown that cellular functions could be influenced by visual light (400-700 nm). Recent evidence indicates that cellular proliferation could be triggered by the interaction of a helium-neon laser (He-Ne laser, 632.8 nm) with the mitochondrial photoacceptor-cytochrome c oxidase. Our previous studies demonstrated that He-Ne irradiation induced an increase in cell proliferation, but not migration, in the melanoma cell line A2058 cell. The aim of this study was to investigate the underlying mechanisms involved in photostimulatory effects induced by an He-Ne laser. Using the A2058 cell as a model for cell proliferation, the photobiologic effects induced by an He-Ne laser were studied. He-Ne irradiation immediately induced an increase in mitochondrial membrane potential (delta psi(mt)), ATP, and cAMP via enhanced cytochrome c oxidase activity and promoted phosphorylation of Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) expressions. He-Ne irradiation-induced A2058 cell proliferation was significantly abrogated by the addition of delta psi(mt) and JNK inhibitors. Moreover, treatment with an He-Ne laser resulted in delayed effects on IL-8 and transforming growth factor-beta1 release from A2058 cells. These results suggest that He-Ne irradiation elicits photostimulatory effects in mitochondria processes, which involve JNK/AP-1 activation and enhanced growth factor release, and ultimately lead to A2058 cell proliferation.

  12. Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling.

    PubMed

    Sena, Laura A; Li, Sha; Jairaman, Amit; Prakriya, Murali; Ezponda, Teresa; Hildeman, David A; Wang, Chyung-Ru; Schumacker, Paul T; Licht, Jonathan D; Perlman, Harris; Bryce, Paul J; Chandel, Navdeep S

    2013-02-21

    It is widely appreciated that T cells increase glycolytic flux during activation, but the role of mitochondrial flux is unclear. Here, we have shown that mitochondrial metabolism in the absence of glucose metabolism is sufficient to support interleukin-2 (IL-2) induction. Furthermore, we used mice with reduced mitochondrial reactive oxygen species (mROS) production in T cells (T-Uqcrfs(-/-) mice) to show that mitochondria are required for T cell activation to produce mROS for activation of nuclear factor of activated T cells (NFAT) and subsequent IL-2 induction. These mice could not induce antigen-specific expansion of T cells in vivo, but Uqcrfs1(-/-) T cells retained the ability to proliferate in vivo under lymphopenic conditions. This suggests that Uqcrfs1(-/-) T cells were not lacking bioenergetically but rather lacked specific ROS-dependent signaling events needed for antigen-specific expansion. Thus, mitochondrial metabolism is a critical component of T cell activation through the production of complex III ROS.

  13. A Cytosolic Network Suppressing Mitochondria-Mediated Proteostatic Stress and Cell Death

    PubMed Central

    Wang, Xiaowen; Chen, Xin Jie

    2015-01-01

    Mitochondria are multifunctional organelles whose dysfunction leads to neuromuscular degeneration and ageing. The multi-functionality poses a great challenge for understanding the mechanisms by which mitochondrial dysfunction causes specific pathologies. Among the leading mitochondrial mediators of cell death are energy depletion, free radical production, defect in iron-sulfur cluster biosynthesis, the release of pro-apoptotic and non-cell-autonomous signaling molecules, and altered stress signaling 1–5. Here, we identified a novel pathway of mitochondria-mediated cell death. This pathway was named mitochondrial Precursor Over-accumulation Stress (mPOS), characterized by aberrant accumulation of mitochondrial precursors in the cytosol. mPOS can be triggered by clinically relevant mitochondrial damage which is not limited to the core machineries of protein import. We also identified a large network of genes that suppress mPOS, by modulating ribosomal biogenesis, mRNA decapping, transcript-specific translation, protein chaperoning and turnover. In response to mPOS, several ribosome-associated proteins were up-regulated including Gis2 and Nog2, which promote cap-independent translation and inhibit the nuclear export of the 60S ribosomal subunit respectively 6, 7. Gis2 and Nog2 up-regulation promotes cell survival, which may be part of a feedback loop that attenuates mPOS. Our data indicate that mitochondrial dysfunction contributes directly to cytosolic proteostatic stress, and provide an explanation for the enigmatic association between these two hallmarks of degenerative diseases and ageing. The results are relevant to understanding diseases (e.g., spinocerebellar ataxia, amyotrophic lateral sclerosis and myotonic dystrophy) that involve mutations within the anti-degenerative network. PMID:26192197

  14. The mitochondria-regulated death pathway mediates asbestos-induced alveolar epithelial cell apoptosis.

    PubMed

    Panduri, Vijayalakshmi; Weitzman, Sigmund A; Chandel, Navdeep; Kamp, David W

    2003-02-01

    The mechanisms underlying asbestos-induced pulmonary toxicity are not fully understood. Alveolar epithelial cell (AEC) apoptosis by iron-derived reactive oxygen species (ROS) is one important mechanism implicated. The two major pathways regulating apoptosis include (i) the mitochondrial death (intrinsic) pathway caused by DNA damage, and (ii) the plasma-membrane death receptor (extrinsic) pathway. However, it is unknown whether asbestos activates either death pathway in AEC. We determined whether asbestos triggers AEC mitochondrial dysfunction by exposing cells (A549 and rat alveolar type II) to amosite asbestos and assessing mitochondrial membrane potential changes (deltapsi(m)) using a fluorometric technique involving tetremethylrhodamine ethyl ester (TMRE) and mitotracker green. Unlike inert particulates (titanium dioxide and glass beads), amosite asbestos caused dose- and time-dependent reductions in deltapsi(m). Asbestos-induced deltapsi(m) was associated with the release of cytochrome c from the mitochondria to the cytoplasm as well as activation of caspase 9, a mitochondrial-activated caspase. In contrast, a lower level of caspase 8, the death receptor-activated caspase, was detected in asbestos-exposed AEC. An iron chelator (phytic acid or deferoxamine) or a hydroxyl radical scavenger (sodium benzoate) each blocked asbestos-induced reductions in deltapsi(m) and caspase 9 activation, suggesting a role for iron-derived ROS. Finally, Bcl-X(L), a mitochondrial antiapoptotic protein that prevents cell death by preserving the outer mitochondrial membrane integrity, blocked asbestos-induced decreases in A549 cell deltapsi(m) and reduced apoptosis as assessed by DNA fragmentation. We conclude that asbestos-induced AEC apoptosis results from mitochondrial dysfunction, in part due to iron-derived ROS, which is followed by the release of cytochrome c and caspase 9 activation. Our findings suggest an important role for the mitochondria-regulated death pathway in the

  15. Transcellular degradation of axonal mitochondria

    PubMed Central

    Davis, Chung-ha O.; Kim, Keun-Young; Bushong, Eric A.; Mills, Elizabeth A.; Boassa, Daniela; Shih, Tiffany; Kinebuchi, Mira; Phan, Sebastien; Zhou, Yi; Bihlmeyer, Nathan A.; Nguyen, Judy V.; Jin, Yunju; Ellisman, Mark H.; Marsh-Armstrong, Nicholas

    2014-01-01

    It is generally accepted that healthy cells degrade their own mitochondria. Here, we report that retinal ganglion cell axons of WT mice shed mitochondria at the optic nerve head (ONH), and that these mitochondria are internalized and degraded by adjacent astrocytes. EM demonstrates that mitochondria are shed through formation of large protrusions that originate from otherwise healthy axons. A virally introduced tandem fluorophore protein reporter of acidified mitochondria reveals that acidified axonal mitochondria originating from the retinal ganglion cell are associated with lysosomes within columns of astrocytes in the ONH. According to this reporter, a greater proportion of retinal ganglion cell mitochondria are degraded at the ONH than in the ganglion cell soma. Consistently, analyses of degrading DNA reveal extensive mtDNA degradation within the optic nerve astrocytes, some of which comes from retinal ganglion cell axons. Together, these results demonstrate that surprisingly large proportions of retinal ganglion cell axonal mitochondria are normally degraded by the astrocytes of the ONH. This transcellular degradation of mitochondria, or transmitophagy, likely occurs elsewhere in the CNS, because structurally similar accumulations of degrading mitochondria are also found along neurites in superficial layers of the cerebral cortex. Thus, the general assumption that neurons or other cells necessarily degrade their own mitochondria should be reconsidered. PMID:24979790

  16. ER-mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem-like cells.

    PubMed

    Bassoy, Esen Yonca; Kasahara, Atsuko; Chiusolo, Valentina; Jacquemin, Guillaume; Boydell, Emma; Zamorano, Sebastian; Riccadonna, Cristina; Pellegatta, Serena; Hulo, Nicolas; Dutoit, Valérie; Derouazi, Madiha; Dietrich, Pierre Yves; Walker, Paul R; Martinvalet, Denis

    2017-03-10

    Glioblastoma is a highly heterogeneous aggressive primary brain tumor, with the glioma stem-like cells (GSC) being more sensitive to cytotoxic lymphocyte-mediated killing than glioma differentiated cells (GDC). However, the mechanism behind this higher sensitivity is unclear. Here, we found that the mitochondrial morphology of GSCs modulates the ER-mitochondria contacts that regulate the surface expression of sialylated glycans and their recognition by cytotoxic T lymphocytes and natural killer cells. GSCs displayed diminished ER-mitochondria contacts compared to GDCs. Forced ER-mitochondria contacts in GSCs increased their cell surface expression of sialylated glycans and reduced their susceptibility to cytotoxic lymphocytes. Therefore, mitochondrial morphology and dynamism dictate the ER-mitochondria contacts in order to regulate the surface expression of certain glycans and thus play a role in GSC recognition and elimination by immune effector cells. Targeting the mitochondrial morphology, dynamism, and contacts with the ER could be an innovative strategy to deplete the cancer stem cell compartment to successfully treat glioblastoma.

  17. L-carnitine attenuates oxidant injury in HK-2 cells via ROS-mitochondria pathway.

    PubMed

    Ye, Junsheng; Li, Juan; Yu, Yuming; Wei, Qiang; Deng, Wenfeng; Yu, Lixin

    2010-04-09

    Oxidative stress has been considered as the possible mechanism of renal ischemia/reperfusion injury. L-carnitine is an endogenous mitochondrial membrane compound and could effectively protect ischemia-reperfusion injury in the kidney. To elucidate the nephroprotective effects of L-carnitine, here we assessed the effect of L-carnitine on hydrogen peroxide (H(2)O(2))-mediated oxidative stress in the human proximal tubule epithelial cell line, HK-2 cells. The results showed that pretreatment with L-carnitine 12h inhibited H(2)O(2)-induced cell viability loss, intracellular reactive oxygen species generation and lipid peroxidation in a concentration-dependent manner. Also L-carnitine promoted endogenous antioxidant defense components including total antioxidative capacity, glutathione peroxidase, catalase and superoxide dismutase. In parallel, cell apoptosis triggered by H(2)O(2) characterized with the DNA fragment and caspase-3 activity were also inhibited by L-carnitine. Furthermore, mitochondrial dysfunction associated with cell apoptosis including membrane potential loss, down-regulation of Bcl-2 and up-regulation of Bax and the release of cytochrome c were abrogated in the presence of L-carnitine. These results suggested that L-carnitine could protect HK-2 cells from H(2)O(2)-induced injury through the inhibition of oxidative damage, mitochondria dysfunction and ultimately inhibition of cell apoptosis, which indicates that L-carnitine may be a promising approach for the treatment of oxidative stress in renal diseases.

  18. Bone Marrow Mesenchymal Stem Cells Attenuate Mitochondria Damage Induced by Hypoxia in Mouse Trophoblasts

    PubMed Central

    Wang, Lingjuan; Xu, Xiaoyan; Kang, Lina

    2016-01-01

    Objective We aimed to observe the change of mitochondrial function and structure as well as the cell function induced by hypoxia in mouse trophoblasts, and moreover, to validate the restoration of these changes after co-culture with bone marrow mesenchymal stem cells (hereinafter referred to as “MSCs”). Further, we explored the mechanism of MSCs attenuating the functional damage of trophoblasts caused by hypoxia. Methods Cells were divided into two groups, trophoblasts and MSCs+trophoblasts respectively, and the two groups of cells were incubated with normoxia or hypoxia. Chemiluminescence was used to assay the β-HCG and progesterone in cell culture supernatants quantitatively. Western blotting and PCR were applied to detect the expression of Mfn2, MMP-2, MMP-9 and integrin β1 in the two groups. The mitochondrial membrane potential of each group of cells was detected with JC-1 dye and the ATP content was measured by the phosphomolybdic acid colorimetric method. We utilized transmission electron microscopy for observing the ultrastructure of mitochondria in trophoblasts. Finally, we assessed the cell apoptosis with flow cytometry (FCM) and analyzed the expression of the apoptosis related genes—Bcl-2, Bax, Caspase3 and Caspase9 by western blotting. Results The results showed that the Mfn2 expression was reduced after 4 h in hypoxia compared with that in normoxia, but increased in the co-culture group when compared with that in the separated-culture group (p<0.05). In addition, compared with the separated-culture group, theβ-HCG and progesterone levels in the co-culture group were significantly enhanced (p<0.05), and so were the expressions of MMP-2, MMP-9 and integrin β1 (p<0.05). Moreover, it exhibited significantly higher in ATP levels and intensified about the mitochondrial membrane potential in the co-culture group. TEM revealed disorders of the mitochondrial cristae and presented short rod-like structure and spheroids in hypoxia, however, in the co

  19. A novel manganese complex selectively induces malignant glioma cell death by targeting mitochondria.

    PubMed

    Geng, Ji; Li, Jing; Huang, Tao; Zhao, Kaidi; Chen, Qiuyun; Guo, Wenjie; Gao, Jing

    2016-09-01

    Despite advances in treatment, malignant glioma commonly exhibits recurrence, subsequently leading to a poor prognosis. As manganese (Mn) compounds can be transported by the transferrin‑transferrin receptor system, the present study synthesized and examined the potential use of Adpa‑Mn as a novel antitumor agent. Adpa‑Mn time and dose‑dependently inhibited U251 and C6 cell proliferation; however, it had little effect on normal astrocytes. Apoptosis was significantly elevated following treatment with Adpa‑Mn, as detected by chromatin condensation, Annexin V/propidium iodide staining, cytochrome c release from mitochondria to the cytoplasm, and the activation of caspases‑9, ‑7 and ‑3 and poly (ADP‑ribose) polymerase. In addition, Adpa‑Mn enhanced fluorescence intensity of monodansylcadaverine and elevated the expression levels of the autophagy‑related protein microtubule‑associated protein 1 light chain 3. Pretreatment with the autophagy inhibitors 3‑methyladenine and chloroquine enhanced Adpa‑Mn‑induced cell inhibition, thus indicating that autophagy has an essential role in this process. Furthermore, evidence of mitochondrial dysfunction was detected in the Adpa‑Mn‑treated group, including disrupted membrane potential, elevated levels of reactive oxygen species (ROS) and depleted adenosine triphosphate. Conversely, treatment with the mitochondrial permeability transition inhibitor cyclosporin A reversed Adpa‑Mn‑induced ROS production, mitochondrial damage and cell apoptosis, thus suggesting that Adpa‑Mn may target the mitochondria. Taken together, these data suggested that Adpa‑Mn may be considered for use as a novel anti‑glioma therapeutic option.

  20. Caffeine increases mitochondrial function and blocks melatonin signaling to mitochondria in Alzheimer's mice and cells.

    PubMed

    Dragicevic, Natasa; Delic, Vedad; Cao, Chuanhai; Copes, Neil; Lin, Xiaoyang; Mamcarz, Maggie; Wang, Li; Arendash, Gary W; Bradshaw, Patrick C

    2012-12-01

    Caffeine and melatonin have been shown to protect the Swedish mutant amyloid precursor protein (APP(sw)) transgenic mouse model of Alzheimer's disease from cognitive dysfunction. But their mechanisms of action remain incompletely understood. These Alzheimer's mice have extensive mitochondrial dysfunction, which likely contributes to their cognitive decline. To further explore the mechanism through which caffeine and melatonin protect cognitive function in these mice, we monitored the function of isolated mitochondria from APP(sw) mice treated with caffeine, melatonin, or both in their drinking water for one month. Melatonin treatment yielded a near complete restoration of mitochondrial function in assays of respiratory rate, membrane potential, reactive oxygen species production, and ATP levels. Caffeine treatment by itself yielded a small increase in mitochondrial function. However, caffeine largely blocked the large enhancement of mitochondrial function provided by melatonin. Studies with N2a neuroblastoma cells stably expressing APP(sw) showed that specific inhibition of cAMP-dependent phosphodiesterase (PDE) 4 or cGMP-dependent PDE5 also blocked melatonin protection of mitochondrial function, but A(2a) and A₁ adenosine receptor antagonists were without effect. Melatonin or caffeine at the concentrations used to modulate mitochondrial function in the cells had no effect on cAMP-dependent PDE activity or cellular cAMP or cGMP levels. Therefore, caffeine and increased cyclic nucleotide levels likely block melatonin signaling to mitochondria by independent mechanisms that do not involve adenosine receptor antagonism. The results of this study indicate that melatonin restores mitochondrial function much more potently than caffeine in APP(sw) transgenic mouse and cell models of Alzheimer's disease.

  1. Siramesine triggers cell death through destabilisation of mitochondria, but not lysosomes

    PubMed Central

    Hafner Česen, M; Repnik, U; Turk, V; Turk, B

    2013-01-01

    A sigma-2 receptor agonist siramesine has been shown to trigger cell death of cancer cells and to exhibit a potent anticancer activity in vivo. However, its mechanism of action is still poorly understood. We show that siramesine can induce rapid cell death in a number of cell lines at concentrations above 20 μM. In HaCaT cells, cell death was accompanied by caspase activation, rapid loss of mitochondrial membrane potential (MMP), cytochrome c release, cardiolipin peroxidation and typical apoptotic morphology, whereas in U-87MG cells most apoptotic hallmarks were not notable, although MMP was rapidly lost. In contrast to the rapid loss of MMP above 20 μM siramesine, a rapid increase in lysosomal pH was observed at all concentrations tested (5–40 μM); however, it was not accompanied by lysosomal membrane permeabilisation (LMP) and the release of lysosomal enzymes into the cytosol. Increased lysosomal pH reduced the lysosomal degradation potential as indicated by the accumulation of immature forms of cysteine cathepsins. The lipophilic antioxidant α-tocopherol, but not the hydrophilic antioxidant N-acetyl-cysteine, considerably reduced cell death and destabilisation of mitochondrial membranes, but did not prevent the increase in lysosomal pH. At concentrations below 15 μM, siramesine triggered cell death after 2 days or later, which seems to be associated with a general metabolic and energy imbalance due to defects in the endocytic pathway, intracellular trafficking and energy production, and not by a specific molecular event. Overall, we show that cell death in siramesine-treated cells is induced by destabilisation of mitochondria and is independent of LMP and the release of cathepsins into the cytosol. Moreover, it is unlikely that siramesine acts exclusively through sigma-2 receptors, but rather through multiple molecular targets inside the cell. Our findings are therefore of significant importance in designing the next generation of siramesine

  2. Histone H1.2 is translocated to mitochondria and associates with Bak in bleomycin-induced apoptotic cells.

    PubMed

    Okamura, Hirohiko; Yoshida, Kaya; Amorim, Bruna Rabelo; Haneji, Tatsuji

    2008-04-01

    Bleomycin induces single- and double-stranded breaks in DNA, with consequent mitochondrial membrane aberrations that lead to the apoptotic cell death. It is poorly understood how DNA damage-inducing apoptotic signals are transmitted to mitochondria, from which apoptotic factors are released into the cytoplasm. Here, we investigated the localization of histone H1.2 in the bleomycin-treated human squamous carcinoma SCCTF cells. The presence of DNA double-strand breaks in the bleomycin-treated cells was examined by Western analysis using antibody against phosphorylated histone H2AX (gamma-H2AX). Incubation of SCCTF cells for 48 h with 10 microM bleomycin induced apoptosis, as determined by cleavage of lamin B1 to 28 kDa fragment and DNA ladder formation. The mitochondrial permeabilization causing apoptotic feature was also detected with MitoCapture in the bleomycin-treated cells. Histone H1.2 was translocated from the nucleus to the mitochondria after treatment with bleomycin and co-localized with Bak in mitochondria. Our present results suggest that histone H1.2 plays an important role in transmitting apoptotic signals from the nucleus to the mitochondria following double-stranded breaks of DNA by bleomycin.

  3. Novel mechanism of elimination of malfunctioning mitochondria (mitoptosis): formation of mitoptotic bodies and extrusion of mitochondrial material from the cell.

    PubMed

    Lyamzaev, Konstantin G; Nepryakhina, Olga K; Saprunova, Valeria B; Bakeeva, Lora E; Pletjushkina, Olga Yu; Chernyak, Boris V; Skulachev, Vladimir P

    2008-01-01

    Energy catastrophe, when mitochondria hydrolyze glycolytic ATP instead of producing respiratory ATP, has been modeled. In highly glycolyzing HeLa cells, 30-50% of the population survived after inhibition of respiration and uncoupling of oxidative phosphorylation for 2-4 days. The survival was accompanied by selective elimination of mitochondria. This type of mitoptosis includes (i) fission of mitochondrial filaments, (ii) clustering of the resulting roundish mitochondria in the perinuclear area, (iii) occlusion of mitochondrial clusters by a membrane (formation of a "mitoptotic body"), (iv) decomposition of mitochondria inside this body to small membrane vesicles, (v) protrusion of the body from the cell, and (vi) disruption of the body boundary membrane. Autophagy was not involved in this mitoptotic program. Increased production of reactive oxygen species (ROS) was necessary for execution of the program, since antioxidants prevent mitoptosis and kill the cells treated with the mitochondrial poisons as if a ROS-linked mitoptosis serves for protection of the cells under conditions of severe mitochondrial stress. It is suggested that exocytosis of mitoptotic bodies may be involved in maturation of reticulocytes and lens fiber cells.

  4. Mitochondria-Mediated Protein Regulation Mechanism of Polymorphs-Dependent Inhibition of Nanoselenium on Cancer Cells

    NASA Astrophysics Data System (ADS)

    Wang, Ge; Guo, Yuming; Yang, Gai; Yang, Lin; Ma, Xiaoming; Wang, Kui; Zhu, Lin; Sun, Jiaojiao; Wang, Xiaobing; Zhang, Hua

    2016-08-01

    The present study was (i) to prepare two types of selenium nanoparticles, namely an amorphous form of selenium quantum dots (A-SeQDs) and a crystalline form of selenium quantum dots (C-SeQDs); and (ii) to investigate the nano-bio interactions of A-SeQDs and C-SeQDs in MCF-7, HepG2, HeLa, NIH/3T3, L929 cells and BRL-3A cells. It was found that A-SeQDs could induce the mitochondria-mediated apoptosis, necrosis and death of cells, while C-SeQDs had much weaker effects. This polymorphs-dependent anti-proliferative activity of nano-selenium was scarcely reported. Further investigation demonstrated that A-SeQDs could differentially regulate 61 proteins and several pathways related to stress response, protein synthesis, cell migration and cell cycle, including “p38 MAPK Signaling”, “p53 Signaling”, “14-3-3-mediated Signaling”, “p70S6K Signaling” and “Protein Ubiquitination Pathway”. This was the first report to demonstrate the involvement of protein synthesis and post-translational modification pathways in the anti-proliferative activity associated with NMs. Compared with previously fragmentary studies, this study use a nanomics approach combining bioinformatics and proteomics to systematically investigate the nano-bio interactions of selenium nanoparticles in cancer cells.

  5. Mitochondria-Mediated Protein Regulation Mechanism of Polymorphs-Dependent Inhibition of Nanoselenium on Cancer Cells

    PubMed Central

    Wang, Ge; Guo, Yuming; Yang, Gai; Yang, Lin; Ma, Xiaoming; Wang, Kui; Zhu, Lin; Sun, Jiaojiao; Wang, Xiaobing; Zhang, Hua

    2016-01-01

    The present study was (i) to prepare two types of selenium nanoparticles, namely an amorphous form of selenium quantum dots (A-SeQDs) and a crystalline form of selenium quantum dots (C-SeQDs); and (ii) to investigate the nano-bio interactions of A-SeQDs and C-SeQDs in MCF-7, HepG2, HeLa, NIH/3T3, L929 cells and BRL-3A cells. It was found that A-SeQDs could induce the mitochondria-mediated apoptosis, necrosis and death of cells, while C-SeQDs had much weaker effects. This polymorphs-dependent anti-proliferative activity of nano-selenium was scarcely reported. Further investigation demonstrated that A-SeQDs could differentially regulate 61 proteins and several pathways related to stress response, protein synthesis, cell migration and cell cycle, including “p38 MAPK Signaling”, “p53 Signaling”, “14-3-3-mediated Signaling”, “p70S6K Signaling” and “Protein Ubiquitination Pathway”. This was the first report to demonstrate the involvement of protein synthesis and post-translational modification pathways in the anti-proliferative activity associated with NMs. Compared with previously fragmentary studies, this study use a nanomics approach combining bioinformatics and proteomics to systematically investigate the nano-bio interactions of selenium nanoparticles in cancer cells. PMID:27514819

  6. Eimeria bovis: an update on parasite-host cell interactions.

    PubMed

    Hermosilla, Carlos; Ruiz, Antonio; Taubert, Anja

    2012-10-01

    Apicomplexan parasites are obligate intracellular protozoans and are well recognized modulators of the host cell machinery on varying levels such as host cell metabolism, MHC expression, cell cycle, or apoptosis in order to guarantee their intracellular development and survival. One of the most thoroughly examined apicomplexan pathogens demonstrating a potent manipulative capacity with respect to various host cell functions is Toxoplasma gondii, a protozoon exhibiting rapid intracellular development with small meronts in any nucleated cell, almost irrespective of the cell type or host origin. In contrast, Eimeria bovis merogony I is host- and cell type-restricted and occurs exclusively in bovine endothelial host cells. Furthermore, as a peculiarity, intracellular E. bovis meront I development is a long-lasting process (up to 3 weeks), leading to the formation of huge macromeronts of up to 300 μm in size, containing up to 120,000 merozoites I as offspring. In consequence, the necessity for intense host cell modulation to support this particular development appears even more pressing than in other apicomplexan parasite cases. Here we review the data currently available on E. bovis-host cell interactions, indicating the intriguing capacity of this protozoan to exploit and utilize its host cell for its own benefit.

  7. 3-Methyl pyruvate enhances radiosensitivity through increasing mitochondria-derived reactive oxygen species in tumor cell lines.

    PubMed

    Nishida, Naoya; Yasui, Hironobu; Nagane, Masaki; Yamamori, Tohru; Inanami, Osamu

    2014-05-01

    Considerable interest has recently been focused on the special characteristics of cancer metabolism, and several drugs designed to modulate cancer metabolism have been tested as potential anticancer agents. To date, however, very few studies have been conducted to investigate the combined effects of anticancer drugs and radiotherapy. In this study, to evaluate the role of mitochondria-derived reactive oxygen species (ROS) in the radiation-induced cell death of tumor cells, we have examined the effect of 3-methyl pyruvate (MP). MP is a membrane-permeable pyruvate derivative that is capable of activating mitochondrial energy metabolism in human lung carcinoma A549 cells and murine squamous carcinoma SCCVII cells. Pretreatment with MP significantly enhanced radiation-induced cell death in both cell lines, and also led to increases in the mitochondrial membrane potential, intracellular adenosine triphosphate content, and mitochondria-derived ROS production following the exposure of the cells to X-rays. In A549 cells, MP-induced radiosensitization was completely abolished by vitamin C. In contrast, it was partially abolished in SCCVII cells. These results therefore suggest that the treatment of the cells with MP induced radiosensitization via the production of excess mitochondria-derived ROS in tumor cells.

  8. The pathways of glutamate and glutamine oxidation by tumor cell mitochondria. Role of mitochondrial NAD(P)+-dependent malic enzyme.

    PubMed

    Moreadith, R W; Lehninger, A L

    1984-05-25

    Little evidence has been available on the oxidative pathways of glutamine and glutamate, the major respiratory substrates of cancer cells. Glutamate formed from glutamine by phosphate-dependent glutaminase undergoes quantitative transamination by aerobic tumor mitochondria to yield aspartate. However, when malate is also added there is a pronounced decrease in aspartate production and a large formation of citrate and alanine, in both state 3 and 4 conditions. In contrast, addition of malate to normal rat heart, liver, or kidney mitochondria oxidizing glutamate causes a marked increase in aspartate production. Further analysis showed that extramitochondrial malate is oxidized almost quantitatively to pyruvate + CO2 by NAD(P)+-linked malic enzyme, present in the mitochondria of all tumors tested, but absent in heart, liver, and kidney mitochondria. On the other hand intramitochondrial malate generated from glutamate is oxidized quantitatively to oxalacetate by mitochondrial malate dehydrogenase of tumors. Acetyl-CoA derived from extramitochondrial malate via pyruvate and oxalacetate derived from glutamate via intramitochondrial malate are quantitatively converted into citrate, which is extruded. No evidence was found that malic enzyme of tumor mitochondria converts glutamate-derived malate into pyruvate as postulated in other reports. Possible mechanisms for the integration of mitochondrial malic enzyme and malate dehydrogenase activities in tumors are discussed.

  9. The position of mitochondria and ER in relation to that of the secretory sites in chromaffin cells.

    PubMed

    Villanueva, José; Viniegra, Salvador; Gimenez-Molina, Yolanda; García-Martinez, Virginia; Expósito-Romero, Giovanna; del Mar Frances, Maria; García-Sancho, Javier; Gutiérrez, Luis M

    2014-12-01

    Knowledge of the distribution of mitochondria and endoplasmic reticulum (ER) in relation to the position of exocytotic sites is relevant to understanding the influence of these organelles in tuning Ca(2+) signals and secretion. Confocal images of probes tagged to mitochondria and the F-actin cytoskeleton revealed the existence of two populations of mitochondria, one that was cortical and one that was perinuclear. This mitochondrial distribution was also confirmed by using electron microscopy. In contrast, ER was sparse in the cortex and more abundant in deep cytoplasmic regions. The mitochondrial distribution might be due to organellar transport, which experiences increasing restrictions in the cell cortex. Further study of organelle distribution in relation to the position of SNARE microdomains and the granule fusion sites revealed that a third of the cortical mitochondria colocalized with exocytotic sites and another third located at a distance closer than two vesicle diameters. ER structures were also present in the vicinity of secretory sites but at a lower density. Therefore, mitochondria and ER have a spatial distribution that suggests a specialized role in modulation of exocytosis that fits with the role of cytosolic Ca(2+) microdomains described previously.

  10. Integrating Enzymatic Self-Assembly and Mitochondria Targeting for Selectively Killing Cancer Cells without Acquired Drug Resistance.

    PubMed

    Wang, Huaimin; Feng, Zhaoqianqi; Wang, Youzhi; Zhou, Rong; Yang, Zhimou; Xu, Bing

    2016-12-14

    Targeting organelles by modulating the redox potential of mitochondria is a promising approach to kill cancer cells that minimizes acquired drug resistance. However, it lacks selectivity because mitochondria perform essential functions for (almost) all cells. We show that enzyme-instructed self-assembly (EISA), a bioinspired molecular process, selectively generates the assemblies of redox modulators (e.g., triphenyl phosphinium (TPP)) in the pericellular space of cancer cells for uptake, which allows selectively targeting the mitochondria of cancer cells. The attachment of TPP to a pair of enantiomeric, phosphorylated tetrapeptides produces the precursors (L-1P or D-1P) that form oligomers. Upon dephosphorylation catalyzed by ectophosphatases (e.g., alkaline phosphatase (ALP)) overexpressed on cancer cells (e.g., Saos2), the oligomers self-assemble to form nanoscale assemblies only on the surface of the cancer cells. The cancer cells thus uptake these assemblies of TPP via endocytosis, mainly via a caveolae/raft-dependent pathway. Inside the cells, the assemblies of TPP-peptide conjugates escape from the lysosome, induce dysfunction of mitochondria to release cytochrome c, and result in cell death, while the controls (i.e., omitting TPP motif, inhibiting ALP, or removing phosphate trigger) hardly kill the Saos2 cells. Most importantly, the repeated stimulation of the cancers by the precursors, unexpectedly, sensitizes the cancer cells to the precursors. As the first example of the integration of subcellular targeting with cell targeting, this study validates the spatial control of the assemblies of nonspecific cytotoxic agents by EISA as a promising molecular process for selectively killing cancer cells without inducing acquired drug resistance.

  11. Acute effects of ACTH on dissociated adrenocortical cells: quantitative changes in mitochondria and lipid droplets.

    PubMed

    Zoller, L C; Malamed, S

    1975-08-01

    To study the role of certain organelles in steroidogenesis, dissociated rat adrenocortical cells were incubated for two hours with ACTH at a concentration that induces a high level of steroid production. Sections of ACTH treated and untreated cells were photographed in the electron microscope, and morphometric analysis was undertaken to assess possible ACTH-induced changes in total cell volume, volume density and numerical denisty of lipid droplets and mitochondria. There was no change in total cell volume. Lipid droplet volume density and numerical density decreased. Mitochondrial volume density did not change, but numerical density increased. The decrease in lipid droplet volume density indicates a rapid depletion of cholesterol for steroid production. This depletion is almost entirely due to the disappearance of lipid droplets, rather than to an overall diminution in their size, as shown by the decrease in lipid droplet numerical density. The mitochondrial data suggest that the adrenocortical cell has an adedquate mitochondrial apparatus to respond to acute ACTH stimulation with increased steroid output without an increase inmitochondrial volume.

  12. Mitochondria as an important target in heavy metal toxicity in rat hepatoma AS-30D cells.

    PubMed

    Belyaeva, Elena A; Dymkowska, Dorota; Wieckowski, Mariusz R; Wojtczak, Lech

    2008-08-15

    The mechanisms of toxic effects of divalent cations of three heavy metals Hg, Cd and Cu in rat ascites hepatoma AS-30D cells cultivated in vitro were compared. It was found that the toxicity of these ions, applied in the micromolar range (10-500 microM), decreased from Hg(2+) (most toxic) to Cu(2+) (least toxic). Hg(2+) and Cd(2+) produced a high percentage of cell death by both necrosis and apoptosis, whereas Cu(2+) at concentrations up to 500 microM was weakly effective. Hg(2+) at concentration of 10 microM appeared slightly uncoupling (i.e., stimulated resting state respiration and decreased the mitochondrial transmembrane potential), whereas it exerted a strong inhibitory effect on the respiratory chain and rapid dissipation of the membrane potential at higher concentrations. Cu(2+) had inhibitory effect on cell respiration only at 500 microM concentration and after incubation of 48 h but produced a significant uncoupling effect at lower concentrations. Cu(2+) induced an early and sharp increase of intracellular production of reactive oxygen species (ROS). The action of Hg(2+) and Cd(2+) on ROS generation was biphasic. They stimulated ROS generation within the cells at low concentrations and at short incubation times but decreased ROS generation at higher concentrations and at longer incubation. It is concluded that mitochondria are an important target for toxic effects of Hg(2+), Cd(2+) and Cu(2+) in AS-30D rat hepatoma cells.

  13. Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair.

    PubMed

    Akbari, Mansour; Keijzers, Guido; Maynard, Scott; Scheibye-Knudsen, Morten; Desler, Claus; Hickson, Ian D; Bohr, Vilhelm A

    2014-04-01

    Base excision repair (BER) is the most prominent DNA repair pathway in human mitochondria. BER also results in a temporary generation of AP-sites, single-strand breaks and nucleotide gaps. Thus, incomplete BER can result in the generation of DNA repair intermediates that can disrupt mitochondrial DNA replication and transcription and generate mutations. We carried out BER analysis in highly purified mitochondrial extracts from human cell lines U2OS and HeLa, and mouse brain using a circular DNA substrate containing a lesion at a specific position. We found that DNA ligation is significantly slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I by rotenone. Our results suggest that the amount of DNA ligase III in mitochondria may be critical for cell survival following prolonged oxidative stress, and demonstrate a functional link between mitochondrial DNA damage and repair, cell survival upon oxidative stress, and removal of dysfunctional mitochondria by autophagy.

  14. The sorting of a small potassium channel in mammalian cells can be shifted between mitochondria and plasma membrane.

    PubMed

    von Charpuis, Charlotte; Meckel, Tobias; Moroni, Anna; Thiel, Gerhard

    2015-07-01

    The two small and similar viral K(+) channels Kcv and Kesv are sorted in mammalian cells and yeast to different destinations. Analysis of the sorting pathways shows that Kcv is trafficking via the secretory pathway to the plasma membrane, while Kesv is inserted via the TIM/TOM complex to the inner membrane of mitochondria. Studies with Kesv mutants show that an N-terminal mitochondrial targeting sequence in this channel is neither necessary nor sufficient for sorting of Kesv the mitochondria. Instead the sorting of Kesv can be redirected from the mitochondria to the plasma membrane by an insertion of ≥2 amino acids in a position sensitive manner into the C-terminal transmembrane domain (TMD2) of this channel. The available data advocate the presence of a C-terminal sorting signal in TMD2 of Kesv channel, which is presumably not determined by the length of this domain.

  15. Microcystin-LR induces mitochondria-mediated apoptosis in human bronchial epithelial cells

    PubMed Central

    Li, Yang; Li, Jinhui; Huang, Hui; Yang, Mingfeng; Zhuang, Donggang; Cheng, Xuemin; Zhang, Huizhen; Fu, Xiaoli

    2016-01-01

    The present study aimed to investigate the toxicity of microcystin-LR (MC-LR) and to explore the mechanism of MC-LR-induced apoptosis in human bronchial epithelial (HBE) cells. HBE cells were treated with MC-LR (1, 10, 20, 30 and 40 µg/ml) alone or with MC-LR (0, 2.5, 5 and 10 µg/ml) and Z-VAD-FMK (0, 10, 20, 40, 60, 80, 100, 120 and 140 µM), which is a caspase inhibitor, for 24 and 48 h. Cell viability was assessed via an MTT assay and the half maximal effective concentration of MC-LR was determined. The optimal concentration of Z-VAD-FMK was established as 50 µm, which was then used in the subsequent experiments. MC-LR significantly inhibited cell viability and induced apoptosis of HBE cells in a dose-dependent manner, as detected by an Annexin V/propidium iodide assay. MC-LR induced cell apoptosis, excess reactive oxygen species production and mitochondrial membrane potential collapse, upregulated Bax expression and downregulated B-cell lymphoma-2 expression in HBE cells. Moreover, western blot analysis demonstrated that MC-LR increased the activity levels of caspase-3 and caspase-9 and induced cytochrome c release into the cytoplasm, suggesting that MC-LR-induced apoptosis is associated with the mitochondrial pathway. Furthermore, pretreatment with Z-VAD-FMK reduced MC-LR-induced apoptosis by blocking caspase activation in HBE cells. Therefore, the results of the present study suggested that MC-LR is capable of significantly inhibiting the viability of HBE cells by inducing apoptosis in a mitochondria-dependent manner. The present study provides a foundation for further understanding the mechanism underlying the toxicity of MC-LR in the respiratory system. PMID:27446254

  16. Bioenergetic and Antiapoptotic Properties of Mitochondria from Cultured Human Prostate Cancer Cell Lines PC-3, DU145 and LNCaP

    PubMed Central

    Panov, Alexander; Orynbayeva, Zulfiya

    2013-01-01

    The purpose of this work was to reveal the metabolic features of mitochondria that might be essential for inhibition of apoptotic potential in prostate cancer cells. We studied mitochondria isolated from normal prostate epithelial cells (PrEC), metastatic prostate cancer cell lines LNCaP, PC-3, DU145; and non-prostate cancer cells - human fibrosarcoma HT1080 cells; and normal human lymphoblastoid cells. PrEC cells contained 2 to 4 times less mitochondria per gram of cells than the three PC cell lines. Respiratory activities of PrEC cell mitochondria were 5-20-fold lower than PC mitochondria, depending on substrates and the metabolic state, due to lower content and lower activity of the respiratory enzyme complexes. Mitochondria from the three metastatic prostate cancer cell lines revealed several features that are distinctive only to these cells: low affinity of Complex I for NADH, 20-30 mV higher electrical membrane potential (ΔΨ). Unprotected with cyclosporine A (CsA) the PC-3 mitochondria required 4 times more Ca2+ to open the permeability transition pore (mPTP) when compared with the PrEC mitochondria, and they did not undergo swelling even in the presence of alamethicin, a large pore forming antibiotic. In the presence of CsA, the PC-3 mitochondria did not open spontaneously the mPTP. We conclude that the low apoptotic potential of the metastatic PC cells may arise from inhibition of the Ca2+-dependent permeability transition due to a very high ΔΨ and higher capacity to sequester Ca2+. We suggest that due to the high ΔΨ, mitochondrial metabolism of the metastatic prostate cancer cells is predominantly based on utilization of glutamate and glutamine, which may promote development of cachexia. PMID:23951286

  17. Validation of a Strategy for Cancer Therapy: Delivering Aminoglycoside Drugs to Mitochondria in HeLa Cells.

    PubMed

    Abe, Jiro; Yamada, Yuma; Harashima, Hideyoshi

    2016-02-01

    Mitochondria in human cancer cells have been implicated in cancer cell proliferation, invasion, metastasis, and even drug-resistance mechanisms, making them a potential target organelle for the treatment of human malignancies. Gentamicin (GM), an aminoglycoside drug (AG), is a small molecule that functions as an antibiotic and has ototoxic and nephrotoxic characteristics. Thus, the delivery of GM to mitochondria in cancer cells would be an innovative anticancer therapeutic strategy. In this study, we attempted mitochondrial delivery of GM in HeLa cells derived from a human cervical cancer. For the mitochondrial delivery, we used MITO-Porter, a liposomal nanocarrier for mitochondrial delivery via membrane fusion. We first encapsulated GM in the aqueous phase of the carrier to construct GM-MITO-Porter. Flow cytometry analysis and fluorescent microscopy observations permitted us to confirm that the GM-MITO-Porter was efficiently taken up by HeLa cells and accumulated in mitochondria, whereas naked GM was not taken up by the cells. Moreover, cell viability assays using HeLa cells showed that the GM-MITO-Porter induced strong cytotoxic effects related to mitochondrial disorder. This finding is the first report of the mitochondrial delivery of an AG to cancer cells for cancer therapeutic strategy.

