Characterization of the Respiration-Induced Yeast Mitochondrial Permeability Transition Pore

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Bradshaw, Patrick C.; Pfeiffer, Douglas R.

2013-01-01

When isolated mitochondria from the yeast Saccharomyces cerevisiae oxidize respiratory substrates in the absence of phosphate and ADP, the yeast mitochondrial unselective channel, also called the yeast permeability transition pore (yPTP), opens in the inner membrane dissipating the electrochemical gradient. ATP also induces yPTP opening. yPTP opening allows mannitol transport into isolated mitochondria of laboratory yeast strains, but mannitol is not readily permeable throug...

How do yeast sense mitochondrial dysfunction?

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Dmitry A. Knorre

2016-09-01

Full Text Available Apart from energy transformation, mitochondria play important signaling roles. In yeast, mitochondrial signaling relies on several molecular cascades. However, it is not clear how a cell detects a particular mitochondrial malfunction. The problem is that there are many possible manifestations of mitochondrial dysfunction. For example, exposure to the specific antibiotics can either decrease (inhibitors of respiratory chain or increase (inhibitors of ATP-synthase mitochondrial transmembrane potential. Moreover, even in the absence of the dysfunctions, a cell needs feedback from mitochondria to coordinate mitochondrial biogenesis and/or removal by mitophagy during the division cycle. To cope with the complexity, only a limited set of compounds is monitored by yeast cells to estimate mitochondrial functionality. The known examples of such compounds are ATP, reactive oxygen species, intermediates of amino acids synthesis, short peptides, Fe-S clusters and heme, and also the precursor proteins which fail to be imported by mitochondria. On one hand, the levels of these molecules depend not only on mitochondria. On the other hand, these substances are recognized by the cytosolic sensors which transmit the signals to the nucleus leading to general, as opposed to mitochondria-specific, transcriptional response. Therefore, we argue that both ways of mitochondria-to-nucleus communication in yeast are mostly (if not completely unspecific, are mediated by the cytosolic signaling machinery and strongly depend on cellular metabolic state.

Saccharomyces cerevisiae mitochondria are required for optimal attractiveness to Drosophila melanogaster

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Schiabor, Kelly M.; Quan, Allison S.; Eisen, Michael B.

2014-01-01

While screening a large collection of wild and laboratory yeast strains for their ability to attract Drosophila melanogaster adults, we noticed a large difference in fly preference for two nearly isogenic strains of Saccharomyces cerevisiae, BY4741 and BY4742. Using standard genetic analyses, we tracked the preference difference to the lack of mitochondria in the BY4742 strain used in the initial experiment. We used gas chromatography coupled with mass spectroscopy to examine the volatile compounds produced by BY4741 and the mitochondria-deficient BY4742, and found that they differed significantly. We observed that several ethyl esters are present at much higher levels in strains with mitochondria, even in fermentative conditions. We found that nitrogen levels in the substrate affect the production of these compounds, and that they are produced at the highest level by strains with mitochondria when fermenting natural fruit substrates. Collectively these observations demonstrate that core metabolic processes mediate the interaction between yeasts and insect vectors, and highlight the importance mitochondrial functions in yeast ecology. PMID:25462617

Rewiring yeast acetate metabolism through MPC1 loss of function leads to mitochondrial damage and decreases chronological lifespan