  18. Beyond mitochondria, what would be the energy source of the cell?

    PubMed

    Herrera, Arturo S; Del C A Esparza, Maria; Md Ashraf, Ghulam; Zamyatnin, Andrey A; Aliev, Gjumrakch

    2015-01-01

    Currently, cell biology is based on glucose as the main source of energy. Cellular bioenergetic pathways have become unnecessarily complex in their eagerness to explain that how the cell is able to generate and use energy from the oxidation of glucose, where mitochondria play an important role through oxidative phosphorylation. During a descriptive study about the three leading causes of blindness in the world, the ability of melanin to transform light energy into chemical energy through the dissociation of water molecule was unraveled. Initially, during 2 or 3 years; we tried to link together our findings with the widely accepted metabolic pathways already described in metabolic pathway databases, which have been developed to collect and organize the current knowledge on metabolism scattered across a multitude of scientific articles. However, firstly, the literature on metabolism is extensive but rarely conclusive evidence is available, and secondly, one would expect these databases to contain largely the same information, but the contrary is true. For the apparently well studied metabolic process Krebs cycle, which was described as early as 1937 and is found in nearly every biology and chemistry curriculum, there is a considerable disagreement between at least five databases. Of the nearly 7000 reactions contained jointly by these five databases, only 199 are described in the same way in all the five databases. Thus to try to integrate chemical energy from melanin with the supposedly well-known bioenergetic pathways is easier said than done; and the lack of consensus about metabolic network constitutes an insurmountable barrier. After years of unsuccessful results, we finally realized that the chemical energy released through the dissociation of water molecule by melanin represents over 90% of cell energy requirements. These findings reveal a new aspect of cell biology, as glucose and ATP have biological functions related mainly to biomass and not so much with

  19. Contribution of mitochondria and lysosomes to photodynamic therapy-induced death in cancer cells

    NASA Astrophysics Data System (ADS)

    Nieminen, Anna-Liisa; Azizuddin, Kashif; Zhang, Ping; Kenney, Malcolm E.; Pediaditakis, Peter; Lemasters, John J.; Oleinick, Nancy L.

    2008-02-01

    In photodynamic therapy (PDT), visible light activates a photosensitizing drug added to a tissue, resulting in singlet oxygen formation and cell death. Employing confocal microscopy, we previously found that the phthalocyanine Pc 4 localized primarily to mitochondrial membranes in various cancer cell lines, resulting in mitochondrial reactive oxygen species (ROS) production, followed by inner membrane permeabilization (mitochondrial permeability transition) with mitochondrial depolarization and swelling, which in turn led to cytochrome c release and apoptotic death. Recently, derivatives of Pc 4 with OH groups added to one of the axial ligands were synthesized. These derivatives appeared to be taken up more avidly by cells and caused more cytotoxicity than the parent compound Pc 4. Using organelle-specific fluorophores, we found that one of these derivatives, Pc 181, accumulated into lysosomes and that PDT with Pc 181 caused rapid disintegration of lysosomes. We hypothesized that chelatable iron released from lysosomes during PDT contributes to mitochondrial damage and subsequent cell death. We monitored cytosolic Fe2+ concentrations after PDT with calcein. Fe2+ binds to calcein causing quenching of calcein fluorescence. After bafilomycin, an inhibitor of the vacuolar proton-translocating ATPase, calcein fluorescence became quenched, an effect prevented by starch desferal s-DFO, an iron chelator that enters cells by endocytosis. After Pc 181-PDT, cytosolic calcein fluorescence also decreased, indicating increased chelatable Fe2+ in the cytosol, and apoptosis occurred. s-DFO decreased Pc 181-PDT-induced apoptosis as measured by a decrease of caspase-3 activation. In isolated mitochondria preparations, Fe2+ induced mitochondrial swelling, which was prevented by Ru360, an inhibitor of the mitochondrial Ca2+ uniporter. The data support a hypothesis of oxidative injury in which Pc 181-PDT disintegrates lysosomes and releases constituents that synergistically promote

  20. Mitochondria of a human multidrug-resistant hepatocellular carcinoma cell line constitutively express inducible nitric oxide synthase in the inner membrane.

    PubMed

    Fantappiè, Ornella; Sassoli, Chiara; Tani, Alessia; Nosi, Daniele; Marchetti, Serena; Formigli, Lucia; Mazzanti, Roberto

    2015-06-01

    Mitochondria play a crucial role in pathways of stress conditions. They can be transported from one cell to another, bringing their features to the cell where they are transported. It has been shown in cancer cells overexpressing multidrug resistance (MDR) that mitochondria express proteins involved in drug resistance such as P-glycoprotein (P-gp), breast cancer resistant protein and multiple resistance protein-1. The MDR phenotype is associated with the constitutive expression of COX-2 and iNOS, whereas celecoxib, a specific inhibitor of COX-2 activity, reverses drug resistance of MDR cells by releasing cytochrome c from mitochondria. It is possible that COX-2 and iNOS are also expressed in mitochondria of cancer cells overexpressing the MDR phenotype. This study involved experiments using the human HCC PLC/PRF/5 cell line with and without MDR phenotype and melanoma A375 cells that do not express the MDR1 phenotype but they do iNOS. Western blot analysis, confocal immunofluorescence and immune electron microscopy showed that iNOS is localized in mitochondria of MDR1-positive cells, whereas COX-2 is not. Low and moderate concentrations of celecoxib modulate the expression of iNOS and P-gp in mitochondria of MDR cancer cells independently from inhibition of COX-2 activity. However, A375 cells that express iNOS also in mitochondria, were not MDR1 positive. In conclusion, iNOS can be localized in mitochondria of HCC cells overexpressing MDR1 phenotype, however this phenomenon appears independent from the MDR1 phenotype occurrence. The presence of iNOS in mitochondria of human HCC cells phenotype probably concurs to a more aggressive behaviour of cancer cells.

  1. Multiple enzymatic defects in mitochondria in hematological cells of patients with primary sideroblastic anemia.

    PubMed

    Aoki, Y

    1980-07-01

    Activities of mitochondrial enzymes in blood cells from 69 patients with primary sideroblastic anemia were determined to elucidate the pathogenesis of the disease. In erythroblasts of patients with primary acquired type the activities of both delta-aminolevulinic acid synthetase and mitochondrial serine protease were inevitably decreased. The susceptibility to the protease of apo-delta-aminolevulinic acid synthetase prepared from erythroblasts of patients with this type was within the normal range, in contrast to that of pyridoxine-responsive anemia. The activities of mitochondrial enzymes such as cytochrome oxidase, serine protease, and oligomycin-sensitive ATPase, except citrate synthetase, were usually decreased in mature granulocytes of the patients. Patients with hereditary sideroblastic anemia also had decreased delta-aminolevulinic acid synthetase activity in erythroblasts, and decreased serine protease activity in both erythroblasts and mature granulocytes. Mature granulocytes obtained from patients with pyridoxine-responsive anemia before therapy had decreased cytochrome oxidase activity, however, the activity increased to a normal level when the patients were in remission. The activities of other mitochondrial enzymes in mature granulocytes were within normal range in these patients before pyridoxine therapy. The activities of these mitochondrial enzymes in lymphocytes were within normal range in all groups of patients with primary sideroblastic anemia. We suggest that patients with primary acquired, and possibly also those with hereditary sideroblastic anemia have impaired mitochondrial function in both erythroblasts and granulocytes. That only anemia is observed in these patients is because a functional abnormality of mitochondria in erythroblasts is most important because of the role of mitochondria in the formation of heme in erythrocyte development. In contrast to these two types of sideroblastic anemia, only delta-aminolevulinic acid synthetase in

  2. Human mitochondrial transcription factor A functions in both nuclei and mitochondria and regulates cancer cell growth

    SciTech Connect

    Han, Bin; Izumi, Hiroto; Yasuniwa, Yoshihiro; Akiyama, Masaki; Yamaguchi, Takahiro; Fujimoto, Naohiro; Matsumoto, Tetsuro; Wu, Bin; Tanimoto, Akihide; Sasaguri, Yasuyuki; Kohno, Kimitoshi

    2011-04-29

    Highlights: {yields} Mitochondrial transcription factor A (mtTFA) localizes in nuclei and binds tightly to the nuclear chromatin. {yields} mtTFA contains two putative nuclear localization signals (NLS) in the HMG-boxes. {yields} Overexpression of mtTFA enhances the growth of cancer cells, whereas downregulation of mtTFA inhibits their growth by regulating mtTFA target genes, such as baculoviral IAP repeat-containing 5 (BIRC5; also known as survivin). {yields} Knockdown of mtTFA expression induces p21-dependent G1 cell cycle arrest. -- Abstract: Mitochondrial transcription factor A (mtTFA) is one of the high mobility group protein family and is required for both transcription from and maintenance of mitochondrial genomes. However, the roles of mtTFA have not been extensively studied in cancer cells. Here, we firstly reported the nuclear localization of mtTFA. The proportion of nuclear-localized mtTFA varied among different cancer cells. Some mtTFA binds tightly to the nuclear chromatin. DNA microarray and chromatin immunoprecipitation assays showed that mtTFA can regulate the expression of nuclear genes. Overexpression of mtTFA enhanced the growth of cancer cell lines, whereas downregulation of mtTFA inhibited their growth by regulating mtTFA target genes, such as baculoviral IAP repeat-containing 5 (BIRC5; also known as survivin). Knockdown of mtTFA expression induced p21-dependent G1 cell cycle arrest. These results imply that mtTFA functions in both nuclei and mitochondria to promote cell growth.

  3. l-Lactate metabolism in HEP G2 cell mitochondria due to the l-lactate dehydrogenase determines the occurrence of the lactate/pyruvate shuttle and the appearance of oxaloacetate, malate and citrate outside mitochondria.

    PubMed

    Pizzuto, Roberto; Paventi, Gianluca; Porcile, Carola; Sarnataro, Daniela; Daniele, Aurora; Passarella, Salvatore

    2012-09-01

    As part of an ongoing study of l-lactate metabolism both in normal and in cancer cells, we investigated whether and how l-lactate metabolism occurs in mitochondria of human hepatocellular carcinoma (Hep G2) cells. We found that Hep G2 cell mitochondria (Hep G2-M) possess an l-lactate dehydrogenase (ml-LDH) restricted to the inner mitochondrial compartments as shown by immunological analysis, confocal microscopy and by assaying ml-LDH activity in solubilized mitochondria. Cytosolic and mitochondrial l-LDHs were found to differ from one another in their saturation kinetics. Having shown that l-lactate itself can enter Hep G2 cells, we found that Hep G2-M swell in ammonium l-lactate, but not in ammonium pyruvate solutions, in a manner inhibited by mersalyl, this showing the occurrence of a carrier-mediated l-lactate transport in these mitochondria. Occurrence of the l-lactate/pyruvate shuttle and the appearance outside mitochondria of oxaloacetate, malate and citrate arising from l-lactate uptake and metabolism together with the low oxygen consumption and membrane potential generation are in favor of an anaplerotic role for l-LAC in Hep G2-M.

  4. Role of mitochondria in apoptotic and necroptotic cell death in the developing brain

    PubMed Central

    Thornton, Claire; Hagberg, Henrik

    2015-01-01

    Hypoxic–ischemic encephalopathy induces secondary brain injury characterized by delayed energy failure. Currently, therapeutic hypothermia is the sole treatment available after severe intrapartum asphyxia in babies and acts to attenuate secondary loss of high energy phosphates improving both short- and long-term outcome. In order to develop the next generation of neuroprotective therapies, we urgently need to understand the underlying molecular mechanisms leading to cell death. Hypoxia–ischemia creates a toxic intracellular environment including accumulation of reactive oxygen/nitrosative species and intracellular calcium after the insult, inducing mitochondrial impairment. More specifically mitochondrial respiration is suppressed and calcium signaling is dysregulated. At a certain threshold, Bax-dependent mitochondrial permeabilization will occur leading to activation of caspase-dependent and apoptosis-inducing factor-dependent apoptotic cell death. In addition, hypoxia–ischemia induces inflammation, which leads to the release of TNF-α, TRAIL, TWEAK, FasL and Toll-like receptor agonists that will activate death receptors on neurons and oligodendroglia. Death receptors trigger apoptotic death via caspase-8 and necroptotic cell death through formation of the necrosome (composed of RIP1, RIP3 and MLKL), both of which converge at the mitochondria. PMID:25661091

  5. Mechanisms of cell signaling by nitric oxide and peroxynitrite: from mitochondria to MAP kinases

    NASA Technical Reports Server (NTRS)

    Levonen, A. L.; Patel, R. P.; Brookes, P.; Go, Y. M.; Jo, H.; Parthasarathy, S.; Anderson, P. G.; Darley-Usmar, V. M.

    2001-01-01

    Many of the biological and pathological effects of nitric oxide (NO) are mediated through cell signaling pathways that are initiated by NO reacting with metalloproteins. More recently, it has been recognized that the reaction of NO with free radicals such as superoxide and the lipid peroxyl radical also has the potential to modulate redox signaling. Although it is clear that NO can exert both cytotoxic and cytoprotective actions, the focus of this overview are those reactions that could lead to protection of the cell against oxidative stress in the vasculature. This will include the induction of antioxidant defenses such as glutathione, activation of mitogen-activated protein kinases in response to blood flow, and modulation of mitochondrial function and its impact on apoptosis. Models are presented that show the increased synthesis of glutathione in response to shear stress and inhibition of cytochrome c release from mitochondria. It appears that in the vasculature NO-dependent signaling pathways are of three types: (i) those involving NO itself, leading to modulation of mitochondrial respiration and soluble guanylate cyclase; (ii) those that involve S-nitrosation, including inhibition of caspases; and (iii) autocrine signaling that involves the intracellular formation of peroxynitrite and the activation of the mitogen-activated protein kinases. Taken together, NO plays a major role in the modulation of redox cell signaling through a number of distinct pathways in a cellular setting.

  6. Environmental regulation of mitochondria-rich cells in Chalcalburnus tarichi (Pallas, 1811) during reproductive migration.

    PubMed

    Oğuz, Ahmet R

    2013-03-01

    Chalcalburnus tarichi is an anadromous cyprinid fish that has adapted to extreme conditions (salinity 22 ‰, pH 9.8 and alkalinity 153 mEq × l⁻¹) in Lake Van in eastern Turkey. Changes in immunoreactivity of Na⁺/K⁺-ATPase in gill tissue and osmolarity and ion levels in plasma were investigated in C. tarichi during reproductive migration. Physicochemical characteristics and ion levels in Lake Van were high compared freshwater. Plasma osmolality and plasma ion concentrations ([Na⁺], [K⁺] and [Cl⁻]) increased after transfer from freshwater to Lake Van. The mitochondria-rich (MR) cells of the gill were stained in both filament and lamellar epithelia of C. tarichi by immunocytochemistry with a specific antiserum for Na⁺/K⁺-ATPase in river fish samples. Density and area of MR cells were decreased in lake-adapted fishes. These results indicated that freshwater acclimation capacity is correlated with the size and distribution of MR cells in C. tarichi, in contrast to many teleost fishes.

  7. Skeletal Muscle Satellite Cells, Mitochondria, and MicroRNAs: Their Involvement in the Pathogenesis of ALS.

    PubMed

    Tsitkanou, Stavroula; Della Gatta, Paul A; Russell, Aaron P

    2016-01-01

    Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND), is a fatal motor neuron disorder. It results in progressive degeneration and death of upper and lower motor neurons, protein aggregation, severe muscle atrophy and respiratory insufficiency. Median survival with ALS is between 2 and 5 years from the onset of symptoms. ALS manifests as either familial ALS (FALS) (~10% of cases) or sporadic ALS (SALS), (~90% of cases). Mutations in the copper/zinc (CuZn) superoxide dismutase (SOD1) gene account for ~20% of FALS cases and the mutant SOD1 mouse model has been used extensively to help understand the ALS pathology. As the precise mechanisms causing ALS are not well understood there is presently no cure. Recent evidence suggests that motor neuron degradation may involve a cell non-autonomous phenomenon involving numerous cell types within various tissues. Skeletal muscle is now considered as an important tissue involved in the pathogenesis of ALS by activating a retrograde signaling cascade that degrades motor neurons. Skeletal muscle heath and function are regulated by numerous factors including satellite cells, mitochondria and microRNAs. Studies demonstrate that in ALS these factors show various levels of dysregulation within the skeletal muscle. This review provides an overview of their dysregulation in various ALS models as well as how they may contribute individually and/or synergistically to the ALS pathogenesis.

  8. Subversion of Cell-Autonomous Host Defense by Chlamydia Infection.

    PubMed

    Fischer, Annette; Rudel, Thomas

    2016-05-13

    Obligate intracellular bacteria entirely depend on the metabolites of their host cell for survival and generation of progeny. Due to their lifestyle inside a eukaryotic cell and the lack of any extracellular niche, they have to perfectly adapt to compartmentalized intracellular environment of the host cell and counteract the numerous defense strategies intrinsically present in all eukaryotic cells. This so-called cell-autonomous defense is present in all cell types encountering Chlamydia infection and is in addition closely linked to the cellular innate immune defense of the mammalian host. Cell type and chlamydial species-restricted mechanisms point a long-term evolutionary adaptation that builds the basis of the currently observed host and cell-type tropism among different Chlamydia species. This review will summarize the current knowledge on the strategies pathogenic Chlamydia species have developed to subvert and overcome the multiple mechanisms by which eukaryotic cells defend themselves against intracellular pathogens.

  9. Fusion of mitochondria in tobacco suspension cultured cells is dependent on the cellular ATP level but not on actin polymerization.

    PubMed

    Wakamatsu, Kairo; Fujimoto, Masaru; Nakazono, Mikio; Arimura, Shin-ichi; Tsutsumi, Nobuhiro

    2010-10-01

    Mitochondria in plant cells undergo fusion and fission frequently. Although the mechanisms and proteins of mitochondrial fusion are well known in yeast and mammalian cells, they remain poorly understood in plant cells. To clarify the physiological requirements for plant mitochondrial fusion, we investigated the fusion frequency of mitochondria in tobacco cultured cells using the photoconvertible fluorescent protein Kaede and some physiological inhibitors. The latter included two uncouplers, 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of mitochondrial ATP synthase, oligomycin, and an actin polymerization inhibitor, latrunculin B (Lat B). The frequency of mitochondrial fusion was clearly reduced by DNP, CCCP and oligomycin, but not by Lat B, although Lat B severely inhibited mitochondrial movement. Moreover, DNP, CCCP and oligomycin evidently lowered the cellular ATP levels. These results indicate that plant mitochondrial fusion depends on the cellular ATP level, but not on actin polymerization.

  10. Mitochondria: a target for bacteria.

    PubMed

    Lobet, Elodie; Letesson, Jean-Jacques; Arnould, Thierry

    2015-04-01

    Eukaryotic cells developed strategies to detect and eradicate infections. The innate immune system, which is the first line of defence against invading pathogens, relies on the recognition of molecular patterns conserved among pathogens. Pathogen associated molecular pattern binding to pattern recognition receptor triggers the activation of several signalling pathways leading to the establishment of a pro-inflammatory state required to control the infection. In addition, pathogens evolved to subvert those responses (with passive and active strategies) allowing their entry and persistence in the host cells and tissues. Indeed, several bacteria actively manipulate immune system or interfere with the cell fate for their own benefit. One can imagine that bacterial effectors can potentially manipulate every single organelle in the cell. However, the multiple functions fulfilled by mitochondria especially their involvement in the regulation of innate immune response, make mitochondria a target of choice for bacterial pathogens as they are not only a key component of the central metabolism through ATP production and synthesis of various biomolecules but they also take part to cell signalling through ROS production and control of calcium homeostasis as well as the control of cell survival/programmed cell death. Furthermore, considering that mitochondria derived from an ancestral bacterial endosymbiosis, it is not surprising that a special connection does exist between this organelle and bacteria. In this review, we will discuss different mitochondrial functions that are affected during bacterial infection as well as different strategies developed by bacterial pathogens to subvert functions related to calcium homeostasis, maintenance of redox status and mitochondrial morphology.

  11. KLP6: a newly identified kinesin that regulates the morphology and transport of mitochondria in neuronal cells.

    PubMed

    Tanaka, Kousuke; Sugiura, Yoshimi; Ichishita, Ryohei; Mihara, Katsuyoshi; Oka, Toshihiko

    2011-07-15

    Mitochondria utilize diverse cytoskeleton-based mechanisms to control their functions and morphology. Here, we report a role for kinesin-like protein KLP6, a newly identified member of the kinesin family, in mitochondrial morphology and dynamics. An RNA interference screen using Caenorhabditis elegans led us to identify a C. elegans KLP-6 involved in maintaining mitochondrial morphology. We cloned a cDNA coding for a rat homolog of C. elegans KLP-6, which is an uncharacterized kinesin in vertebrates. A rat KLP6 mutant protein lacking the motor domain induced changes in mitochondrial morphology and significantly decreased mitochondrial motility in HeLa cells, but did not affect the morphology of other organelles. In addition, the KLP6 mutant inhibited transport of mitochondria during anterograde movement in differentiated neuro 2a cells. To date, two kinesins, KIF1Bα and kinesin heavy chain (KHC; also known as KIF5) have been shown to be involved in the distribution of mitochondria in neurons. Expression of the kinesin heavy chain/KIF5 mutant prevented mitochondria from entering into neurites, whereas both the KLP6 and KIF1Bα mutants decreased mitochondrial transport in axonal neurites. Furthermore, both KLP6 and KIF1Bα bind to KBP, a KIF1-binding protein required for axonal outgrowth and mitochondrial distribution. Thus, KLP6 is a newly identified kinesin family member that regulates mitochondrial morphology and transport.

  12. Novel Mitochondria-Targeted Heat-Soluble Proteins Identified in the Anhydrobiotic Tardigrade Improve Osmotic Tolerance of Human Cells

    PubMed Central

    Tanaka, Sae; Tanaka, Junko; Miwa, Yoshihiro; Horikawa, Daiki D.; Katayama, Toshiaki; Arakawa, Kazuharu; Toyoda, Atsushi; Kubo, Takeo; Kunieda, Takekazu

    2015-01-01

    Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called “anhydrobiosis”. Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble) and SAHS (Secretory Abundant Heat Soluble) proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble), as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments. PMID:25675104

  13. Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic Tardigrade improve osmotic tolerance of human cells.

    PubMed

    Tanaka, Sae; Tanaka, Junko; Miwa, Yoshihiro; Horikawa, Daiki D; Katayama, Toshiaki; Arakawa, Kazuharu; Toyoda, Atsushi; Kubo, Takeo; Kunieda, Takekazu

    2015-01-01

    Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called "anhydrobiosis". Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble) and SAHS (Secretory Abundant Heat Soluble) proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble), as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.

  14. Recombinant Buckwheat Trypsin Inhibitor Induces Mitophagy by Directly Targeting Mitochondria and Causes Mitochondrial Dysfunction in Hep G2 Cells.

    PubMed

    Wang, Zhuanhua; Li, Shanshan; Ren, Rong; Li, Jiao; Cui, Xiaodong

    2015-09-09

    Mitochondria are essential targets for cancer chemotherapy and other disease treatments. Recombinant buckwheat trypsin inhibitor (rBTI), a member of the potato type I proteinase inhibitor family, was derived from tartary buckwheat extracts. Our results showed that rBTI directly targeted mitochondria and induced mitochondrial fragmentation and mitophagy. This occurs through enhanced depolarization of the mitochondrial membrane potential, increasing reactive oxygen species (ROS) generation associated with the rise of the superoxide dismutase and catalase activity and glutathione peroxidase (GSH) content, and changes in the GSH/oxidized glutathione ratio. Mild and transient ROS induced by rBTI were shown to be important signaling molecules required to induce Hep G2 mitophagy to remove dysfunctional mitochondria. Furthermore, rBTI could directly induce mitochondrial fragmentation. It was also noted that rBTI highly increased colocalization of mitochondria in treated cells compared to nontreated cells. Tom 20, a subunit of the translocase of the mitochondrial outer membrane complex responsible for recognizing mitochondrial presequences, may be the direct target of rBTI.

  15. Steroidogenesis in MA-10 Mouse Leydig Cells Is Altered via Fatty Acid Import into the Mitochondria1

    PubMed Central

    Rone, Malena B.; Midzak, Andrew S.; Martinez-Arguelles, Daniel B.; Fan, Jinjiang; Ye, Xiaoying; Blonder, Josip; Papadopoulos, Vassilios

    2014-01-01

    ABSTRACT Mitochondria are home to many cellular processes, including oxidative phosphorylation and fatty acid metabolism, and in steroid-synthesizing cells, they are involved in cholesterol import and metabolism, which is the initiating step in steroidogenesis. The formation of macromolecular protein complexes aids in the regulation and efficiency of these mitochondrial functions, though because of their dynamic nature, they are hard to identify. To overcome this problem, we used Blue-Native PAGE with whole-gel mass spectrometry on isolated mitochondria from control and hormone-treated MA-10 mouse tumor Leydig cells. The presence of multiple mitochondrial protein complexes was shown. Although these were qualitatively similar under control and human chorionic gonadotropin (hCG)-stimulated conditions, quantitative differences in the components of the complexes emerged after hCG treatment. A prominent decrease was observed with proteins involved in fatty acid import into the mitochondria, implying that mitochondrial beta-oxidation is not essential for steroidogenesis. To confirm this observation, we inhibited fatty acid import utilizing the CPT1a inhibitor etomoxir, resulting in increased steroid production. Conversely, stimulation of mitochondrial beta-oxidation with metformin resulted in a dose-dependent reduction in steroidogenesis. These changes were accompanied by changes in mitochondrial respiration and in the lactic acid formed during glycolysis. Taken together, these results suggest that upon hormonal stimulation, mitochondria efficiently import cholesterol for steroid production at the expense of other lipids necessary for energy production, specifically fatty acids required for beta-oxidation. PMID:25210128

  16. Interaction of chlamydiae and host cells in vitro.

    PubMed Central

    Moulder, J W

    1991-01-01

    The obligately intracellular bacteria of the genus Chlamydia, which is only remotely related to other eubacterial genera, cause many diseases of humans, nonhuman mammals, and birds. Interaction of chlamydiae with host cells in vitro has been studied as a model of infection in natural hosts and as an example of the adaptation of an organism to an unusual environment, the inside of another living cell. Among the novel adaptations made by chlamydiae have been the substitution of disulfide-bond-cross-linked polypeptides for peptidoglycans and the use of host-generated nucleotide triphosphates as sources of metabolic energy. The effect of contact between chlamydiae and host cells in culture varies from no effect at all to rapid destruction of either chlamydiae or host cells. When successful infection occurs, it is usually followed by production of large numbers of progeny and destruction of host cells. However, host cells containing chlamydiae sometimes continue to divide, with or without overt signs of infection, and chlamydiae may persist indefinitely in cell cultures. Some of the many factors that influence the outcome of chlamydia-host cell interaction are kind of chlamydiae, kind of host cells, mode of chlamydial entry, nutritional adequacy of the culture medium, presence of antimicrobial agents, and presence of immune cells and soluble immune factors. General characteristics of chlamydial multiplication in cells of their natural hosts are reproduced in established cell lines, but reproduction in vitro of the subtle differences in chlamydial behavior responsible for the individuality of the different chlamydial diseases will require better in vitro models. PMID:2030670

  17. Host cells and methods for production of isobutanol

    DOEpatents

    Anthony, Larry Cameron; He, Hongxian; Huang, Lixuan Lisa; Okeefe, Daniel P.; Kruckeberg, Arthur Leo; Li, Yougen; Maggio-Hall, Lori Ann; McElvain, Jessica; Nelson, Mark J.; Patnaik, Ranjan; Rothman, Steven Cary

    2016-08-23

    Provided herein are recombinant yeast host cells and methods for their use for production of isobutanol. Yeast host cells provided comprise an isobutanol biosynthetic pathway and at least one of reduced or eliminated aldehyde dehydrogenase activity, reduced or eliminated acetolactate reductase activity; or a heterologous polynucleotide encoding a polypeptide having ketol-acid reductoisomerase activity.

  18. Methods for production of proteins in host cells

    DOEpatents

    Donnelly, Mark; Joachimiak, Andrzej

    2004-01-13

    The present invention provides methods for the production of proteins, particularly toxic proteins, in host cells. The invention provides methods which use a fusion protein comprising a chaperonin binding domain in host cells induced or regulated to have increased levels of chaperonin which binds the chaperonin binding domain.

  19. Apoptosis Induced by Manganese on Neuronal SK-N-MC Cell Line: Endoplasmic Reticulum (ER) Stress and Mitochondria Dysfunction

    PubMed Central

    Yoon, Hyonok; Kim, Do-Sung; Lee, Geum-Hwa; Kim, Kee-Won; Kim, Hyung-Ryong

    2011-01-01

    Objectives Manganese chloride (MnCl2) is one of heavy metals for causing neurogenerative dysfunction like Manganism. The purpose of this study was to determine the acute toxicity of MnCl2 using different times and various concentrations including whether manganese toxicity may involve in two intrinsic pathways, endoplasmic reticulum (ER) stress and mitochondria dysfunction and lead to neuronal apoptosis mediated by organelle disorders in neuroblastoma cell line SK-N-MC. Methods In the acute toxicity test, five concentrations (200, 400, 600, 800, 1,000 uM) of MnCl2 with 3, 6, 12, 24, 48 hours exposure were selected to analyze cell viability. In addition, to better understand their toxicity, acute toxicity was examined with 1,000 uM MnCl2 for 24 hours exposure via reactive oxygen species (ROS), mitochondria membrane potential, western blotting and mitochondrial complex activities. Results Our results showed that both increments of dose and time prompt the increments in the number of dead cells. Cells treated by 1,000 µM MnCl2 activated 265% (±8.1) caspase-3 compared to control cell. MnCl2 induced intracellular ROS produced 168% (±2.3%) compared to that of the control cells and MnCl2 induced neurotoxicity significantly dissipated 48.9% of mitochondria membrane potential compared to the control cells. Conclusions This study indicated that MnCl2 induced apoptosis via ER stress and mitochondria dysfunction. In addition, MnCl2 affected only complex I except complex II, III or IV activities. PMID:22232721

  20. Mitochondria mediate cell membrane repair and contribute to Duchenne muscular dystrophy

    PubMed Central

    Vila, Maria C; Rayavarapu, Sree; Hogarth, Marshall W; Van der Meulen, Jack H; Horn, Adam; Defour, Aurelia; Takeda, Shin'ichi; Brown, Kristy J; Hathout, Yetrib; Nagaraju, Kanneboyina; Jaiswal, Jyoti K

    2017-01-01

    Dystrophin deficiency is the genetic basis for Duchenne muscular dystrophy (DMD), but the cellular basis of progressive myofiber death in DMD is not fully understood. Using two dystrophin-deficient mdx mouse models, we find that the mitochondrial dysfunction is among the earliest cellular deficits of mdx muscles. Mitochondria in dystrophic myofibers also respond poorly to sarcolemmal injury. These mitochondrial deficits reduce the ability of dystrophic muscle cell membranes to repair and are associated with a compensatory increase in dysferlin-mediated membrane repair proteins. Dysferlin deficit in mdx mice further compromises myofiber cell membrane repair and enhances the muscle pathology at an asymptomatic age for dysferlin-deficient mice. Restoring partial dystrophin expression by exon skipping improves mitochondrial function and offers potential to improve myofiber repair. These findings identify that mitochondrial deficit in muscular dystrophy compromises the repair of injured myofibers and show that this repair mechanism is distinct from and complimentary to the dysferlin-mediated repair of injured myofibers. PMID:27834955

  1. Toxicological assessment of multi-walled carbon nanotubes in vitro: potential mitochondria effects on male reproductive cells

    PubMed Central

    Xu, Cheng; Liu, Qian; Liu, Hui; Zhang, Chunlan; Shao, Wentao; Gu, Aihua

    2016-01-01

    Multi-walled carbon nanotubes (MWCNTs) have been widely used in many fields and were reported to cause reversible testis damage in mice at high-dose. However the reproductive effects of low dose MWCNTs remained elusive. Herein, we used the mice spermatocyte cell line (GC-2spd) to assess the reproductive effects of MWCNTs. Size distribution, zeta potential, and intensity of MWCNTs were characterized. A maximal concentration of 0.5 μg/mL MWCNTs was found to be nonlethal to GC-2spd. At this dose, cell cycles and the ROS levels were in normal status. We also found MWCNTs accumulated in mitochondria, which caused potential mitochondrial DNA damage in spermatocyte. Furthermore, the expression level of mitochondria-related genes, the oxygen consumption rate, and cellular ATP content were declined compared to controls, even at the nonlethal dose. Our results suggested for the first time that, in germ cells, mitochondrion was a cellular organelle that accumulated MWCNTs. PMID:27248475

  2. Label-free and noninvasive optical detection of the distribution of nanometer-size mitochondria in single cells

    NASA Astrophysics Data System (ADS)

    Su, Xuantao; Qiu, Yuanyuan; Marquez-Curtis, Leah; Gupta, Manisha; Capjack, Clarence E.; Rozmus, Wojciech; Janowska-Wieczorek, Anna; Tsui, Ying Y.

    2011-06-01

    A microfluidic flow cytometric technique capable of obtaining information on nanometer-sized organelles in single cells in a label-free, noninvasive optical manner was developed. Experimental two-dimensional (2D) light scattering patterns from malignant lymphoid cells (Jurkat cell line) and normal hematopoietic stem cells (cord blood CD34+ cells) were compared with those obtained from finite-difference time-domain simulations. In the simulations, we assumed that the mitochondria were randomly distributed throughout a Jurkat cell, and aggregated in a CD34+ cell. Comparison of the experimental and simulated light scattering patterns led us to conclude that distinction from these two types of cells may be due to different mitochondrial distributions. This observation was confirmed by conventional confocal fluorescence microscopy. A method for potential cell discrimination was developed based on analysis of the 2D light scattering patterns. Potential clinical applications using mitochondria as intrinsic biological markers in single cells were discussed in terms of normal cells (CD34+ cell and lymphocytes) versus malignant cells (THP-1 and Jurkat cell lines).

  3. Autophagy inhibition enhances isorhamnetin-induced mitochondria-dependent apoptosis in non-small cell lung cancer cells

    PubMed Central

    RUAN, YUSHU; HU, KE; CHEN, HONGBO

    2015-01-01

    Isorhamnetin (ISO) is a flavonoid from plants of the Polygonaceae family and is also an immediate metabolite of quercetin in mammals. To date, the anti-tumor effects of ISO and the underlying mechanisms have not been elucidated in lung cancer cells. The present study investigated the inhibitory effects of ISO on the growth of human lung cancer A549 cells. Treatment of the lung cancer cells with ISO significantly suppressed cell proliferation and colony formation. ISO treatment also resulted in a significant increase in apoptotic cell death of A549 cells in a time- and dose-dependent manner. Further investigation showed that the apoptosis proceeded via the mitochondria-dependent pathway as indicated by alteration of the mitochondrial membrane potential, the release of cytochrome C and caspase activation. Of note, treatment with ISO also induced the formation of autophagosomes and light chain 3-II protein in A549 cells. Furthermore, co-treatment with autophagy inhibitors 3-methyladenine and hydroxychloroquine significantly inhibited the ISO-induced autophagy and enhanced the ISO-induced apoptotic cell death in vitro as well as in vivo. Thus, the results of the present study suggested that ISO is a potential anti-lung cancer agent. In addition, the results indicated that the inhibition of autophagy may be a useful strategy for enhancing the chemotherapeutic effect of ISO on lung cancer cells. PMID:26238746

  4. Control of respiration and ATP synthesis in mammalian mitochondria and cells.

    PubMed

    Brown, G C

    1992-05-15

    We have seen that there is no simple answer to the question 'what controls respiration?' The answer varies with (a) the size of the system examined (mitochondria, cell or organ), (b) the conditions (rate of ATP use, level of hormonal stimulation), and (c) the particular organ examined. Of the various theories of control of respiration outlined in the introduction the ideas of Chance & Williams (1955, 1956) give the basic mechanism of how respiration is regulated. Increased ATP usage can cause increased respiration and ATP synthesis by mass action in all the main tissues. Superimposed on this basic mechanism is calcium control of matrix dehydrogenases (at least in heart and liver), and possibly also of the respiratory chain (at least in liver) and ATP synthase (at least in heart). In many tissues calcium also stimulates ATP usage directly; thus calcium may stimulate energy metabolism at (at least) four possible sites, the importance of each regulation varying with tissue. Regulation of multiple sites may occur (from a teleological point of view) because: (a) energy metabolism is branched and thus proportionate regulation of branches is required in order to maintain constant fluxes to branches (e.g. to proton leak or different ATP uses); and/or (b) control over fluxes is shared by a number of reactions, so that large increases in flux requires stimulation at multiple sites because each site has relatively little control. Control may be distributed throughout energy metabolism, possibly due to the necessity of minimizing cell protein levels (see Brown, 1991). The idea that energy metabolism is regulated by energy charge (as proposed by Atkinson, 1968, 1977) is misleading in mammals. Neither mitochondrial ATP synthesis nor cellular ATP usage is a unique function of energy charge as AMP is not a significant regulator (see for example Erecinska et al., 1977). The near-equilibrium hypothesis of Klingenberg (1961) and Erecinska & Wilson (1982) is partially correct in that

  5. Control of respiration and ATP synthesis in mammalian mitochondria and cells.

    PubMed Central

    Brown, G C

    1992-01-01

    We have seen that there is no simple answer to the question 'what controls respiration?' The answer varies with (a) the size of the system examined (mitochondria, cell or organ), (b) the conditions (rate of ATP use, level of hormonal stimulation), and (c) the particular organ examined. Of the various theories of control of respiration outlined in the introduction the ideas of Chance & Williams (1955, 1956) give the basic mechanism of how respiration is regulated. Increased ATP usage can cause increased respiration and ATP synthesis by mass action in all the main tissues. Superimposed on this basic mechanism is calcium control of matrix dehydrogenases (at least in heart and liver), and possibly also of the respiratory chain (at least in liver) and ATP synthase (at least in heart). In many tissues calcium also stimulates ATP usage directly; thus calcium may stimulate energy metabolism at (at least) four possible sites, the importance of each regulation varying with tissue. Regulation of multiple sites may occur (from a teleological point of view) because: (a) energy metabolism is branched and thus proportionate regulation of branches is required in order to maintain constant fluxes to branches (e.g. to proton leak or different ATP uses); and/or (b) control over fluxes is shared by a number of reactions, so that large increases in flux requires stimulation at multiple sites because each site has relatively little control. Control may be distributed throughout energy metabolism, possibly due to the necessity of minimizing cell protein levels (see Brown, 1991). The idea that energy metabolism is regulated by energy charge (as proposed by Atkinson, 1968, 1977) is misleading in mammals. Neither mitochondrial ATP synthesis nor cellular ATP usage is a unique function of energy charge as AMP is not a significant regulator (see for example Erecinska et al., 1977). The near-equilibrium hypothesis of Klingenberg (1961) and Erecinska & Wilson (1982) is partially correct in that

  6. Zinc pyrithione induces cellular stress signaling and apoptosis in Hep-2 cervical tumor cells: the role of mitochondria and lysosomes.

    PubMed

    Rudolf, Emil; Cervinka, Miroslav

    2010-04-01

    Increased intracellular free zinc concentrations are associated with activation of several stress signaling pathways, specific organelle injury and final cell death. In the present work we examined the involvement of mitochondria and lysosomes and their crosstalk in free zinc-induced cell demise. We report that treatment of cervical tumor Hep-2 cells with zinc pyrithione leads to an early appearance of cytoplasmic zinc-specific foci with corresponding accumulation of zinc first in mitochondria and later in lysosomes. Concomitant with these changes, upregulation of expression of metallothionein II A gene as well as the increased abundance of its protein occurs. Moreover, zinc activates p53 and its dependent genes including Puma and Bax and they contribute to an observed loss of mitochondrial membrane potential and activation of apoptosis. Conversely, lysosomal membrane permeabilization and its promoted cleavage of Bid occurs in a delayed manner in treated cells and their effect on decrease of mitochondrial membrane potential is limited. The use of specific inhibitors as well as siRNA technology suggest a crucial role of MT-IIA in trafficking of free zinc into mitochondria or lysosomes and regulation of apoptotic or necrotic cell demise.

  7. Paragonimus westermani possesses aerobic and anaerobic mitochondria in different tissues, adapting to fluctuating oxygen tension in microaerobic habitats.

    PubMed

    Takamiya, Shinzaburo; Fukuda, Koich; Nakamura, Takeshi; Aoki, Takashi; Sugiyama, Hiromu

    2010-12-01

    We previously showed that adult Paragonimus westermani, the causative agent of paragonimiasis and whose habitat is the host lung, possesses both aerobic and anaerobic respiratory chains, i.e., cyanide-sensitive succinate oxidase and NADH-fumarate reductase systems, in isolated mitochondria (Takamiya et al., 1994). This finding raises the intriguing question as to whether adult Paragonimus worms possess two different populations of mitochondria, one having an aerobic succinate oxidase system and the other an anaerobic fumarate reductase system, or whether the worms possess a single population of mitochondria possessing both respiratory chains (i.e., mixed-functional mitochondria). Staining of trematode tissues for cytochrome c oxidase activity showed three types of mitochondrial populations: small, strongly stained mitochondria with many cristae, localised in the tegument and tegumental cells; and two larger parenchymal cell mitochondria, one with developed cristae and the other with few cristae. The tegumental and parenchymal mitochondria could be separated by isopycnic density-gradient centrifugation and showed different morphological characteristics and respiratory activities, with low-density tegumental mitochondria having cytochrome c oxidase activity and high-density parenchymal mitochondria having fumarate reductase activity. These results indicate that Paragonimus worms possess three different populations of mitochondria, which are distributed throughout trematode tissues and function facultatively, rather than having mixed-functional mitochondria.

  8. Dithiothreitol abrogates the effect of arsenic trioxide on normal rat liver mitochondria and human hepatocellular carcinoma cells

    SciTech Connect

    Paul, Manash K. Kumar, Rajinder; Mukhopadhyay, Anup K.

    2008-01-15

    Arsenic trioxide (ATO) is a known environmental toxicant and a potent chemotherapeutic agent. Significant correlation has been reported between consumption of arsenic-contaminated water and occurrence of liver cancer; moreover, ATO-treated leukemia patients also suffers from liver toxicity. Hence, modulation of ATO action may help to prevent populations suffering from arsenic toxicity as well as help reduce the drug-related side effects. Dithiothreitol (DTT) is a well-known dithiol agent reported to modulate the action of ATO. Controversial reports exist regarding the effect of DTT on ATO-induced apoptosis in leukemia cells. To the best of our knowledge, no report illustrates the modulatory effect of DTT on ATO-induced liver toxicity, the prime target for arsenic. Mitochondria serve as the doorway to apoptosis and have been implicated in ATO-induced cell death. Hence, we attempted to study the modulatory effect of DTT on ATO-induced dysfunction of mammalian liver mitochondria and human hepatocellular carcinoma cell line (Hep3B). We, for the first time, report that ATO produces complex I-mediated electron transfer inhibition, reactive oxygen species (ROS) generation, respiration inhibition, and ATO-induced ROS-mediated mitochondrial permeability transition (MPT) opening. DTT at low concentration (100 {mu}M and less) prevents the effect of ATO-induced complex I-malfunctions. DTT protects mitochondria from ATO-mediated opening of MPT and membrane potential depolarization. DTT also prevented ATO-induced Hep3B cell death. Thus, at low concentrations DTT abrogates the effect of ATO on rat liver mitochondria and Hep3B cell line. Therefore, the present result suggests, that use of low concentration of dithiols as food supplement may prevent arsenic toxicity in affected population.

  9. ABT737 reverses cisplatin resistance by regulating ER-mitochondria Ca2+ signal transduction in human ovarian cancer cells.

    PubMed

    Xie, Qi; Su, Jing; Jiao, Bingxuan; Shen, Luyan; Ma, Liwei; Qu, Xianzhi; Yu, Chunyan; Jiang, Xianrui; Xu, Ye; Sun, Liankun

    2016-12-01

    Bcl-2, which belongs to the Bcl-2 family, is frequently overexpressed in various types of cancer cells and contributes to drug resistance. However, the function of Bcl-2 in cisplatin resistance in human ovarian cancer cells is not fully understood. In this study, we found that the pharmacological inhibitor ABT737 or genetic knockdown of Bcl-2 increased cisplatin cytotoxicity in cisplatin-resistant ovarian cancer cells. Additionally, treatment with ABT737 or Bcl-2 siRNA increased cisplatin-induced free Ca2+ levels in the cytosol and mitochondria, which increased endoplasmic reticulum (ER)-associated and mitochondria-mediated apoptosis. In addition, ABT737 or Bcl-2 siRNA increased the ER-mitochondria contact sites induced by cisplatin in cisplatin-resistant SKOV3/DDP ovarian cancer cells. Consistently with the in vitro results, ABT737 potently synergized with cisplatin in inhibiting the growth of human ovarian cancer xenografts in nude mice. Collectively, these results indicate that pharmacological inhibitors or genetic knockdown of Bcl-2 may be a potential strategy for improving cisplatin treatment of ovarian cancer.

  10. Inhibition of host cell translation elongation by Legionella pneumophila blocks the host cell unfolded protein response.

    PubMed

    Hempstead, Andrew D; Isberg, Ralph R

    2015-12-08

    Cells of the innate immune system recognize bacterial pathogens by detecting common microbial patterns as well as pathogen-specific activities. One system that responds to these stimuli is the IRE1 branch of the unfolded protein response (UPR), a sensor of endoplasmic reticulum (ER) stress. Activation of IRE1, in the context of Toll-like receptor (TLR) signaling, induces strong proinflammatory cytokine induction. We show here that Legionella pneumophila, an intravacuolar pathogen that replicates in an ER-associated compartment, blocks activation of the IRE1 pathway despite presenting pathogen products that stimulate this response. L. pneumophila TLR ligands induced the splicing of mRNA encoding XBP1s, the main target of IRE1 activity. L. pneumophila was able to inhibit both chemical and bacterial induction of XBP1 splicing via bacterial translocated proteins that interfere with host protein translation. A strain lacking five translocated translation elongation inhibitors was unable to block XBP1 splicing, but this could be rescued by expression of a single such inhibitor, consistent with limitation of the response by translation elongation inhibitors. Chemical inhibition of translation elongation blocked pattern recognition receptor-mediated XBP1 splicing, mimicking the effects of the bacterial translation inhibitors. In contrast, host cell-promoted inhibition of translation initiation in response to the pathogen was ineffective in blocking XBP1 splicing, demonstrating the need for the elongation inhibitors for protection from the UPR. The inhibition of host translation elongation may be a common strategy used by pathogens to limit the innate immune response by interfering with signaling via the UPR.

  11. Targeting microbiota-mitochondria inter-talk: Microbiota control mitochondria metabolism.

    PubMed

    Saint-Georges-Chaumet, Y; Attaf, D; Pelletier, E; Edeas, M

    2015-09-26

    Our aim is to highlight the subtle relationship that exists between microbiota and mitochondria. Microbiota targets mitochondria by modulating the Reactive Oxygen Species (ROS) production and the mitochondrial activity through interactions with toxins, proteins or other metabolites released by gut microbiota. The intriguing relationship that exists between mitochondria and microbiota is strengthened by the probable prokaryotic origin of mitochondria. Emerging data implicates a role for ROS, nitric oxide, Short Chain Fatty Acids and hydrogen sulfide in the cross-talk between microbiota - mitochondria and REDOX signaling. Several studies have shown that microbiota act and modulate mitochondrial activity, and use it as a relay to strengthen host-microbiotal interaction. This modulation depends on the gut bacterial strain quality and diversity to increase its pathogenic versus beneficial effects. Furthermore, based on conclusions from new studies, it is possible that microbiota can directly interact with the host cell gene expression by favoring bacterial and mitochondrial DNA insertion in the nuclear genome. The emerging knowledge of mitochondria-microbiota interaction may be of great importance to better understand the mechanism of mitochondrial and metabolic diseases, and the syndromes associated with change in quality and quantity of microbiotal species. We suggest that microbiota via mitochondrial modulation influence cell homeostasis and metabolism. The challenge will be to find strategies to modulate the quality and diversity of microbiota rather than acting on microbiota metabolites and microbiota related factors. The medicine of tomorrow will be completely personalized. Firstly there will be a test to show the quality, quantity and diversity of microbiota, and secondly a preventive or therapeutic strategy will be administrated (probiotics, diet, prodrug or fecal transplantation). The era of digital medicine is here.

  12. Ultrastructure of germ cells, Sertoli cells and mitochondria during spermatogenesis in mature testis of the Chinese Taihang black goats (Capra hircus).

    PubMed

    Shi, Liguang; Xun, Wenjuan; Zhou, Hanlin; Hou, Guanyu; Yue, Wenbin; Zhang, Chunxiang; Ren, Youshe; Yang, Rujie

    2013-07-01

    The objective of this study was to describe the ultrastructure of germ cells, Sertoli cells and mitochondria in mature testis of the Chinese Taihang black goat. The characteristics of germ cell nucleus and mitochondria changing during spermatogenesis were investigated by transmission electron microscopy (TEM). The results showed that the spermatogonium was elliptical, and its nucleus was about 4-5 μm. The round mitochondria can be observed throughout the cytoplasm around the nucleus. Small patches of heterochromatin were distributed throughout the nucleus. Spermatocyte was oval-shaped with a nucleus of about 4-4.5 μm in diameter. The heterochromatin began to attach to the inner surface of the nuclear membrane. Spermatid was about 4 μm and oval in shape. Its nucleus was oval or round and approximately 2-3 μm in diameter. The borderline between nucleus membrane and karyoplasm was distinct. During spermiogenesis, spermatid nucleus was condensed and elongated, and chromatin reached the highest condensation in the mature spermatozoon. The mid-piece was surrounded by mitochondria at the neck region. The sperm tail showed the typical "9+2″ structure, contained axoneme and central singlet microtubules. The nuclei of the Sertoli cells were irregular shaped and showed indentations in the membrane. In the mature testes of goat bucks, abundant mitochondria were around the germ cells and Sertoli cells. The scattered mitochondria were aggregated around the base of the flagellum (axoneme) during the spermatid differentiation stage. In conclusion, the present study showed that the spermatogenic process of Taihang black goat followed the pattern of mammals with some specific.

  13. Calcium Handling by Endoplasmic Reticulum and Mitochondria in a Cell Model of Huntington's Disease.

    PubMed

    De Mario, Agnese; Scarlatti, Chiara; Costiniti, Veronica; Primerano, Simona; Lopreiato, Raffaele; Calì, Tito; Brini, Marisa; Giacomello, Marta; Carafoli, Ernesto

    2016-01-06

    Huntington disease (HD) is caused by the CAG (Q) expansion in exon 1 of the IT15 gene encoding a polyglutamine (poly-Q) stretch of the Huntingtin protein (Htt). In the wild type protein, the repeats specify a stretch of up 34 Q in the N-terminal portion of Htt. In the pathological protein (mHtt) the poly-Q tract is longer. Proteolytic cleavage of the protein liberates an N-terminal fragment containing the expanded poly-Q tract becomes harmful to cells, in particular to striatal neurons. The fragments cause the transcriptional dysfunction of genes that are essential for neuronal survival. Htt, however, could also have non-transcriptional effects, e.g. it could directly alter Ca2+ homeostasis and/or mitochondrial morphology and function. Ca2+ dyshomeostasis and mitochondrial dysfunction are considered important in the molecular aetiology of the disease. Here we have analyzed the effect of the overexpression of Htt fragments (18Q, wild type form, wtHtt and 150Q mutated form, mHtt) on Ca2+ homeostasis in striatal neuronal precursor cells (Q7/7). We have found that the transient overexpression of the Htt fragments increases Ca2+ transients in the mitochondria of cells stimulated with Ca2+-mobilizing agonists. The bulk Ca2+ transients in the cytosol were unaffected, but the Ca2+ content of the endoplasmic reticulum was significantly decreased in the case of mHtt expression. To rule out possible transcriptional effects due to the presence of mHtt, we have measured the mRNA level of a subunit of the respiratory chain complex II, whose expression is commonly altered in many HD models. No effects on the mRNA level was found suggesting that, in our experimental condition, transcriptional action of Htt is not occurring and that the effects on Ca2+ homeostasis were dependent to non-transcriptional mechanisms.

  14. Hitting the Bull’s-Eye in Metastatic Cancers—NSAIDs Elevate ROS in Mitochondria, Inducing Malignant Cell Death

    PubMed Central

    Ralph, Stephen John; Pritchard, Rhys; Rodríguez-Enríquez, Sara; Moreno-Sánchez, Rafael; Ralph, Raymond Keith

    2015-01-01

    Tumor metastases that impede the function of vital organs are a major cause of cancer related mortality. Mitochondrial oxidative stress induced by hypoxia, low nutrient levels, or other stresses, such as genotoxic events, act as key drivers of the malignant changes in primary tumors to enhance their progression to metastasis. Emerging evidence now indicates that mitochondrial modifications and mutations resulting from oxidative stress, and leading to OxPhos stimulation and/or enhanced reactive oxygen species (ROS) production, are essential for promoting and sustaining the highly metastatic phenotype. Moreover, the modified mitochondria in emerging or existing metastatic cancer cells, by their irreversible differences, provide opportunities for selectively targeting their mitochondrial functions with a one-two punch. The first blow would block their anti-oxidative defense, followed by the knockout blow—promoting production of excess ROS, capitulating the terminal stage—activation of the mitochondrial permeability transition pore (mPTP), specifically killing metastatic cancer cells or their precursors. This review links a wide area of research relevant to cellular mechanisms that affect mitochondria activity as a major source of ROS production driving the pro-oxidative state in metastatic cancer cells. Each of the important aspects affecting mitochondrial function are discussed including: hypoxia, HIFs and PGC1 induced metabolic changes, increased ROS production to induce a more pro-oxidative state with reduced antioxidant defenses. It then focuses on how the mitochondria, as a major source of ROS in metastatic cancer cells driving the pro-oxidative state of malignancy enables targeting drugs affecting many of these altered processes and why the NSAIDs are an excellent example of mitochondria-targeted agents that provide a one-two knockout activating the mPTP and their efficacy as selective anticancer metastasis drugs. PMID:25688484

  15. Effect of selected NAD+ analogues on mitochondria activity and proliferation of endothelial EA.hy926 cells.

    PubMed

    Nowak, Robert; Wrzosek, Antoni; Łukasiak, Agnieszka; Rutkowska, Magdalena; Adamus, Jan; Gebicki, Jerzy; Dołowy, Krzysztof; Szewczyk, Adam; Tarasiuk, Jolanta

    2010-08-25

    The aim of the study was to examine the effect of 1-methylnicotinamide (MNA) and 1-methyl-3-nitropyridine (MNP) on mitochondria activity and proliferation of endothelial EA.hy926 cells. The activity of MNA was also referred to nicotinamide (NAM) being MNA metabolic precursor. NAM and MNA used at high concentrations (up to 1 mM) had no effect on mitochondria metabolism and proliferation of EA.hy926 cells. It could be related to the fact that these compounds hardly cross the cell membrane. It supports the results of our previous study suggesting that anti-inflammatory and anti-thrombotic effects of MNA could be associated with its ability to bind to glycosaminoglycans, especially heparins, located on the endothelium membrane without entering into target cells. In contrast, MNP caused substantial changes in mitochondria activity and proliferation of EA.hy926 cells. This compound used at low concentrations (below 100 microM) blocked the cell cycle of EA.hy926 cells in G1 phase and was very effective in inhibiting cell growth (IC50=13.8+/-2.4 microM). At higher concentrations (0.1-1 mM) MNP caused a significant reduction of cell survival. The observed effects of MNP could be related, at least in part, to its ability to influence the ATP and NAD+ intracellular levels. MNP caused also important changes in Ca2+ intracellular concentration, significant decrease in inner mitochondrial membrane potential and high increase in mitochondrial respiration of EA.hy926 cells. The observed effects of MNP may be related in part to its cellular metabolites detected after 45 min incubation with 250 microM MNP.

  16. Host manipulation by cancer cells: Expectations, facts, and therapeutic implications.

    PubMed

    Tissot, Tazzio; Arnal, Audrey; Jacqueline, Camille; Poulin, Robert; Lefèvre, Thierry; Mery, Frédéric; Renaud, François; Roche, Benjamin; Massol, François; Salzet, Michel; Ewald, Paul; Tasiemski, Aurélie; Ujvari, Beata; Thomas, Frédéric

    2016-03-01

    Similar to parasites, cancer cells depend on their hosts for sustenance, proliferation and reproduction, exploiting the hosts for energy and resources, and thereby impairing their health and fitness. Because of this lifestyle similarity, it is predicted that cancer cells could, like numerous parasitic organisms, evolve the capacity to manipulate the phenotype of their hosts to increase their own fitness. We claim that the extent of this phenomenon and its therapeutic implications are, however, underappreciated. Here, we review and discuss what can be regarded as cases of host manipulation in the context of cancer development and progression. We elaborate on how acknowledging the applicability of these principles can offer novel therapeutic and preventive strategies. The manipulation of host phenotype by cancer cells is one more reason to adopt a Darwinian approach in cancer research.

  17. Mitochondria are the primary target in the induction of apoptosis by chiral ruthenium(II) polypyridyl complexes in cancer cells.

    PubMed

    Wang, Jin-Quan; Zhang, Ping-Yu; Qian, Chen; Hou, Xiao-Juan; Ji, Liang-Nian; Chao, Hui

    2014-03-01

    A series of novel chiral ruthenium(II) polypyridyl complexes (Δ-Ru1, Λ-Ru1, Δ-Ru2, Λ-Ru2, Δ-Ru3, Λ-Ru3) were synthesized and evaluated to determine their antiproliferative activities. Colocalization, inductively coupled plasma mass spectrometry, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay studies showed that these ruthenium(II) complexes accumulated preferentially in the mitochondria and exhibited cytotoxicity against various cancer cells in vitro. The complex Δ-Ru1 is of particular interest because it was found to have half-maximal inhibitory concentrations comparable to those of cisplatin and better activity than cisplatin against a cisplatin-resistant cell line, A549-CP/R. Δ-Ru1 induced alterations in the mitochondrial membrane potential and triggered intrinsic mitochondria-mediated apoptosis in HeLa cells, which involved the regulation of Bcl-2 family members and the activation of caspases. Taken together, these data suggest that Δ-Ru1 may be a novel mitochondria-targeting anticancer agent.

  18. Transferring Xenogenic Mitochondria Provides Neural Protection Against Ischemic Stress in Ischemic Rat Brains.

    PubMed

    Huang, Po-Jui; Kuo, Chi-Chung; Lee, Hsiu-Chin; Shen, Ching-I; Cheng, Fu-Chou; Wu, Shih-Fang; Chang, Jui-Chih; Pan, Hung-Chuan; Lin, Shinn-Zong; Liu, Chin-San; Su, Hong-Lin

    2016-01-01

    Transferring exogenous mitochondria has therapeutic effects on damaged heart, liver, and lung tissues. Whether this protective effect requires the symbiosis of exogenous mitochondria in host cells remains unknown. Here xenogenic mitochondria derived from a hamster cell line were applied to ischemic rat brains and rat primary cortical neurons. Isolated hamster mitochondria, either through local intracerebral or systemic intra-arterial injection, significantly restored the motor performance of brain-ischemic rats. The brain infarct area and neuronal cell death were both attenuated by the exogenous mitochondria. Although internalized mitochondria could be observed in neurons and astrocytes, the low efficacy of mitochondrial internalization could not completely account for the high rate of rescue of the treated neural cells. We further illustrated that disrupting electron transport or ATPase synthase in mitochondria significantly attenuated the protective effect, suggesting that intact respiratory activity is essential for the mitochondrial potency on neural protection. These results emphasize that nonsymbiotic extracellular mitochondria can provide an effective cell defense against acute injurious ischemic stress in the central nervous system.

  19. Metabolic adaptation of Chlamydia trachomatis to mammalian host cells.

    PubMed

    Mehlitz, Adrian; Eylert, Eva; Huber, Claudia; Lindner, Buko; Vollmuth, Nadine; Karunakaran, Karthika; Goebel, Werner; Eisenreich, Wolfgang; Rudel, Thomas

    2017-03-01

    Metabolic adaptation is a key feature for the virulence of pathogenic intracellular bacteria. Nevertheless, little is known about the pathways in adapting the bacterial metabolism to multiple carbon sources available from the host cell. To analyze the metabolic adaptation of the obligate intracellular human pathogen Chlamydia trachomatis, we labeled infected HeLa or Caco-2 cells with (13) C-marked glucose, glutamine, malate or a mix of amino acids as tracers. Comparative GC-MS-based isotopologue analysis of protein-derived amino acids from the host cell and the bacterial fraction showed that C. trachomatis efficiently imported amino acids from the host cell for protein biosynthesis. FT-ICR-MS analyses also demonstrated that label from exogenous (13) C-glucose was efficiently shuffled into chlamydial lipopolysaccharide probably via glucose 6-phosphate of the host cell. Minor fractions of bacterial Ala, Asp, and Glu were made de novo probably using dicarboxylates from the citrate cycle of the host cell. Indeed, exogenous (13) C-malate was efficiently taken up by C. trachomatis and metabolized into fumarate and succinate when the bacteria were kept in axenic medium containing the malate tracer. Together, the data indicate co-substrate usage of intracellular C. trachomatis in a stream-lined bipartite metabolism with host cell-supplied amino acids for protein biosynthesis, host cell-provided glucose 6-phosphate for cell wall biosynthesis, and, to some extent, one or more host cell-derived dicarboxylates, e.g. malate, feeding the partial TCA cycle of the bacterium. The latter flux could also support the biosynthesis of meso-2,6-diaminopimelate required for the formation of chlamydial peptidoglycan.

  20. Morphological responses of mitochondria-rich cells to hypersaline environment in the Australian mudskipper, Periophthalmus minutus.

    PubMed

    Itoki, Naoko; Sakamoto, Tatsuya; Hayashi, Masahiro; Takeda, Tatsusuke; Ishimatsu, Atsushi

    2012-07-01

    A population of the Australian mudskipper, Periophthalmus minutus, was found to inhabit mudflat that remained uncovered by tide for more than 20 days in some neap tides. During these prolonged emersion periods, P. minutus retreated into burrows containing little water, with a highest recorded salinity of 84 ± 7.4 psu (practical salinity unit). To explore the mechanical basis for this salinity tolerance in P. minutus, we determined the densities of mitochondria-rich cells (MRCs) in the inner and outer opercula and the pectoral fin skin, in comparison with P. takita, [corrected] from an adjacent lower intertidal habitat, and studied morphological responses of MRCs to exposure to freshwater (FW), and 100% (34-35 psu) and 200% seawater (SW). Periophthalmus minutus showed a higher density of MRCs in the inner operculum (3365 ± 821 cells mm(-2)) than in the pectoral fin skin (1428 ± 161) or the outer operculum (1100 ± 986), all of which were higher than the MRC densities in p. takita. [corrected]. No mortality occurred in 100% or 200% SW, but half of the fish died within four days in FW. Neither 200% SW nor FW exposure affected MRC density. Transfer to 200% SW doubled MRC size after 9-14 days with no change in the proportion of MRCs with apical pits or plasma sodium concentration. In contrast, transfer to FW resulted in a rapid closing of pits and a significant reduction in plasma sodium concentration. These results suggest that P. minutus has evolved morphological and physiological mechanisms to withstand hypersaline conditions that they may encounter in their habitat.

  1. Inhibition of host cell apoptosis by Eimeria bovis sporozoites.

    PubMed

    Lang, Mirjam; Kann, Michael; Zahner, Horst; Taubert, Anja; Hermosilla, Carlos

    2009-03-09

    Sophisticated evasion strategies of obligate intracellular parasites, in particular prevention of host cell apoptosis, are necessary to ensure successful replication. To study the ability of Eimeria bovis in this regard, in vitro experiments were performed applying bovine foetal gastrointestinal cells (BFGC), bovine umbilical vein endothelial cells (BUVEC) and African green monkey kidney cells (VERO) as host cells. BUVEC and BFGC allow maturation of sporozoites to macromeronts, in VERO cells sporozoites survive for weeks without showing further development. In highly infected BUVEC monolayers, infected cells survived until merozoite release whereas uninfected cells underwent apoptosis. Light microscopy and TUNEL assays performed 3-10 days p.i. showed that, within infected BFGC and VERO cell monolayers, uninfected cells underwent programmed cell death after application of various inducers of apoptosis, whereas infected cells survived. Incidentally, the anti-apoptotic efficacies in infected cells were independent of the drugs and the host cell type. We could not demonstrate significant differences between infected and uninfected cells after colchicin treatment in terms of translation of phosphatidylserines to the host cell surface, caspase 3 activity and cytochrome c release, probably since obtainable infection rates were too low. However, we could show by laser scanning confocal microscopy on single cell levels that the expression of the anti-apoptotic factors cellular Flice inhibitory protein (c-FLIP) and cellular inhibition of apoptosis protein 1 (c-IAP1) were enhanced in E. bovis infected cells after application of colchicin, in the latter case also in non-infected cells directly neighbouring infected ones. Our data show that E. bovis protects its host cell from apoptosis by increasing expression of c-IAP1 and c-FLIP.

  2. Respiration characteristics of mitochondria in parental and giant transformed cells of the murine Nemeth-Kellner lymphoma.

    PubMed

    Horbay, Rostyslav O; Manko, Bohdan O; Manko, Volodymyr V; Lootsik, Maxim D; Stoika, Rostyslav S

    2012-01-01

    Respiration characteristics of mitochondria of the parental and giant cells of murine NK/Ly (Nemeth-Kellner lymphoma) were studied. The giant cell-enriched ascites were obtained by serial intraperitoneal injections of vinblastine in tumour-bearing mice. Ascites containing >70% giant cells were used. Their diameter of was over 17 μm (~2800 μm(3)), while the diameter of the parental cells was 12.7 μm (1100 μm(3)). The respiration rate of mitochondria in situ was measured by oxygen consumption in intact and digitonin-permeabilized NK/Ly cells. Endogenous respiration of intact giant NK/Ly cells was three times higher compared to the parental ones, roughly in agreement with the volume change. The giant NK/Ly cells were far more resistant to permeabilization with digitonin than the parental cells, as shown by Trypan Blue and LDH (lactate dehydrogenase) release tests. After digitonin permeabilization, oxygen consumption was reduced to a minimal level (0.06 ng atom O/(s × 106 cells) in both types of cells. Addition of α-ketoglutarate or succinate to the incubation medium increased oxygen consumption in the parental cells by 46 and 164% respectively. In the giant NK/Ly cells, the corresponding increases were 164 and 276%. Addition of ADP to α-ketoglutarate- or succinate-supplemented medium further stimulated oxygen consumption of the permeabilized NK/Ly cells; however, the effect of ADP was more pronounced in the giant cells. In addition, indices of respiratory control were significantly higher in the giant cells. Oligomycin suppressed considerably the respiration of the intact giant cells but had a much weaker effect on parental cells. Thus, giant NK/Ly cells possess much higher respiration rates and show tighter coupling between the respiration and oxidative phosphorylation compared with parental cells.

  3. Large-conductance Ca²⁺-activated potassium channel in mitochondria of endothelial EA.hy926 cells.

    PubMed

    Bednarczyk, Piotr; Koziel, Agnieszka; Jarmuszkiewicz, Wieslawa; Szewczyk, Adam

    2013-06-01

    In the present study, we describe the existence of a large-conductance Ca²⁺-activated potassium (BKCa) channel in the mitochondria of the human endothelial cell line EA.hy926. A single-channel current was recorded from endothelial mitoplasts (i.e., inner mitochondrial membrane) using the patch-clamp technique in the mitoplast-attached mode. A potassium-selective current was recorded with a mean conductance equal to 270 ± 10 pS in a symmetrical 150/150 mM KCl isotonic solution. The channel activity, which was determined as the open probability, increased with the addition of calcium ions and the potassium channel opener NS1619. Conversely, the activity of the channel was irreversibly blocked by paxilline and iberiotoxin, BKCa channel inhibitors. The open-state probability was found to be voltage dependent. The substances known to modulate BKCa channel activity influenced the bioenergetics of mitochondria isolated from human endothelial EA.hy926 cells. In isolated mitochondria, 100 μM Ca²⁺, 10 μM NS1619, and 0.5 μM NS11021 depolarized the mitochondrial membrane potential and stimulated nonphosphorylating respiration. These effects were blocked by iberiotoxin and paxilline in a potassium-dependent manner. Under phosphorylating conditions, NS1619-induced, iberiotoxin-sensitive uncoupling diverted energy from ATP synthesis during the phosphorylating respiration of the endothelial mitochondria. Immunological analysis with antibodies raised against proteins of the plasma membrane BKCa channel identified a pore-forming α-subunit and an auxiliary β₂-subunit of the channel in the endothelial mitochondrial inner membrane. In conclusion, we show for the first time that the inner mitochondrial membrane in human endothelial EA.hy926 cells contains a large-conductance calcium-dependent potassium channel with properties similar to those of the surface membrane BKCa channel.

  4. Coral host cells acidify symbiotic algal microenvironment to promote photosynthesis.

    PubMed

    Barott, Katie L; Venn, Alexander A; Perez, Sidney O; Tambutté, Sylvie; Tresguerres, Martin

    2015-01-13

    Symbiotic dinoflagellate algae residing inside coral tissues supply the host with the majority of their energy requirements through the translocation of photosynthetically fixed carbon. The algae, in turn, rely on the host for the supply of inorganic carbon. Carbon must be concentrated as CO2 in order for photosynthesis to proceed, and here we show that the coral host plays an active role in this process. The host-derived symbiosome membrane surrounding the algae abundantly expresses vacuolar H(+)-ATPase (VHA), which acidifies the symbiosome space down to pH ∼ 4. Inhibition of VHA results in a significant decrease in average H(+) activity in the symbiosome of up to 75% and a significant reduction in O2 production rate, a measure of photosynthetic activity. These results suggest that host VHA is part of a previously unidentified carbon concentrating mechanism for algal photosynthesis and provide mechanistic evidence that coral host cells can actively modulate the physiology of their symbionts.

  5. Coral host cells acidify symbiotic algal microenvironment to promote photosynthesis

    PubMed Central

    Barott, Katie L.; Venn, Alexander A.; Perez, Sidney O.; Tambutté, Sylvie; Tresguerres, Martin

    2015-01-01

    Symbiotic dinoflagellate algae residing inside coral tissues supply the host with the majority of their energy requirements through the translocation of photosynthetically fixed carbon. The algae, in turn, rely on the host for the supply of inorganic carbon. Carbon must be concentrated as CO2 in order for photosynthesis to proceed, and here we show that the coral host plays an active role in this process. The host-derived symbiosome membrane surrounding the algae abundantly expresses vacuolar H+-ATPase (VHA), which acidifies the symbiosome space down to pH ∼4. Inhibition of VHA results in a significant decrease in average H+ activity in the symbiosome of up to 75% and a significant reduction in O2 production rate, a measure of photosynthetic activity. These results suggest that host VHA is part of a previously unidentified carbon concentrating mechanism for algal photosynthesis and provide mechanistic evidence that coral host cells can actively modulate the physiology of their symbionts. PMID:25548188

  6. Capacitive immunosensor for the detection of host cell proteins.

    PubMed

    Teeparuksapun, Kosin; Hedström, Martin; Kanatharana, Proespichaya; Thavarungkul, Panote; Mattiasson, Bo

    2012-01-01

    A new analysis for monitoring host cell proteins in preparations of transgenically produced protein pharmaceuticals is described. A capacitive biosensor with a very high sensitivity is used to monitor trace amounts of host cell proteins. The sensor consists of a gold electrode, the surface of which is well insulated and on which a preparation of a population of polyclonal antibodies raised against the complete protein set-up of the host cell are immobilized. Host cell proteins are present at very low concentrations during the production of a transgenic protein. The system studied here is a model system with an enzyme expressed in Escherichia coli (E. coli). Due to the high sensitivity, it may even be possible to dilute the samples to be analyzed, thereby reducing a negative influence from non-specific binding to the sensor surface.

  7. Fungal invasion of normally non-phagocytic host cells.

    PubMed

    Filler, Scott G; Sheppard, Donald C

    2006-12-01

    Many fungi that cause invasive disease invade host epithelial cells during mucosal and respiratory infection, and subsequently invade endothelial cells during hematogenous infection. Most fungi invade these normally non-phagocytic host cells by inducing their own uptake. Candida albicans hyphae interact with endothelial cells in vitro by binding to N-cadherin on the endothelial cell surface. This binding induces rearrangement of endothelial cell microfilaments, which results in the endocytosis of the organism. The capsule of Cryptococcus neoformans is composed of glucuronoxylomannan, which binds specifically to brain endothelial cells, and appears to mediate both adherence and induction of endocytosis. The mechanisms by which other fungal pathogens induce their own uptake are largely unknown. Some angioinvasive fungi, such as Aspergillus species and the Zygomycetes, invade endothelial cells from the abluminal surface during the initiation of invasive disease, and subsequently invade the luminal surface of endothelial cells during hematogenous dissemination. Invasion of normally non-phagocytic host cells has different consequences, depending on the type of invading fungus. Aspergillus fumigatus blocks apoptosis of pulmonary epithelial cells, whereas Paracoccidioides brasiliensis induces apoptosis of epithelial cells. This review summarizes the mechanisms by which diverse fungal pathogens invade normally non-phagocytic host cells and discusses gaps in our knowledge that provide opportunities for future research.

  8. Fungal Invasion of Normally Non-Phagocytic Host Cells

    PubMed Central

    Filler, Scott G; Sheppard, Donald C

    2006-01-01

    Many fungi that cause invasive disease invade host epithelial cells during mucosal and respiratory infection, and subsequently invade endothelial cells during hematogenous infection. Most fungi invade these normally non-phagocytic host cells by inducing their own uptake. Candida albicans hyphae interact with endothelial cells in vitro by binding to N-cadherin on the endothelial cell surface. This binding induces rearrangement of endothelial cell microfilaments, which results in the endocytosis of the organism. The capsule of Cryptococcus neoformans is composed of glucuronoxylomannan, which binds specifically to brain endothelial cells, and appears to mediate both adherence and induction of endocytosis. The mechanisms by which other fungal pathogens induce their own uptake are largely unknown. Some angioinvasive fungi, such as Aspergillus species and the Zygomycetes, invade endothelial cells from the abluminal surface during the initiation of invasive disease, and subsequently invade the luminal surface of endothelial cells during hematogenous dissemination. Invasion of normally non-phagocytic host cells has different consequences, depending on the type of invading fungus. Aspergillus fumigatus blocks apoptosis of pulmonary epithelial cells, whereas Paracoccidioides brasiliensis induces apoptosis of epithelial cells. This review summarizes the mechanisms by which diverse fungal pathogens invade normally non-phagocytic host cells and discusses gaps in our knowledge that provide opportunities for future research. PMID:17196036

  9. Segregation of sperm mitochondria in two- and four-cell embryos of the blue mussel Mytilus edulis: Implications for the mechanism of doubly uniparental inheritance of mitochondrial DNA.

    PubMed

    Cogswell, Andrew T; Kenchington, Ellen L R; Zouros, Eleftherios

    2006-07-01

    Species of the family Mytilidae have 2 mitochondrial genomes, one that is transmitted through the egg and one that is transmitted through the sperm. In the Mytilus edulis species complex (M. edulis, M. galloprovincialis, and M. trossulus) there is also a strong mother-dependent sex-ratio bias in favor of one or the other sex among progeny from pair matings. In a previous study, we have shown that sperm mitochondria enter the egg and that their behavior during cell division is different depending on whether the egg originated from a female- or male-biased mother. Specifically, in eggs from females that produce mostly or exclusively daughters, sperm mitochondria disperse randomly among cells after egg division. In eggs from females that produce predominantly sons, sperm mitochondria tend to stay together in the same cell. Here, we extend these observations and show that in 2- and 4-cell embryos from male-biased mothers most sperm mitochondria are located near or at the cleavage furrow of the major cell, in contrast to embryos from female-biased mothers where there is no preferential association of sperm mitochondria with the cleavage furrow. This observation provides evidence for an early developmental mechanism through which sperm mitochondria are preferentially channeled into the primordial cells of male embryos, thus making the paternal mitochondrial genome the dominant mtDNA component of the male germ line.

  10. Host cells and methods for producing isoprenyl alkanoates

    SciTech Connect

    Lee, Taek Soon; Fortman, Jeffrey L.; Keasling, Jay D.

    2015-12-01

    The invention provides for a method of producing an isoprenyl alkanoate in a genetically modified host cell. In one embodiment, the method comprises culturing a genetically modified host cell which expresses an enzyme capable of catalyzing the esterification of an isoprenol and a straight-chain fatty acid, such as an alcohol acetyltransferase (AAT), wax ester synthase/diacylglycerol acyltransferase (WS/DGAT) or lipase, under a suitable condition so that the isoprenyl alkanoate is produced.

  11. In self-defence: hexokinase promotes voltage-dependent anion channel closure and prevents mitochondria-mediated apoptotic cell death.

    PubMed

    Azoulay-Zohar, Heftsi; Israelson, Adrian; Abu-Hamad, Salah; Shoshan-Barmatz, Varda

    2004-01-15

    In tumour cells, elevated levels of mitochondria-bound isoforms of hexokinase (HK-I and HK-II) result in the evasion of apoptosis, thereby allowing the cells to continue proliferating. The molecular mechanisms by which bound HK promotes cell survival are not yet fully understood. Our studies relying on the purified mitochondrial outer membrane protein VDAC (voltage-dependent anion channel), isolated mitochondria or cells in culture suggested that the anti-apoptotic activity of HK-I occurs via modulation of the mitochondrial phase of apoptosis. In the present paper, a direct interaction of HK-I with bilayer-reconstituted purified VDAC, inducing channel closure, is demonstrated for the first time. Moreover, HK-I prevented the Ca(2+)-dependent opening of the mitochondrial PTP (permeability transition pore) and release of the pro-apoptotic protein cytochrome c. The effects of HK-I on VDAC activity and PTP opening were prevented by the HK reaction product glucose 6-phosphate, a metabolic intermediate in most biosynthetic pathways. Furthermore, glucose 6-phosphate re-opened both the VDAC and the PTP closed by HK-I. The HK-I-mediated effects on VDAC and PTP were not observed using either yeast HK or HK-I lacking the N-terminal hydrophobic peptide responsible for binding to mitochondria, or in the presence of an antibody specific for the N-terminus of HK-I. Finally, HK-I overexpression in leukaemia-derived U-937 or vascular smooth muscle cells protected against staurosporine-induced apoptosis, with a decrease of up to 70% in cell death. These results offer insight into the mechanisms by which bound HK promotes tumour cell survival, and suggests that its overexpression not only ensures supplies of energy and phosphometabolites, but also reflects an anti-apoptotic defence mechanism.

  12. Rhein Elicits In Vitro Cytotoxicity in Primary Human Liver HL-7702 Cells by Inducing Apoptosis through Mitochondria-Mediated Pathway

    PubMed Central

    Bounda, Guy-Armel; Zhou, Wang; Wang, Dan-dan; Yu, Feng

    2015-01-01

    Objective. To study rhein-induced apoptosis signaling pathway and to investigate its molecular mechanisms in primary human hepatic cells. Results. Cell viability of HL-7702 cells treated with rhein showed significant decrease in dose-dependent manner. Following rhein treatment (25 μM, 50 μM, and 100 μM) for 12 h, the detection of apoptotic cells was significantly analyzed by flow cytometry and nuclear morphological changes by Hoechst 33258, respectively. Fatty degeneration studies showed upregulation level of the relevant hepatic markers (P < 0.01). Caspase activities expressed significant upregulation of caspase-3, caspase-9, and caspase-8. Moreover, apoptotic cells by rhein were significantly inhibited by Z-LEHD-FMK and Z-DEVD-FMK, caspase-9 inhibitor, and caspase-3 inhibitor, respectively. Overproduction of reactive oxygen species, lipid peroxidation, and loss of mitochondrial membrane potential were detected by fluorometry. Additionally, NAC, a ROS scavenger, significantly attenuated rhein-induced oxidative damage in HL-7702 cells. Furthermore, real-time qPCR results showed significant upregulation of p53, PUMA, Apaf-1, and Casp-9 and Casp-3 mRNA, with no significant changes of Fas and Cytochrome-c. Immunoblotting revealed significant Cytochrome-c release from mitochondria into cytosol and no change in Fas expression. Conclusion. Taken together, these observations suggested that rhein could induce apoptosis in HL-7702 cells via mitochondria-mediated signal pathway with involvement of oxidative stress mechanism. PMID:26221172

  13. The effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca2+]i in the cells

    NASA Astrophysics Data System (ADS)

    Ji, YuBin; Gao, ShiYong; Ji, ChenFeng; Zou, Xiang

    2008-12-01

    To observe the effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca2+]i in the cells, and to uncover the mechanism by which solanine induces apoptosis. HepG2 cells are double stained with and Fluo-3/AM, and both the change in membrane potential of mitochondria and that of [Ca2+]i in the cells are observed using LCSM. The results of double staining with TMRE and Fluo-3/AM show that solanine can lower membrane potential and increase the concentration of Ca2+ in the cells Solanine opens up the PT channels in the membrane by lowering the membrane potential, leading to Ca2+ being transported down its concentration gradient, which in turn leads to the rise of the concentration of Ca2+ in the cell, turning on the mechanism for apoptosis.

  14. Listeria monocytogenes induces host DNA damage and delays the host cell cycle to promote infection

    PubMed Central

    Leitão, Elsa; Costa, Ana Catarina; Brito, Cláudia; Costa, Lionel; Pombinho, Rita; Cabanes, Didier; Sousa, Sandra

    2014-01-01

    Listeria monocytogenes (Lm) is a human intracellular pathogen widely used to uncover the mechanisms evolved by pathogens to establish infection. However, its capacity to perturb the host cell cycle was never reported. We show that Lm infection affects the host cell cycle progression, increasing its overall duration but allowing consecutive rounds of division. A complete Lm infectious cycle induces a S-phase delay accompanied by a slower rate of DNA synthesis and increased levels of host DNA strand breaks. Additionally, DNA damage/replication checkpoint responses are triggered in an Lm dose-dependent manner through the phosphorylation of DNA-PK, H2A.X, and CDC25A and independently from ATM/ATR. While host DNA damage induced exogenously favors Lm dissemination, the override of checkpoint pathways limits infection. We propose that host DNA replication disturbed by Lm infection culminates in DNA strand breaks, triggering DNA damage/replication responses, and ensuring a cell cycle delay that favors Lm propagation. PMID:24552813

  15. Effects of cumene hydroperoxide on the Ca(2+)-induced Ca2+ efflux from mitochondria and on the viability of hepatoma cells.

    PubMed

    Teplova, V V; Kudin, A P; Evtodienko YuV

    1998-01-01

    Effects of cumene hydroperoxide on the Ca(2+)-induced Ca2+ efflux from mitochondria isolated from rat liver and Zaidelja hepatoma were compared. Cumene hydroperoxide at micromolar concentrations (0.3-10 microM) prevented the closing of the permeability transition pore in the inner mitochondrial membrane and, therefore, potentiated the Ca(2+)-induced Ca2+ efflux. This response was 10-100 times greater in hepatoma mitochondria than in rat liver mitochondria. Micromolar concentrations of cumene hydroperoxide induced the death of the hepatoma cells in vitro.