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Ivan Orlandi

2014-11-01

Full Text Available During growth on fermentable substrates, such as glucose, pyruvate, which is the end-product of glycolysis, can be used to generate acetyl-CoA in the cytosol via acetaldehyde and acetate, or in mitochondria by direct oxidative decarboxylation. In the latter case, the mitochondrial pyruvate carrier (MPC is responsible for pyruvate transport into mitochondrial matrix space. During chronological aging, yeast cells which lack the major structural subunit Mpc1 display a reduced lifespan accompanied by an age-dependent loss of autophagy. Here, we show that the impairment of pyruvate import into mitochondria linked to Mpc1 loss is compensated by a flux redirection of TCA cycle intermediates through the malic enzyme-dependent alternative route. In such a way, the TCA cycle operates in a “branched” fashion to generate pyruvate and is depleted of intermediates. Mutant cells cope with this depletion by increasing the activity of glyoxylate cycle and of the pathway which provides the nucleocytosolic acetyl-CoA. Moreover, cellular respiration decreases and ROS accumulate in the mitochondria which, in turn, undergo severe damage. These acquired traits in concert with the reduced autophagy restrict cell survival of the mpc1∆ mutant during chronological aging. Conversely, the activation of the carnitine shuttle by supplying acetyl-CoA to the mitochondria is sufficient to abrogate the short-lived phenotype of the mutant.

Lack of HXK2 Induces Localization of Active Ras in Mitochondria and Triggers Apoptosis in the Yeast Saccharomyces cerevisiae

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Loredana Amigoni

2013-01-01

Full Text Available We recently showed that activated Ras proteins are localized to the plasma membrane and in the nucleus in wild-type cells growing exponentially on glucose, while in the hxk2Δ strain they accumulated mainly in mitochondria. An aberrant accumulation of activated Ras in these organelles was previously reported and correlated to mitochondrial dysfunction, accumulation of ROS, and cell death. Here we show that addition of acetic acid to wild-type cells results in a rapid recruitment of Ras-GTP from the nucleus and the plasma membrane to the mitochondria, providing a further proof that Ras proteins might be involved in programmed cell death. Moreover, we show that Hxk2 protects against apoptosis in S. cerevisiae. In particular, cells lacking HXK2 and showing a constitutive accumulation of activated Ras at the mitochondria are more sensitive to acetic-acid-induced programmed cell death compared to the wild type strain. Indeed, deletion of HXK2 causes an increase of apoptotic cells with several morphological and biochemical changes that are typical of apoptosis, including DNA fragmentation, externalization of phosphatidylserine, and ROS production. Finally, our results suggest that apoptosis induced by lack of Hxk2 may not require the activation of Yca1, the metacaspase homologue identified in yeast.

Lack of HXK2 induces localization of active Ras in mitochondria and triggers apoptosis in the yeast Saccharomyces cerevisiae.

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Amigoni, Loredana; Martegani, Enzo; Colombo, Sonia

2013-01-01

We recently showed that activated Ras proteins are localized to the plasma membrane and in the nucleus in wild-type cells growing exponentially on glucose, while in the hxk2Δ strain they accumulated mainly in mitochondria. An aberrant accumulation of activated Ras in these organelles was previously reported and correlated to mitochondrial dysfunction, accumulation of ROS, and cell death. Here we show that addition of acetic acid to wild-type cells results in a rapid recruitment of Ras-GTP from the nucleus and the plasma membrane to the mitochondria, providing a further proof that Ras proteins might be involved in programmed cell death. Moreover, we show that Hxk2 protects against apoptosis in S. cerevisiae. In particular, cells lacking HXK2 and showing a constitutive accumulation of activated Ras at the mitochondria are more sensitive to acetic-acid-induced programmed cell death compared to the wild type strain. Indeed, deletion of HXK2 causes an increase of apoptotic cells with several morphological and biochemical changes that are typical of apoptosis, including DNA fragmentation, externalization of phosphatidylserine, and ROS production. Finally, our results suggest that apoptosis induced by lack of Hxk2 may not require the activation of Yca1, the metacaspase homologue identified in yeast.

A proteomic and metabolomic approach for understanding the role of the flor yeast mitochondria in the velum formation.