  16. Side effects of antibiotics during bacterial infection: mitochondria, the main target in host cell.

    PubMed

    Singh, Rochika; Sripada, Lakshmi; Singh, Rajesh

    2014-05-01

    Antibiotics are frontline therapy against microbial infectious diseases. Many antibiotics are known to cause several side effects in humans. Ribosomal RNA (rRNA) is the main target of antibiotics that inhibit protein synthesis. According to the endosymbiont theory, mitochondrion is of bacterial origin and their molecular and structural components of the protein expression system are almost similar. It has been observed that the rate of mutations in mitochondrial rRNA is higher as compared to that of nuclear rRNA. The presence of these mutations may mimic prokaryotic rRNA structure and bind to antibiotics targeted to ribosomes of bacteria. Mitochondrial functions are compromised hence may be one of the major causes of side effects observed during antibiotic therapy. The current review had summarized the studies on the role of antibiotics on mitochondrial functions and its relevance to the observed side effects in physiological and pathological conditions.

  17. Role of mitochondria in parvovirus pathology.

    PubMed

    Nykky, Jonna; Vuento, Matti; Gilbert, Leona

    2014-01-01

    Proper functioning of the mitochondria is crucial for the survival of the cell. Viruses are able to interfere with mitochondrial functions as they infect the host cell. Parvoviruses are known to induce apoptosis in infected cells, but the role of the mitochondria in parvovirus induced cytopathy is only partially known. Here we demonstrate with confocal and electron microscopy that canine parvovirus (CPV) associated with the mitochondrial outer membrane from the onset of infection. During viral entry a transient depolarization of the mitochondrial transmembrane potential and increase in ROS level was detected. Subsequently, mitochondrial homeostasis was normalized shortly, as detected by repolarization of the mitochondrial membrane and decrease of ROS. Indeed, activation of cell survival signalling through ERK1/2 cascade was observed early in CPV infected cells. At 12 hours post infection, concurrent with the expression of viral non-structural protein 1, damage to the mitochondrial structure and depolarization of its membrane were apparent. Results of this study provide additional insight of parvovirus pathology and also more general information of virus-mitochondria association.

  18. Role of Mitochondria in Parvovirus Pathology

    PubMed Central

    Nykky, Jonna; Vuento, Matti; Gilbert, Leona

    2014-01-01

    Proper functioning of the mitochondria is crucial for the survival of the cell. Viruses are able to interfere with mitochondrial functions as they infect the host cell. Parvoviruses are known to induce apoptosis in infected cells, but the role of the mitochondria in parvovirus induced cytopathy is only partially known. Here we demonstrate with confocal and electron microscopy that canine parvovirus (CPV) associated with the mitochondrial outer membrane from the onset of infection. During viral entry a transient depolarization of the mitochondrial transmembrane potential and increase in ROS level was detected. Subsequently, mitochondrial homeostasis was normalized shortly, as detected by repolarization of the mitochondrial membrane and decrease of ROS. Indeed, activation of cell survival signalling through ERK1/2 cascade was observed early in CPV infected cells. At 12 hours post infection, concurrent with the expression of viral non-structural protein 1, damage to the mitochondrial structure and depolarization of its membrane were apparent. Results of this study provide additional insight of parvovirus pathology and also more general information of virus-mitochondria association. PMID:24465910

  19. Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

    SciTech Connect

    Wang, Yi-Fen; Shyu, Huey-Wen; Chang, Yi-Chuang; Tseng, Wei-Chang; Huang, Yeou-Lih; Lin, Kuan-Hua; Chou, Miao-Chen; Liu, Heng-Ling; Chen, Chang-Yu

    2012-03-01

    Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

  20. Why translation counts for mitochondria - retrograde signalling links mitochondrial protein synthesis to mitochondrial biogenesis and cell proliferation.

    PubMed

    Battersby, Brendan J; Richter, Uwe

    2013-10-01

    Organelle biosynthesis is a key requirement for cell growth and division. The regulation of mitochondrial biosynthesis exhibits additional layers of complexity compared with that of other organelles because they contain their own genome and dedicated ribosomes. Maintaining these components requires gene expression to be coordinated between the nucleo-cytoplasmic compartment and mitochondria in order to monitor organelle homeostasis and to integrate the responses to the physiological and developmental demands of the cell. Surprisingly, the parameters that are used to monitor or count mitochondrial abundance are not known, nor are the signalling pathways. Inhibiting the translation on mito-ribosomes genetically or with antibiotics can impair cell proliferation and has been attributed to defects in aerobic energy metabolism, even though proliferating cells rely primarily on glycolysis to fuel their metabolic demands. However, a recent study indicates that mitochondrial translational stress and the rescue mechanisms that relieve this stress cause the defect in cell proliferation and occur before any impairment of oxidative phosphorylation. Therefore, the process of mitochondrial translation in itself appears to be an important checkpoint for the monitoring of mitochondrial homeostasis and might have a role in establishing mitochondrial abundance within a cell. This hypothesis article will explore the evidence supporting a role for mito-ribosomes and translation in a mitochondria-counting mechanism.

  1. Asiatic acid uncouples respiration in isolated mouse liver mitochondria and induces HepG2 cells death.

    PubMed

    Lu, Yapeng; Liu, Siyuan; Wang, Ying; Wang, Dang; Gao, Jing; Zhu, Li

    2016-09-05

    Asiatic acid, one of the triterpenoid components isolated from Centella asiatica, has received increasing attention due to a wide variety of biological activities. To date, little is known about its mechanisms of action. Here we examined the cytotoxic effect of asiatic acid on HepG2 cells and elucidated some of the underlying mechanisms. Asiatic acid induced rapid cell death, as well as mitochondrial membrane potential (MMP) dissipation, ATP depletion and cytochrome c release from mitochondria to the cytosol in HepG2 cells. In mitochondria isolated from mouse liver, asiatic acid treatment significantly stimulated the succinate-supported state 4 respiration rate, dissipated the MMP, increased Ca(2+) release from Ca(2+)-loaded mitochondria, decreased ATP content and promoted cytochrome c release, indicating the uncoupling effect of asiatic acid. Hydrogen peroxide (H2O2) produced by succinate-supported mitochondrial respiration was also significantly inhibited by asiatic acid. In addition, asiatic acid inhibited Ca(2+)-induced mitochondrial swelling but did not induce mitochondrial swelling in hyposmotic potassium acetate medium which suggested that asiatic acid may not act as a protonophoric uncoupler. Inhibition of uncoupling proteins (UCPs) or blockade of adenine nucleotide transporter (ANT) attenuated the effect of asiatic acid on MMP dissipation, Ca(2+) release, mitochondrial respiration and HepG2 cell death. When combined inhibition of UCPs and ANT, asiatic acid-mediated uncoupling effect was noticeably alleviated. These results suggested that both UCPs and ANT partially contribute to the uncoupling properties of asiatic acid. In conclusion, asiatic acid is a novel mitochondrial uncoupler and this property is potentially involved in its toxicity on HepG2 cells.

  2. Host Cell Factors in Filovirus Entry: Novel Players, New Insights

    PubMed Central

    Hofmann-Winkler, Heike; Kaup, Franziska; Pöhlmann, Stefan

    2012-01-01

    Filoviruses cause severe hemorrhagic fever in humans with high case-fatality rates. The cellular factors exploited by filoviruses for their spread constitute potential targets for intervention, but are incompletely defined. The viral glycoprotein (GP) mediates filovirus entry into host cells. Recent studies revealed important insights into the host cell molecules engaged by GP for cellular entry. The binding of GP to cellular lectins was found to concentrate virions onto susceptible cells and might contribute to the early and sustained infection of macrophages and dendritic cells, important viral targets. Tyrosine kinase receptors were shown to promote macropinocytic uptake of filoviruses into a subset of susceptible cells without binding to GP, while interactions between GP and human T cell Ig mucin 1 (TIM-1) might contribute to filovirus infection of mucosal epithelial cells. Moreover, GP engagement of the cholesterol transporter Niemann-Pick C1 was demonstrated to be essential for GP-mediated fusion of the viral envelope with a host cell membrane. Finally, mutagenic and structural analyses defined GP domains which interact with these host cell factors. Here, we will review the recent progress in elucidating the molecular interactions underlying filovirus entry and discuss their implications for our understanding of the viral cell tropism. PMID:23342362

  3. Light-induced translocation of Pyronine G from mitochondria to nucleoli in monkey kidney CV-1 cells

    NASA Astrophysics Data System (ADS)

    Geze, Marc; Dellinger, M.; Bazin, M.; Santus, Rene C.

    1996-12-01

    Pyronine G (3,6-bis-N,N-dimethylaminoxanthylium chloride; PG) is a cationic dye that concentrates in mitochondria of living cells due to the high membrane potential of these organelles, similarly to rhodamine 123 and many other cationic dyes. Pyronine G also shows a preferential affinity for RNA. Upon light irradiation PG has been shown to induce cell death, but the photosensitizing properties of this molecule and the mechanism of cell death are not well understood. Microfluorometry and most particularly microspectrofluorometry are now powerful non-invasive techniques for quantitative studies of single living cells in real time which allow, for example, knowing how living cells are affected by photosensitization. To demonstrate the usefulness of image acquisition with high resolution and high sensitive camera, we present data on photosensitizer relocalization during illumination leading to functional and structural damage in the cells.

  4. Hijacking Host Cell Highways: Manipulation of the Host Actin Cytoskeleton by Obligate Intracellular Bacterial Pathogens

    PubMed Central

    Colonne, Punsiri M.; Winchell, Caylin G.; Voth, Daniel E.

    2016-01-01

    Intracellular bacterial pathogens replicate within eukaryotic cells and display unique adaptations that support key infection events including invasion, replication, immune evasion, and dissemination. From invasion to dissemination, all stages of the intracellular bacterial life cycle share the same three-dimensional cytosolic space containing the host cytoskeleton. For successful infection and replication, many pathogens hijack the cytoskeleton using effector proteins introduced into the host cytosol by specialized secretion systems. A subset of effectors contains eukaryotic-like motifs that mimic host proteins to exploit signaling and modify specific cytoskeletal components such as actin and microtubules. Cytoskeletal rearrangement promotes numerous events that are beneficial to the pathogen, including internalization of bacteria, structural support for bacteria-containing vacuoles, altered vesicular trafficking, actin-dependent bacterial movement, and pathogen dissemination. This review highlights a diverse group of obligate intracellular bacterial pathogens that manipulate the host cytoskeleton to thrive within eukaryotic cells and discusses underlying molecular mechanisms that promote these dynamic host-pathogen interactions. PMID:27713866

  5. Mitochondria and Cancer.

    PubMed

    Zong, Wei-Xing; Rabinowitz, Joshua D; White, Eileen

    2016-03-03

    Decades ago, Otto Warburg observed that cancers ferment glucose in the presence of oxygen, suggesting that defects in mitochondrial respiration may be the underlying cause of cancer. We now know that the genetic events that drive aberrant cancer cell proliferation also alter biochemical metabolism, including promoting aerobic glycolysis, but do not typically impair mitochondrial function. Mitochondria supply energy; provide building blocks for new cells; and control redox homeostasis, oncogenic signaling, innate immunity, and apoptosis. Indeed, mitochondrial biogenesis and quality control are often upregulated in cancers. While some cancers have mutations in nuclear-encoded mitochondrial tricarboxylic acid (TCA) cycle enzymes that produce oncogenic metabolites, there is negative selection for pathogenic mitochondrial genome mutations. Eliminating mtDNA limits tumorigenesis, and rare human tumors with mutant mitochondrial genomes are relatively benign. Thus, mitochondria play a central and multifunctional role in malignant tumor progression, and targeting mitochondria provides therapeutic opportunities.

  6. Aloe emodin-induced apoptosis in t-HSC/Cl-6 cells involves a mitochondria-mediated pathway.

    PubMed

    Lian, Li-Hua; Park, Eun-Jeon; Piao, Hui-Shan; Zhao, Yu-Zhe; Sohn, Dong Hwan

    2005-06-01

    The aim of our study was to clarify the apoptosis pathway induced by aloe emodin, an hydroxyanthraquinone present in aloe vera leaves, in rat hepatic stellate cells transformed by simian virus 40 (t-HSC/Cl-6), which retain the features of activated rat stellate cells. Apoptosis was determined by DNA fragmentation, caspase activity assay and western blotting analysis. Treatment of t-HSC/Cl-6 cells with 12.5, 25, or 50 microM aloe emodin inhibited t-HSC/Cl-6 cell viability in a dose- and time-dependent manner. The induction of apoptosis by aloe emodin was confirmed by typical DNA ladder formation and annexin v-propidium iodide flow-cytometric analysis. Aloe emodin treatment of t-HSC/Cl-6 cells caused activation of caspase-3 and caspase-9, detected with a caspase activity assay, although no change was observed in caspase-8 activity. Western blotting showed caspase-3 and caspase-9 active forms and the subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. Aloe emodin induced mitochondrial membrane depolarization. Our data also show that cytochrome c increased in the cytosol but decreased in the mitochondria in a time-dependent manner. Increased Bax and unchanged Bcl-2 levels resulted in an increased Bax/Bcl-2 ratio. Thus, our research provides evidence that aloe emodin-induced apoptosis involves a mitochondria-associated apoptosis pathway.

  7. The acylphloroglucinols hyperforin and myrtucommulone A cause mitochondrial dysfunctions in leukemic cells by direct interference with mitochondria.

    PubMed

    Wiechmann, Katja; Müller, Hans; Fischer, Dagmar; Jauch, Johann; Werz, Oliver

    2015-11-01

    The acylphloroglucinols hyperforin (Hypf) and myrtucommulone A (MC A) induce death of cancer cells by triggering the intrinsic/mitochondrial pathway of apoptosis, accompanied by a loss of the mitochondrial membrane potential and release of cytochrome c. However, the upstream targets and mechanisms leading to these mitochondrial events in cancer cells remain elusive. Here we show that Hypf and MC A directly act on mitochondria derived from human leukemic HL-60 cells and thus, disrupt mitochondrial functions. In isolated mitochondria, Hypf and MC A efficiently impaired mitochondrial viability (EC50 = 0.2 and 0.9 µM, respectively), caused loss of the mitochondrial membrane potential (at 0.03 and 0.1 µM, respectively), and suppressed mitochondrial ATP synthesis (IC50 = 0.2 and 0.5 µM, respectively). Consequently, the compounds activated the adenosine monophosphate-activated protein kinase (AMPK) in HL-60 cells, a cellular energy sensor involved in apoptosis of cancer cells. Side by side comparison with the protonophore CCCP and the ATP synthase inhibitor oligomycin suggest that Hypf and MC A act as protonophores that primarily dissipate the mitochondrial membrane potential by direct interaction with the mitochondrial membrane. Together, Hypf and MC A abolish the mitochondrial proton motive force that on one hand impairs mitochondrial viability and on the other cause activation of AMPK due to lowered ATP levels which may further facilitate the intrinsic mitochondrial pathway of apoptosis.

  8. Oxidant stress, mitochondria, and cell death mechanisms in drug-induced liver injury: lessons learned from acetaminophen hepatotoxicity.

    PubMed

    Jaeschke, Hartmut; McGill, Mitchell R; Ramachandran, Anup

    2012-02-01

    Hepatotoxicity is a serious problem during drug development and for the use of many established drugs. For example, acetaminophen overdose is currently the most frequent cause of acute liver failure in the United States and Great Britain. Evaluation of the mechanisms of drug-induced liver injury indicates that mitochondria are critical targets for drug toxicity, either directly or indirectly through the formation of reactive metabolites. The consequence of these modifications is generally a mitochondrial oxidant stress and peroxynitrite formation, which leads to structural alterations of proteins and mitochondrial DNA and, eventually, to the opening of mitochondrial membrane permeability transition (MPT) pores. MPT pore formation results in a collapse of mitochondrial membrane potential and cessation of adenosine triphosphate synthesis. In addition, the release of intermembrane proteins, such as apoptosis-inducing factor and endonuclease G, and their translocation to the nucleus, leads to nuclear DNA fragmentation. Together, these events trigger necrotic cell death. Alternatively, the release of cytochrome c and other proapoptotic factors from mitochondria can promote caspase activation and apoptotic cell death. Drug toxicity can also induce an inflammatory response with the formation of reactive oxygen species by Kupffer cells and neutrophils. If not properly detoxified, these extracellularly generated oxidants can diffuse into hepatocytes and trigger mitochondrial dysfunction and oxidant stress, which then induces MPT and necrotic cell death. This review addresses the formation of oxidants and the defense mechanisms available for cells and applies this knowledge to better understand mechanisms of drug hepatotoxicity, especially acetaminophen-induced liver injury.

  9. Bovine lactoferricin causes apoptosis in Jurkat T-leukemia cells by sequential permeabilization of the cell membrane and targeting of mitochondria

    SciTech Connect

    Mader, Jamie S.; Richardson, Angela; Salsman, Jayme; Top, Deniz; Antueno, Roberto de; Duncan, Roy; Hoskin, David W. . E-mail: d.w.hoskin@dal.ca

    2007-07-15

    Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that kills Jurkat T-leukemia cells by the mitochondrial pathway of apoptosis. However, the process by which LfcinB triggers mitochondria-dependent apoptosis is not well understood. Here, we show that LfcinB-induced apoptosis in Jurkat T-leukemia cells was preceded by LfcinB binding to, and progressive permeabilization of the cell membrane. Colloidal gold electron microscopy revealed that LfcinB entered the cytoplasm of Jurkat T-leukemia cells prior to the onset of mitochondrial depolarization. LfcinB was not internalized by endocytosis because endocytosis inhibitors did not prevent LfcinB-induced cytotoxicity. Furthermore, intracellular delivery of LfcinB via fusogenic liposomes caused the death of Jurkat T-leukemia cells, as well as normal human fibroblasts. Collectively, these findings suggest that LfcinB caused damage to the cell membrane that allowed LfcinB to enter the cytoplasm of Jurkat T-leukemia cells and mediate cytotoxicity. In addition, confocal microscopy showed that intracellular LfcinB co-localized with mitochondria in Jurkat T-leukemia cells, while flow cytometry and colloidal gold electron microscopy showed that LfcinB rapidly associated with purified mitochondria. Furthermore, purified mitochondria treated with LfcinB rapidly lost transmembrane potential and released cytochrome c. We conclude that LfcinB-induced apoptosis in Jurkat T-leukemia cells resulted from cell membrane damage and the subsequent disruption of mitochondrial membranes by internalized LfcinB.

  10. Bystander Host Cell Killing Effects of Clostridium perfringens Enterotoxin

    PubMed Central

    Shrestha, Archana; Hendricks, Matthew R.; Bomberger, Jennifer M.

    2016-01-01

    ABSTRACT Clostridium perfringens enterotoxin (CPE) binds to claudin receptors, e.g., claudin-4, and then forms a pore that triggers cell death. Pure cultures of host cells that do not express claudin receptors, e.g., fibroblasts, are unaffected by pathophysiologically relevant CPE concentrations in vitro. However, both CPE-insensitive and CPE-sensitive host cells are present in vivo. Therefore, this study tested whether CPE treatment might affect fibroblasts when cocultured with CPE-sensitive claudin-4 fibroblast transfectants or Caco-2 cells. Under these conditions, immunofluorescence microscopy detected increased death of fibroblasts. This cytotoxic effect involved release of a toxic factor from the dying CPE-sensitive cells, since it could be reproduced using culture supernatants from CPE-treated sensitive cells. Supernatants from CPE-treated sensitive cells, particularly Caco-2 cells, were found to contain high levels of membrane vesicles, often containing a CPE species. However, most cytotoxic activity remained in those supernatants even after membrane vesicle depletion, and CPE was not detected in fibroblasts treated with supernatants from CPE-treated sensitive cells. Instead, characterization studies suggest that a major cytotoxic factor present in supernatants from CPE-treated sensitive cells may be a 10- to 30-kDa host serine protease or require the action of that host serine protease. Induction of caspase-3-mediated apoptosis was found to be important for triggering release of the cytotoxic factor(s) from CPE-treated sensitive host cells. Furthermore, the cytotoxic factor(s) in these supernatants was shown to induce a caspase-3-mediated killing of fibroblasts. This bystander killing effect due to release of cytotoxic factors from CPE-treated sensitive cells could contribute to CPE-mediated disease. PMID:27965452

  11. Involvement of PI 3 kinase/Akt-dependent Bad phosphorylation in Toxoplasma gondii-mediated inhibition of host cell apoptosis.

    PubMed

    Quan, Juan-Hua; Cha, Guang-Ho; Zhou, Wei; Chu, Jia-Qi; Nishikawa, Yoshifumi; Lee, Young-Ha

    2013-04-01

    Toxoplasma gondii-infected cells are resistant to various apoptotic stimuli, however, the role of the pro-apoptotic BH3-only Bad protein in T. gondii-imposed inhibition of host cell apoptosis in connection with the phosphoinositide 3-kinase (PI3K)-PKB/Akt pathway was not well delineated. Here, we investigated the signaling patterns of Bad, Bax and PKB/Akt in T. gondii-infected and uninfected THP-1 cells treated with staurosporine (STS) or PI3K inhibitors. STS treatment, without T. gondii infection, reduced the viability of THP-1 cells in proportion to STS concentration and triggered many cellular death events such as caspase-3 and -9 activation, Bax translocation, cytochrome c release from host cell mitochondria into cytosol, and PARP cleavage in the host cell. However, T. gondii infection eliminated the STS-triggered mitochondrial apoptotic events described above. Additionally, T. gondii infection in vitro and in vivo induced the phosphorylation of PKB/Akt and Bad in a parasite-load-dependent manner which subsequently inhibited Bax translocation. The PI3K inhibitors, LY294002 and Wortmannin, both blocked parasite-induced phosphorylation of PKB/Akt and Bad. Furthermore, THP-1 cells pretreated with these PI3K inhibitors showed reduced phosphorylation of Bad in a dose-dependent manner and subsequently failed to inhibit the Bax translocation, also these cells also failed to overcome the T. gondii-imposed inhibition of host cell apoptosis. These data demonstrate that the PI3K-PKB/Akt pathway may be one of the major route for T. gondii in the prevention of host cell apoptosis and T. gondii phosphorylates the pro-apoptotic Bad protein to prevent apoptosis.

  12. Alterations of host cell ubiquitination machinery by pathogenic bacteria.

    PubMed

    Alomairi, Jaafar; Bonacci, Thomas; Ghigo, Eric; Soubeyran, Philippe

    2015-01-01

    Response of immune and non-immune cells to pathogens infections is a very dynamic process. It involves the activation/modulation of many pathways leading to actin remodeling, membrane engulfing, phagocytosis, vesicle trafficking, phagolysosome formation, aiming at the destruction of the intruder. These sophisticated and rapid mechanisms rely on post-translational modifications (PTMs) of key host cells' factors, and bacteria have developed various strategies to manipulate them to favor their survival. Among these important PTMs, ubiquitination has emerged as a major mediator/modulator/regulator of host cells response to infections that pathogens have also learned to use for their own benefit. In this mini-review, we summarize our current knowledge about the normal functions of ubiquitination during host cell infection, and we detail its hijacking by model pathogens to escape clearance and to proliferate.

  13. Viroporins Customize Host Cells for Efficient Viral Propagation

    PubMed Central

    Giorda, Kristina M.

    2013-01-01

    Viruses are intracellular parasites that must access the host cell machinery to propagate. Viruses hijack the host cell machinery to help with entry, replication, packaging, and release of progeny to infect new cells. To carry out these diverse functions, viruses often transform the cellular environment using viroporins, a growing class of viral-encoded membrane proteins that promote viral proliferation. Viroporins modify the integrity of host membranes, thereby stimulating the maturation of viral infection, and are critical for virus production and dissemination. Significant advances in molecular and cell biological approaches have helped to uncover some of the roles that viroporins serve in the various stages of the viral life cycle. In this study, the ability of viroporins to modify the cellular environment will be discussed, with particular emphasis on their role in the stepwise progression of the viral life cycle. PMID:23945006

  14. Isoimperatorin induces apoptosis of the SGC-7901 human gastric cancer cell line via the mitochondria-mediated pathway

    PubMed Central

    Tong, Kehui; Xin, Chang; Chen, Wenzhong

    2017-01-01

    The present study was designed to investigate the antiproliferative activity of isoimperatorin against SGC-7901 cells and to examine the possible mechanisms. The antiproliferative activity of isoimperatorin against SGC-7901 cells was evaluated using an MTT assay, and the mechanisms were investigated using flow cytometry and western blot assays, which were used to determine the apoptotic rate and expression levels of mitochondria-mediated apoptosis-associated proteins, including Survivin, myeloid leukemia cell-1 (Mcl-1), B cell lymphoma-extra large (Bcl-xl), B cell lymphoma 2 (Bcl-2), second mitochondria-derived activator of caspase (Smac), Bcl-2-associated X factor (Bax), cleaved (c)-caspase-3 and c-caspase-9 in SGC-7901 cells. Additionally, a xenograft assay was used to confirm whether isoimperatorin had an inhibitory effect on SGC-7901 cell-induced tumors in vivo. The results of the MTT assay suggested that isoimperatorin significantly inhibited the proliferation of SGC-7901 cells in a dose- and time-dependent manner, and the half maximal inhibitory concentration was 18.75 µg/ml. The results of the flow cytometric analysis indicated that, following treatment with isoimperatorin, the apoptotic rate of SGC-7901 cells was significantly increased, compared with that of cells in the control group. The results of the western blot analysis indicated that, following treatment with isoimperatorin, the expression levels of the pro-apoptotic proteins, Bax, c-caspase-3 and c-caspase-9, were significantly increased and the expression levels of the anti-apoptotic proteins, Survivin and Bcl-2, were significantly reduced, compared with the control group. No alterations in expression were found in the other apoptosis-associated proteins, including Mcl-1, Bcl-xl and Smac. The results of the xenograft assay indicated that isoimperatorin significantly inhibited the growth of SGC-7901 cell-induced tumor in vivo by increasing the expression levels of pro-apoptotic proteins (Bax, c

  15. Targeting mitochondria in cancer cells using gold nanoparticle-enhanced radiotherapy: A Monte Carlo study

    SciTech Connect

    Kirkby, Charles Ghasroddashti, Esmaeel

    2015-02-15

    Purpose: Radiation damage to mitochondria has been shown to alter cellular processes and even lead to apoptosis. Gold nanoparticles (AuNPs) may be used to enhance these effects in scenarios where they collect on the outer membranes of mitochondria. A Monte Carlo (MC) approach is used to estimate mitochondrial dose enhancement under a variety of conditions. Methods: The PENELOPE MC code was used to generate dose distributions resulting from photons striking a 13 nm diameter AuNP with various thicknesses of water-equivalent coatings. Similar dose distributions were generated with the AuNP replaced by water so as to estimate the gain in dose on a microscopic scale due to the presence of AuNPs within an irradiated volume. Models of mitochondria with AuNPs affixed to their outer membrane were then generated—considering variation in mitochondrial size and shape, number of affixed AuNPs, and AuNP coating thickness—and exposed (in a dose calculation sense) to source spectra ranging from 6 MV to 90 kVp. Subsequently dose enhancement ratios (DERs), or the dose with the AuNPs present to that for no AuNPs, for the entire mitochondrion and its components were tallied under these scenarios. Results: For a representative case of a 1000 nm diameter mitochondrion affixed with 565 AuNPs, each with a 13 nm thick coating, the mean DER over the whole organelle ranged from roughly 1.1 to 1.6 for the kilovoltage sources, but was generally less than 1.01 for the megavoltage sources. The outer membrane DERs remained less than 1.01 for the megavoltage sources, but rose to 2.3 for 90 kVp. The voxel maximum DER values were as high as 8.2 for the 90 kVp source and increased further when the particles clustered together. The DER exhibited dependence on the mitochondrion dimensions, number of AuNPs, and the AuNP coating thickness. Conclusions: Substantial dose enhancement directly to the mitochondria can be achieved under the conditions modeled. If the mitochondrion dose can be directly

  16. Invasion of Host Cells and Tissues by Uropathogenic Bacteria

    PubMed Central

    Lewis, Adam J.; Richards, Amanda C.; Mulvey, Matthew A.

    2016-01-01

    Within the mammalian urinary tract uropathogenic bacteria face many challenges, including the shearing flow of urine, numerous antibacterial molecules, the bactericidal effects of phagocytes, and a scarcity of nutrients. These problems may be circumvented in part by the ability of uropathogenic Escherichia coli (UPEC) and several other uropathogens to invade the epithelial cells that line the urinary tract. By entering host cells, uropathogens can gain access to additional nutrients and protection from both host defenses and antibiotic treatments. Translocation through host cells can facilitate bacterial dissemination within the urinary tract, while the establishment of stable intracellular bacterial populations may create reservoirs for relapsing and chronic urinary tract infections (UTIs). Here we review the mechanisms and consequences of host cell invasion by uropathogenic bacteria, with consideration of the defenses that are brought to bear against facultative intracellular pathogens within the urinary tract. The relevance of host cell invasion to the pathogenesis of UTIs in human patients is also assessed, along with some of the emerging treatment options that build upon our growing understanding of the infectious life cycle of UPEC and other uropathogenic bacteria. PMID:28087946

  17. Role of the Mitochondria in Immune-Mediated Apoptotic Death of the Human Pancreatic β Cell Line βLox5

    PubMed Central

    Lightfoot, Yaíma L.; Chen, Jing; Mathews, Clayton E.

    2011-01-01

    Mitochondria are indispensable in the life and death of many types of eukaryotic cells. In pancreatic beta cells, mitochondria play an essential role in the secretion of insulin, a hormone that regulates blood glucose levels. Unregulated blood glucose is a hallmark symptom of diabetes. The onset of Type 1 diabetes is preceded by autoimmune-mediated destruction of beta cells. However, the exact role of mitochondria has not been assessed in beta cell death. In this study, we examine the role of mitochondria in both Fas- and proinflammatory cytokine-mediated destruction of the human beta cell line, βLox5. IFNγ primed βLox5 cells for apoptosis by elevating cell surface Fas. Consequently, βLox5 cells were killed by caspase-dependent apoptosis by agonistic activation of Fas, but only after priming with IFNγ. This beta cell line undergoes both apoptotic and necrotic cell death after incubation with the combination of the proinflammatory cytokines IFNγ and TNFα. Additionally, both caspase-dependent and -independent mechanisms that require proper mitochondrial function are involved. Mitochondrial contributions to βLox5 cell death were analyzed using mitochondrial DNA (mtDNA) depleted βLox5 cells, or βLox5 ρ0 cells. βLox5 ρ0 cells are not sensitive to IFNγ and TNFα killing, indicating a direct role for the mitochondria in cytokine-induced cell death of the parental cell line. However, βLox5 ρ0 cells are susceptible to Fas killing, implicating caspase-dependent extrinsic apoptotic death is the mechanism by which these human beta cells die after Fas ligation. These data support the hypothesis that immune mediators kill βLox5 cells by both mitochondrial-dependent intrinsic and caspase-dependent extrinsic pathways. PMID:21738580

  18. Novel role for mitochondria: protein kinase Ctheta-dependent oxidative signaling organelles in activation-induced T-cell death.

    PubMed

    Kaminski, Marcin; Kiessling, Michael; Süss, Dorothee; Krammer, Peter H; Gülow, Karsten

    2007-05-01

    Reactive oxygen species (ROS) play a key role in regulation of activation-induced T-cell death (AICD) by induction of CD95L expression. However, the molecular source and the signaling steps necessary for ROS production are largely unknown. Here, we show that the proximal T-cell receptor-signaling machinery, including ZAP70 (zeta chain-associated protein kinase 70), LAT (linker of activated T cells), SLP76 (SH2 domain-containing leukocyte protein of 76 kDa), PLCgamma1 (phospholipase Cgamma1), and PKCtheta (protein kinase Ctheta), are crucial for ROS production. PKCtheta is translocated to the mitochondria. By using cells depleted of mitochondrial DNA, we identified the mitochondria as the source of activation-induced ROS. Inhibition of mitochondrial electron transport complex I assembly by small interfering RNA (siRNA)-mediated knockdown of the chaperone NDUFAF1 resulted in a block of ROS production. Complex I-derived ROS are converted into a hydrogen peroxide signal by the mitochondrial superoxide dismutase. This signal is essential for CD95L expression, as inhibition of complex I assembly by NDUFAF1-specific siRNA prevents AICD. Similar results were obtained when metformin, an antidiabetic drug and mild complex I inhibitor, was used. Thus, we demonstrate for the first time that PKCtheta-dependent ROS generation by mitochondrial complex I is essential for AICD.

  19. Taurine inhibits 2,5-hexanedione-induced oxidative stress and mitochondria-dependent apoptosis in PC12 cells

    PubMed Central

    LI, Shuangyue; GUAN, Huai; QIAN, Zhiqiang; SUN, Yijie; GAO, Chenxue; LI, Guixin; YANG, Yi; PIAO, Fengyuan; HU, Shuhai

    2016-01-01

    2,5-hexanedione (HD) is the ultimate neurotoxic metabolite of hexane, causing the progression of nerve diseases in human. It was reported that HD induced apoptosis and oxidative stress. Taurine has been shown to be a potent antioxidant. In the present study, we investigated the protection of taurine against HD-induced apoptosis in PC12 cells and the underlying mechanism. Our results showed the decreased viability and increased apoptosis in HD-exposed PC12 cells. HD also induced the disturbance of Bax and Bcl-2 expression, the loss of MMP, the release of mitochondrial cytochrome c and caspase-3 activation in PC12 cells. Moreover, HD resulted in an increase in reactive oxygen species (ROS) level and a decline in the activities of superoxidedismutase and catalase in PC12 cells. However, taurine pretreatment ameliorated the increased apoptosis and the alterations in key regulators of mitochondria-dependent pathway in PC12 exposed to HD. The increased ROS level and the decreased activities of the antioxidant enzymes in HD group were attenuated by taurine. These results indicate that pretreatment of taurine may, at least partly, prevent HD-induced apoptosis via inhibiting mitochondria-dependent pathway. It is also suggested that the potential of taurine against HD-induced apoptosis may benefit from its anti-oxidative property. PMID:27840369

  20. Mitochondria present in excised patches from pancreatic B-cells may form microcompartments with ATP-dependent potassium channels.

    PubMed

    Rustenbeck, I; Dickel, C; Herrmann, C; Grimmsmann, T

    1999-04-01

    Experiments with inside-out patches excised from pancreatic B-cells have yielded evidence that mitochondria are often contained in the cytoplasmic plug protruding into the tip of patch pipette. When intact B-cells were loaded with the fluorescent mitochondrial stain, rhodamine 123, and membrane patches excised from these cells, a green fluorescence could be observed in the lumen at the tip of the patch pipette. The same result was obtained with the mitochondrial stain, MitoTracker Green FM, which is only fluorescent in a membrane-bound state. Furthermore, the open probability of ATP-dependent potassium (K(ATP)) channels in inside-out patches was influenced by mitochondrial fuels and inhibitors. Respiratory substrates like tetramethyl phenylene diamine (2 mM) plus ascorbate (5 mM) or alpha-ketoisocaproic acid (10 mM) reduced the open probability of K(ATP) channels in inside-out patches significantly (down to 57% or 65% of control, respectively). This effect was antagonized by the inhibitor of cytochrome oxidase, sodium azide (5 mM). Likewise, the inhibitor of succinate dehydrogenase, malonate (5 mM), increased the open probability of K(ATP) channels in the presence of succinate (1 mM). However, oligomycin in combination with antimycin and rotenone did not increase open probability. Although it cannot be excluded that these effects result from a direct interaction with the K(ATP) channels, the presence of mitochondria in the close vicinity permits the hypothesis that changes in mitochondrial metabolism are involved, mitochondria and K(ATP) channels thus forming functional microcompartments.

  1. Specific imaging and tracking of mitochondria in live cells by a photostable AIE luminogen.

    PubMed

    Leung, Chris W T; Hong, Yuning; Tang, Ben Zhong

    2015-01-01

    Tracking the dynamics of mitochondrial morphology has attracted much research interest because of its involvement in early stage apoptosis and degenerative conditions. To follow this process, highly specific and photostable fluorescent probes are in demand. Commercially available mitochondria trackers, however, suffer from poor photostability. To overcome this limitation, we have designed and synthesized a fluorescent agent, tetraphenylethene-triphenylphosphonium (TPE-TPP), for mitochondrial imaging. Inherent from the mitochondrial-targeting ability of TPP groups and the aggregation-induced emission (AIE) characteristics of the TPE core, TPE-TPP possesses high specificity to mitochondria, superior photostability, and appreciable tolerance to environmental change, allowing imaging and tracking of the mitochondrial morphological changes in a long period of time.

  2. On Cellular Darwinism: Mitochondria.

    PubMed

    Bull, Larry

    2016-01-01

    The significant role of mitochondria within cells is becoming increasingly clear. This letter uses the NKCS model of coupled fitness landscapes to explore aspects of organelle-nucleus coevolution. The phenomenon of mitochondrial diversity is allowed to emerge under a simple intracellular evolutionary process, including varying the relative rate of evolution by the organelle. It is shown how the conditions for the maintenance of more than one genetic variant of mitochondria are similar to those previously suggested as needed for the original symbiotic origins of the relationship using the NKCS model.

  3. Host NKT cells can prevent graft-versus-host disease and permit graft antitumor activity after bone marrow transplantation.

    PubMed

    Pillai, Asha B; George, Tracy I; Dutt, Suparna; Teo, Pearline; Strober, Samuel

    2007-05-15

    Allogeneic bone marrow transplantation is a curative treatment for leukemia and lymphoma, but graft-vs-host disease (GVHD) remains a major complication. Using a GVHD protective nonmyeloablative conditioning regimen of total lymphoid irradiation and antithymocyte serum (TLI/ATS) in mice that has been recently adapted to clinical studies, we show that regulatory host NKT cells prevent the expansion and tissue inflammation induced by donor T cells, but allow retention of the killing activity of donor T cells against the BCL1 B cell lymphoma. Whereas wild-type hosts given transplants from wild-type donors were protected against progressive tumor growth and lethal GVHD, NKT cell-deficient CD1d-/- and Jalpha-18-/- host mice given wild-type transplants cleared the tumor cells but died of GVHD. In contrast, wild-type hosts given transplants from CD8-/- or perforin-/- donors had progressive tumor growth without GVHD. Injection of host-type NKT cells into Jalpha-18-/- host mice conditioned with TLI/ATS markedly reduced the early expansion and colon injury induced by donor T cells. In conclusion, after TLI/ATS host conditioning and allogeneic bone marrow transplantation, host NKT cells can separate the proinflammatory and tumor cytolytic functions of donor T cells.

  4. Modification of Bacterial Effector Proteins Inside Eukaryotic Host Cells

    PubMed Central

    Popa, Crina M.; Tabuchi, Mitsuaki; Valls, Marc

    2016-01-01

    Pathogenic bacteria manipulate their hosts by delivering a number of virulence proteins -called effectors- directly into the plant or animal cells. Recent findings have shown that such effectors can suffer covalent modifications inside the eukaryotic cells. Here, we summarize the recent reports where effector modifications by the eukaryotic machinery have been described. We restrict our focus on proteins secreted by the type III or type IV systems, excluding other bacterial toxins. We describe the known examples of effectors whose enzymatic activity is triggered by interaction with plant and animal cell factors, including GTPases, E2-Ubiquitin conjugates, cyclophilin and thioredoxins. We focus on the structural interactions with these factors and their influence on effector function. We also review the described examples of host-mediated post-translational effector modifications which are required for proper subcellular location and function. These host-specific covalent modifications include phosphorylation, ubiquitination, SUMOylation, and lipidations such as prenylation, fatty acylation and phospholipid binding. PMID:27489796

  5. Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum.

    PubMed

    Lampe, Elisabeth O; Brenz, Yannick; Herrmann, Lydia; Repnik, Urska; Griffiths, Gareth; Zingmark, Carl; Sjöstedt, Anders; Winther-Larsen, Hanne C; Hagedorn, Monica

    2015-12-28

    Francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. Mammalian cell lines have mainly been used to study the mechanisms by which Francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. Here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba Dictyostelium discoideum combined with the fish pathogen Francisella noatunensis subsp. noatunensis. Phagocytosed F. noatunensis subsp. noatunensis interacts with the endosomal pathway and escapes further phagosomal maturation by translocating into the host cell cytosol. F. noatunensis subsp. noatunensis lacking IglC, a known virulence determinant required for Francisella intracellular replication, follows the normal phagosomal maturation and does not grow in Dictyostelium. The attenuation of the F. noatunensis subsp. noatunensis ΔiglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis ΔiglC was restricted. In Dictyostelium, F. noatunensis subsp. noatunensis interacts with the autophagic machinery. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Δatg1), F. noatunensis subsp. noatunensis accumulates within Dictyostelium cells. Altogether, the Dictyostelium-F. noatunensis subsp. noatunensis infection model recapitulates the course of infection described in other host systems. The genetic and biochemical tractability of the system allows new approaches to elucidate the dynamic interactions between pathogenic Francisella and its host organism.

  6. Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum

    PubMed Central

    Lampe, Elisabeth O.; Brenz, Yannick; Herrmann, Lydia; Repnik, Urska; Griffiths, Gareth; Zingmark, Carl; Sjöstedt, Anders; Winther-Larsen, Hanne C.

    2015-01-01

    Francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. Mammalian cell lines have mainly been used to study the mechanisms by which Francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. Here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba Dictyostelium discoideum combined with the fish pathogen Francisella noatunensis subsp. noatunensis. Phagocytosed F. noatunensis subsp. noatunensis interacts with the endosomal pathway and escapes further phagosomal maturation by translocating into the host cell cytosol. F. noatunensis subsp. noatunensis lacking IglC, a known virulence determinant required for Francisella intracellular replication, follows the normal phagosomal maturation and does not grow in Dictyostelium. The attenuation of the F. noatunensis subsp. noatunensis ΔiglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis ΔiglC was restricted. In Dictyostelium, F. noatunensis subsp. noatunensis interacts with the autophagic machinery. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Δatg1), F. noatunensis subsp. noatunensis accumulates within Dictyostelium cells. Altogether, the Dictyostelium-F. noatunensis subsp. noatunensis infection model recapitulates the course of infection described in other host systems. The genetic and biochemical tractability of the system allows new approaches to elucidate the dynamic interactions between pathogenic Francisella and its host organism. PMID:26712555

  7. Mitochondria in lung disease.

    PubMed

    Cloonan, Suzanne M; Choi, Augustine M K

    2016-03-01

    Mitochondria are a distinguishing feature of eukaryotic cells. Best known for their critical function in energy production via oxidative phosphorylation (OXPHOS), mitochondria are essential for nutrient and oxygen sensing and for the regulation of critical cellular processes, including cell death and inflammation. Such diverse functional roles for organelles that were once thought to be simple may be attributed to their distinct heteroplasmic genome, exclusive maternal lineage of inheritance, and ability to generate signals to communicate with other cellular organelles. Mitochondria are now thought of as one of the cell's most sophisticated and dynamic responsive sensing systems. Specific signatures of mitochondrial dysfunction that are associated with disease pathogenesis and/or progression are becoming increasingly important. In particular, the centrality of mitochondria in the pathological processes and clinical phenotypes associated with a range of lung diseases is emerging. Understanding the molecular mechanisms regulating the mitochondrial processes of lung cells will help to better define phenotypes and clinical manifestations associated with respiratory disease and to identify potential diagnostic and therapeutic targets.

  8. Neuroglobin upregulation induced by 17β-estradiol sequesters cytocrome c in the mitochondria preventing H2O2-induced apoptosis of neuroblastoma cells

    PubMed Central

    De Marinis, E; Fiocchetti, M; Acconcia, F; Ascenzi, P; Marino, M

    2013-01-01

    The sex steroid hormone 17β-estradiol (E2) upregulates the levels of neuroglobin (NGB), a new neuroprotectant globin, to elicit its neuroprotective effect against H2O2-induced apoptosis. Several mechanisms could be proposed to justify the NGB involvement in E2 prevention of stress-induced apoptotic cell death. Here, we evaluate the ability of E2 to modulate the intracellular NGB localization and the NGB interaction with mitochondrial cytochrome c following the H2O2-induced toxicity. Present results demonstrate that NGB is expressed in the nuclei, mitochondria, and cytosol of human neuroblastoma SK-N-BE cells. E2, but not H2O2 treatment of SK-N-BE cells, reallocates NGB mainly at the mitochondria and contemporarily reduces the number of apoptotic nuclei and the levels of cleaved caspase-3. Remarkably, the E2 treatment strongly increases NGB–cytochrome c association into mitochondria and reduces the levels of cytochrome c into the cytosol of SK-N-BE cells. Although both estrogen receptors (ERα and ERβ) are expressed in the nucleus, mitochondria, and cytosol of SK-N-BE cells, this E2 effect specifically requires the mitochondrial ERβ activity. As a whole, these data demonstrate that the interception of the intrinsic apoptotic pathway into mitochondria (i.e., the prevention of cytochrome c release) is one of the pivotal mechanisms underlying E2-dependent NGB neuroprotection against H2O2 toxicity. PMID:23429294

  9. Reactive biomolecular divergence in genetically altered yeast cells and isolated mitochondria as measured by biocavity laser spectroscopy: rapid diagnostic method for studying cellular responses to stress and disease.

    PubMed

    Gourley, Paul L; Hendricks, Judy K; McDonald, Anthony E; Copeland, R Guild; Yaffe, Michael P; Naviaux, Robert K

    2007-01-01

    We report an analysis of four strains of baker's yeast (Saccharomyces cerevisiae) using biocavity laser spectroscopy. The four strains are grouped in two pairs (wild type and altered), in which one strain differs genetically at a single locus, affecting mitochondrial function. In one pair, the wild-type rho+ and a rho0 strain differ by complete removal of mitochondrial DNA (mtDNA). In the second pair, the wild-type rho+ and a rho- strain differ by knock-out of the nuclear gene encoding Cox4, an essential subunit of cytochrome c oxidase. The biocavity laser is used to measure the biophysical optic parameter Deltalambda, a laser wavelength shift relating to the optical density of cell or mitochondria that uniquely reflects its size and biomolecular composition. As such, Deltalambda is a powerful parameter that rapidly interrogates the biomolecular state of single cells and mitochondria. Wild-type cells and mitochondria produce Gaussian-like distributions with a single peak. In contrast, mutant cells and mitochondria produce leptokurtotic distributions that are asymmetric and highly skewed to the right. These distribution changes could be self-consistently modeled with a single, log-normal distribution undergoing a thousand-fold increase in variance of biomolecular composition. These features reflect a new state of stressed or diseased cells that we call a reactive biomolecular divergence (RBD) that reflects the vital interdependence of mitochondria and the nucleus.

  10. Host cell infiltration into PDT-treated tumor

    NASA Astrophysics Data System (ADS)

    Korbelik, Mladen; Krosl, Gorazd; Dougherty, Graeme J.; Chaplin, David J.

    1992-06-01

    C3H mice bearing SCCVII squamous cell carcinoma were treated with photodynamic therapy (PDT) 24 hours after receiving Photofrin (25 mg/kg, i.v.). Single cell suspensions obtained by the enzymatic digestion of tumors excised either 30 minutes or 4 hours after PDT were analyzed for the content of host immune cells and colony forming ability of malignant cells. The results were compared to the data obtained with non-treated tumors. It is shown that there is a marked increase in the content of cells expressing Mac-1 (monocytes/macrophages or granulocytes) in the tumor 30 minutes post PDT, while a high level of other leucocytes are found within the tumors by 4 hours after PDT. As elaborated in Discussion, the infiltration rate of host immune cells, dying of malignant tumor cells, and yet unknown death rate of host cells originally present in PDT treated tumor occurring concomitantly during this time period complicates this analysis. The results of this study suggest a massive infiltration of macrophages and other leucocytes in PDT treated SCCVII tumor, supporting the suggestion that a potent immune reaction is one of the main characteristics of PDT action in solid tumors. It remains to be determined to what extent is the activity of tumor infiltrating immune cells responsible for its eradication by PDT.

  11. Early Bunyavirus-Host Cell Interactions.

    PubMed

    Albornoz, Amelina; Hoffmann, Anja B; Lozach, Pierre-Yves; Tischler, Nicole D

    2016-05-24

    The Bunyaviridae is the largest family of RNA viruses, with over 350 members worldwide. Several of these viruses cause severe diseases in livestock and humans. With an increasing number and frequency of outbreaks, bunyaviruses represent a growing threat to public health and agricultural productivity globally. Yet, the receptors, cellular factors and endocytic pathways used by these emerging pathogens to infect cells remain largely uncharacterized. The focus of this review is on the early steps of bunyavirus infection, from virus binding to penetration from endosomes. We address current knowledge and advances for members from each genus in the Bunyaviridae family regarding virus receptors, uptake, intracellular trafficking and fusion.

  12. Early Bunyavirus-Host Cell Interactions

    PubMed Central

    Albornoz, Amelina; Hoffmann, Anja B.; Lozach, Pierre-Yves; Tischler, Nicole D.

    2016-01-01

    The Bunyaviridae is the largest family of RNA viruses, with over 350 members worldwide. Several of these viruses cause severe diseases in livestock and humans. With an increasing number and frequency of outbreaks, bunyaviruses represent a growing threat to public health and agricultural productivity globally. Yet, the receptors, cellular factors and endocytic pathways used by these emerging pathogens to infect cells remain largely uncharacterized. The focus of this review is on the early steps of bunyavirus infection, from virus binding to penetration from endosomes. We address current knowledge and advances for members from each genus in the Bunyaviridae family regarding virus receptors, uptake, intracellular trafficking and fusion. PMID:27213430

  13. Silencing of Pokemon Enhances Caspase-Dependent Apoptosis via Fas- and Mitochondria-Mediated Pathways in Hepatocellular Carcinoma Cells

    PubMed Central

    Lin, Bi-Yun; Shi, Ying; Liu, Yun-Peng; Liu, Jing-Jing; Guleng, Bayasi; Ren, Jian-Lin

    2013-01-01

    The role of Pokemon (POK erythroid myeloid ontogenic actor), a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC) and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma) as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy. PMID:23874836

  14. Mitochondria in relation to cancer metastasis.

    PubMed

    Bhandary, Bidur; Marahatta, Anu; Kim, Hyung-Ryong; Chae, Han-Jung

    2012-12-01

    Mitochondria, also known as "Power House of cell," are crucial organelles, regulating energy metabolism. Recently, an involvement of mitochondria in cancer occurrence and metastasis has been proposed. The roles of mitochondria in cancer progression/metastasis include alteration of glycolysis, regulation of ROS and suppression of intrinsic apoptosis. This mini-review explains the specific mitochondrial characteristics during cancer metastasis with past and recent findings. It may contribute to understanding mitochondria-related mechanisms of cancer metastasis.

  15. Toxoplasma gondii inhibits cytochrome c-induced caspase activation in its host cell by interference with holo-apoptosome assembly

    PubMed Central

    Graumann, Kristin; Schaumburg, Frieder; Reubold, Thomas F.; Hippe, Diana; Eschenburg, Susanne; Lüder, Carsten G. K.

    2015-01-01

    Inhibition of programmed cell death pathways of mammalian cells often facilitates the sustained survival of intracellular microorganisms. The apicomplexan parasite Toxoplasma gondii is a master regulator of host cell apoptotic pathways. Here, we have characterized a novel anti-apoptotic activity of T. gondii. Using a cell-free cytosolic extract model, we show that T. gondii interferes with the activities of caspase 9 and caspase 3/7 which have been induced by exogenous cytochrome c and dATP. Proteolytic cleavage of caspases 9 and 3 is also diminished suggesting inhibition of holo-apoptosome function. Parasite infection of Jurkat T cells and subsequent triggering of apoptosome formation by exogenous cytochrome c in vitro and in vivo indicated that T. gondii also interferes with caspase activation in infected cells. Importantly, parasite inhibition of cytochrome c-induced caspase activation considerably contributes to the overall anti-apoptotic activity of T. gondii as observed in staurosporine-treated cells. Co-immunoprecipitation showed that T. gondii abolishes binding of caspase 9 to Apaf-1 whereas the interaction of cytochrome c with Apaf-1 remains unchanged. Finally, T. gondii lysate mimics the effect of viable parasites and prevents holo-apoptosome functionality in a reconstituted in vitro system comprising recombinant Apaf-1 and caspase 9. Beside inhibition of cytochrome c release from host cell mitochondria, T. gondii thus also targets the holo-apoptosome assembly as a second mean to efficiently inhibit the caspase-dependent intrinsic cell death pathway. PMID:28357287

  16. Mitochondria in lung disease

    PubMed Central

    Cloonan, Suzanne M.; Choi, Augustine M.K.

    2016-01-01

    Mitochondria are a distinguishing feature of eukaryotic cells. Best known for their critical function in energy production via oxidative phosphorylation (OXPHOS), mitochondria are essential for nutrient and oxygen sensing and for the regulation of critical cellular processes, including cell death and inflammation. Such diverse functional roles for organelles that were once thought to be simple may be attributed to their distinct heteroplasmic genome, exclusive maternal lineage of inheritance, and ability to generate signals to communicate with other cellular organelles. Mitochondria are now thought of as one of the cell’s most sophisticated and dynamic responsive sensing systems. Specific signatures of mitochondrial dysfunction that are associated with disease pathogenesis and/or progression are becoming increasingly important. In particular, the centrality of mitochondria in the pathological processes and clinical phenotypes associated with a range of lung diseases is emerging. Understanding the molecular mechanisms regulating the mitochondrial processes of lung cells will help to better define phenotypes and clinical manifestations associated with respiratory disease and to identify potential diagnostic and therapeutic targets. PMID:26928034

  17. ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

    SciTech Connect

    Vieira da Silva, Claudio; Alves da Silva, Erika; Costa Cruz, Mario; Chavrier, Philippe; Arruda Mortara, Renato

    2009-01-16

    Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP{sub 2} and PIP{sub 3} to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.

  18. Mechanisms of outer membrane vesicle entry into host cells

    PubMed Central

    O'Donoghue, Eloise J.

    2016-01-01

    Abstract Bacterial outer membrane vesicles (OMVs) are nano‐sized compartments consisting of a lipid bilayer that encapsulates periplasm‐derived, luminal content. OMVs, which pinch off of Gram‐negative bacteria, are now recognized as a generalized secretion pathway which provides a means to transfer cargo to other bacterial cells as well as eukaryotic cells. Compared with other secretion systems, OMVs can transfer a chemically extremely diverse range of cargo, including small molecules, nucleic acids, proteins, and lipids to proximal cells. Although it is well recognized that OMVs can enter and release cargo inside host cells during infection, the mechanisms of host association and uptake are not well understood. This review highlights existing studies focusing on OMV‐host cell interactions and entry mechanisms, and how these entry routes affect cargo processing within the host. It further compares the wide range of methods currently used to dissect uptake mechanisms, and discusses potential sources of discrepancy regarding the mechanism of OMV uptake across different studies. PMID:27529760

  19. Toxoplasma Co-opts Host Cells It Does Not Invade

    PubMed Central

    Koshy, Anita A.; Dietrich, Hans K.; Christian, David A.; Melehani, Jason H.; Shastri, Anjali J.; Hunter, Christopher A.; Boothroyd, John C.

    2012-01-01

    Like many intracellular microbes, the protozoan parasite Toxoplasma gondii injects effector proteins into cells it invades. One group of these effector proteins is injected from specialized organelles called the rhoptries, which have previously been described to discharge their contents only during successful invasion of a host cell. In this report, using several reporter systems, we show that in vitro the parasite injects rhoptry proteins into cells it does not productively invade and that the rhoptry effector proteins can manipulate the uninfected cell in a similar manner to infected cells. In addition, as one of the reporter systems uses a rhoptry:Cre recombinase fusion protein, we show that in Cre-reporter mice infected with an encysting Toxoplasma-Cre strain, uninfected-injected cells, which could be derived from aborted invasion or cell-intrinsic killing after invasion, are actually more common than infected-injected cells, especially in the mouse brain, where Toxoplasma encysts and persists. This phenomenon has important implications for how Toxoplasma globally affects its host and opens a new avenue for how other intracellular microbes may similarly manipulate the host environment at large. PMID:22910631

  20. Human transporters, PEPT1/2, facilitate melatonin transportation into mitochondria of cancer cells: an implication of the therapeutic potential.

    PubMed

    Huo, Xiaokui; Wang, Chao; Yu, Zhenlong; Peng, Yulin; Wang, Shumei; Feng, Shengnan; Zhang, Shouji; Tian, Xiangge; Sun, Chengpeng; Liu, Kexin; Deng, Sa; Ma, Xiaochi

    2017-01-18

    Melatonin is present in virtually all organisms from bacteria to mammals, and it exhibits a broad spectrum of biological functions, including synchronization of circadian rhythms and oncostatic activity. Several functions of melatonin are mediated by its membrane receptors but others are receptor-independent. For the latter, melatonin is required to penetrate membrane and enters intracellular compartments. However, the mechanism by which melatonin enters cells remains debatable. In the current study, it was identified that melatonin and its sulfation metabolites were the substrates of oligopeptide transporter (PEPT) 1/2 and organic anion transporter (OAT) 3, respectively. The docking analysis showed that the binding of melatonin to PEPT1/2 was attributed to their low binding energy and suitable binding conformation in which melatonin was embedded in the active site of PEPT1/2 and fitted well with the cavity in three-dimensional space. PEPT1/2 transporters play a pivotal role in melatonin uptake in cells. Melatonin's membrane transportation via PEPT1/2 renders its oncostatic effect in malignant cells. For the first time, PEPT1/2 were identified to localize in the mitochondrial membrane of human cancer cell lines of PC3 and U118. PEPT1/2 facilitated the transportation of melatonin into mitochondria. Melatonin accumulation in mitochondria induced apoptosis of PC3 and U118 cells. Thus, PEPT1/2 can potentially be used as a cancer cell-targeted melatonin delivery system to improve the therapeutic effects of melatonin in cancer treatment. This article is protected by copyright. All rights reserved.

  1. Th9 Cells Drive Host Immunity against Gastrointestinal Worm Infection.

    PubMed

    Licona-Limón, Paula; Henao-Mejia, Jorge; Temann, Angela U; Gagliani, Nicola; Licona-Limón, Ileana; Ishigame, Harumichi; Hao, Liming; Herbert, De'broski R; Flavell, Richard A

    2013-10-17

    Type 2 inflammatory cytokines, including interleukin-4 (IL-4), IL-5, IL-9, and IL-13, drive the characteristic features of immunity against parasitic worms and allergens. Whether IL-9 serves an essential role in the initiation of host-protective responses is controversial, and the importance of IL-9- versus IL-4-producing CD4⁺ effector T cells in type 2 immunity is incompletely defined. Herein, we generated IL-9-deficient and IL-9-fluorescent reporter mice that demonstrated an essential role for this cytokine in the early type 2 immunity against Nippostrongylus brasiliensis. Whereas T helper 9 (Th9) cells and type 2 innate lymphoid cells (ILC2s) were major sources of infection-induced IL-9 production, the adoptive transfer of Th9 cells, but not Th2 cells, caused rapid worm expulsion, marked basophilia, and increased mast cell numbers in Rag2-deficient hosts. Taken together, our data show a critical and nonredundant role for Th9 cells and IL-9 in host-protective type 2 immunity against parasitic worm infection.

  2. Photosensitization of phycocyanin extracted from Microcystis in human hepatocellular carcinoma cells: implication of mitochondria-dependent apoptosis.

    PubMed

    Wang, Chun-yan; Wang, Xinyan; Wang, Yu; Zhou, Tao; Bai, Yu; Li, Yu-cheng; Huang, Bei

    2012-12-05

    The aim of this study was to explore the possibility that Microcystis phycocyanin (MC-PC) functions as a photosensitizer and to investigate the mechanism for the apoptosis induced by Microcystis phycocyanin-mediated photodynamic therapy (MC-PC-PDT) in human hepatocellular carcinoma cells (HepG2). After incubation with MC-PC, HepG2 cells were exposed to a He-Ne Laser beam and the cell survival rate was detected by MTT and Colony forming assay. The mechanism of apoptosis was determined by ultrastructural observation, reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) assay, activity detection of caspase-3 and cytosol cytochrome c and flow cytometry (FCM) for cell cycle analysis. Our results demonstrated that MC-PC-PDT effectively inhibits HepG2 proliferation with a half-lethal dose of 100 μg/mL and induces apoptosis at 24h with a dose of 200 μg/mL MC-PC. MC-PC was found to localized in mitochondria, it could induce a high level of ROS accumulation at 16 h after PDT treatment, cause mitochondrial damage and the release of mitochondrial cytochrome c into the cytosol. These cellular changes are accompanied by a reduction of the Δψm, activation of caspase-3 and G2/M phase arrest, finally leading to apoptosis through a mitochondria-dependent pathway after 24h. Meanwhile, necrosis was also contributed to cell death in MC-PC PDT process. The present study also identified a new source of phycocyanin from Microcystis as a safe and effective photosensitizer.

  3. Increased pancreatic beta-cell apoptosis following fetal and neonatal exposure to nicotine is mediated via the mitochondria.

    PubMed

    Bruin, Jennifer E; Gerstein, Hertzel C; Morrison, Katherine M; Holloway, Alison C

    2008-06-01

    In Canada, nicotine replacement therapy is recommended as a safe smoking cessation aid for pregnant women. However, we have shown in an animal model that fetal and neonatal nicotine exposure causes increased beta-cell apoptosis and loss of beta-cell mass, which leads to the development of postnatal dysglycemia and obesity. The goal of this study was to determine whether the observed beta-cell apoptosis is mediated via the mitochondrial and/or death receptor pathway. Female Wistar rats were given saline (control) or nicotine bitartrate (1 mg/kg/day) via sc injection for 2 weeks prior to mating until weaning (postnatal day 21). At weaning, pancreas tissue was collected for Western blotting, electron microscopy (EM), and immunohistochemistry. Key markers of each apoptotic pathway were examined in whole pancreas homogenates and mitochondrial/cytosolic pancreas fractions. In the death receptor pathway, Fas and soluble Fas ligand (FasL) protein were significantly increased in the nicotine-exposed offspring compared to control animals; there was no difference in the ratio of inactive/active caspase-8 or membrane-bound FasL expression. In the mitochondrial pathway, there was a significant increase in the ratio of Bcl2/Bax, Bax translocation to the mitochondria, cytochrome c release to the cytosol, and the ratio of active/inactive caspase-3 in nicotine-exposed offspring relative to control animals. Furthermore, increased mitochondrial swelling was observed by EM in the pancreatic beta cells of nicotine-exposed offspring. Taken together, these data suggest that beta-cell apoptosis following developmental nicotine exposure is mediated via the mitochondria.

  4. Resveratrol protects against hyperglycemia-induced oxidative damage to mitochondria by activating SIRT1 in rat mesangial cells

    SciTech Connect

    Xu, Ying; Nie, Ling; Yin, Yang-Guang; Tang, Jian-Lin; Zhou, Ji-Yin; Li, Dan-Dan; Zhou, Shi-Wen

    2012-03-15

    Oxidative stress and mitochondrial dysfunction are involved in the pathogenesis of diabetic nephropathy (DN). Resveratrol has potent protective effects on diabetes and diabetic complications including diabetic nephropathy. We aimed to investigate the protective effects of resveratrol on mitochondria and the underlying mechanisms by using an in vitro model of hyperglycemia. We exposed primary cultured rat mesangial cells to high glucose (30 mM) for 48 h. We found that pretreatment with resveratrol (10 μM) 6 h prior to high glucose treatment significantly reduced hyperglycemia-induced increase in reactive oxygen species (ROS) production and mitochondrial superoxide generation, as well as stimulated MnSOD activity. In addition, resveratrol pretreatment significantly reversed the decrease of mitochondrial complex III activity in glucose-treated mesangial cells, which is considered to be the major source of mitochondrial oxidative stress in glucose-treated cells. Furthermore, resveratrol pretreatment efficiently restored the hyperpolarization of ∆Ψm, increased ATP production and preserved the mtDNA content. All of these protective effects of resveratrol were successfully blocked by siRNA targeting SIRT1 and EX-527, a specific inhibitor of SIRT1 activity. Our results indicated that resveratrol efficiently reduced oxidative stress and maintained mitochondrial function related with activating SIRT1 in glucose-treated mesangial cells. It suggested that resveratrol is pharmacologically promising for treating diabetic nephropathy. -- Highlights: ► We treat mesangial cells with glucose as an in vitro model of diabetic nephropathy. ► We find that the nephroprotective effects of resveratrol relate with mitochondria. ► The beneficial effect of resveratrol was prevented by siRNA SIRT1 or its inhibitor.

  5. On the Origin of Mitochondrial Mutants: Evidence for Intracellular Selection of Mitochondria in the Origin of Antibiotic-Resistant Cells in Yeast

    PubMed Central

    Birky, C. W.

    1973-01-01

    In wild-type Saccharomyces cerevisiae, erythromycin and certain other antibacterial antibiotics inhibit the formation of respiratory enzymes in mitochondria by inhibiting translation on mitochondrial ribosomes. This paper is concerned with the origin of mutant cells, resistant to erythromycin by virtue of having a homogeneous population of mutant mitochondrial DNA molecules. Such mutant cells are obtained by plating wild-type (sensitive) cells on a nonfermentable substrate plus the antibiotic. Colonies of mutant cells appear first about four days after the time of appearance of established mutant cells; new colonies continue to appear, often at a constant rate, for many days. Application of the Newcombe respreading experiment demonstrates that most or all of the mutant cells which form the resistant colonies on selective medium arise only after exposure of the population to erythromycin. It is suggested that this result is most probably due to intracellular selection for mitochondrial genomes. Resistant mitochondria arising from spontaneous mutation are postulated to be at a selective disadvantage in the absence of erythromycin; reproducing more slowly than wild-type sensitive mitochondria, they cannot easily accumulate in sufficient numbers in a cell to render it resistant as a whole. In the presence of erythromycin, resistant mitochondria can continue to reproduce while sensitive mitochondria cannot, until there is a sufficient number to make the cell resistant, i.e. to permit normal cell growth. The same phenomenon is seen with respect to chloramphenicol resistance. Intracellular selection is considered more likely than direct induction of mutation by the antibiotic, since mutant cells do not accumulate in the presence of erythromycin if the mitochondrial genome is rendered non-essential by growth on glucose or nontranslatable by chloramphenicol. Intra-cellular selection provides a mechanism for direct adaptation at the cell level, compatible with currently

  6. Mesenchymal stem cells transfer mitochondria to the cells with virtually no mitochondrial function but not with pathogenic mtDNA mutations.

    PubMed

    Cho, Young Min; Kim, Ju Han; Kim, Mingoo; Park, Su Jin; Koh, Sang Hyeok; Ahn, Hyo Seop; Kang, Gyeong Hoon; Lee, Jung-Bin; Park, Kyong Soo; Lee, Hong Kyu

    2012-01-01

    It has been reported that human mesenchymal stem cells (MSCs) can transfer mitochondria to the cells with severely compromised mitochondrial function. We tested whether the reported intercellular mitochondrial transfer could be replicated in different types of cells or under different experimental conditions, and tried to elucidate possible mechanism. Using biochemical selection methods, we found exponentially growing cells in restrictive media (uridine(-) and bromodeoxyuridine [BrdU](+)) during the coculture of MSCs (uridine-independent and BrdU-sensitive) and 143B-derived cells with severe mitochondrial dysfunction induced by either long-term ethidium bromide treatment or short-term rhodamine 6G (R6G) treatment (uridine-dependent but BrdU-resistant). The exponentially growing cells had nuclear DNA fingerprint patterns identical to 143B, and a sequence of mitochondrial DNA (mtDNA) identical to the MSCs. Since R6G causes rapid and irreversible damage to mitochondria without the removal of mtDNA, the mitochondrial function appears to be restored through a direct transfer of mitochondria rather than mtDNA alone. Conditioned media, which were prepared by treating mtDNA-less 143B ρ(0) cells under uridine-free condition, induced increased chemotaxis in MSC, which was also supported by transcriptome analysis. Cytochalasin B, an inhibitor of chemotaxis and cytoskeletal assembly, blocked mitochondrial transfer phenomenon in the above condition. However, we could not find any evidence of mitochondrial transfer to the cells harboring human pathogenic mtDNA mutations (A3243G mutation or 4,977 bp deletion). Thus, the mitochondrial transfer is limited to the condition of a near total absence of mitochondrial function. Elucidation of the mechanism of mitochondrial transfer will help us create a potential cell therapy-based mitochondrial restoration or mitochondrial gene therapy for human diseases caused by mitochondrial dysfunction.

  7. Leaf mitochondria modulate whole cell redox homeostasis, set antioxidant capacity, and determine stress resistance through altered signaling and diurnal regulation.

    PubMed

    Dutilleul, Christelle; Garmier, Marie; Noctor, Graham; Mathieu, Chantal; Chétrit, Philippe; Foyer, Christine H; de Paepe, Rosine

    2003-05-01

    To explore the role of plant mitochondria in the regulation of cellular redox homeostasis and stress resistance, we exploited a Nicotiana sylvestris mitochondrial mutant. The cytoplasmic male-sterile mutant (CMSII) is impaired in complex I function and displays enhanced nonphosphorylating rotenone-insensitive [NAD(P)H dehydrogenases] and cyanide-insensitive (alternative oxidase) respiration. Loss of complex I function is not associated with increased oxidative stress, as shown by decreased leaf H(2)O(2) and the maintenance of glutathione and ascorbate content and redox state. However, the expression and activity of several antioxidant enzymes are modified in CMSII. In particular, diurnal patterns of alternative oxidase expression are lost, the relative importance of the different catalase isoforms is modified, and the transcripts, protein, and activity of cytosolic ascorbate peroxidase are enhanced markedly. Thus, loss of complex I function reveals effective antioxidant crosstalk and acclimation between the mitochondria and other organelles to maintain whole cell redox balance. This reorchestration of the cellular antioxidative system is associated with higher tolerance to ozone and Tobacco mosaic virus.

  8. Transport and metabolism of L-lactate occur in mitochondria from cerebellar granule cells and are modified in cells undergoing low potassium dependent apoptosis.

    PubMed

    Atlante, Anna; de Bari, Lidia; Bobba, Antonella; Marra, Ersilia; Passarella, Salvatore

    2007-11-01

    Having confirmed that externally added L-lactate can enter cerebellar granule cells, we investigated whether and how L-lactate is metabolized by mitochondria from these cells under normal or apoptotic conditions. (1) L-lactate enters mitochondria, perhaps via an L-lactate/H+ symporter, and is oxidized in a manner stimulated by ADP. The existence of an L-lactate dehydrogenase, located in the inner mitochondrial compartment, was shown by immunological analysis. Neither the protein level nor the Km and Vmax values changed en route to apoptosis. (2) In both normal and apoptotic cell homogenates, externally added L-lactate caused reduction of the intramitochondrial pyridine cofactors, inhibited by phenylsuccinate. This process mirrored L-lactate uptake by mitochondria and occurred with a hyperbolic dependence on L-lactate concentrations. Pyruvate appeared outside mitochondria as a result of external addition of L-lactate. The rate of the process depended on L-lactate concentration and showed saturation characteristics. This shows the occurrence of an intracellular L-lactate/pyruvate shuttle, whose activity was limited by the putative L-lactate/pyruvate antiporter. Both the carriers were different from the monocarboxylate carrier. (3) L-lactate transport changed en route to apoptosis. Uptake increased in the early phase of apoptosis, but decreased in the late phase with characteristics of a non-competitive like inhibition. In contrast, the putative L-lactate/pyruvate antiport decreased en route to apoptosis with characteristics of a competitive like inhibition in early apoptosis, and a mixed non-competitive like inhibition in late apoptosis.

  9. Lactate Metabolism is Associated with Mammalian Mitochondria

    PubMed Central

    Chen, Ying-Jr; Mahieu, Nathaniel G.; Huang, Xiaojing; Singh, Manmilan; Crawford, Peter A; Johnson, Stephen L.; Gross, Richard W.; Schaefer, Jacob

    2016-01-01

    It is well established that lactate secreted by fermenting cells can be oxidized or used as a gluconeogenic substrate by other cells and tissues. Within the fermenting cell itself, however, it is generally assumed that lactate is produced to replenish NAD+ and then is secreted. Here we explored the possibility that cytosolic lactate is metabolized by the mitochondria of fermenting mammalian cells. We found that fermenting HeLa and H460 cells utilize exogenous lactate carbon to synthesize a large percentage of their lipids. With high-resolution mass spectrometry, we found that both 13C and 2-2H labels from enriched lactate enter the mitochondria. The lactate dehydrogenase (LDH) inhibitor oxamate decreased respiration of isolated mitochondria incubated in lactate, but not isolated mitochondria incubated in pyruvate. Additionally, transmission electron microscopy (TEM) showed that LDHB localizes to the mitochondria. Taken together, our results demonstrate a link between lactate metabolism and the mitochondria of fermenting mammalian cells. PMID:27618187

  10. Host cell factors as antiviral targets in arenavirus infection.

    PubMed

    Linero, Florencia N; Sepúlveda, Claudia S; Giovannoni, Federico; Castilla, Viviana; García, Cybele C; Scolaro, Luis A; Damonte, Elsa B

    2012-09-01

    Among the members of the Arenaviridae family, Lassa virus and Junin virus generate periodic annual outbreaks of severe human hemorrhagic fever (HF) in endemic areas of West Africa and Argentina, respectively. Given the human health threat that arenaviruses represent and the lack of a specific and safe chemotherapy, the search for effective antiviral compounds is a continuous demanding effort. Since diverse host cell pathways and enzymes are used by RNA viruses to fulfill their replicative cycle, the targeting of a host process has turned an attractive antiviral approach in the last years for many unrelated virus types. This strategy has the additional benefit to reduce the serious challenge for therapy of RNA viruses to escape from drug effects through selection of resistant variants triggered by their high mutation rate. This article focuses on novel strategies to identify inhibitors for arenavirus therapy, analyzing the potential for antiviral developments of diverse host factors essential for virus infection.

  11. Toxoplasma exports dense granule proteins beyond the vacuole to the host cell nucleus and rewires the host genome expression.

    PubMed

    Bougdour, Alexandre; Tardieux, Isabelle; Hakimi, Mohamed-Ali

    2014-03-01

    Toxoplasma gondii is the most widespread apicomplexan parasite and occupies a large spectrum of niches by infecting virtually any warm-blooded animals. As an obligate intracellular parasite, Toxoplasma has evolved a repertoire of strategies to fine-tune the cellular environment in an optimal way to promote growth and persistence in host tissues hence increasing the chance to be transmitted to new hosts. Short and long-term intracellular survival is associated with Toxoplasma ability to both evade the host deleterious immune defences and to stimulate a beneficial immune balance by governing host cell gene expression. It is only recently that parasite proteins responsible for driving these transcriptional changes have been identified. While proteins contained in the apical secretory Rhoptry organelle have already been identified as bona fide secreted effectors that divert host signalling pathways, recent findings revealed that dense granule proteins should be added to the growing list of effectors as they reach the host cell cytoplasm and nucleus and target various host cell pathways in the course of cell infection. Herein, we emphasize on a novel subfamily of dense granule residentproteins, exemplified with the GRA16 and GRA24 members we recently discovered as both are exported beyond the vacuole-containing parasites and reach the host cell nucleus to reshape the host genome expression.

  12. The Rice Dynamin-Related Protein OsDRP1E Negatively Regulates Programmed Cell Death by Controlling the Release of Cytochrome c from Mitochondria

    PubMed Central

    Zhou, Xueping

    2017-01-01

    Programmed cell death (PCD) mediated by mitochondrial processes has emerged as an important mechanism for plant development and responses to abiotic and biotic stresses. However, the role of translocation of cytochrome c from the mitochondria to the cytosol during PCD remains unclear. Here, we demonstrate that the rice dynamin-related protein 1E (OsDRP1E) negatively regulates PCD by controlling mitochondrial structure and cytochrome c release. We used a map-based cloning strategy to isolate OsDRP1E from the lesion mimic mutant dj-lm and confirmed that the E409V mutation in OsDRP1E causes spontaneous cell death in rice. Pathogen inoculation showed that dj-lm significantly enhances resistance to fungal and bacterial pathogens. Functional analysis of the E409V mutation showed that the mutant protein impairs OsDRP1E self-association and formation of a higher-order complex; this in turn reduces the GTPase activity of OsDRP1E. Furthermore, confocal microscopy showed that the E409V mutation impairs localization of OsDRP1E to the mitochondria. The E409V mutation significantly affects the morphogenesis of cristae in mitochondria and causes the abnormal release of cytochrome c from mitochondria into cytoplasm. Taken together, our results demonstrate that the mitochondria-localized protein OsDRP1E functions as a negative regulator of cytochrome c release and PCD in plants. PMID:28081268

  13. Permeabilization of mitochondria and red blood cells by polycationic peptides BTM-P1 and retro-BTM-P1.

    PubMed

    Lemeshko, Victor V

    2011-10-01

    Mitochondrial and plasma membrane permeabilization by polycationic peptides BTM-P1 and retro-BTM-P1 were studied. BTM-P1 was more active than its retro-analog. In the sucrose medium, the capacity of BTM-P1 to permeabilize mitochondria was lower than in salt media. In contrast, retro-BTM-P1 showed the lowest activity in the KCl medium. The efficacy of both peptides to permeabilize red blood cells was higher in the sucrose medium and depended on the nature of salt in high ionic strength media. BTM-P1, but not retro-BTM-P1, induced biphasic change in light dispersion of red blood cells with artificially generated high transmembrane potential: the initial phase of fast cell shrinkage preceded the subsequent phase of cell swelling. The shrunken red blood cells demonstrated increased sensitivity to BTM-P1 that might be explained by the cell suicide mechanism via phosphatidylserine exposure at the cell surface. As a working hypothesis, we assume that some peptide topology characteristics, such as the orientation and values of the total and local electrical dipole moments, interacting with the membrane dipole potential, as well as the asymmetric distribution of polar and non-polar side chains are important factors affecting the membrane-permeabilizing activity of polycationic peptides.

  14. Sendai virus utilizes specific sialyloligosaccharides as host cell receptor determinants.

    PubMed Central

    Markwell, M A; Paulson, J C

    1980-01-01

    Purified sialyltransferases (CMP-N-acetyl-neuraminate:D-galactosyl-glycoprotein N-acetylneuraminyl-transferase, EC 2.4.99.1) in conjunction with neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) were used to produce cell surface sialyloligosaccharides of defined sequence to investigate their role in paramyxovirus infection of host cells. Infection of Madin-Darby bovine kidney cells by Sendai virus was monitored by hemagglutination titer of the virus produced and by changes in morphological characteristics. By either criterion, treatment of the cells with Vibrio cholerae neuraminidase to remove cell surface sialic acids rendered them resistant to infection by Sendai virus. Endogenous replacement of receptors by the cell occurred slowly but supported maximal levels of infection within 6 hr. In contrast, sialylation during a 20-min incubation with CMP-sialic acid and beta-galactoside alpha 2,3-sialytransferase restored full susceptibility to infection. This enzyme elaborates the NeuAc alpha 2,3Gal beta 1,3GalNAc (NeuAc, N-acetylneuraminic acid) sequence on glycoproteins and glycolipids. No restoration of infectivity was observed when neuraminidase-treated cells were sialylated by using beta-galactoside alpha 2,6-sialytransferase, which elaborates the NeuAc-alpha 2,6Gal beta 1,4GlcNAc sequence. These results suggest that sialyloligosaccharide receptor determinants of defined sequence are required for Sendai virus infection of host cells. Images PMID:6255459

  15. Wheat Mitochondria

    PubMed Central

    Raison, John K.; Chapman, Elza A.; White, P. Y.

    1977-01-01

    Mitochondrial oxidative activity and membrane lipid structure of two wheat (Triticum aestivum L.) cultivars were measured as a function of temperature. The Arrhenius activation energy for the oxidation of both succinate and α-ketoglutarate was constant over the temperature range of 3 to 27 C. The activation energy for succinate-cytochrome c oxidoreductase activity was also constant over the same temperature range. The concentration of mitochondria in the reaction, the degree of initial inhibition of state 3 respiration, and the time after isolation of mitochondria were each shown to be capable of causing a disproportionate decrease in the rate of oxidation at low temperatures which resulted in an apparent increase in the activation energy of oxidative activity. Using three spin-labeling techniques, wheat membrane lipids were shown to undergo phase changes at about 0 C and 30 C. It is concluded that the membrane lipids of wheat, a chillingresistant plant, undergo a phase transition similar to the transition observed in the membrane lipids of chilling-sensitive plants. For wheat, however, the transition is initiated at a lower temperature and extends over a wider temperature range. PMID:16659906

  16. Novel HIV-1 Therapeutics through Targeting Altered Host Cell Pathways

    PubMed Central

    Coley, William; Kehn-Hall, Kylene; Van Duyne, Rachel; Kashanchi, Fatah

    2009-01-01

    The emergence of drug-resistant human immunodeficiency virus type I (HIV-1) strains presents a challenge for the design of new drugs. Anti-HIV compounds currently in use are the subject of advanced clinical trials using either HIV-1 reverse-transcriptase, viral protease, or integrase inhibitors. Recent studies show an increase in the number of HIV-1 variants resistant to anti-retroviral agents in newly infected individuals. Targeting host cell factors involved in the regulation of HIV-1 replication might be one way to combat HIV-1 resistance to the currently available anti-viral agents. A specific inhibition of HIV-1 gene expression could be expected from the development of compounds targeting host cell factors that participate in the activation of the HIV-1 LTR promoter. Here we will discuss how targeting the host can be accomplished either by using small molecules to alter the function of the host’s proteins such as p53 or cdk9, or by utilizing new advances in siRNA therapies to knock down essential host factors such as CCR5 and CXCR4. Finally, we will discuss how the viral protein interactomes should be performed to better design therapeutics against HIV-1. PMID:19732026

  17. Recombinant host cells and media for ethanol production

    DOEpatents

    Wood, Brent E; Ingram, Lonnie O; Yomano, Lorraine P; York, Sean W

    2014-02-18

    Disclosed are recombinant host cells suitable for degrading an oligosaccharide that have been optimized for growth and production of high yields of ethanol, and methods of making and using these cells. The invention further provides minimal media comprising urea-like compounds for economical production of ethanol by recombinant microorganisms. Recombinant host cells in accordance with the invention are modified by gene mutation to eliminate genes responsible for the production of unwanted products other than ethanol, thereby increasing the yield of ethanol produced from the oligosaccharides, relative to unmutated parent strains. The new and improved strains of recombinant bacteria are capable of superior ethanol productivity and yield when grown under conditions suitable for fermentation in minimal growth media containing inexpensive reagents. Systems optimized for ethanol production combine a selected optimized minimal medium with a recombinant host cell optimized for use in the selected medium. Preferred systems are suitable for efficient ethanol production by simultaneous saccharification and fermentation (SSF) using lignocellulose as an oligosaccharide source. The invention also provides novel isolated polynucleotide sequences, polypeptide sequences, vectors and antibodies.