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Moreno-García, Jaime; García-Martínez, Teresa; Moreno, Juan; Millán, M Carmen; Mauricio, Juan Carlos

2014-02-17

Saccharomyces cerevisiae "flor" yeast shows a strong tolerance to high ethanol concentrations and develops a velum (biofilm) on the wine surface after the alcoholic fermentation of grape must. This velum remains along several years during the so called "biological aging" process in the elaboration of some special wines carried out in specific regions around the world and it contributes to the typical organoleptic characteristics of these wines. In order to grow in this condition, flor yeast has to elaborate a response where the mitochondrial function is essential. The objective of this study is to elucidate the role of the mitochondria in the response of a flor yeast, S. cerevisiae G1, growing in a controlled velum formation condition. For this purpose, proteome and metabolome were characterized by comparing data with those from an initial fermentative condition used as reference. The obtained proteomic profiles show more mitochondrial proteins related with the ethanol resistance (13), cell respiration (18), mitochondrial genome maintenance (13), and apoptosis (2) detected under the velum formation condition. Also, the finger-printing obtained by means of the exo-metabolites directly related with the quality of fermented beverages and quantified in the velum condition shows important differences from those obtained in the reference condition. Copyright © 2013 Elsevier B.V. All rights reserved.

Immobilization of Mitochondria on Graphene

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2013-08-29

poly-L-lysine has also been reported for immobilization of yeast mitochondria. Coating was performed by repetitive washing of cover slips with 0.02...of Poly-L-lysine Applications of PLL PLL is a production of bacterial fermentation and is used as a food preservative. In biology, PLL is used in

Differential Proteome Analysis of a Flor Yeast Strain under Biofilm Formation.

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Moreno-García, Jaime; Mauricio, Juan Carlos; Moreno, Juan; García-Martínez, Teresa

2017-03-28

Several Saccharomyces cerevisiae strains (flor yeasts) form a biofilm (flor velum) on the surface of Sherry wines after fermentation, when glucose is depleted. This flor velum is fundamental to biological aging of these particular wines. In this study, we identify abundant proteins in the formation of the biofilm of an industrial flor yeast strain. A database search to enrich flor yeast "biological process" and "cellular component" according to Gene Ontology Terminology (GO Terms) and, "pathways" was carried out. The most abundant proteins detected were largely involved in respiration, translation, stress damage prevention and repair, amino acid metabolism (glycine, isoleucine, leucine and arginine), glycolysis/gluconeogenesis and biosynthesis of vitamin B9 (folate). These proteins were located in cellular components as in the peroxisome, mitochondria, vacuole, cell wall and extracellular region; being these two last directly related with the flor formation. Proteins like Bgl2p, Gcv3p, Hyp2p, Mdh1p, Suc2p and Ygp1p were quantified in very high levels. This study reveals some expected processes and provides new and important information for the design of conditions and genetic constructions of flor yeasts for improving the cellular survival and, thus, to optimize biological aging of Sherry wine production.

Nanolaser Spectroscopy of Genetically Engineered Yeast: New Tool for a Better Brew?

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Gourley, Paul L.; Hendricks, Judy K.; Naviaux, Robert K.; Yaffe, Michael P.

2006-03-01

A basic function of the cell membrane is to selectively uptake ions or molecules from its environment to concentrate them into the interior. This concentration difference results in an osmostic pressure difference across the membrane. Ultimately, this pressure and its fluctuation from cell to cell will be limited by the availability and fluctuations of the solute concentrations in solution, the extent of inter-cell communication, and the state of respiring intracellular mitochondria that fuel the process. To measure these fluctuations, we have employed a high-speed nanolaser technique that samples the osmotic pressure in individual yeast cells and isolated mitochondria. We analyzed 2 yeast cell strains, normal baker’s yeast and a genetically-altered version, that differ only by the presence of mitochondrial DNA. 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. These cells have mitochondria, but the mitochondria lack most normal respiratory chain complexes. The frequency distributions in the nanolaser spectra produced by wild-type and modified cells and mitochondria show a striking shift from Gaussian to Poissonian distributions, revealing a powerful novel method for studying statistical physics of yeast.