  18. Hsp105 family proteins suppress staurosporine-induced apoptosis by inhibiting the translocation of Bax to mitochondria in HeLa cells

    SciTech Connect

    Yamagishi, Nobuyuki; Ishihara, Keiichi; Saito, Youhei; Hatayama, Takumi . E-mail: hatayama@mb.kyoto-phu.ac.jp

    2006-10-15

    Hsp105 (Hsp105{alpha} and Hsp105{beta}), major heat shock proteins in mammalian cells, belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105{alpha} has completely different effects on stress-induced apoptosis depending on cell type. However, the molecular mechanisms by which Hsp105{alpha} regulates stress-induced apoptosis are not fully understood. Here, we established HeLa cells that overexpress either Hsp105{alpha} or Hsp105{beta} by removing doxycycline and examined how Hsp105 modifies staurosporine (STS)-induced apoptosis in HeLa cells. Apoptotic features such as the externalization of phosphatidylserine on the plasma membrane and nuclear morphological changes were induced by the treatment with STS, and the STS-induced apoptosis was suppressed by overexpression of Hsp105{alpha} or Hsp105{beta}. In addition, we found that overexpression of Hsp105{alpha} or Hsp105{beta} suppressed the activation of caspase-3 and caspase-9 by preventing the release of cytochrome c from mitochondria. Furthermore, the translocation of Bax to mitochondria, which results in the release of cytochrome c from the mitochondria, was also suppressed by the overexpression of Hsp105{alpha} or Hsp105{beta}. Thus, it is suggested that Hsp105 suppresses the stress-induced apoptosis at its initial step, the translocation of Bax to mitochondria in HeLa cells.

  19. Trichostatin A induces apoptosis of p815 mastocytoma cells in histone acetylation- and mitochondria-dependent fashion.

    PubMed

    Yee, Su-Bog; Kim, Myung Soo; Baek, Soo Jin; Kim, Gyoo Cheon; Yoo, Ki Soo; Yoo, Young Hyun; Park, Bong Soo

    2004-11-01

    Although inhibition of histone deacetylase has been demonstrated to induce apoptosis of various cancer cells, there is no report on its effect on mast cell demise to date. Here we studied whether a histone deacetylase inhibitor Trichostatin A (TSA) produces apoptosis in p815 mastocytoma cells. TSA prominently increased the amount of acetylated histones, H3, H4, H2A and H2B, in p815 mastocytoma cells. TSA reduced the viability of p815 mastocytoma cells, and many apoptotic manifestations such as generation of DNA fragmentation, activation of caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), and increase of DNA hypoploidy proved that the reduction of viability resulted from apoptosis. Whereas TSA treatment increased the expression level of Bad, it decreased the level of Bcl-2, Bcl-xL, and X-linked inhibitor of apoptosis protein. The reduction of mitochondrial membrane potential, the release of cytochrome c and Smac/DIABLO to cytosol, and mitochondrial localization of Bad were also shown. Taken together, TSA induces apoptosis on p815 mastocytoma cells in histone acetylation- and mitochondria-dependent fashion. Our data therefore provide the possibility that TSA could be considered as a novel therapeutic strategy for mastocytoma from its apoptosis-inducing activity.

  20. Glutaredoxin 2 Prevents H2O2-Induced Cell Apoptosis by Protecting Complex I Activity in the Mitochondria*

    PubMed Central

    Wu, Hongli; Xing, Kuiyi; Lou, Marjorie F.

    2010-01-01

    Glutaredoxin 2 (Grx2) belongs to the oxidoreductase family and is an isozyme of glutaredoxin 1 (Grx1) present in the mitochondria, however its function is not well understood. The purpose of this study is to evaluate the potential anti-apoptotic function of Grx2 by examining its ability to protect complex I in the mitochondrial electron transport system using human lens epithelial cells as a model. We found that cells treated with 200 μM hydrogen peroxide (H2O2) for 24 h exhibited decreased viability and became apoptotic with corresponding Bax up-regulation, Bcl-2 down-regulation, caspase 3 activation and mitochondrial cytochrome c leakage. Grx2 over-expression (OE) could protect cells against H2O2-induced damage while Grx2 knockdown (KD) showed the opposite effect. Under the same conditions, H2O2 treatment caused 50% inactivation of complex I activity in control cells (vector only), 75% in Grx2 KD cells but only 20% in Grx2 OE cells. This antiapoptotic function of Grx2 is specific as rotenone, a complex I specific inhibitor, could block this Grx2-mediated protection of complex I activity. Immunoprecipitation study also revealed that Grx2 co-precipitated with complex I in the mitochondrial lysate. Thus, the mechanism of Grx2 protection against H2O2-induced apoptosis is likely associated with its ability to preserve complex I. PMID:20547138

  1. Mitochondria and neuroplasticity

    PubMed Central

    Cheng, Aiwu; Hou, Yan; Mattson, Mark P

    2010-01-01

    The production of neurons from neural progenitor cells, the growth of axons and dendrites and the formation and reorganization of synapses are examples of neuroplasticity. These processes are regulated by cell-autonomous and intercellular (paracrine and endocrine) programs that mediate responses of neural cells to environmental input. Mitochondria are highly mobile and move within and between subcellular compartments involved in neuroplasticity (synaptic terminals, dendrites, cell body and the axon). By generating energy (ATP and NAD+), and regulating subcellular Ca2+ and redox homoeostasis, mitochondria may play important roles in controlling fundamental processes in neuroplasticity, including neural differentiation, neurite outgrowth, neurotransmitter release and dendritic remodelling. Particularly intriguing is emerging data suggesting that mitochondria emit molecular signals (e.g. reactive oxygen species, proteins and lipid mediators) that can act locally or travel to distant targets including the nucleus. Disturbances in mitochondrial functions and signalling may play roles in impaired neuroplasticity and neuronal degeneration in Alzheimer's disease, Parkinson's disease, psychiatric disorders and stroke. PMID:20957078

  2. RELATION OF TOBACCO MOSAIC VIRUS TO THE HOST CELLS

    PubMed Central

    Esau, Katherine; Cronshaw, James

    1967-01-01

    The relation of tobacco mosaic virus (TMV) to host cells was studied in leaves of Nicotiana tabacum L. systemically infected with the virus. The typical TMV inclusions, striate or crystalline material and ameboid or X-bodies, which are discernible with the light microscope, and/or particles of virus, which are identifiable with the electron microscope, were observed in epidermal cells, mesophyll cells, parenchyma cells of the vascular bundles, differentiating and mature tracheary elements, and immature and mature sieve elements. Virus particles were observed in the nuclei and the chloroplasts of parenchyma cells as well as in the ground cytoplasm, the vacuole, and between the plasma membrane and the cell wall. The nature of the conformations of the particle aggregates in the chloroplasts was compatible with the concept that some virus particles may be assembled in these organelles. The virus particles in the nuclei appeared to be complete particles. Under the electron microscope the X-body constitutes a membraneless assemblage of endoplasmic reticulum, ribosomes, virus particles, and of virus-related material in the form of wide filaments indistinctly resolvable as bundles of tubules. Some parenchyma cells contained aggregates of discrete tubules in parallel arrangement. These groups of tubules were relatively free from components of host protoplasts. PMID:6036529

  3. Giant mitochondria do not fuse and exchange their contents with normal mitochondria

    SciTech Connect

    Navratil, Marian; Terman, Alexei; Arriaga, Edgar A.

    2008-01-01

    Giant mitochondria accumulate within aged or diseased postmitotic cells as a consequence of insufficient autophagy, which is normally responsible for mitochondrial degradation. We report that giant mitochondria accumulating in cultured rat myoblasts due to inhibition of autophagy have low inner membrane potential and do not fuse with each other or with normal mitochondria. In addition to the low inner mitochondrial membrane potential in giant mitochondria, the quantity of the OPA1 mitochondrial fusion protein in these mitochondria was low, but the abundance of mitofusin-2 (Mfn2) remained unchanged. The combination of these factors may explain the lack of mitochondrial fusion in giant mitochondria and imply that the dysfunctional giant mitochondria cannot restore their function by fusing and exchanging their contents with fully functional mitochondria. These findings have important implications for understanding the mechanisms of accumulation of age-related mitochondrial damage in postmitotic cells.

  4. Ecdysteroid receptor (EcR) is associated with microtubules and with mitochondria in the cytoplasm of prothoracic gland cells of Rhodnius prolixus (Hemiptera).

    PubMed

    Vafopoulou, Xanthe

    2009-12-01

    We have shown previously that EcR in larval Rhodnius is present in the cytoplasm of various cell types and undergoes daily cycling in abundance in the cytoplasm (Vafopoulou and Steel, 2006. Cell Tissue Res 323:443-455). It is unknown which organelles are associated with EcR. Here, we report that cytoplasmic EcR in prothoracic gland cells is associated with both microtubules and mitochondria, and discuss the implications for both nuclear and non-genomic actions of EcR. EcR was localized immunohistochemically using several antibodies to EcR of Manduca and Drosophila and a confocal laser scanning microscope. Double labels were made to visualize EcR and (1) microtubules (using an antibody to tyrosylated alpha-tubulin) and (2) mitochondria (using a fluorescent MitoTracker probe), both after stabilization of microtubules with taxol. EcR co-localized with both tubulin and mitochondria. All the different EcR antibodies produced similar co-localization patterns. EcR was seen in the perinuclear aggregation of mitochondria, indicating that mitochondria are targets of ecdysone, which could influence mitochondrial gene transcription. EcR was also distributed throughout the microtubule network. Co-localization of EcR with tubulin or mitochondria was maintained after depolymerization of microtubules with colchicine. Treatment with taxol resulted in accumulation of EcR in the cytoplasm and simultaneous depletion of EcR from the nucleus, suggesting that microtubules may be involved in targeted intracellular transport of EcR to the nucleus (genomic action) or may play a role in rapid ecdysone signal transduction in the extranuclear compartment, i.e., in non-genomic actions of ecdysone. These findings align EcR more closely with steroid hormone receptors in vertebrates.

  5. Translation and Assembly of Radiolabeled Mitochondrial DNA-Encoded Protein Subunits from Cultured Cells and Isolated Mitochondria.

    PubMed

    Formosa, Luke E; Hofer, Annette; Tischner, Christin; Wenz, Tina; Ryan, Michael T

    2016-01-01

    In higher eukaryotes, the mitochondrial electron transport chain consists of five multi-subunit membrane complexes responsible for the generation of cellular ATP. Of these, four complexes are under dual genetic control as they contain subunits encoded by both the mitochondrial and nuclear genomes, thereby adding another layer of complexity to the puzzle of respiratory complex biogenesis. These subunits must be synthesized and assembled in a coordinated manner in order to ensure correct biogenesis of different respiratory complexes. Here, we describe techniques to (1) specifically radiolabel proteins encoded by mtDNA to monitor the rate of synthesis using pulse labeling methods, and (2) analyze the stability, assembly, and turnover of subunits using pulse-chase methods in cultured cells and isolated mitochondria.

  6. [Mitochondria inheritance in yeast saccharomyces cerevisiae].

    PubMed

    Fizikova, A Iu

    2011-01-01

    The review is devoted to the main mechanisms of mitochondria inheritance in yeast Saccharonmyces cerevisiae. The genetic mechanisms of functionally active mitochondria inheritance in eukaryotic cells is one of the most relevant in modem researches. A great number of genetic diseases are associated with mitochondria dysfunction. Plasticity of eukaryotic cell metabolism according to the environmental changes is ensured by adequate mitochondria functioning by means of ATP synthesis coordination, reactive oxygen species accumulation, apoptosis regulation and is an important factor of cell adaptation to stress. Mitochondria participation in important for cell vitality processes masters the presence of accurate mechanisms of mitochondria functions regulation according to environment fluctuations. The mechanisms of mitochondria division and distribution are highly conserved. Baker yeast S. cerevisiae is an ideal model object for mitochondria researches due to energetic metabolism lability, ability to switch over respiration to fermentation, and petite-positive phenotype. Correction of metabolism according to the environmental changes is necessary for cell vitality. The influence of respiratory, carbon, amino acid and phosphate metabolism on mitochondria functions was shown. As far as the mechanisms that stabilize functions of mitochondria and mtDNA are highly conserve, we can project yeast regularities on higher eukaryotes systems. This makes it possible to approximate understanding the etiology and pathogenesis of a great number of human diseases.

  7. Infection Strategies of Intestinal Parasite Pathogens and Host Cell Responses

    PubMed Central

    Di Genova, Bruno M.; Tonelli, Renata R.

    2016-01-01

    Giardia lamblia, Cryptosporidium sp., and Entamoeba histolytica are important pathogenic intestinal parasites and are amongst the leading causes worldwide of diarrheal illness in humans. Diseases caused by these organisms, giardiasis, cryptosporidiosis, and amoebiasis, respectively, are characterized by self-limited diarrhea but can evolve to long-term complications. The cellular and molecular mechanisms underlying the pathogenesis of diarrhea associated with these three pathogens are being unraveled, with knowledge of both the strategies explored by the parasites to establish infection and the methods evolved by hosts to avoid it. Special attention is being given to molecules participating in parasite–host interaction and in the mechanisms implicated in the diseases’ pathophysiologic processes. This review focuses on cell mechanisms that are modulated during infection, including gene transcription, cytoskeleton rearrangements, signal transduction pathways, and cell death. PMID:26973630

  8. Metal binding proteins, recombinant host cells and methods

    DOEpatents

    Summers, Anne O.; Caguiat, Jonathan J.

    2004-06-15

    The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.

  9. Brucella T4SS: the VIP pass inside host cells.

    PubMed

    Lacerda, Thais Lourdes Santos; Salcedo, Suzana Pinto; Gorvel, Jean-Pierre

    2013-02-01

    For many Gram-negative bacteria, like Brucella, the type IV secretion system (T4SS) has a critical role in bacterial virulence. In Brucella, the VirB T4SS permits the injection of bacterial effectors inside host cells, leading to subversion of signaling pathways and favoring bacterial growth and pathogenesis. The virB operon promoter is tightly regulated by a combination of transcriptional activators and repressors that are expressed according to the environmental conditions encountered by Brucella. Recent advances have shed light on the Brucella T4SS regulatory mechanisms and also its substrates. Characterization of the targets and functions of these translocated effectors is underway and will help understand the role of the T4SS in the establishment of a replication niche inside host cells.

  10. Host cell autophagy in immune response to zoonotic infections.

    PubMed

    Skendros, Panagiotis; Mitroulis, Ioannis

    2012-01-01

    Autophagy is a fundamental homeostatic process in which cytoplasmic targets are sequestered within double-membraned autophagosomes and subsequently delivered to lysosomes for degradation. Accumulating evidence supports the pivotal role of autophagy in host defense against intracellular pathogens implicating both innate and adaptive immunity. Many of these pathogens cause common zoonotic infections worldwide. The induction of the autophagic machinery by innate immune receptors signaling, such as TLRs, NOD1/2, and p62/SQSTM1 in antigen-presenting cells results in inhibition of survival and elimination of invading pathogens. Furthermore, Th1 cytokines induce the autophagic process, whereas autophagy also contributes to antigen processing and MHC class II presentation, linking innate to adaptive immunity. However, several pathogens have developed strategies to avoid autophagy or exploit autophagic machinery to their advantage. This paper focuses on the role of host cell autophagy in the regulation of immune response against intracellular pathogens, emphasizing on selected bacterial and protozoan zoonoses.

  11. Cytotoxicity of withasteroids: withametelin induces cell cycle arrest at G2/M phase and mitochondria-mediated apoptosis in non-small cell lung cancer A549 cells.

    PubMed

    Rao, Poorna Chandra; Begum, Sajeli; Jahromi, Mohammad Ali Farboodniay; Jahromi, Zahra Hosseini; Sriram, Saketh; Sahai, Mahendra

    2016-09-01

    Considerable interest has been gained by withasteroids because of their structural uniqueness and wide spectrum of biological activities. However, limited systematic studies for proving their cytotoxic potential have so far been reported. Hence, an attempt was made to test the cytotoxicity of six withasteroids viz., withametelin (WM), withaphysalin D, withaphysalin E, 12-deoxywithastramonolide, Withaperuvin B, and physalolactone against A549, HT-29, and MDA-MB-231 cancer cell lines. Significant cytotoxic effect of WM against A549 cells (IC50 value of 6.0 μM), MDA-MB-231 cells (IC50 value of 7.6 μM), and HT-29 cells (IC50 value of 8.2 μM) was observed. Withaperuvin B and physalolactone were found to be effective against MDA-MB-231 cells. The significantly active WM arrested the A549 cells at G2/M phase and downregulated the expression of G2/M regulatory proteins such as cdc2, cyclin B1, and cdc25C. Apoptosis induced by WM in A549 cells was associated with the generation of ROS and depletion of MMP. Furthermore, WM treatment resulted in Bax upregulation, Bcl-2 downregulation, translocation of cytochrome c to mitochondria, activation of caspase-9 and -3, and PARP cleavage corroborating the apoptosis induction through intrinsic apoptotic pathway. Thus, WM possessing broader cytotoxic effect is a promising lead molecule which has the potential to be developed as a new therapeutic agent for NSCLC.

  12. Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease

    PubMed Central

    Muratovska, Aleksandra; Lightowlers, Robert N.; Taylor, Robert W.; Turnbull, Douglass M.; Smith, Robin A. J.; Wilce, Jacqueline A.; Martin, Stephen W.; Murphy, Michael P.

    2001-01-01

    The selective manipulation of mitochondrial DNA (mtDNA) replication and expression within mammalian cells has proven difficult. One promising approach is to use peptide nucleic acid (PNA) oligomers, nucleic acid analogues that bind selectively to complementary DNA or RNA sequences inhibiting replication and translation. However, the potential of PNAs is restricted by the difficulties of delivering them to mitochondria within cells. To overcome this problem we conjugated a PNA 11mer to a lipophilic phosphonium cation. Such cations are taken up by mitochondria through the lipid bilayer driven by the membrane potential across the inner membrane. As anticipated, phosphonium–PNA (ph–PNA) conjugates of 3.4–4 kDa were imported into both isolated mitochondria and mitochondria within human cells in culture. This was confirmed by using an ion-selective electrode to measure uptake of the ph–PNA conjugates; by cell fractionation in conjunction with immunoblotting; by confocal microscopy; by immunogold-electron microscopy; and by crosslinking ph–PNA conjugates to mitochondrial matrix proteins. In all cases dissipating the mitochondrial membrane potential with an uncoupler prevented ph–PNA uptake. The ph–PNA conjugate selectively inhibited the in vitro replication of DNA containing the A8344G point mutation that causes the human mtDNA disease ‘myoclonic epilepsy and ragged red fibres’ (MERRF) but not the wild-type sequence that differs at a single nucleotide position. Therefore these modified PNA oligomers retain their selective binding to DNA and the lipophilic cation delivers them to mitochondria within cells. When MERRF cells were incubated with the ph–PNA conjugate the ratio of MERRF to wild-type mtDNA was unaffected, even though the ph–PNA content of the mitochondria was sufficient to inhibit MERRF mtDNA replication in a cell-free system. This unexpected finding suggests that nucleic acid derivatives cannot bind their complementary sequences during mt

  13. Cathepsin D protects colorectal cancer cells from acetate-induced apoptosis through autophagy-independent degradation of damaged mitochondria.

    PubMed

    Oliveira, C S F; Pereira, H; Alves, S; Castro, L; Baltazar, F; Chaves, S R; Preto, A; Côrte-Real, M

    2015-06-18

    Acetate is a short-chain fatty acid secreted by Propionibacteria from the human intestine, known to induce mitochondrial apoptotic death in colorectal cancer (CRC) cells. We previously established that acetate also induces lysosome membrane permeabilization in CRC cells, associated with release of the lysosomal protease cathepsin D (CatD), which has a well-established role in the mitochondrial apoptotic cascade. Unexpectedly, we showed that CatD has an antiapoptotic role in this process, as pepstatin A (a CatD inhibitor) increased acetate-induced apoptosis. These results mimicked our previous data in the yeast system showing that acetic acid activates a mitochondria-dependent apoptosis process associated with vacuolar membrane permeabilization and release of the vacuolar protease Pep4p, ortholog of mammalian CatD. Indeed, this protease was required for cell survival in a manner dependent on its catalytic activity and for efficient mitochondrial degradation independently of autophagy. In this study, we therefore assessed the role of CatD in acetate-induced mitochondrial alterations. We found that, similar to acetic acid in yeast, acetate-induced apoptosis is not associated with autophagy induction in CRC cells. Moreover, inhibition of CatD with small interfering RNA or pepstatin A enhanced apoptosis associated with higher mitochondrial dysfunction and increased mitochondrial mass. This effect seems to be specific, as inhibition of CatB and CatL with E-64d had no effect, nor were these proteases significantly released to the cytosol during acetate-induced apoptosis. Using yeast cells, we further show that the role of Pep4p in mitochondrial degradation depends on its protease activity and is complemented by CatD, indicating that this mechanism is conserved. In summary, the clues provided by the yeast model unveiled a novel CatD function in the degradation of damaged mitochondria when autophagy is impaired, which protects CRC cells from acetate-induced apoptosis. Cat

  14. Baculovirus Infection Influences Host Protein Expression in Two Established Insect Cell Lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We identified host proteins that changed in response to host cell susceptibility to baculovirus infection. We used three baculovirus–host cell systems utilizing two cell lines derived from pupal ovaries, Hz-AM1 (from Helicoverpa zea) and Hv-AM1 (from Heliothis virescens). Hv-AM1 cells are permissive...

  15. Viral degradasome hijacks mitochondria to suppress innate immunity

    PubMed Central

    Goswami, Ramansu; Majumdar, Tanmay; Dhar, Jayeeta; Chattopadhyay, Saurabh; Bandyopadhyay, Sudip K; Verbovetskaya, Valentina; Sen, Ganes C; Barik, Sailen

    2013-01-01

    The balance between the innate immunity of the host and the ability of a pathogen to evade it strongly influences pathogenesis and virulence. The two nonstructural (NS) proteins, NS1 and NS2, of respiratory syncytial virus (RSV) are critically required for RSV virulence. Together, they strongly suppress the type I interferon (IFN)-mediated innate immunity of the host cells by degrading or inhibiting multiple cellular factors required for either IFN induction or response pathways, including RIG-I, IRF3, IRF7, TBK1 and STAT2. Here, we provide evidence for the existence of a large and heterogeneous degradative complex assembled by the NS proteins, which we named “NS-degradasome” (NSD). The NSD is roughly ∼300-750 kD in size, and its degradative activity was enhanced by the addition of purified mitochondria in vitro. Inside the cell, the majority of the NS proteins and the substrates of the NSD translocated to the mitochondria upon RSV infection. Genetic and pharmacological evidence shows that optimal suppression of innate immunity requires mitochondrial MAVS and mitochondrial motility. Together, we propose a novel paradigm in which the mitochondria, known to be important for the innate immune activation of the host, are also important for viral suppression of the innate immunity. PMID:23877405

  16. Resveratrol Specifically Kills Cancer Cells by a Devastating Increase in the Ca2+ Coupling Between the Greatly Tethered Endoplasmic Reticulum and Mitochondria

    PubMed Central

    Madreiter-Sokolowski, Corina T.; Gottschalk, Benjamin; Parichatikanond, Warisara; Eroglu, Emrah; Klec, Christiane; Waldeck-Weiermair, Markus; Malli, Roland; Graier, Wolfgang F.

    2017-01-01

    Background/Aims Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol. Methods The two cancer cell lines EA.hy926 and HeLa, and somatic short-term cultured HUVEC were used. Cell viability and caspase 3/7 activity were tested. Mitochondrial, cytosolic and endoplasmic reticulum Ca2+ as well as cytosolic and mitochondrial ATP levels were measured using single cell fluorescence microscopy and respective genetically-encoded sensors. Mitochondria-ER junctions were analyzed applying super-resolution SIM and ImageJ-based image analysis. Results Resveratrol and piceatannol selectively trigger death in cancer but not somatic cells. Hence, these polyphenols strongly enhanced mitochondrial Ca2+ uptake in cancer exclusively. Resveratrol and piceatannol predominantly affect mitochondrial but not cytosolic ATP content that yields in a reduced SERCA activity. Decreased SERCA activity and the strongly enriched tethering of the ER and mitochondria in cancer cells result in an enhanced MCU/Letm1-dependent mitochondrial Ca2+ uptake upon intracellular Ca2+ release exclusively in cancer cells. Accordingly, resveratrol/piceatannol-induced cancer cell death could be prevented by siRNA-mediated knock-down of MCU and Letm1. Conclusions Because their greatly enriched ER-mitochondria tethering, cancer cells are highly susceptible for resveratrol/piceatannol-induced reduction of SERCA activity to yield mitochondrial Ca2+ overload and subsequent cancer cell death. PMID:27606689

  17. Nitric oxide scavenging causes remodeling of the endoplasmic reticulum, Golgi apparatus and mitochondria in pulmonary arterial endothelial cells.

    PubMed

    Lee, Jason E; Yuan, Huijuan; Liang, Feng-Xia; Sehgal, Pravin B

    2013-09-01

    The dependence of the structure and function of cytoplasmic organelles in endothelial cells on constitutively produced intracellular nitric oxide (NO) remains largely unexplored. We previously reported fragmentation of the Golgi apparatus in cells exposed to NO scavengers or after siRNA-mediated knockdown of eNOS. Others have reported increased mitochondrial fission in response to an NO donor. Functionally, we previously reported that bovine pulmonary arterial endothelial cells (PAECs) exposed to the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) developed a prosecretory phenotype characterized by prolonged secretion of soluble proteins. In the present study, we investigated whether NO scavenging led to remodeling of the endoplasmic reticulum (ER). Live-cell DAF-2DA imaging confirmed the presence of intracellular NO in association with the BODIPY C5-ceramide-labeled Golgi apparatus. Untreated human PAECs displayed a pattern of peripheral tubulo-reticular ER with a juxtanuclear accumulation of ER sheets. Cells exposed to c-PTIO showed a dramatic increase in ER sheets as assayed using immunofluorescence for the ER structural protein reticulon-4b/Nogo-B and the ER-resident GTPase atlastin-3, live-cell fluorescence assays using RTN4-GFP and KDEL-mCherry, and electron microscopy methods. These ER changes were inhibited by the NO donor diethylamine NONOate, and also produced by L-NAME, but not D-NAME or 8-br-cGMP. This ER remodeling was accompanied by Golgi fragmentation and increased fibrillarity and function of mitochondria (uptake of tetramethyl-rhodamine, TMRE). Despite Golgi fragmentation the functional ER/Golgi trafficking unit was preserved as seen by the accumulation of Sec31A ER exit sites adjacent to the dispersed Golgi elements and a 1.8-fold increase in secretion of soluble cargo. Western blotting and immunopanning data showed that RTN4b was increasingly ubiquitinated following c-PTIO exposure, especially in the

  18. Nitric oxide scavenging causes remodeling of the endoplasmic reticulum, Golgi apparatus and mitochondria in pulmonary arterial endothelial cells

    PubMed Central

    Lee, Jason E.; Yuan, Huijuan; Liang, Feng-Xia; Sehgal, Pravin B.

    2013-01-01

    The dependence of the structure and function of cytoplasmic organelles in endothelial cells on constitutively produced intracellular nitric oxide (NO) remains largely unexplored. We previously reported fragmentation of the Golgi apparatus in cells exposed to NO scavengers or after siRNA-mediated knockdown of eNOS. Others have reported increased mitochondrial fission in response to an NO donor. Functionally, we previously reported that bovine pulmonary arterial endothelial cells (PAECs) exposed to the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) developed a prosecretory phenotype characterized by prolonged secretion of soluble proteins. In the present study, we investigated whether NO scavenging led to remodeling of the endoplasmic reticulum (ER). Live-cell DAF-2DA imaging confirmed the presence of intracellular NO in association with the BODIPY C5- ceramide-labelled Golgi apparatus. Untreated human PAECs displayed a pattern of peripheral tubulo-reticular ER with a juxtanuclear accumulation of ER sheets. Cells exposed to c-PTIO showed a dramatic increase in ER sheets as assayed using immunofluoresence for the ER structural protein reticulon-4b/Nogo-B and the ER-resident GTPase atlastin-3, live-cell fluorescence assays using RTN4-GFP and KDEL-mCherry, and electron microscopy methods. These ER changes were inhibited by the NO donor diethylamine NONOate, and also produced by L-NAME, but not D-NAME or 8-br-cGMP. This ER remodeling was accompanied by Golgi fragmentation and increased fibrillarity and function of mitochondria (uptake of tetramethyl- rhodamine, TMRE). Despite Golgi fragmentation the functional ER/Golgi trafficking unit was preserved as seen by the accumulation of Sec31A ER exit sites adjacent to the dispersed Golgi elements and a 1.8-fold increase in secretion of soluble cargo. Western blotting and immunopanning data showed that RTN4b was increasingly ubiquitinated following c-PTIO exposure, especially in the

  19. Metabolic Pathways in Anopheles stephensi mitochondria

    PubMed Central

    Giulivi, Cecilia; Ross-Inta, Catherine; Horton, Ashley A.; Luckhart, Shirley

    2017-01-01

    No studies have been performed on mitochondria of malaria vector mosquitoes. This information would be valuable in understanding mosquito aging and detoxification of insecticides, two parameters that significantly impact malaria parasite transmission in endemic regions. Here, we report the analyses of respiration and oxidative phosphorylation in mitochondria of cultured cells (ASE line) from Anopheles stephensi, a major vector of malaria in India, Southeast Asia and parts of the Middle East. ASE cell mitochondria shared many features in common with mammalian muscle mitochondria, despite the fact that these cells have a larval origin. However, two major differences with mammalian mitochondria were apparent. One, the glycerol-phosphate shuttle plays a major role in NADH oxidation in ASE cell mitochondria as it does in insect muscle mitochondria. In contrast, mammalian white muscle mitochondria depend primarily on lactate dehydrogenase, whereas red muscle mitochondria depend on the malate-oxaloacetate shuttle. Two, ASE mitochondria were able to oxidize Pro at a rate comparable with that of α-glycerophosphate. However, the Pro pathway appeared to differ from the currently accepted pathway, in that ketoglutarate could be catabolyzed completely by the Krebs cycle or via transamination depending on the ATP need. PMID:18588503

  20. Legionella pneumophila type IV effectors hijack the transcription and translation machinery of the host cell.

    PubMed

    Rolando, Monica; Buchrieser, Carmen

    2014-12-01

    Intracellular bacterial pathogens modulate the host response to persist and replicate inside a eukaryotic cell and cause disease. Legionella pneumophila, the causative agent of Legionnaires' disease, is present in freshwater environments and represents one of these pathogens. During coevolution with protozoan cells, L. pneumophila has acquired highly sophisticated and diverse strategies to hijack host cell processes. It secretes hundreds of effectors into the host cell, and these manipulate host signaling pathways and key cellular processes. Recently it has been shown that L. pneumophila is also able to alter the transcription and translation machinery of the host and to exploit epigenetic mechanisms in the cells it resides in to counteract host responses.

  1. A novel class of mitochondria-targeted soft electrophiles modifies mitochondrial proteins and inhibits mitochondrial metabolism in breast cancer cells through redox mechanisms.

    PubMed

    Vayalil, Praveen K; Oh, Joo-Yeun; Zhou, Fen; Diers, Anne R; Smith, M Ryan; Golzarian, Hafez; Oliver, Patsy G; Smith, Robin A J; Murphy, Michael P; Velu, Sadanandan E; Landar, Aimee

    2015-01-01

    Despite advances in screening and treatment over the past several years, breast cancer remains a leading cause of cancer-related death among women in the United States. A major goal in breast cancer treatment is to develop safe and clinically useful therapeutic agents that will prevent the recurrence of breast cancers after front-line therapeutics have failed. Ideally, these agents would have relatively low toxicity against normal cells, and will specifically inhibit the growth and proliferation of cancer cells. Our group and others have previously demonstrated that breast cancer cells exhibit increased mitochondrial oxygen consumption compared with non-tumorigenic breast epithelial cells. This suggests that it may be possible to deliver redox active compounds to the mitochondria to selectively inhibit cancer cell metabolism. To demonstrate proof-of-principle, a series of mitochondria-targeted soft electrophiles (MTSEs) has been designed which selectively accumulate within the mitochondria of highly energetic breast cancer cells and modify mitochondrial proteins. A prototype MTSE, IBTP, significantly inhibits mitochondrial oxidative phosphorylation, resulting in decreased breast cancer cell proliferation, cell attachment, and migration in vitro. These results suggest MTSEs may represent a novel class of anti-cancer agents that prevent cancer cell growth by modification of specific mitochondrial proteins.

  2. A Novel Class of Mitochondria-Targeted Soft Electrophiles Modifies Mitochondrial Proteins and Inhibits Mitochondrial Metabolism in Breast Cancer Cells through Redox Mechanisms

    PubMed Central

    Vayalil, Praveen K.; Oh, Joo-Yeun; Zhou, Fen; Diers, Anne R.; Smith, M. Ryan; Golzarian, Hafez; Oliver, Patsy G.; Smith, Robin A. J.; Murphy, Michael P.; Velu, Sadanandan E.; Landar, Aimee

    2015-01-01

    Despite advances in screening and treatment over the past several years, breast cancer remains a leading cause of cancer-related death among women in the United States. A major goal in breast cancer treatment is to develop safe and clinically useful therapeutic agents that will prevent the recurrence of breast cancers after front-line therapeutics have failed. Ideally, these agents would have relatively low toxicity against normal cells, and will specifically inhibit the growth and proliferation of cancer cells. Our group and others have previously demonstrated that breast cancer cells exhibit increased mitochondrial oxygen consumption compared with non-tumorigenic breast epithelial cells. This suggests that it may be possible to deliver redox active compounds to the mitochondria to selectively inhibit cancer cell metabolism. To demonstrate proof-of-principle, a series of mitochondria-targeted soft electrophiles (MTSEs) has been designed which selectively accumulate within the mitochondria of highly energetic breast cancer cells and modify mitochondrial proteins. A prototype MTSE, IBTP, significantly inhibits mitochondrial oxidative phosphorylation, resulting in decreased breast cancer cell proliferation, cell attachment, and migration in vitro. These results suggest MTSEs may represent a novel class of anti-cancer agents that prevent cancer cell growth by modification of specific mitochondrial proteins. PMID:25785718

  3. VHL-deficient renal cancer cells gain resistance to mitochondria-activating apoptosis inducers by activating AKT through the IGF1R-PI3K pathway.

    PubMed

    Yamaguchi, Ryuji; Harada, Hiroshi; Hirota, Kiichi

    2016-10-01

    We previously developed (2-deoxyglucose)-(ABT-263) combination therapy (2DG-ABT), which induces apoptosis by activating Bak in the mitochondria of highly glycolytic cells with varied genetic backgrounds. However, the rates of apoptosis induced by 2DG-ABT were lower in von Hippel-Lindau (VHL)-deficient cancer cells. The re-expression of VHL protein in these cells lowered IGF1R expression in a manner independent of oxygen concentration. Lowering IGF1R expression via small interfering RNA (siRNA) sensitized the cells to 2DG-ABT, suggesting that IGF1R interfered with the activation of apoptosis by the mitochondria. To determine which of the two pathways activated by IGF1R, the Ras-ERK pathway or the PI3K-AKT pathway, was involved in the impairment of mitochondria activation, the cells were treated with a specific inhibitor of either PI3K or ERK, and 2DG-ABT was added to activate the mitochondria. The apoptotic rates resulting from 2DG-ABT treatment were higher in the cells treated with the PI3K inhibitor, while the rates remained approximately the same in the cells treated with the ERK inhibitor. In 2DG-ABT-sensitive cells, a 4-h 2DG treatment caused the dissociation of Mcl-1 from Bak, while ABT treatment alone caused the dissociation of Bcl-xL from Bak without substantially reducing Mcl-1 levels. In 2DG-ABT-resistant cells, Mcl-1 dissociated from Bak only when AKT activity was inhibited during the 4-h 2DG treatment. Thus, in VHL-deficient cells, IGF1R activated AKT and stabilized the Bak-Mcl-1 complex, thereby conferring cell resistance to apoptosis.

  4. Variation in RNA Virus Mutation Rates across Host Cells

    PubMed Central

    Combe, Marine; Sanjuán, Rafael

    2014-01-01

    It is well established that RNA viruses exhibit higher rates of spontaneous mutation than DNA viruses and microorganisms. However, their mutation rates vary amply, from 10−6 to 10−4 substitutions per nucleotide per round of copying (s/n/r) and the causes of this variability remain poorly understood. In addition to differences in intrinsic fidelity or error correction capability, viral mutation rates may be dependent on host factors. Here, we assessed the effect of the cellular environment on the rate of spontaneous mutation of the vesicular stomatitis virus (VSV), which has a broad host range and cell tropism. Luria-Delbrück fluctuation tests and sequencing showed that VSV mutated similarly in baby hamster kidney, murine embryonic fibroblasts, colon cancer, and neuroblastoma cells (approx. 10−5 s/n/r). Cell immortalization through p53 inactivation and oxygen levels (1–21%) did not have a significant impact on viral replication fidelity. This shows that previously published mutation rates can be considered reliable despite being based on a narrow and artificial set of laboratory conditions. Interestingly, we also found that VSV mutated approximately four times more slowly in various insect cells compared with mammalian cells. This may contribute to explaining the relatively slow evolution of VSV and other arthropod-borne viruses in nature. PMID:24465205

  5. Bid mediates fission, membrane permeabilization and peri-nuclear accumulation of mitochondria as a prerequisite for oxidative neuronal cell death.

    PubMed

    Grohm, Julia; Plesnila, Nikolaus; Culmsee, Carsten

    2010-07-01

    Mitochondria are highly dynamic organelles that undergo permanent fusion and fission, a process that is important for mitochondrial function and cellular survival. Emerging evidence suggests that oxidative stress disturbs mitochondrial morphology dynamics, resulting in detrimental mitochondrial fragmentation. In particular, such fatal mitochondrial fission has been detected in neurons exposed to oxidative stress, suggesting mitochondrial dynamics as a key feature in intrinsic death pathways. However, the regulation of mitochondrial fission in neurons exposed to lethal stress is largely unknown. Here, we used a model of glutamate toxicity in HT-22 cells for investigating mitochondrial fission and fusion in neurons exposed to oxidative stress. In these immortalized hippocampal neurons, glutamate induces glutathione depletion and increased formation of reactive oxygen species (ROS). Glutamate toxicity resulted in mitochondrial fragmentation and peri-nuclear accumulation of the organelles. Further, mitochondrial fission was associated with loss of mitochondrial outer membrane potential (MOMP). The Bid-inhibitor BI-6c9 prevented MOMP and mitochondrial fission, and protected the cells from cell death. In conclusion, oxidative stress induced by glutamate causes mitochondrial translocation of Bid thereby inducing mitochondrial fission and associated mitochondrial cell death pathways. Inhibiting regulators of pathological mitochondrial fragmentation is proposed as an efficient strategy of neuroprotection.

  6. Chitosan attenuates dibutyltin-induced apoptosis in PC12 cells through inhibition of the mitochondria-dependent pathway.

    PubMed

    Wang, Xiaorui; Miao, Junqiu; Yan, Chaoqun; Ge, Rui; Liang, Taigang; Liu, Enli; Li, Qingshan

    2016-10-20

    Dibutyltin (DBT) which was widely used as biocide and plastic stabilizer has been described as a potent neurotoxicant. Chitosan (CS), a natural nontoxic biopolymer, possesses a variety of biological activities including antibacterial, antifungal, free radical scavenging and neuroprotective activities. The present study was undertaken to investigate the protective effects of CS against DBT-induced apoptosis in rat pheochromocytoma (PC12) cells and the underlying mechanisms in vitro. Our results demonstrated that pretreatment with CS significantly increased the cell viability and decreased lactate dehydrogenase (LDH) release induced by DBT in a dose-dependent manner. Meanwhile, DBT-induced cell apoptosis, mitochondrial membrane potential (MMP) disruption, and generation of intracellular reactive oxygen species (ROS) were attenuated by CS. Real-time PCR assay showed that DBT markedly enhanced the mRNA levels of Bax, Bad, cytochrome-c and Apaf-1, reduced the Bcl-2 and Bcl-xL mRNA levels, while these genes expression alteration could be partially reversed by CS treatment. Furthermore, CS also inhibited the DBT-inducted activation of caspase-9, and -3 at mRNA and protein expression levels. Taken together, these results suggested that CS could protect the PC12 cells from apoptosis induced by DBT through inhibition of the mitochondria-dependent pathway.

  7. Central role of mitochondria and p53 in PUVA-induced apoptosis in human keratinocytes cell line NCTC-2544

    SciTech Connect

    Viola, Giampietro Fortunato, Elena; Cecconet, Laura; Del Giudice, Laura; Dall'Acqua, Francesco; Basso, Giuseppe

    2008-02-15

    Despite strong evidence concerning the high efficiency of PUVA therapy (psoralen plus UVA light), its mechanism of action has not yet been fully elucidated. In this study, we have evaluated in a cell line of human keratinocytes (NCTC-2544) the effects of two linear psoralen derivatives, 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen (5-MOP), that are widely used in PUVA therapy and two angular derivatives, Angelicin (ANG) and 4,6,4'-trymetyl angelicin (TMA). All derivatives photoinduce cellular death, TMA being the most active compound. The cell cycle analysis showed that the four derivatives induce, 24 h after irradiation, a cell cycle arrest in G1 phase later followed by massive apoptosis. The G1 arrest is correlated to an increase in the expression of p21{sup Waf1/Cip1}, a protein associated with the cell cycle block and apoptosis. Furthermore, treatment of NCTC-2544 resulted in p53 activation by 5-MOP, 8-MOP, and ANG but not TMA and its phosphorylation at serine-15. The levels of p21{sup Waf1/Cip1} paralleled p53 protein staining pattern suggesting that p53 activation correlated with p21{sup Waf1/Cip1} induction. Simultaneous to p53 activation, psoralens induced mitochondrial depolarization, cytochrome c release, mitochondrial production of reactive oxygen species, as well as caspase-3 and -9 activation. Thus these results strongly indicate the necessity of p53 activation and the induction of the apoptotic machinery downstream of mitochondria.

  8. Mitochondria and mitochondrial DNA in porcine oocytes and cumulus cells--A search for developmental competence marker.

    PubMed

    Pawlak, Piotr; Chabowska, Agnieszka; Malyszka, Natalia; Lechniak, Dorota

    2016-03-01

    The development of mammalian oocytes is dependent on bidirectional signaling with the surrounding cumulus cells. Among the numerous factors that contribute to oocyte developmental competence, the mitochondria and the mitochondrial DNA play pivotal roles. Although these highly abundant organelles have been well-studied in oocytes, their roles, abundance and metabolism remain elusive in cumulus cells. Therefore, the aim of our study was to analyze the correlation between the mtDNA copy number in cumulus cells and oocytes, as well as the mitochondrial distribution patterns in oocytes, using two groups of animals that differ in terms of the developmental competence of their oocytes. We determined a positive correlation between the mtDNA copy number in the cumulus cells and mtDNA copy number in oocytes of prepubertal pigs and negative correlation in cyclic gilts. These opposing correlations may reflect the differences in the developmental competence of the prepubertal and cyclic oocytes. We also hypothesize that observed differences may reflect different metabolism and energy requirements of the cumulus-oocyte complexes from prepubertal and cyclic gilts. The mitochondrial distribution patterns in the prepubertal and cyclic gilts were not different.

  9. Animal mitochondria: evolution, function, and disease.

    PubMed

    Tao, M; You, C-P; Zhao, R-R; Liu, S-J; Zhang, Z-H; Zhang, C; Liu, Y

    2014-01-01

    Mitochondria are sub-cellular organelles responsible for producing the majority of cellular energy through the process of oxidative phosphorylation (OXPHOS), and are found in nearly all eukaryotic cells. Mitochondria have a unique genetic system, mitochondrial DNA (mtDNA), which is a small, self-replicating and diverse genome. In the past 30 years, mtDNA has made significant contribution to molecular ecology and phylogeography. Mitochondria also represent a unique system of mitochondrial-nuclear genomic cooperation. Additionally, mitochondrial dysfunction can be fatal. In this paper, we review several aspects of mitochondria, including evolution and the origin of mitochondria, energy supply and the central role of mitochondria in apoptosis, and mitochondrial dysfunction. It is shown that mitochondria play a critical role in many aspects of life.

  10. Cytomegalovirus cell death suppressor vMIA blocks Bax- but not Bak-mediated apoptosis by binding and sequestering Bax at mitochondria.

    PubMed

    Arnoult, Damien; Bartle, Laura M; Skaletskaya, Anna; Poncet, Delphine; Zamzami, Naoufal; Park, Peter U; Sharpe, Juanita; Youle, Richard J; Goldmacher, Victor S

    2004-05-25

    We report that the cytomegalovirus-encoded cell death suppressor vMIA binds Bax and prevents Bax-mediated mitochondrial membrane permeabilization by sequestering Bax at mitochondria in the form of a vMIA-Bax complex. vMIA mutants with a defective mitochondria-targeting domain retain their Bax-binding function but not their ability to suppress mitochondrial membrane permeabilization or cell death. vMIA does not seem to either specifically associate with Bak or suppress Bak-mediated mitochondrial membrane permeabilization. Recent evidence suggests that the contribution of Bax and Bak in the mitochondrial apoptotic signaling pathway depends on the distinct phenotypes of cells, and it appears from our data that vMIA is capable of suppressing apoptosis in cells in which this pathway is dominated by Bax, but not in cells where Bak also plays a role.

  11. Overexpression of ALTERNATIVE OXIDASE1a alleviates mitochondria-dependent programmed cell death induced by aluminium phytotoxicity in Arabidopsis.

    PubMed

    Liu, Jian; Li, Zhe; Wang, Yongqiang; Xing, Da

    2014-08-01

    Alternative oxidase (AOX) is a terminal oxidase found in all plants, and functions to maintain the electron flux and reduce the production of reactive oxygen species (ROS). Our previous study demonstrated that aluminium (Al) treatment could induce increased expression of the AOX1a gene, but the mechanism of how AOX1a participates in the regulation of Al-induced programmed cell death (PCD) is still not clear. To investigate the possible mechanism, mitochondrial ROS production and the behaviour of mitochondria, as well as caspase-3-like activation were monitored under Al treatment in wild-type (WT), AOX1a-lacking (aox1a), and AOX1a-overexpressing (AOX1a-OE) Arabidopsis. Our results showed that Al treatment increased the expression of AOX1a at both the transcriptional and translational levels. Overexpression of AOX1a reduced mitochondrial ROS production by maintaining the mitochondrial electron flux, and alleviated subsequent mitochondrial dysfunction and caspase-3-like activation in Al-induced PCD. Moreover, it was found that a change in AOX1a level could influence the expression levels of downstream functional genes that play protective roles in Al-induced PCD. Experiments using mutants and inhibitors demonstrated that superoxide anion (O2 (-)) derived from mitochondria was involved in Al-induced upregulation of AOX1a gene expression. Taken together, these results indicated that overexpression of AOX1a alleviated Al-induced PCD by maintaining mitochondrial function and promoting the expression of protective functional genes, providing new insights into the signalling cascades that modulate the Al phytotoxicity mechanism.

  12. Studies of the significance of functional and structural changes in mitochondria in PhotofrinTM-photodynamic-therapy-resistant cells

    NASA Astrophysics Data System (ADS)

    Wilson, Brian C.; Olivo, Malini; Moorehead, Roger; Singh, Gurmit

    1994-07-01

    This paper reports further studies of Radiation Induced Fibrosarcoma (RIF-1) tumor cells which have been made resistant to Photodynamic Therapy by multiple treatment and regrowth in vitro using the hematoporphyrin derivative photosensitizer Photofrin. Previous work has shown both structural and functional changes in the mitochondria of the resistant (RIF-8A) cells. Colocalization of Photofrin and the mitochondrial localizer Rhodamine-123 was assessed by double-label confocal fluorescence microscopy (CFM). At 18h Photofrin incubation, there was strong correlation in discrete subcellular sites between Photofrin and Rhodamine fluorescence. However, in RIF-8A cells there were also discrete regions of Rhodamine localization which showed weak or no Photofrin fluorescence. This was not observed in RIF-1 cells. CFM measurements also showed that the Photofrin fluorescence after 18h incubation was reduced by increasing concentration of Rhodamine (30 min. incubation), and that this dependence was different for the two cell types. The RIF-8A cells were also shown to be cross-resistant to cisplatin and to have an associated reduced level of Pt-DNA adducts, suggesting the possibility of increased repair capacity. Cross-resistance was not observed, however, with a ruthenium phthalocyanine photosensitizer nor, as previously reported, with other chemotherapeutic agents such as Adriamcyin. Thus, there is a complex pattern of cross-resistance with these cells. Preliminary observations of the effects of a respiratory chain inhibitor (oligomycin) and an uncoupler of oxidative phosphorylation (FCCP) indicate differences between RIF-8A and RIF-1 which may be related to the condensed mitochondrial structure of the RIF-8A cells.

  13. Cytokine toxicity in insulin-producing cells is mediated by nitro-oxidative stress-induced hydroxyl radical formation in mitochondria.

    PubMed

    Gurgul-Convey, Ewa; Mehmeti, Ilir; Lortz, Stephan; Lenzen, Sigurd

    2011-08-01

    Although nitric oxide (NO) and oxidative stress both contribute to proinflammatory cytokine toxicity in pancreatic β-cells during type 1 diabetes mellitus (T1DM) development, the interactions between NO and reactive oxygen species (ROS) in cytokine-mediated β-cell death have not been clarified. Exposure of insulin-producing RINm5F cells to IL-1β generated NO, while exposure to a combination of IL-1β, TNF-α, and IFN-γ, which simulates T1DM conditions, generated both NO and ROS. In theory, two reactions between NO and ROS are possible, one with the superoxide radical yielding peroxynitrite, and the other with hydrogen peroxide (H(2)O(2)) yielding hydroxyl radicals. Results of the present work exclude peroxynitrite involvement in cytokine toxicity to β-cells because its generation did not correlate with the toxic action of cytokines. On the other hand, we show that H(2)O(2), produced upon exposure of insulin-producing cell clones and primary rat islet cells to cytokines almost exclusively in the mitochondria, reacted in the presence of trace metal (Fe(++)) with NO forming highly toxic hydroxyl radicals, thus explaining the severe toxicity that causes apoptotic β-cell death. Expression of the H(2)O(2)-inactivating enzyme catalase in mitochondria protected against cytokine toxicity by preventing hydroxyl radical formation. We therefore conclude that proinflammatory cytokine-mediated β-cell death is due to nitro-oxidative stress-mediated hydroxyl radical formation in the mitochondria.

  14. The novel mitochondria-targeted hydrogen sulfide (H2S) donors AP123 and AP39 protect against hyperglycemic injury in microvascular endothelial cells in vitro.

    PubMed

    Gerő, Domokos; Torregrossa, Roberta; Perry, Alexis; Waters, Alicia; Le-Trionnaire, Sophie; Whatmore, Jacqueline L; Wood, Mark; Whiteman, Matthew

    2016-11-01

    The development of diabetic vascular complications is initiated, at least in part, by mitochondrial reactive oxygen species (ROS) production in endothelial cells. Hyperglycemia induces superoxide production in the mitochondria and initiates changes in the mitochondrial membrane potential that leads to mitochondrial dysfunction. Hydrogen sulfide (H2S) supplementation has been shown to reduce the mitochondrial oxidant production and shows efficacy against diabetic vascular damage in vivo. However, the half-life of H2S is very short and it is not specific for the mitochondria. We have therefore evaluated two novel mitochondria-targeted anethole dithiolethione and hydroxythiobenzamide H2S donors (AP39 and AP123 respectively) at preventing hyperglycemia-induced oxidative stress and metabolic changes in microvascular endothelial cells in vitro. Hyperglycemia (HG) induced significant increase in the activity of the citric acid cycle and led to elevated mitochondrial membrane potential. Mitochondrial oxidant production was increased and the mitochondrial electron transport decreased in hyperglycemic cells. AP39 and AP123 (30-300nM) decreased HG-induced hyperpolarisation of the mitochondrial membrane and inhibited the mitochondrial oxidant production. Both H2S donors (30-300nM) increased the electron transport at respiratory complex III and improved the cellular metabolism. Targeting H2S to mitochondria retained the cytoprotective effect of H2S against glucose-induced damage in endothelial cells suggesting that the molecular target of H2S action is within the mitochondria. Mitochondrial targeting of H2S also induced >1000-fold increase in the potency of H2S against hyperglycemia-induced injury. The high potency and long-lasting effect elicited by these H2S donors strongly suggests that these compounds could be useful against diabetic vascular complications.

  15. Identifying Francisella tularensis genes required for growth in host cells.

    PubMed

    Brunton, J; Steele, S; Miller, C; Lovullo, E; Taft-Benz, S; Kawula, T

    2015-08-01

    Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The ΔFTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence.

  16. Identifying Francisella tularensis Genes Required for Growth in Host Cells

    PubMed Central

    Brunton, J.; Steele, S.; Miller, C.; Lovullo, E.; Taft-Benz, S.

    2015-01-01

    Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The ΔFTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence. PMID:25987704

  17. Coenzyme Q10 Protects Astrocytes from ROS-Induced Damage through Inhibition of Mitochondria-Mediated Cell Death Pathway

    PubMed Central

    Jing, Li; He, Mao-Tao; Chang, Yue; Mehta, Suresh L.; He, Qing-Ping; Zhang, Jian-Zhong; Li, P. Andy

    2015-01-01

    Coenzyme Q10 (CoQ10) acts by scavenging reactive oxygen species to protect neuronal cells against oxidative stress in neurodegenerative diseases. The present study was designed to examine whether CoQ10 was capable of protecting astrocytes from reactive oxygen species (ROS) mediated damage. For this purpose, ultraviolet B (UVB) irradiation was used as a tool to induce ROS stress to cultured astrocytes. The cells were treated with 10 and 25 μg/ml of CoQ10 for 3 or 24 h prior to the cells being exposed to UVB irradiation and maintained for 24 h post UVB exposure. Cell viability was assessed by MTT conversion assay. Mitochondrial respiration was assessed by respirometer. While superoxide production and mitochondrial membrane potential were measured using fluorescent probes, levels of cytochrome C (cyto-c), cleaved caspase-9, and caspase-8 were detected using Western blotting and/or immunocytochemistry. The results showed that UVB irradiation decreased cell viability and this damaging effect was associated with superoxide accumulation, mitochondrial membrane potential hyperpolarization, mitochondrial respiration suppression, cyto-c release, and the activation of both caspase-9 and -8. Treatment with CoQ10 at two different concentrations started 24 h before UVB exposure significantly increased the cell viability. The protective effect of CoQ10 was associated with reduction in superoxide, normalization of mitochondrial membrane potential, improvement of mitochondrial respiration, inhibition of cyto-c release, suppression of caspase-9. Furthermore, CoQ10 enhanced mitochondrial biogenesis. It is concluded that CoQ10 may protect astrocytes through suppression of oxidative stress, prevention of mitochondrial dysfunction, blockade of mitochondria-mediated cell death pathway, and enhancement of mitochondrial biogenesis. PMID:25552930

  18. Cissus quadrangularis Linn. Stem Ethanolic Extract Liberates Reactive Oxygen Species and Induces Mitochondria Mediated Apoptosis in KB Cells

    PubMed Central

    Sheikh, Saba; Siddiqui, Sahabjada; Dhasmana, Anupam; Safia; Haque, Ejazul; Kamil, Mohammed; Lohani, Mohtashim; Arshad, Mohammad; Mir, Snober Shabnam

    2015-01-01

    Background: Cissus quadrangularis Linn. (CQ) commonly known as Hadjod (Family: Vitaceae) is usually distributed in India and Sri Lanka and contains several bioactive compounds responsible for various metabolic and physiologic effects. Objective: In this study, the biological effects of CQ ethanolic extract were evaluated by in vitro and supported by in silico analysis on KB oral epidermoid cancer cell line. Materials and Methods: Anti-cancer potential of ethanolic extract of CQ stem against KB oral epidermoid cancer cells was evaluated in terms of morphological analysis, nuclei staining, liberation of reactive oxygen species (ROS), cell cycle arrest, mitochondrial membrane potential (MMP) and p53 and Bcl-2 protein expression which reveal the induction of apoptosis along with supporting in silico analysis. Results: Ethanolic extract of CQ stem contains various bioactive compounds responsible for cancer cell morphological alterations, liberation of ROS, G1 phase cell cycle arrest and decreased MMP along with up-regulation of p53 and down-regulation of Bcl-2. By employing in silico approach, we have also postulated that the CQ extract active constituents sequester Bcl-2 with higher affinity as compared to p53, which may be the reason for induction of growth arrest and apoptosis in KB cells. Conclusion: Our data indicate that the CQ extract has a remarkable apoptotic effect that suggests that it could be a viable treatment option for specific types of cancers. SUMMARY Cissus quadrangularis stem ethanolic extract induces apoptosis and cell cycle arrest at G1 phaseIt liberates (ROS) and mitochondria mediated apoptosisIt upregulates p53 and down-regulates Bcl-2 protein expressionIn silico studies indicates that the active constituents of CQ binds Bcl-2 with higher affinity as compared to p53. PMID:26929569

  19. Interaction theory of mammalian mitochondria.

    PubMed

    Nakada, K; Inoue, K; Hayashi, J

    2001-11-09

    We generated mice with deletion mutant mtDNA by its introduction from somatic cells into mouse zygotes. Expressions of disease phenotypes are limited to tissues expressing mitochondrial dysfunction. Considering that all these mice share the same nuclear background, these observations suggest that accumulation of the mutant mtDNA and resultant expressions of mitochondrial dysfunction are responsible for expression of disease phenotypes. On the other hand, mitochondrial dysfunction and expression of clinical abnormalities were not observed until the mutant mtDNA accumulated predominantly. This protection is due to the presence of extensive and continuous interaction between exogenous mitochondria from cybrids and recipient mitochondria from embryos. Thus, we would like to propose a new hypothesis on mitochondrial biogenesis, interaction theory of mitochondria: mammalian mitochondria exchange genetic contents, and thus lost the individuality and function as a single dynamic cellular unit.

  20. Stepwise adaptation of murine cytomegalovirus to cells of a foreign host for identification of host range determinants.

    PubMed

    Ostermann, Eleonore; Pawletko, Kerstin; Indenbirken, Daniela; Schumacher, Uwe; Brune, Wolfram

    2015-06-01

    Ever since their first isolation 60 years ago, cytomegaloviruses have been recognized as being highly species specific. They replicate only in cells of their own or a closely related host species, while cells of phylogenetically more distant hosts are usually not permissive for viral replication. For instance, human cytomegalovirus replicates in human and chimpanzee fibroblasts but not in rodent cells, and murine cytomegalovirus (MCMV) replicates in cells of mice and rats but not in primate cells. However, the viral and cellular factors determining the narrow host range of cytomegaloviruses have remained largely unknown. We show that MCMV can be adapted stepwise to replicate in cultured human retinal pigment epithelial (RPE-1) cells and human fibroblasts. The human RPE-1 cells used for the initial adaptation step showed a pronounced contact inhibition and produced very low level of interferon-β transcripts upon cytomegalovirus infection, suggesting that these cells provide a particularly favorable environment for adaptation. By whole genome sequencing of the 230 kbp viral genomes of several adapted mutants, a limited number of mutations were detected. Comparison of several human cell-adapted MCMV clones and introduction of specific mutations into the wild-type MCMV genome by site-directed mutagenesis allows for the identification of viral host range determinants and provides the basis for elucidating the molecular basis of the cytomegalovirus host species specificity.

  1. Mitochondria in teleost spermatozoa.

    PubMed

    Ulloa-Rodríguez, Patricio; Figueroa, Elías; Díaz, Rommy; Lee-Estevez, Manuel; Short, Stefania; Farías, Jorge G

    2017-01-06

    There is an extraordinary diversity of reproductive modes in teleost and this variability is related to the phylogenetic relationships and adaption to very different biotopes. As in all vertebrates, sperm is produced as the end product of the process of spermatogenesis, and regarding teleost the spermatozoa lack an acrosome in almost all species and motility is activated as a response to osmolarity and ion content of the aquatic medium where the sperm is released. In this context, mitochondria possess a fundamental role for fish spermatozoa motility and integrity, hence, fertilizing potential; they are the energy supplier that allows flagellar movement and their dysfunction could play a main role in structural and functional damage to the spermatozoa. The ATP production through oxidative phosphorylation provides not only energy for cell activities, which includes Na(+)/K(+) ATPase pump, endocytosis, protein synthesis and many other cell processes; but also produces reactive oxygen species, that under mitochondrial dysfunction causes oxidative stress. The assessment of mitochondrial function (e.g. through measurement of mitochondrial membrane potential) as well as ATP content (mostly supplied by mitochondrial respiration) can be useful as quality markers of fish spermatozoa. Also quantification of ROS and antioxidant status, strongly influenced by mitochondria, are used as complementary measurements. There is much information about sperm mitochondria and their function but studies of these aspects on fish reproduction are still required for applications in aquaculture. The real role of fish sperm mitochondria under short and long term storage and in vitro manipulation is not fully understood yet. Thus future research should focus on these matters.

  2. Mitochondria are the source of hydrogen peroxide for dynamic brain-cell signaling

    PubMed Central

    Bao, Li; Avshalumov, Marat V.; Patel, Jyoti C.; Lee, Christian R.; Miller, Evan W.; Chang, Christopher J.; Rice, Margaret E.

    2010-01-01

    Hydrogen peroxide (H2O2) is emerging as a ubiquitous small-molecule messenger in biology, particularly in the brain, but underlying mechanisms of peroxide signaling remain an open frontier for study. For example, dynamic dopamine transmission in dorsolateral striatum is regulated on a subsecond timescale by glutamate via H2O2 signaling, which activates ATP-sensitive potassium (KATP) channels to inhibit dopamine release. However, the origin of this modulatory H2O2 has been elusive. Here we addressed three possible sources of H2O2 produced for rapid neuronal signaling in striatum: mitochondrial respiration; monoamine oxidase (MAO); and NADPH oxidase (Nox). Evoked dopamine release in guinea-pig striatal slices was monitored with carbon-fiber microelectrodes and fast-scan cyclic voltammetry. Using direct fluorescence imaging of H2O2 and tissue analysis of ATP, we found that co-application of rotenone (50 nM), a mitochondrial complex I inhibitor, and succinate (5 mM), a complex II substrate, limited H2O2 production, but maintained tissue ATP content. Strikingly, co-application of rotenone and succinate also prevented glutamate-dependent regulation of dopamine release, implicating mitochondrial H2O2 in release modulation. By contrast, inhibitors of MAO or Nox had no effect on dopamine release, suggesting a limited role for these metabolic enzymes in rapid H2O2 production in the striatum. These data provide the first demonstration that respiring mitochondria are the primary source of H2O2 generation for dynamic neuronal signaling. PMID:19605638

  3. Mitochondria are the source of hydrogen peroxide for dynamic brain-cell signaling.

    PubMed

    Bao, Li; Avshalumov, Marat V; Patel, Jyoti C; Lee, Christian R; Miller, Evan W; Chang, Christopher J; Rice, Margaret E

    2009-07-15

    Hydrogen peroxide (H(2)O(2)) is emerging as a ubiquitous small-molecule messenger in biology, particularly in the brain, but underlying mechanisms of peroxide signaling remain an open frontier for study. For example, dynamic dopamine transmission in dorsolateral striatum is regulated on a subsecond timescale by glutamate via H(2)O(2) signaling, which activates ATP-sensitive potassium (K(ATP)) channels to inhibit dopamine release. However, the origin of this modulatory H(2)O(2) has been elusive. Here we addressed three possible sources of H(2)O(2) produced for rapid neuronal signaling in striatum: mitochondrial respiration, monoamine oxidase (MAO), and NADPH oxidase (Nox). Evoked dopamine release in guinea-pig striatal slices was monitored with carbon-fiber microelectrodes and fast-scan cyclic voltammetry. Using direct fluorescence imaging of H(2)O(2) and tissue analysis of ATP, we found that coapplication of rotenone (50 nM), a mitochondrial complex I inhibitor, and succinate (5 mM), a complex II substrate, limited H(2)O(2) production, but maintained tissue ATP content. Strikingly, coapplication of rotenone and succinate also prevented glutamate-dependent regulation of dopamine release, implicating mitochondrial H(2)O(2) in release modulation. In contrast, inhibitors of MAO or Nox had no effect on dopamine release, suggesting a limited role for these metabolic enzymes in rapid H(2)O(2) production in the striatum. These data provide the first demonstration that respiring mitochondria are the primary source of H(2)O(2) generation for dynamic neuronal signaling.

  4. Ca2+ Homeostasis in the Agonist-sensitive Internal Store: Functional Interactions Between Mitochondria and the ER Measured In Situ in Intact Cells

    PubMed Central

    Landolfi, Barbara; Curci, Silvana; Debellis, Lucantonio; Pozzan, Tullio; Hofer, Aldebaran M.

    1998-01-01

    Mitochondria have a well-established capacity to detect cytoplasmic Ca2+ signals resulting from the discharge of ER Ca2+ stores. Conversely, both the buffering of released Ca2+ and ATP production by mitochondria are predicted to influence ER Ca2+ handling, but this complex exchange has been difficult to assess in situ using conventional measurement techniques. Here we have examined this interaction in single intact BHK-21 cells by monitoring intraluminal ER [Ca2+] directly using trapped fluorescent low-affinity Ca2+ indicators. Treatment with mitochondrial inhibitors (FCCP, antimycin A, oligomycin, and rotenone) dramatically prolonged the refilling of stores after release with bradykinin. This effect was largely due to inhibition of Ca2+ entry pathways at the plasma membrane, but a significant component appears to arise from reduction of SERCA-mediated Ca2+ uptake, possibly as a consequence of ATP depletions in a localized subcellular domain. The rate of bradykinin-induced Ca2+ release was reduced to 51% of control by FCCP. This effect was largely overcome by loading cells with BAPTA-AM, highlighting the importance of mitochondrial Ca2+ buffering in shaping the release kinetics. However, mitochondria-specific ATP production was also a significant determinant of the release dynamic. Our data emphasize the localized nature of the interaction between these organelles, and show that competent mitochondria are essential for generating explosive Ca2+ signals. PMID:9732284

  5. Mitochondria-dependent apoptogenic activity of the aqueous root extract of Croton membranaceus against human BPH-1 cells.

    PubMed

    Afriyie, D K; Asare, G A; Bugyei, K; Lin, J; Peng, J; Hong, Z

    2015-01-15

    Croton membranaceus aqueous root extract (CMARE) is among the widely used phytotherapeutics in Ghana for the management of benign prostatic hyperplasia (BPH) and prostate cancer. However, the mechanism of action of CMARE remains to be elucidated. This study aimed to establish whether apoptosis is involved in the antiproliferative effect of CMARE on human BPH-1 cells. We determined the effect of treatment with 0, 1, 3, and 5 mg/mL CMARE for 24, 48, and 72 h on the viability and morphology of BPH-1 cells using the MMT assay and phase-contrast microscopy, respectively. We examined the apoptosis-inducing effects of CMARE after 48 h at the cellular level using Hoescht 33258 and JC-1 dye staining and flow cytometry analysis. We performed reverse transcription polymerase chain reaction and Western blotting to confirm the apoptotic effects of CMARE at the molecular level. CMARE induced a significant dose-dependent inhibition in the proliferation of BPH-1 cells (P < 0.05) and an alteration in their morphology and a reduction their density. Furthermore, CMARE induced dose-dependent staining of the nuclear chromatin, significant DNA fragmentation with G₀/G₁ sub-diploid cells (P < 0.01), and loss of the mitochondrial membrane potential in the treated cells compared to the controls after 48 h (P < 0.01). Additionally, while CMARE induced a significant upregulation of the mRNA and protein levels of Bax, those of Bcl2 did not change significantly. Therefore, induction of mitochondria-dependent apoptosis of BPH-1 cells may be a possible mechanism of action of CMARE.

  6. Inkjet printing of silk nest arrays for cell hosting.

    PubMed

    Suntivich, Rattanon; Drachuk, Irina; Calabrese, Rossella; Kaplan, David L; Tsukruk, Vladimir V

    2014-04-14

    An inkjet printing approach is presented for the facile fabrication of microscopic arrays of biocompatible silk "nests" capable of hosting live cells for prospective biosensors. The patterning of silk fibroin nests were constructed by the layer-by-layer (LbL) assembly of silk polyelectrolytes chemically modified with poly-(l-lysine) and poly-(l-glutamic acid) side chains. The inkjet-printed silk circular regions with a characteristic "nest" shape had diameters of 70-100 μm and a thickness several hundred nanometers were stabilized by ionic pairing and by the formation of the silk II crystalline secondary structure. These "locked-in" silk nests remained anchored to the substrate during incubation in cell growth media to provide a biotemplated platform for printing-in, immobilization, encapsulation and growth of cells. The process of inkjet-assisted printing is versatile and can be applied on any type of substrate, including rigid and flexible, with scalability and facile formation.

  7. Depletion of host cell riboflavin reduces Wolbachia levels in cultured mosquito cells.

    PubMed

    Fallon, Ann M; Baldridge, Gerald D; Carroll, Elissa M; Kurtz, Cassandra M

    2014-09-01

    Wolbachia is an obligate intracellular alphaproteobacterium that occurs in arthropod and nematode hosts. Wolbachia presumably provides a fitness benefit to its hosts, but the basis for its retention and spread in host populations remains unclear. Wolbachia genomes retain biosynthetic pathways for some vitamins, and the possibility that these vitamins benefit host cells provides a potential means of selecting for Wolbachia-infected cell lines. To explore whether riboflavin produced by Wolbachia is available to its host cell, we established that growth of uninfected C7-10 mosquito cells decreases in riboflavin-depleted culture medium. A well-studied inhibitor of riboflavin uptake, lumiflavin, further inhibits growth of uninfected C7-10 cells with an LC50 of approximately 12 μg/ml. Growth of C/wStr1 mosquito cells, infected with Wolbachia from the planthopper, Laodelphax striatellus, was enhanced in medium containing low levels of lumiflavin, but Wolbachia levels decreased. Lumiflavin-enhanced growth thus resembled the improved growth that accompanies treatment with antibiotics that deplete Wolbachia, rather than a metabolic advantage provided by the Wolbachia infection. We used the polymerase chain reaction to validate the decrease in Wolbachia abundance and evaluated our results in the context of a proteomic analysis in which we detected nearly 800 wStr proteins. Our data indicate that Wolbachia converts riboflavin to FMN and FAD for its own metabolic needs, and does not provide a source of riboflavin for its host cell.

  8. Depletion of host cell riboflavin reduces Wolbachia levels in cultured mosquito cells

    PubMed Central

    Baldridge, Gerald D.; Carroll, Elissa M.; Kurtz, Cassandra M.

    2015-01-01

    Wolbachia is an obligate intracellular alphaproteobacterium that occurs in arthropod and nematode hosts. Wolbachia presumably provides a fitness benefit to its hosts, but the basis for its retention and spread in host populations remains unclear. Wolbachia genomes retain biosynthetic pathways for some vitamins, and the possibility that these vitamins benefit host cells provides a potential means of selecting for Wolbachia-infected cell lines. To explore whether riboflavin produced by Wolbachia is available to its host cell, we established that growth of uninfected C7–10 mosquito cells decreases in riboflavin-depleted culture medium. A well studied inhibitor of riboflavin uptake, lumiflavin, further inhibits growth of uninfected C7–10 cells with an LC50 of approximately 12 µg/ml. Growth of C/wStr1 mosquito cells, infected with Wolbachia from the planthopper, Laodelphax striatellus, was enhanced in medium containing low levels of lumiflavin, but Wolbachia levels decreased. Lumiflavin-enhanced growth thus resembled the improved growth that accompanies treatment with antibiotics that deplete Wolbachia, rather than a metabolic advantage provided by the Wolbachia infection. We used the polymerase chain reaction to validate the decrease in Wolbachia abundance and evaluated our results in the context of a proteomic analysis in which we detected nearly 800 wStr proteins. Our data indicate that Wolbachia converts riboflavin to FMN and FAD for its own metabolic needs, and does not provide a source of riboflavin for its host cell. PMID:24789726

  9. Exosomes Secreted by Toxoplasma gondii-Infected L6 Cells: Their Effects on Host Cell Proliferation and Cell Cycle Changes.

    PubMed

    Kim, Min Jae; Jung, Bong-Kwang; Cho, Jaeeun; Song, Hyemi; Pyo, Kyung-Ho; Lee, Ji Min; Kim, Min-Kyung; Chai, Jong-Yil

    2016-04-01

    Toxoplasma gondii infection induces alteration of the host cell cycle and cell proliferation. These changes are not only seen in directly invaded host cells but also in neighboring cells. We tried to identify whether this alteration can be mediated by exosomes secreted by T. gondii-infected host cells. L6 cells, a rat myoblast cell line, and RH strain of T. gondii were selected for this study. L6 cells were infected with or without T. gondii to isolate exosomes. The cellular growth patterns were identified by cell counting with trypan blue under confocal microscopy, and cell cycle changes were investigated by flow cytometry. L6 cells infected with T. gondii showed decreased proliferation compared to uninfected L6 cells and revealed a tendency to stay at S or G2/M cell phase. The treatment of exosomes isolated from T. gondii-infected cells showed attenuation of cell proliferation and slight enhancement of S phase in L6 cells. The cell cycle alteration was not as obvious as reduction of the cell proliferation by the exosome treatment. These changes were transient and disappeared at 48 hr after the exosome treatment. Microarray analysis and web-based tools indicated that various exosomal miRNAs were crucial for the regulation of target genes related to cell proliferation. Collectively, our study demonstrated that the exosomes originating from T. gondii could change the host cell proliferation and alter the host cell cycle.

  10. Transgenic plant cells lacking mitochondrial alternative oxidase have increased susceptibility to mitochondria-dependent and -independent pathways of programmed cell death.

    PubMed

    Robson, Christine A; Vanlerberghe, Greg C

    2002-08-01

    The plant mitochondrial electron transport chain is branched such that electrons at ubiquinol can be diverted to oxygen via the alternative oxidase (AOX). This pathway does not contribute to ATP synthesis but can dampen the mitochondrial generation of reactive oxygen species. Here, we establish that transgenic tobacco (Nicotiana tabacum L. cv Petit Havana SR1) cells lacking AOX (AS8 cells) show increased susceptibility to three different death-inducing compounds (H(2)O(2), salicylic acid [SA], and the protein phosphatase inhibitor cantharidin) in comparison with wild-type cells. The timing and extent of AS8 cell death are very similar among the three treatments and, in each case, are accompanied by the accumulation of oligonucleosomal fragments of DNA, indicative of programmed cell death. Death induced by H(2)O(2) or SA occurs by a mitochondria-dependent pathway characterized by cytochrome c release from the mitochondrion. Conversely, death induced by cantharidin occurs by a pathway without any obvious mitochondrial involvement. The ability of AOX to attenuate these death pathways may relate to its ability to maintain mitochondrial function after insult with a death-inducing compound or may relate to its ability to prevent chronic oxidative stress within the mitochondrion. In support of the latter, long-term treatment of AS8 cells with an antioxidant compound increased the resistance of AS8 cells to SA- or cantharidin-induced death. The results indicate that plants maintain both mitochondria-dependent and -independent pathways of programmed cell death and that AOX may act as an important mitochondrial "survival protein" against such death.

  11. Fisetin-induced apoptosis of human oral cancer SCC-4 cells through reactive oxygen species production, endoplasmic reticulum stress, caspase-, and mitochondria-dependent signaling pathways.

    PubMed

    Su, Chen-Hsuan; Kuo, Chao-Lin; Lu, Kung-Wen; Yu, Fu-Shun; Ma, Yi-Shih; Yang, Jiun-Long; Chu, Yung-Lin; Chueh, Fu-Shin; Liu, Kuo-Ching; Chung, Jing-Gung

    2017-02-09

    Oral cancer is one of the cancer-related diseases in human populations and its incidence rates are rising worldwide. Fisetin, a flavonoid from natural products, has been shown to exhibit anticancer activities in many human cancer cell lines but the molecular mechanism of fisetin-induced apoptosis in human oral cancer cells is still unclear; thus, in this study, we investigated fisetin-induced cell death and associated signal pathways on human oral cancer SCC-4 cells in vitro. We examined cell morphological changes, total viable cells, and cell cycle distribution by phase contrast microscopy and flow cytometry assays. Reactive oxygen species (ROS), Ca(2+) , mitochondria membrane potential (ΔΨm ), and caspase-8, -9, and -3 activities were also measured by flow cytometer. Results indicate that fisetin induced cell death through the cell morphological changes, caused G2/M phase arrest, induction of apoptosis, promoted ROS and Ca(2+) production, and decreased the level of ΔΨm and increased caspase-3, -8, and -9 activities in SCC-4 cells. DAPI staining and DNA gel electrophoresis were also used to confirm fisetin-induced cell apoptosis in SCC-4 cells. Western blotting also found out that Fisetin increased the proapoptotic proteins such as Bax and Bid and decreased the antiapoptotic proteins such as Bcl-2. Furthermore, results also showed that Fisetin increased the cytochrome c, AIF, and Endo G release from mitochondria in SCC-4 cells. We also used ATF-6α, ATF-6β, GADD153, and GRP78 which indicated that fisetin induced cell death through ER stress. Based on those observations, we suggest that fisetin induced cell apoptosis through ER stress, mitochondria-, and caspase-dependent pathways.

  12. Lassa Virus Cell Entry Reveals New Aspects of Virus-Host Cell Interaction.

    PubMed

    Torriani, Giulia; Galan-Navarro, Clara; Kunz, Stefan

    2017-02-15

    Viral entry represents the first step of every viral infection and is a determinant for the host range and disease potential of a virus. Here, we review the latest developments on cell entry of the highly pathogenic Old World arenavirus Lassa virus, providing novel insights into the complex virus-host cell interaction of this important human pathogen. We will cover new discoveries on the molecular mechanisms of receptor recognition, endocytosis, and the use of late endosomal entry factors.

  13. Fisetin induces apoptosis in human nonsmall lung cancer cells via a mitochondria-mediated pathway.

    PubMed

    Kang, Kyoung Ah; Piao, Mei Jing; Hyun, Jin Won

    2015-03-01

    The present study investigated the apoptotic effects of fisetin, a phenolic compound, against the human nonsmall cell lung cancer cell line, NCI-H460. Fisetin showed dose-dependent cytotoxic activity against NCI-H460 cells, with 50% inhibition of cell viability occurring at a concentration of 75 μg/mL. Fisetin induced both the production of intracellular reactive oxygen species and apoptosis, as evidenced by apoptotic body formation, DNA fragmentation, an increase in the number of sub-G1 phase cells, and mitochondrial membrane depolarization. Moreover, fisetin significantly modulated the expression of apoptosis-associated proteins, resulting in reduced expression of B cell lymphoma-2, increased expression of Bcl-2-associated X protein, and activation of caspase-9 and caspase-3. In addition, pretreatment with a caspase inhibitor blocked fisetin-induced cell death.

  14. GENESIS OF MITOCHONDRIA IN INSECT FAT BODY

    PubMed Central

    Larsen, W. J.

    1970-01-01

    Electron microscopy and stereological methods have been used to study the time course and mechanism of mitochondrial genesis in the adult fat body of Calpodes ethlius, (Lepidoptera, Hesperiidae). Most of the larval mitochondria are destroyed during a phase of autolysis shortly before pupation, so that pupal and early adult fat body cells have few mitochondria. The number of mitochondria per cell increases rapidly at the end of the 1st day after the adult emerges. Characteristic partitioned mitochondria appear during the period when the number is rapidly increasing. This evidence, coupled with the results of morphometric analyses of mitochondrial diameter, volume, and surface area, confirms the view that the genesis of adult mitochondria involves the growth and division of mitochondria surviving from the larva. PMID:19866737

  15. Reactive oxygen species-provoked mitochondria-dependent cell death during ageing of elm (Ulmus pumila L.) seeds.

    PubMed

    Wang, Yu; Li, Ying; Xue, Hua; Pritchard, Hugh W; Wang, Xiaofeng

    2015-02-01

    Previous studies have shown that controlled deterioration treatment (CDT) induces programmed cell death in elm (Ulmus pumila L.) seeds, which undergo certain fundamental processes that are comparable to apoptosis in animals. In this study, the essential characteristics of mitochondrial physiology in elm seeds during CDT were identified by cellular ultrastructural analysis, whole-body optical imaging, Western blotting and semi-quantitative RT-PCR. The alteration in mitochondrial morphology was an early event during CDT, as indicated by progressive dynamic mitochondrial changes and rupture of the mitochondrial outer membrane; loss of mitochondrial transmembrane potential (Δψ(m)) ensued, and mitochondrial ATP levels decreased. The mitochondrial permeability transition pore inhibitor cyclosporine A effectively suppressed these changes during ageing. The in situ localization of production of reactive oxygen species (ROS), and evaluation of the expression of voltage-dependent anion-selective channel and cyclophilin D indicated that the levels of mitochondrial permeability transition pore components were positively correlated with ROS production, leading to an imbalance of the cellular redox potential and ultimately to programmed cell death. Pre-incubation with ascorbic acid slowed loss of mitochondrial Δψ(m), and decreased the effect of CDT on seed viability. However, there were no significant changes in multiple antioxidant elements or chaperones in the mitochondria during early stages of ageing. Our results indicate that CDT induces dynamic changes in mitochondrial physiology via increased ROS production, ultimately resulting in an irreversible loss of seed viability.

  16. Mitochondria and metazoan epigenesis

    PubMed Central

    Coffman, James A.

    2009-01-01

    In eukaryotes, mitochondrial activity controls ATP production, calcium dynamics, and redox state, thereby establishing physiological parameters governing the transduction of biochemical signals that regulate nuclear gene expression. However, these activities are commonly assumed to fulfill a ‘housekeeping’ function: necessary for life, but an epiphenomenon devoid of causal agency in the developmental flow of genetic information. Moreover, it is difficult to perturb mitochondrial function without generally affecting cell viability. For these reasons little is known about the extent of mitochondrial influence on gene activity in early development. Recent discoveries pertaining to the redox regulation of key developmental signaling systems together with the fact that mitochondria are often asymmetrically distributed in animal embryos suggests that they may contribute spatial information underlying differential specification of cell fate. In many cases such asymmetries correlate with localization of genetic determinants (i.e., mRNAs or proteins), particularly in embryos that rely heavily on cell-autonomous means of cell fate specification. In such embryos the localized genetic determinants play a dominant role, and any developmental information contributed by the mitochondria themselves is likely to be less obvious and more difficult to isolate experimentally. Hence, ‘regulative’ embryos that make more extensive use of conditional cell fate specification are better suited to experimental investigation of mitochondrial impacts on developmental gene regulation. Recent studies of the sea urchin embryo, which is a paradigmatic example of such a system, suggest that anisotropic distribution of mitochondria provides a source gradient of spatial information that directs epigenetic specification of the secondary axis via Nodal-Lefty signaling. PMID:19429498

  17. TPGS2k/PLGA nanoparticles for overcoming multidrug resistance by interfering mitochondria of human alveolar adenocarcinoma cells.