Assessment of the role of oxygen and mitochondria in heat shock induction of radiation and thermal resistance in Saccharomyces cerevisiae

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Mitchel, R.E.J.; Morrison, D.P.

1983-01-01

In response to a heat shock, the yeast Saccharomyces cerevisiae undergoes a large increase in its resistance to heat and, by the induction of its recombinational DNA repair capacity, a corresponding increase in resistance to radiation. Yeast which lack mitochondrial DNA, mitochondria-controlled protein synthetic apparatus, aerobic respiration, and electron transport (rho 0 strain) were used to assess the role of O 2 , mitochondria, and oxidative processes controlled by mitochondria in the induction of these resistances. We have found that rho 0 yeast grown and heat shocked in either the presence or absence of O 2 are capable of developing both radiation and heat resistance. We conclude that neither the stress signal nor its cellular consequences of induced heat and radiation resistance are directly dependent on O 2 , mitochondrial DNA, or mitochondria-controlled protein synthetic or oxidative processes

Depletion of eIF4G from yeast cells narrows the range of translational efficiencies genome-wide

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Hinnebusch Alan G

2011-01-01

Full Text Available Abstract Background Eukaryotic translation initiation factor 4G (eIF4G is thought to influence the translational efficiencies of cellular mRNAs by its roles in forming an eIF4F-mRNA-PABP mRNP that is competent for attachment of the 43S preinitiation complex, and in scanning through structured 5' UTR sequences. We have tested this hypothesis by determining the effects of genetically depleting eIF4G from yeast cells on global translational efficiencies (TEs, using gene expression microarrays to measure the abundance of mRNA in polysomes relative to total mRNA for ~5900 genes. Results Although depletion of eIF4G is lethal and reduces protein synthesis by ~75%, it had small effects (less than a factor of 1.5 on the relative TE of most genes. Within these limits, however, depleting eIF4G narrowed the range of translational efficiencies genome-wide, with mRNAs of better than average TE being translated relatively worse, and mRNAs with lower than average TE being translated relatively better. Surprisingly, the fraction of mRNAs most dependent on eIF4G display an average 5' UTR length at or below the mean for all yeast genes. Conclusions This finding suggests that eIF4G is more critical for ribosome attachment to mRNAs than for scanning long, structured 5' UTRs. Our results also indicate that eIF4G, and the closed-loop mRNP it assembles with the m7 G cap- and poly(A-binding factors (eIF4E and PABP, is not essential for translation of most (if not all mRNAs but enhances the differentiation of translational efficiencies genome-wide.

Study on ionizing radiosensitivity of respiratory deficiency yeast mutants

International Nuclear Information System (INIS)

Mao Shuhong; Chinese Academy of Sciences, Beijing; Jin Genming; Wei Zengquan; Xie Hongmei

2006-01-01

The radiosensitivity of respiratory deficiency yeast mutants has been studied in this work. The mutants which were screened from the yeasts after ionizing irradiation were irradiated with 12 C 6+ at different doses. Because of the great change in its mitochondria and mitochondrial DNA, the respiratory deficiency yeast mutants show radio-sensitivity at dose less than 1 Gy and radioresistance at doses higher than 1 Gy. (authors)

The enrichment of TATA box and the scarcity of depleted proximal nucleosome in the promoters of duplicated yeast genes.

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Kim, Yuseob; Lee, Jang H; Babbitt, Gregory A

2010-01-01

Population genetic theory of gene duplication suggests that the preservation of duplicate copies requires functional divergence upon duplication. Genes that can be readily modified to produce new gene expression patterns may thus be duplicated often. In yeast, genes exhibit dichotomous expression patterns based on their promoter architectures. The expression of genes that contain TATA box or occupied proximal nucleosome (OPN) tends to be variable and respond to external signals. On the other hand, genes without TATA box or with depleted proximal nucleosome (DPN) are expressed constitutively. We find that recent duplicates in the yeast genome are heavily biased to be TATA box containing genes and not to be DPN genes. This suggests that variably expressed genes, due to the functional organization in their promoters, have higher duplicability than constitutively expressed genes.

GABA transaminases from Saccharomyces cerevisiae and Arabidopsis thaliana complement function in cytosol and mitochondria.

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Cao, Juxiang; Barbosa, Jose M; Singh, Narendra; Locy, Robert D

2013-07-01

GABA transaminase (GABA-T) catalyses the conversion of GABA to succinate semialdehyde (SSA) in the GABA shunt pathway. The GABA-T from Saccharomyces cerevisiae (ScGABA-TKG) is an α-ketoglutarate-dependent enzyme encoded by the UGA1 gene, while higher plant GABA-T is a pyruvate/glyoxylate-dependent enzyme encoded by POP2 in Arabidopsis thaliana (AtGABA-T). The GABA-T from A. thaliana is localized in mitochondria and mediated by an 18-amino acid N-terminal mitochondrial targeting peptide predicated by both web-based utilities TargetP 1.1 and PSORT. Yeast UGA1 appears to lack a mitochondrial targeting peptide and is localized in the cytosol. To verify this bioinformatic analysis and examine the significance of ScGABA-TKG and AtGABA-T compartmentation and substrate specificity on physiological function, expression vectors were constructed to modify both ScGABA-TKG and AtGABA-T, so that they express in yeast mitochondria and cytosol. Physiological function was evaluated by complementing yeast ScGABA-TKG deletion mutant Δuga1 with AtGABA-T or ScGABA-TKG targeted to the cytosol or mitochondria for the phenotypes of GABA growth defect, thermosensitivity and heat-induced production of reactive oxygen species (ROS). This study demonstrates that AtGABA-T is functionally interchangeable with ScGABA-TKG for GABA growth, thermotolerance and limiting production of ROS, regardless of location in mitochondria or cytosol of yeast cells, but AtGABA-T is about half as efficient in doing so as ScGABA-TKG. These results are consistent with the hypothesis that pyruvate/glyoxylate-limited production of NADPH mediates the effect of the GABA shunt in moderating heat stress in Saccharomyces. Copyright © 2013 John Wiley & Sons, Ltd.

Characterization of the respiration-induced yeast mitochondrial permeability transition pore.

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Bradshaw, Patrick C; Pfeiffer, Douglas R

2013-12-01

When isolated mitochondria from the yeast Saccharomyces cerevisiae oxidize respiratory substrates in the absence of phosphate and ADP, the yeast mitochondrial unselective channel, also called the yeast permeability transition pore (yPTP), opens in the inner membrane, dissipating the electrochemical gradient. ATP also induces yPTP opening. yPTP opening allows mannitol transport into isolated mitochondria of laboratory yeast strains, but mannitol is not readily permeable through the yPTP in an industrial yeast strain, Yeast Foam. The presence of oligomycin, an inhibitor of ATP synthase, allowed for respiration-induced mannitol permeability in mitochondria from this strain. Potassium (K+) had varied effects on the respiration-induced yPTP, depending on the concentration of the respiratory substrate added. At low respiratory substrate concentrations K+ inhibited respiration-induced yPTP opening, while at high substrate concentrations this effect diminished. However, at the high respiratory substrate concentrations, the presence of K+ partially prevented phosphate inhibition of yPTP opening. Phosphate was found to inhibit respiration-induced yPTP opening by binding a site on the matrix space side of the inner membrane in addition to its known inhibitory effect of donating protons to the matrix space to prevent the pH change necessary for yPTP opening. The respiration-induced yPTP was also inhibited by NAD, Mg2+, NH4 + or the oxyanion vanadate polymerized to decavanadate. The results demonstrate similar effectors of the respiration-induced yPTP as those previously described for the ATP-induced yPTP and reconcile previous strain-dependent differences in yPTP solute selectivity. Copyright © 2013 John Wiley & Sons, Ltd.