    PubMed

    Wang, Dong-Fang; Rong, Wen-Ting; Lu, Yu; Hou, Jie; Qi, Shan-Shan; Xiao, Qing; Zhang, Jue; You, Jin; Yu, Shu-Qin; Xu, Qian

    2015-02-25

    In this study, we successfully synthesized d-α-tocopheryl polyethylene glycol 2000 succinate (TPGS2k) and prepared TPGS2k-modified poly(lactic-co-glycolic acid) nanoparticles (TPGS2k/PLGA NPs) loaded with 7-ethyl-10-hydroxycamptothecin (SN-38), designated TPGS2k/PLGA/SN-38 NPs. Characterization measurements showed that TPGS2k/PLGA/SN-38 NPs displayed flat and spheroidal particles with diameters of 80-104 nm. SN-38 was encapsulated in TPGS2k emulsified PLGA NPs with the entrapment efficiency and loading rates of SN-38 83.6 and 7.85%, respectively. SN-38 could release constantly from TPGS2k/PLGA/SN-38 NPs in vitro. TPGS2k/PLGA/SN-38 NPs induced significantly higher cytotoxicity on A549 cells and the multidrug resistance (MDR) cell line (A549/DDP cells and A549/Taxol cells) compared with free SN-38. Further studies on the mechanism of the NPs in increasing the death of MDR cells showed that following the SN-38 releasing into cytoplasm the remaining TPGS2k/PLGA NPs could reverse the P-gp mediated MDR via interfering with the structure and function of mitochondria and rather than directly inhibiting the enzymatic activity of P-gp ATPase. Therefore, TPGS2k/PLGA NPs can reduce the generation of ATP and the release of energy for the requisite of P-gp efflux transporters. The results indicated that TPGS2k/PLGA NPs could become the nanopharmaceutical materials with the capability to reversal MDR and improve anticancer effects of some chemotherapy drugs as P-gp substrates.

  18. The role of mitochondria in the radiation-induced bystander effect in human lymphoblastoid cells.

    PubMed

    Rajendran, Sountharia; Harrison, Scott H; Thomas, Robert A; Tucker, James D

    2011-02-01

    Cells without intact mitochondrial DNA have been shown to lack the bystander effect, which is an energy-dependent process. We hypothesized that cells harboring mutations in mitochondrial genes responsible for ATP synthesis would show a decreased bystander effect compared to normal cells. Radiation-induced bystander effects were analyzed in two normal and four mitochondrial mutant human lymphoblastoid cells. Medium from previously irradiated cells (conditioned medium) was transferred to unirradiated cells from the respective cell lines and evaluated for the bystander effect using the cytokinesis-block micronucleus assay. Unlike normal cells that were used as a control, mitochondrial mutant cells neither generated nor responded to the bystander signals. The bystander effect was inhibited in normal cells by adding the mitochondrial inhibitors rotenone and oligomycin to the culture medium. Time-controlled blocking of the bystander effect by inhibitors was found to occur either for prolonged exposure to the inhibitor prior to irradiation with an immediate and subsequent removal of the inhibitors or immediate post-application of the inhibitor. Adding the inhibitors just prior to irradiation and removing them immediately after irradiation was uneventful. Fully functional mitochondrial metabolic capability may therefore be essential for the bystander effect.

  19. Interaction of Human Tumor Viruses with Host Cell Surface Receptors and Cell Entry

    PubMed Central

    Schäfer, Georgia; Blumenthal, Melissa J.; Katz, Arieh A.

    2015-01-01

    Currently, seven viruses, namely Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpes virus (KSHV), high-risk human papillomaviruses (HPVs), Merkel cell polyomavirus (MCPyV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T cell lymphotropic virus type 1 (HTLV-1), have been described to be consistently associated with different types of human cancer. These oncogenic viruses belong to distinct viral families, display diverse cell tropism and cause different malignancies. A key to their pathogenicity is attachment to the host cell and entry in order to replicate and complete their life cycle. Interaction with the host cell during viral entry is characterized by a sequence of events, involving viral envelope and/or capsid molecules as well as cellular entry factors that are critical in target cell recognition, thereby determining cell tropism. Most oncogenic viruses initially attach to cell surface heparan sulfate proteoglycans, followed by conformational change and transfer of the viral particle to secondary high-affinity cell- and virus-specific receptors. This review summarizes the current knowledge of the host cell surface factors and molecular mechanisms underlying oncogenic virus binding and uptake by their cognate host cell(s) with the aim to provide a concise overview of potential target molecules for prevention and/or treatment of oncogenic virus infection. PMID:26008702

  20. Interaction of human tumor viruses with host cell surface receptors and cell entry.

    PubMed

    Schäfer, Georgia; Blumenthal, Melissa J; Katz, Arieh A

    2015-05-22

    Currently, seven viruses, namely Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpes virus (KSHV), high-risk human papillomaviruses (HPVs), Merkel cell polyomavirus (MCPyV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T cell lymphotropic virus type 1 (HTLV-1), have been described to be consistently associated with different types of human cancer. These oncogenic viruses belong to distinct viral families, display diverse cell tropism and cause different malignancies. A key to their pathogenicity is attachment to the host cell and entry in order to replicate and complete their life cycle. Interaction with the host cell during viral entry is characterized by a sequence of events, involving viral envelope and/or capsid molecules as well as cellular entry factors that are critical in target cell recognition, thereby determining cell tropism. Most oncogenic viruses initially attach to cell surface heparan sulfate proteoglycans, followed by conformational change and transfer of the viral particle to secondary high-affinity cell- and virus-specific receptors. This review summarizes the current knowledge of the host cell surface factors and molecular mechanisms underlying oncogenic virus binding and uptake by their cognate host cell(s) with the aim to provide a concise overview of potential target molecules for prevention and/or treatment of oncogenic virus infection.

  1. Access of viral proteins to mitochondria via mitochondria-associated membranes.

    PubMed

    Williamson, Chad D; Colberg-Poley, Anamaris M

    2009-05-01

    By exploiting host cell machineries, viruses provide powerful tools for gaining insight into cellular pathways. Proteins from two unrelated viruses, human CMV (HCMV) and HCV, are documented to traffic sequentially from the ER into mitochondria, probably through the mitochondria-associated membrane (MAM) compartment. The MAM are sites of ER-mitochondrial contact enabling the direct transfer of membrane bound lipids and the generation of high calcium (Ca2+) microdomains for mitochondria signalling and responses to cellular stress. Both HCV core protein and HCMV UL37 proteins are associated with Ca2+ regulation and apoptotic signals. Trafficking of viral proteins to the MAM may allow viruses to manipulate a variety of fundamental cellular processes, which converge at the MAM, including Ca2+ signalling, lipid synthesis and transfer, bioenergetics, metabolic flow, and apoptosis. Because of their distinct topologies and targeted MAM sub-domains, mitochondrial trafficking (albeit it through the MAM) of the HCMV and HCV proteins predictably involves alternative pathways and, hence, distinct targeting signals. Indeed, we found that multiple cellular and viral proteins, which target the MAM, showed no apparent consensus primary targeting sequences. Nonetheless, these viral proteins provide us with valuable tools to access the poorly characterised MAM compartment, to define its cellular constituents and describe how virus infection alters these to its own end. Furthermore, because proper trafficking of viral proteins is necessary for their function, discovering the requirements for MAM to mitochondrial trafficking of essential viral proteins may provide novel targets for the rational design of anti-viral drugs.

  2. Role of myeloid cells in HIV-1-host interplay.

    PubMed

    Stevenson, Mario

    2015-06-01

    The AIDS research field has embarked on a bold mission to cure HIV-1-infected individuals of the virus. To do so, scientists are attempting to identify the reservoirs that support viral persistence in patients on therapy, to understand how viral persistence is regulated and to come up with strategies that interrupt viral persistence and that eliminate the viral reservoirs. Most of the attention regarding the cure of HIV-1 infection has focused on the CD4+ T cell reservoir. Investigators are developing tools to probe the CD4+ T cell reservoirs as well as in vitro systems that provide clues on how to perturb them. By comparison, the myeloid cell, and in particular, the macrophage has received far less attention. As a consequence, there is very little understanding as to the role played by myeloid cells in viral persistence in HIV-1-infected individuals on suppressive therapy. As such, should myeloid cells constitute a viral reservoir, unique strategies may be required for their elimination. This article will overview research that is examining the role of macrophage in virus-host interplay and will discuss features of this interplay that could impact efforts to eliminate myeloid cell reservoirs.

  3. Carvacrol induces mitochondria-mediated apoptosis in HL-60 promyelocytic and Jurkat T lymphoma cells.

    PubMed

    Bhakkiyalakshmi, Elango; Suganya, Natarajan; Sireesh, Dornadula; Krishnamurthi, Kannan; Saravana Devi, Sivanesan; Rajaguru, Palanisamy; Ramkumar, Kunka Mohanram

    2016-02-05

    The aim of the present study was to investigate the effect of carvacrol, a phenolic monoterpenoid on the induction of apoptosis in HL-60 (Human acute promyelocytic leukemia cells) and Jurkat (human T lymphocyte cells) cells. Carvacrol showed a potent cytotoxic effect on both cells with dose-dependent increase in the level of free radical formation as measured by an oxidation sensitive fluorescent dye, 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) levels. The reduction in the level of antioxidants such as catalase (CAT) and superoxide dismutase (SOD) (P<0.05) was observed in carvacrol-treated cells. The major cytotoxic effect appears to be intervened by the induction of apoptotic cell death as assessed by annexin-V labeling assay using flow cytometry. Western blot analysis showed that Bax expression was increased, whereas Bcl-2 expression was significantly decreased in carvacrol exposed HL-60 cells and Jurkat cells. Further studies revealed that the dissipation of mitochondrial membrane potential of intact cells was accompanied by the activation of caspase-3. Our results found that the potential mechanism of cellular apoptosis induced by carvacrol is mediated by caspase-3 and is associated with the collapse of mitochondrial membrane potential, generation of free radicals, and depletion of the intracellular antioxidant pool.

  4. Pathogen Cell-to-cell Variability Drives Heterogeneity In Host Immune Responses

    PubMed Central

    Avraham, Roi; Haseley, Nathan; Brown, Douglas; Penaranda, Cristina; Jijon, Humberto B; Trombetta, John J; Satija, Rahul; Shalek, Alex K; Xavier, Ramnik; Regev, Aviv; Hung, Deborah T

    2015-01-01

    Summary Encounters between immune cells and invading bacteria ultimately determine the course of infection. These interactions are usually measured in populations of cells, masking cell-to-cell variation that may be important for infection outcome. To characterize gene expression variation that underlies distinct infection outcomes, we developed an experimental system that combines single-cell RNA-seq with fluorescent markers, monitoring infection phenotypes. Probing the responses of individual macrophages to invading Salmonella, we find that variation between individual infected host cells is determined by the heterogeneous activity of bacterial factors in individual infecting bacteria. We illustrate how variable PhoPQ activity in the population of invading bacteria drives variable host Type I IFN responses by modifying LPS in a subset of bacteria. This work demonstrates a causative link between host and bacterial variability, with cell-to-cell variation between different bacteria being sufficient to drive radically different host immune responses. This co-variation has implications for host-pathogen dynamics in vivo. PMID:26343579

  5. Functional comparison of chronological and in vitro aging: differential role of the cytoskeleton and mitochondria in mesenchymal stromal cells.

    PubMed

    Geissler, Sven; Textor, Martin; Kühnisch, Jirko; Könnig, Delia; Klein, Oliver; Ode, Andrea; Pfitzner, Tilman; Adjaye, James; Kasper, Grit; Duda, Georg N

    2012-01-01

    Mesenchymal stromal cells (MSCs) are of high relevance for the regeneration of mesenchymal tissues such as bone and cartilage. The promising role of MSCs in cell-based therapies and tissue engineering appears to be limited due to a decline of their regenerative potential with increasing donor age, their limited availability in human tissues and the need of in vitro expansion prior to treatment. We therefore aimed to determine to which degree in vitro aging and chronological aging may be similar processes or if in vitro culture-related changes at the cellular and molecular level are at least altered as a function of donor age. For that purpose we established MSCs cultures from young (yMSCs) and aged (aMSCs) rats that were cultured for more than 100 passages. These long-term MSCs cultures were non-tumorigenic and exhibited similar surface marker patterns as primary MSCs of passage 2. During in vitro expansion, but not during chronological aging, MSCs progressively lose their progenitor characteristics, e.g., complete loss of osteogenic differentiation potential, diminished adipogenic differentiation, altered cell morphology and increased susceptibility towards senescence. Transcriptome analysis revealed that long-term in vitro MSCs cultivation leads to down-regulation of genes involved in cell differentiation, focal adhesion organization, cytoskeleton turnover and mitochondria function. Accordingly, functional analysis demonstrated altered mitochondrial morphology, decreased antioxidant capacities and elevated ROS levels in long-term cultivated yMSCs as well as aMSCs. Notably, only the MSC migration potential and their antioxidative capacity were altered by in vitro as well as chronological aging. Based on specific differences observed between the impact of chronological and in vitro MSC aging we conclude that both are distinct processes.

  6. Aluminium based adjuvants and their effects on mitochondria and lysosomes of phagocytosing cells.

    PubMed

    Ohlsson, Lars; Exley, Christopher; Darabi, Anna; Sandén, Emma; Siesjö, Peter; Eriksson, Håkan

    2013-11-01

    Aluminium oxyhydroxide, Al(OH)3 is one of few compounds approved as an adjuvant in human vaccines. However, the mechanism behind its immune stimulating properties is still poorly understood. In vitro co-culture of an aluminium adjuvant and the human monocytic cell line THP-1 resulted in reduced cell proliferation. Inhibition occurred at concentrations of adjuvant several times lower than would be found at the injection site using a vaccine formulation containing an aluminium adjuvant. Based on evaluation of the mitochondrial membrane potential, THP-1 cells showed no mitochondrial rupture after co-culture with the aluminium adjuvant, instead an increase in mitochondrial activity was seen. The THP-1 cells are phagocytosing cells and after co-culture with the aluminium adjuvant the phagosomal pathway was obstructed. Primary or early phagosomes mature into phagolysosomes with an internal pH of 4.5 - 5 and carry a wide variety of hydrolysing enzymes. Co-culture with the aluminium adjuvant yielded a reduced level of acidic vesicles and cathepsin L activity, a proteolytic enzyme of the phagolysosomes, was almost completely inhibited. THP-1 cells are an appropriate in vitro model in order to investigate the mechanism behind the induction of a phagocytosing antigen presenting cell into an inflammatory cell by aluminium adjuvants. Much information will be gained by investigating the phagosomal pathway and what occurs inside the phagosomes and to elucidate the ultimate fate of phagocytosed aluminium particles.

  7. Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro.

    PubMed

    Yang, Zi-Rong; Liu, Meng; Peng, Xiu-Lan; Lei, Xiao-Fei; Zhang, Ji-Xiang; Dong, Wei-Guo

    2012-05-11

    Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC(50)=75 μM). This cytotoxicity was reflected by cell cycle arrest at G(2)/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.

  8. Amorfrutin C Induces Apoptosis and Inhibits Proliferation in Colon Cancer Cells through Targeting Mitochondria.

    PubMed

    Weidner, Christopher; Rousseau, Morten; Micikas, Robert J; Fischer, Cornelius; Plauth, Annabell; Wowro, Sylvia J; Siems, Karsten; Hetterling, Gregor; Kliem, Magdalena; Schroeder, Frank C; Sauer, Sascha

    2016-01-22

    A known (1) and a structurally related new natural product (2), both belonging to the amorfrutin benzoic acid class, were isolated from the roots of Glycyrrhiza foetida. Compound 1 (amorfrutin B) is an efficient agonist of the nuclear peroxisome proliferator activated receptor (PPAR) gamma and of other PPAR subtypes. Compound 2 (amorfrutin C) showed comparably lower PPAR activation potential. Amorfrutin C exhibited striking antiproliferative effects for human colorectal cancer cells (HT-29 and T84), prostate cancer (PC-3), and breast cancer (MCF7) cells (IC50 values ranging from 8 to 16 μM in these cancer cell lines). Notably, amorfrutin C (2) showed less potent antiproliferative effects in primary colon cells. For HT-29 cells, compound 2 induced G0/G1 cell cycle arrest and modulated protein expression of key cell cycle modulators. Amorfrutin C further induced apoptotic events in HT-29 cells, including caspase activation, DNA fragmentation, PARP cleavage, phosphatidylserine externalization, and formation of reactive oxygen species. Mechanistic studies revealed that 2 disrupts the mitochondrial integrity by depolarization of the mitochondrial membrane (IC50 0.6 μM) and permanent opening of the mitochondrial permeability transition pore, leading to increased mitochondrial oxygen consumption and extracellular acidification. Structure-activity-relationship experiments revealed the carboxylic acid and the hydroxy group residues of 2 as fundamental structural requirements for inducing these apoptotic effects. Synergy analyses demonstrated stimulation of the death receptor signaling pathway. Taken together, amorfrutin C (2) represents a promising lead for the development of anticancer drugs.

  9. Energization of sodium absorption by the H(+)-ATPase pump in mitochondria-rich cells of frog skin.

    PubMed

    Harvey, B J

    1992-11-01

    The frog skin in vivo is capable of active transepithelial H+ secretion (JH) which is matched by Na+ absorption (JNa). Studies in vitro demonstrate that JH is generated by an H(+)-ATPase pump localized in apical membranes of mitochondria-rich (MR) cells, whereas JNa occurs through an amiloride-sensitive pathway in principal (P) cells. The H+ pump is sensitive to inhibitors of carbonic anhydrase (e.g. acetazolamide) and to specific inhibitors of mitochondrial F1F0 H(+)-ATPase (oligomycin) and vacuolar (V)-type H(+)-ATPase (N-ethylmaleimide) and to inhibitors of both these types of H(+)-ATPases (dicyclohexylcarbodiimide, DCCD). JH is independent of external K+, which differentiates it from gastric H+/K(+)-ATPase and is strictly dependent on aerobic metabolism. The proton pump is primarily implicated in whole-body acid-base regulation. Acute stimulation of JH in response (seconds-minutes) to an acid load involves insertion of H+ pumps (exocytosis) from a cytosolic pool into the apical membrane. The chronic response (days) to metabolic acid load involves morphological changes (increased apical membrane surface area and number of MR cells). Whole-cell patch-clamp recordings of membrane capacitance and current fluctuations from MR cells demonstrate that a respiratory acid load and aldosterone produce rapid exocytotic insertion of DCCD-sensitive conductive membrane. A secondary role of the H+ pump is to energize sodium absorption (JNa) via principal cells from dilute solutions in the absence of a permeant anion under open-circuit conditions. The apparent 1:1 stoichiometry between JH and JNa is a result of transepithelial electrical coupling between these electrogenic fluxes. The H+ pump in MR cells generates a transepithelial current (serosa to apical) which acts as a physiological voltage-clamp to hyperpolarize the apical membrane of P cells. This hyperpolarization can facilitate passive Na+ entry across the apical membrane against a threefold chemical gradient. Since

  10. Role of mitochondria in the switch mechanism of the cell death mode from apoptosis to necrosis--studies on rho0 cells.

    PubMed

    Wochna, Agnieszka; Niemczyk, Edyta; Kurono, Chieko; Masaoka, Makoto; Majczak, Anna; Kedzior, Jakub; Slominska, Ewa; Lipinski, Marcin; Wakabayashi, Takashi

    2005-04-01

    Detailed mechanisms of the switch of the cell death mode from apoptosis to necrosis remain to be solved, although the intracellular level of ATP and that of free radicals have been postulated to be the major factors involved in the mechanisms. In the present study menadione (MEN)-induced cell injury processes were studied using rho0 cells derived from human osteosarcoma 143B cells and parental rho+ cells co-treated with inhibitors of electron transfer chain of mitochondria or oligomycin, an inhibitor of ATP synthesis. Treatment of rho+ cells with 100 microM MEN induced apoptosis, which reached the maximum at 6 h, and was followed by an abrupt decrease thereafter, while necrotic cells (NC) increased continuously when they were judged by Annexin V and PI double staining. On the other hand, MEN induced apoptotic and necrotic changes much faster in rho0 cells compared to rho+ cells. The frequency to find apoptotic cells (AP) in the former cells was distinctly smaller than that to find NC judged by Annexin V and PI double staining. Electron microscopically, a major population of rho0 cells treated with MEN for 6 h consisted of intermediate cells, and a small number of AP co-existed. At 9 h of the treatment intermediate cells were exclusively seen, and AP were hardly detected. When parental rho+ cells were treated with MEN in the presence of oligomycin or oligomycin plus antimycin A both apoptotic and necrotic changes of the cells were distinctly accelerated. The intracellular level of superoxide in rho0 cells continuously increased after the MEN treatment, whereas that of ATP remained distinctly low before and after the MEN treatment compared to that in rho+ cells. These data suggest that the intracellular level of superoxide may be a key factor controlling the switch from apoptosis to necrosis.

  11. Why to compare absolute numbers of mitochondria.

    PubMed

    Schmitt, Sabine; Schulz, Sabine; Schropp, Eva-Maria; Eberhagen, Carola; Simmons, Alisha; Beisker, Wolfgang; Aichler, Michaela; Zischka, Hans

    2014-11-01

    Prompted by pronounced structural differences between rat liver and rat hepatocellular carcinoma mitochondria, we suspected these mitochondrial populations to differ massively in their molecular composition. Aiming to reveal these mitochondrial differences, we came across the issue on how to normalize such comparisons and decided to focus on the absolute number of mitochondria. To this end, fluorescently stained mitochondria were quantified by flow cytometry. For rat liver mitochondria, this approach resulted in mitochondrial protein contents comparable to earlier reports using alternative methods. We determined similar protein contents for rat liver, heart and kidney mitochondria. In contrast, however, lower protein contents were determined for rat brain mitochondria and for mitochondria from the rat hepatocellular carcinoma cell line McA 7777. This result challenges mitochondrial comparisons that rely on equal protein amounts as a typical normalization method. Exemplarily, we therefore compared the activity and susceptibility toward inhibition of complex II of rat liver and hepatocellular carcinoma mitochondria and obtained significant discrepancies by either normalizing to protein amount or to absolute mitochondrial number. Importantly, the latter normalization, in contrast to the former, demonstrated a lower complex II activity and higher susceptibility toward inhibition in hepatocellular carcinoma mitochondria compared to liver mitochondria. These findings demonstrate that solely normalizing to protein amount may obscure essential molecular differences between mitochondrial populations.

  12. Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro

    SciTech Connect

    Yang, Zi-Rong; Liu, Meng; Peng, Xiu-Lan; Lei, Xiao-Fei; Zhang, Ji-Xiang; Dong, Wei-Guo

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Noscapine inhibited cell viability of colon cancer in a time- and dose- dependent manner. Black-Right-Pointing-Pointer G{sub 2}/M phase arrest and chromatin condensation and nuclear fragmentation were induced. Black-Right-Pointing-Pointer Noscapine promoted apoptosis via mitochondrial pathways. Black-Right-Pointing-Pointer Tumorigenicity was inhibited by noscapine. -- Abstract: Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC{sub 50} = 75 {mu}M). This cytotoxicity was reflected by cell cycle arrest at G{sub 2}/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.

  13. Cantharidin induces apoptosis of H460 human lung cancer cells through mitochondria-dependent pathways.

    PubMed

    Hsia, Te-Chun; Yu, Chien-Chih; Hsu, Shu-Chun; Tang, Nou-Ying; Lu, Hsu-Feng; Huang, Yi-Ping; Wu, Shin-Hwar; Lin, Jaung-Geng; Chung, Jing-Gung

    2014-07-01

    Lung cancer is one of the leading causes of death in cancer-related diseases. Cantharidin (CTD) is one of the components of natural mylabris (Mylabris phalerata Pallas). Numerous studies have shown that CTD induced cytotoxic effects on cancer cells. However, there is no report to demonstrate that CTD induced apoptosis in human lung cancer cells. Herein, we investigated the effect of CTD on the cell death via the induction of apoptosis in H460 human lung cancer cells. Flow cytometry assay was used for examining the percentage of cell viability, sub-G1 phase of the cell cycle, reactive oxygen species (ROS) and Ca²⁺ productions and the levels of mitochondrial membrane potential (∆Ψm). Annexin V/PI staining and DNA gel electrophoresis were also used for examining cell apoptosis. Western blot analysis was used to examine the changes of apoptosis associated protein expression and confocal microscopy for examining the translocation apoptosis associated protein. Results indicated that CTD significantly induced cell morphological changes and decreased the percentage of viable H460 cells. CTD induced apoptosis based on the occurrence of sub-G1 phase and DNA fragmentation. We found that CTD increased gene expression (mRNA) of caspase-3 and -8. Moreover, CTD increased ROS and Ca2+ production and decreased the levels of ∆Ψm. Western blot analysis results showed that CTD increased the expression of cleavage caspase-3 and -8, cytochrome c, Bax and AIF but inhibited the levels of Bcl-xL. CTD promoted ER stress associated protein expression such as GRP78, IRE1α, IRE1β, ATF6α and caspase-4 and it also promoted the expression of calpain 2 and XBP-1, but inhibited calpain 1 that is associated with apoptosis pathways. Based on those observations, we suggest that CTD may be used as a novel anticancer agent for the treatment of lung cancer in the future.

  14. Mitochondria-targeted DsRed2 protein expression during the early stage of bovine somatic cell nuclear transfer embryo development.

    PubMed

    Park, Hyo-Jin; Min, Sung-Hun; Choi, Hoonsung; Park, Junghyung; Kim, Sun-Uk; Lee, Seunghoon; Lee, Sang-Rae; Kong, Il-Keun; Chang, Kyu-Tae; Koo, Deog-Bon; Lee, Dong-Seok

    2016-09-01

    Somatic cell nuclear transfer (SCNT) has been widely used as an efficient tool in biomedical research for the generation of transgenic animals from somatic cells with genetic modifications. Although remarkable advances in SCNT techniques have been reported in a variety of mammals, the cloning efficiency in domestic animals is still low due to the developmental defects of SCNT embryos. In particular, recent evidence has revealed that mitochondrial dysfunction is detected during the early development of SCNT embryos. However, there have been relatively few or no studies regarding the development of a system for evaluating mitochondrial behavior or dynamics. For the first time, in mitochondria of bovine SCNT embryos, we developed a method for the visualization of mitochondria and expression of fluorescence proteins. To express red fluorescence in mitochondria of cloned embryos, bovine ear skin fibroblasts, nuclear donor, were stably transfected with a vector carrying mitochondria-targeting DsRed2 gene tagged with V5 epitope (mito-DsRed2-V5 tag) using lentivirus-mediated gene transfer because of its ability to integrate in the cell genome and the potential for long-term transgene expression in the transduced cells and their dividing cells. From western blotting analysis of V5 tag protein using mitochondrial fraction and confocal microscopy of red fluorescence using SCNT embryos, we found that the mitochondrial expression of the mito-DsRed2 protein was detected until the blastocyst stage. In addition, according to image analysis, it may be suggested possible use of the system for visualization of mitochondrial localization and evaluation of mitochondrial behaviors or dynamics in early development of bovine SCNT embryos.

  15. Targeting mitochondria by Zn(II)N-alkylpyridylporphyrins: the impact of compound sub-mitochondrial partition on cell respiration and overall photodynamic efficacy.

    PubMed

    Odeh, Ahmad M; Craik, James D; Ezzeddine, Rima; Tovmasyan, Artak; Batinic-Haberle, Ines; Benov, Ludmil T

    2014-01-01

    Mitochondria play a key role in aerobic ATP production and redox control. They harness crucial metabolic pathways and control cell death mechanisms, properties that make these organelles essential for survival of most eukaryotic cells. Cancer cells have altered cell death pathways and typically show a shift towards anaerobic glycolysis for energy production, factors which point to mitochondria as potential culprits in cancer development. Targeting mitochondria is an attractive approach to tumor control, but design of pharmaceutical agents based on rational approaches is still not well established. The aim of this study was to investigate which structural features of specially designed Zn(II)N-alkylpyridylporphyrins would direct them to mitochondria and to particular mitochondrial targets. Since Zn(II)N-alkylpyridylporphyrins can act as highly efficient photosensitizers, their localization can be confirmed by photodamage to particular mitochondrial components. Using cultured LS174T adenocarcinoma cells, we found that subcellular distribution of Zn-porphyrins is directed by the nature of the substituents attached to the meso pyridyl nitrogens at the porphyrin ring. Increasing the length of the aliphatic chain from one carbon (methyl) to six carbons (hexyl) increased mitochondrial uptake of the compounds. Such modifications also affected sub-mitochondrial distribution of the Zn-porphyrins. The amphiphilic hexyl derivative (ZnTnHex-2-PyP) localized in the vicinity of cytochrome c oxidase complex, causing its inactivation during illumination. Photoinactivation of critical cellular targets explains the superior efficiency of the hexyl derivative in causing mitochondrial photodamage, and suppressing cellular respiration and survival. Design of potent photosensitizers and redox-active scavengers of free radicals should take into consideration not only selective organelle uptake and localization, but also selective targeting of critical macromolecular structures.

  16. Targeting Mitochondria by Zn(II)N-Alkylpyridylporphyrins: The Impact of Compound Sub-Mitochondrial Partition on Cell Respiration and Overall Photodynamic Efficacy

    PubMed Central

    Odeh, Ahmad M.; Craik, James D.; Ezzeddine, Rima; Tovmasyan, Artak; Batinic-Haberle, Ines; Benov, Ludmil T.

    2014-01-01

    Mitochondria play a key role in aerobic ATP production and redox control. They harness crucial metabolic pathways and control cell death mechanisms, properties that make these organelles essential for survival of most eukaryotic cells. Cancer cells have altered cell death pathways and typically show a shift towards anaerobic glycolysis for energy production, factors which point to mitochondria as potential culprits in cancer development. Targeting mitochondria is an attractive approach to tumor control, but design of pharmaceutical agents based on rational approaches is still not well established. The aim of this study was to investigate which structural features of specially designed Zn(II)N-alkylpyridylporphyrins would direct them to mitochondria and to particular mitochondrial targets. Since Zn(II)N-alkylpyridylporphyrins can act as highly efficient photosensitizers, their localization can be confirmed by photodamage to particular mitochondrial components. Using cultured LS174T adenocarcinoma cells, we found that subcellular distribution of Zn-porphyrins is directed by the nature of the substituents attached to the meso pyridyl nitrogens at the porphyrin ring. Increasing the length of the aliphatic chain from one carbon (methyl) to six carbons (hexyl) increased mitochondrial uptake of the compounds. Such modifications also affected sub-mitochondrial distribution of the Zn-porphyrins. The amphiphilic hexyl derivative (ZnTnHex-2-PyP) localized in the vicinity of cytochrome c oxidase complex, causing its inactivation during illumination. Photoinactivation of critical cellular targets explains the superior efficiency of the hexyl derivative in causing mitochondrial photodamage, and suppressing cellular respiration and survival. Design of potent photosensitizers and redox-active scavengers of free radicals should take into consideration not only selective organelle uptake and localization, but also selective targeting of critical macromolecular structures. PMID

  17. Regulation of Mitochondria Function by TRAF3 in B Lymphocytes and B Cell Malignancies

    DTIC Science & Technology

    2015-10-01

    This DoD grant also allows Dr. Xie to better integrate her educational and research efforts, including the undergraduate courses of Immunology and... Immunology Lab, and the graduate courses of Current Concepts in Immunology and Advanced Topics in Immunology . Taken together, this DoD award has been...1, 2014. 2. Chair, Block Symposia of Innate Immune Responses in Monocytes/Macrophages, Dendritic Cells, and Myeloid Cells, the Annual Meeting of

  18. Chloride conductance across toad skin: effects of ionic acclimations and cyclic AMP and relationship to mitochondria-rich cell density.

    PubMed

    Rozman, A; Gabbay, S; Katz, U

    2000-07-01

    The anionic conductance across toad (Bufo viridis) skin was studied using the voltage-clamp technique following long-term (more than 10 days) acclimation to NaCl and KCl solutions. The non-specific baseline conductance was approximately 0.6 mS cm(-)(2) and was similar in skins from all acclimation conditions. The voltage-activated Cl(-) conductance (G(Cl)) was maximal in skins from distilled-water- and KCl-acclimated toads (>3 mS cm(-)(2)) and was greatly reduced following acclimation to NaCl solutions. Cyclic AMP (EC(50)=13 micromol l(-)(1)) and isobutylmethyl xanthine (IBMX) (EC(50)=69 micromol l(-)(1)) exerted different effects on the activated conductance. IBMX only sensitized the activated conductance, whereas cyclic AMP (CPTcAMP) at high concentrations induced an increase in anionic conductance that was insensitive to electrical potential. Furthermore, external Cl(-) was not required for the stimulatory effect of cyclic AMP, and the conductive pathway had low selectivity. The effects of the two agonists were reversible and depended on the acclimation conditions. Following electrical measurements, the skin of the toads was removed and stained with silver to measure mitochondria-rich cell density (D(mrc)). There was no correlation between D(mrc) and Cl(-) conductance in the present study.

  19. Chloride conductance and mitochondria-rich cell density in isolated skin of Rana catesbeiana acclimated to various environments.

    PubMed

    Claro de Toledo, Manuel; Malheiros Lopes Sanioto, Sonia

    2002-08-01

    The Cl- conductance in isolated skin of frogs (Rana catesbeiana) acclimated to 30 mM solutions of NaCl, Na2SO4, MgCl2 and distilled water (DW) was studied. Transepithelial potential difference (PDtrans), short-circuit current (ISC) and total conductance (Gt) were measured under conditions such that there was Cl- flux in the presence and absence of Na+ transport. The Cl- content of the mucosal solution was acutely replaced with SO42- or gluconate to evaluate the effect of removal of Cl- conductance on electrophysiological parameters. Mitochondria-rich cell density (DMRC) was also measured. Skins from frogs acclimated to NaCl and Na2SO4 showed the lowest and the highest D(MRC), respectively, but no difference could be found between the skins from frogs acclimated to DW and MgCl2 indicating that DMRC is not unconditionally dependent on environmental Cl- in this species. Frogs acclimated to NaCl showed marked differences when compared to the other groups: the highest Gt, probably represented by a higher paracellular conductance; the lowest transepithelial electrical potential difference which remained invariant after replacement of mucosal Cl- with SO42- or replacement of mucosal Cl- with gluconate and an inwardly oriented positive current in the absence of bilateral Na+.

  20. Hyperglycemia magnifies bupivacaine-induced cell apoptosis triggered by mitochondria dysfunction and endoplasmic reticulum stress.

    PubMed

    Li, Le; Ye, Xiao-ping; Lu, Ai-zhu; Zhou, Shu-qin; Liu, Hui; Liu, Zhong-jie; Jiang, Shan; Xu, Shi-yuan

    2013-06-01

    Nerve cell injury associated with apoptosis plays an important role in the development of diabetic peripheral neuropathy (DPN). However, it remains unclear whether preexisting or potential neurocyte damage induced by hyperglycemia increases sensitivity to local anesthetics. SH-SY5Y cells were pretreated with a high concentration of glucose in vitro, to imitate DPN prior to administration of bupivacaine or placebo. Cell viability and apoptosis were investigated with a CCK-8 assay and flow cytometry, respectively. In addition, mitochondrial membrane potential, reactive oxygen species (ROS), mitochondrially generated ROS, and activity of mitochondrial complexes I and III were studied to explore the molecular mechanism of bupivacaine-induced mitochondrial injury. Grp78 and caspase-12 expression were measured by qRT-PCR and Western blot, representing endoplasmic reticulum (ER) stress. Cell structure was also assessed via transmission electron microscopy. Incubation with bupivacaine decreased the activity of mitochondrial complexes I and III; increased ROS production at cell and mitochondrial levels, mitochondrial potential depolarization, and Grp78 and caspase-12 expression at both transcription and translation levels; and affected cell structure, which could be enhanced by glucose pretreatment. These findings indicate that mitochondrial dysfunction and ER stress related to ROS are involved in bupivacaine-induced apoptosis and may be enhanced by glucose administration.

  1. Mitochondria-Derived Reactive Oxygen Species Mediate Heme Oxygenase-1 Expression in Sheared Endothelial CellsS⃞

    PubMed Central

    Han, Zhaosheng; Varadharaj, Saradhadevi; Giedt, Randy J.; Zweier, Jay L.; Szeto, Hazel H.; Alevriadou, B. Rita

    2009-01-01

    Bovine aortic endothelial cells (ECs) respond to nitric oxide (NO) donors by activating the redox-sensitive NF-E2-related factor 2/antioxidant response element pathway and up-regulating heme oxygenase (HO)-1 expression. EC exposure to steady laminar shear stress causes a sustained increase in NO, a transient increase in reactive oxygen species (ROS), and activation of the HO-1 gene. Because steady laminar flow increases the mitochondrial superoxide (\\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\mathrm{O}}_{2}^{\\overline{.}}\\end{equation*}\\end{document}) production, we hypothesized that mitochondria-derived ROS play a role in shear-induced HO-1 expression. Flow (10 dynes/cm2, 6 h)-induced expression of HO-1 protein was abolished when BAECs were preincubated and sheared in the presence of either NG-nitro-l-arginine methyl ester or N-acetyl-l-cysteine, suggesting that either NO or ROS up-regulates HO-1. Ebselen and diphenylene iodonium blocked HO-1 expression, and uric acid had no effect. The mitochondrial electron transport chain inhibitors, myxothiazol, rotenone, or antimycin A, and the mitochondria-targeted antioxidant peptide, Szeto-Schiller (SS)-31, which scavenges \\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\mathrm{O}}_{2}^{\\overline{.}}\\end{equation*}\\end{document}, hydrogen peroxide (H2O2), peroxynitrite, and hydroxyl radicals, markedly inhibited the increase in HO-1 expression. These data collectively suggest that mitochondrial H2O2 mediates the HO-1 induction. Mito

  2. Morphological and quantitative changes in mitochondria, plastids, and peroxisomes during the log-to-stationary transition of the growth phase in cultured tobacco BY-2 cells

    PubMed Central

    Toyooka, Kiminori; Sato, Mayuko; Wakazaki, Mayumi; Matsuoka, Ken

    2016-01-01

    ABSTRACT We developed a wide-range and high-resolution transmission electron microscope acquisition system and obtained giga-pixel images of tobacco BY-2 cells during the log and stationary phases of cell growth. We demonstrated that the distribution and ultrastructure of compartments involved in membrane traffic (i.e., Golgi apparatus, multivesicular body, and vesicle cluster) change during the log-to-stationary transition. Mitochondria, peroxisomes, and plastids were also enumerated. Electron densities of mitochondria and peroxisomes were altered during the growth-phase shift, while their numbers were reduced by nearly half. Plastid structure dramatically changed from atypical to spherical with starch granules. Nearly the same number of plastids was observed in both log and stationary phases. These results indicate that mechanisms regulating organelle populations differ from organelle to organelle. PMID:26855065

  3. Ultrasensitive label-free photothermal imaging, spectral identification, and quantification of cytochrome c in mitochondria, live cells, and solutions

    PubMed Central

    Brusnichkin, Anton V.; Nedosekin, Dmitry A.; Galanzha, Ekaterina I.; Vladimirov, Yuri A.; Shevtsova, Elena F.; Proskurnin, Mikhail A.; Zharov, Vladimir P.

    2012-01-01

    Light-absorbing endogenous cellular proteins, in particular cytochrome c, are used as intrinsic biomarkers for studies of cell biology and environment impacts. To sense cytochrome c against real biological backgrounds, we combined photothermal (PT) thermal-lens single channel schematic in a back-synchronized measurement mode and a multiplex thermal-lens schematic in a transient high resolution (ca. 350 nm) imaging mode. These multifunctional PT techniques using continuous-wave (cw) Ar+ laser and a nanosecond pulsed optical parametric oscillator in the visible range demonstrated the capability for label-free spectral identification and quantification of trace amounts of cytochrome c in a single mitochondrion alone or within a single live cell. PT imaging data were verified in parallel by molecular targeting and fluorescent imaging of cellular cytochrome c. The detection limit of cytochrome c in a cw mode was 5 × 10−9 mol/L (80 attomols in the signal-generation zone); that is ca. 103 lower than conventional absorption spectroscopy. Pulsed fast PT microscopy provided the detection limit for cytochrome c at the level of 13 zmol (13 × 10−21 mol) in the ultra-small irradiated volumes limited by optical diffraction effects. For the first time, we demonstrate a combination of high resolution PT imaging with PT spectral identification and ultrasensitive quantitative PT characterization of cytochrome c within individual mitochondria in single live cells. A potential of far-field PT microscopy to sub-zeptomol detection thresholds, resolution beyond diffraction limit, PT Raman spectroscopy, and 3D imaging are further highlighted. PMID:20572284

  4. Connective tissue diseases: Mitochondria drive NETosis and inflammation in SLE.

    PubMed

    Boilard, Eric; Fortin, Paul R

    2016-04-01

    Mitochondria are the powerhouses of the cell, providing energy through oxidative respiration. Possibly owing to their similarities with bacteria, however, mitochondria extruded from cells promote inflammation. New research demonstrates that in systemic lupus erythematosus, mitochondrial respiration is critical in neutrophil extracellular trap formation, and that mitochondria released by neutrophils induce inflammatory cytokine production.

  5. Fisetin inhibits cellular proliferation and induces mitochondria-dependent apoptosis in human gastric cancer cells.

    PubMed

    Sabarwal, Akash; Agarwal, Rajesh; Singh, Rana P

    2017-02-01

    The anticancer effects of fisetin, a dietary agent, are largely unknown against human gastric cancer. Herein, we investigated the mechanisms of fisetin-induced inhibition of growth and survival of human gastric carcinoma AGS and SNU-1 cells. Fisetin (25-100 μM) caused significant decrease in the levels of G1 phase cyclins and CDKs, and increased the levels of p53 and its S15 phosphorylation in gastric cancer cells. We also observed that growth suppression and death of non-neoplastic human intestinal FHs74int cells were minimally affected by fisetin. Fisetin strongly increased apoptotic cells and showed mitochondrial membrane depolarization in gastric cancer cells. DNA damage was observed as early as 3 h after fisetin treatment which was accompanied with gamma-H2A.X(S139) phosphorylation and cleavage of PARP. Fisetin-induced apoptosis was observed to be independent of p53. DCFDA and MitoSOX analyses showed an increase in mitochondrial ROS generation in time- and dose-dependent fashion. It also increased cellular nitrite and superoxide generation.