Crosstalk between mitochondrial stress signals regulates yeast chronological lifespan.

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Schroeder, Elizabeth A; Shadel, Gerald S

2014-01-01

Mitochondrial DNA (mtDNA) exists in multiple copies per cell and is essential for oxidative phosphorylation. Depleted or mutated mtDNA promotes numerous human diseases and may contribute to aging. Reduced TORC1 signaling in the budding yeast, Saccharomyces cerevisiae, extends chronological lifespan (CLS) in part by generating a mitochondrial ROS (mtROS) signal that epigenetically alters nuclear gene expression. To address the potential requirement for mtDNA maintenance in this response, we analyzed strains lacking the mitochondrial base-excision repair enzyme Ntg1p. Extension of CLS by mtROS signaling and reduced TORC1 activity, but not caloric restriction, was abrogated in ntg1Δ strains that exhibited mtDNA depletion without defects in respiration. The DNA damage response (DDR) kinase Rad53p, which transduces pro-longevity mtROS signals, is also activated in ntg1Δ strains. Restoring mtDNA copy number alleviated Rad53p activation and re-established CLS extension following mtROS signaling, indicating that Rad53p senses mtDNA depletion directly. Finally, DDR kinases regulate nucleus-mitochondria localization dynamics of Ntg1p. From these results, we conclude that the DDR pathway senses and may regulate Ntg1p-dependent mtDNA stability. Furthermore, Rad53p senses multiple mitochondrial stresses in a hierarchical manner to elicit specific physiological outcomes, exemplified by mtDNA depletion overriding the ability of Rad53p to transduce an adaptive mtROS longevity signal. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Overexpression of mitochondrial oxodicarboxylate carrier (ODC1 preserves oxidative phosphorylation in a yeast model of Barth syndrome

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Maxence de Taffin de Tilques

2017-04-01

Full Text Available Cardiolipin (CL is a diglycerol phospholipid mostly found in mitochondria where it optimizes numerous processes, including oxidative phosphorylation (OXPHOS. To function properly, CL needs to be unsaturated, which requires the acyltransferase tafazzin. Loss-of-function mutations in this protein are responsible for Barth syndrome (BTHS, presumably because of a diminished OXPHOS capacity. Here, we show that overexpressing Odc1p, a conserved oxodicarboxylic acid carrier located in the mitochondrial inner membrane, fully restores oxidative phosphorylation in a yeast model (taz1Δ of BTHS. The rescuing activity involves the recovery of normal expression of key components that sustain oxidative phosphorylation, including cytochrome c and electron transport chain complexes IV and III, which are strongly downregulated in taz1Δ yeast. Interestingly, overexpression of Odc1p was also shown previously to rescue yeast models of mitochondrial diseases caused by defects in the assembly of ATP synthase and by mutations in the MPV17 protein that result in hepatocerebral mitochondrial DNA depletion syndrome. These findings define the transport of oxodicarboxylic acids across the inner membrane as a potential therapeutic target for a large spectrum of mitochondrial diseases, including BTHS.

A Powerful Mitochondria-Targeted Iron Chelator Affords High Photoprotection against Solar Ultraviolet A Radiation.

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Reelfs, Olivier; Abbate, Vincenzo; Hider, Robert C; Pourzand, Charareh

2016-08-01

Mitochondria are the principal destination for labile iron, making these organelles particularly susceptible to oxidative damage on exposure to ultraviolet A (UVA, 320-400 nm), the oxidizing component of sunlight. The labile iron-mediated oxidative damage caused by UVA to mitochondria leads to necrotic cell death via adenosine triphosphate depletion. Therefore, targeted removal of mitochondrial labile iron via highly specific tools from these organelles may be an effective approach to protect the skin cells against the harmful effects of UVA. In this work, we designed a mitochondria-targeted hexadentate (tricatechol-based) iron chelator linked to mitochondria-homing SS-like peptides. The photoprotective potential of this compound against UVA-induced oxidative damage and cell death was evaluated in cultured primary skin fibroblasts. Our results show that this compound provides unprecedented protection against UVA-induced mitochondrial damage, adenosine triphosphate depletion, and the ensuing necrotic cell death in skin fibroblasts, and this effect is fully related to its potent iron-chelating property in the organelle. This mitochondria-targeted iron chelator has therefore promising potential for skin photoprotection against the deleterious effects of the UVA component of sunlight. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Desmin common mutation is associated with multi-systemic disease manifestations and depletion of mitochondria and mitochondrial DNA

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Elizabeth eMcCormick

2015-06-01

Full Text Available Desmin (DES is a major muscle scaffolding protein that also functions to anchor mitochondria. Pathogenic DES mutations, however, have not previously been recognized as a cause of multi-systemic mitochondrial disease. Here, we describe a 45-year-old man who presented to The Children’s Hospital of Philadelphia Mitochondrial-Genetics Diagnostic Clinic for evaluation of progressive cardiac, neuromuscular, gastrointestinal, and mood disorders. Muscle biopsy at age 45 was remarkable for cytoplasmic bodies, as well as ragged red fibers and SDH positive/COX negative fibers that were suggestive of a mitochondrial myopathy. Muscle also showed significant reductions in mitochondrial content (16% of control mean for citrate synthase activity and mitochondrial DNA (35% of control mean. His family history was significant for cardiac conduction defects and myopathy in multiple maternal relatives. Multiple single gene and panel-based sequencing studies were unrevealing. Whole exome sequencing identified a known pathogenic p.S13F mutation in DES that had previously been associated with desmin-related myopathy. Desmin-related myopathy is an autosomal dominant disorder characterized by right ventricular hypertrophic cardiomyopathy, myopathy, and arrhythmias. However, neuropathy, gastrointestinal dysfunction, and depletion of both mitochondria and mitochondrial DNA have not previously been widely recognized in this disorder. Recognition that mitochondrial dysfunction occurs in desmin-related myopathy clarifies the basis for the multi-systemic manifestations, as are typical of primary mitochondrial disorders. Understanding the mitochondrial pathophysiology of desmin-related myopathy highlights the possibility of new therapies for the otherwise untreatable and often fatal class of disease. We postulate that drug treatments aimed at improving mitochondrial biogenesis or reducing oxidative stress may be effective therapies to ameliorate the effects of desmin

The role of mitochondria in carbon catabolite repression in yeast.

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Haussmann, P; Zimmermann, F K

1976-10-18

The role of mitochondria in carbon catabolite repression in Saccharomyces cerevisiae was investigated by comparing normal, respiratory competent (RHO) strains with their mitochondrially inherited, respiratory deficient mutant derivatives (rho). Formation of maltase and invertase was used as an indicator system for the effect of carbon catabolite repression on carbon catabolic reactions. Fermentation rates for glucose, maltose and sucrose were the same in RHO and rho strains. Specific activities of maltase and invertase were usually higher in the rho-mutants. A very pronounced difference in invertase levels was observed when cells were grown on maltose; rho-mutants had around 30 times more invertase than their RHO parent strains. The fact that rho-mutants were much less sensitive to carbon catabolite repression of invertase synthesis than their RHO parents was used to search for the mitochondrial factor(s) or function(s) involved in carbon catabolite repression. A possible metabolic influence of mitochondria on this system of regulation was tested after growth of RHO strains under anaerobic conditions (no respiration nor oxidative phosphorylation), in the presence of KCN (respiration inhibited), dinitrophenol (uncoupling of oxidative phosphorylation) and of both inhibitors anaerobic conditions and dinitrophenol had no effect on the extent of invertase repression. KCN reduced the degree of repression but not to the level found in rho-mutants. A combination of both inhibitors gave the same results as with KCN alone. Erythromycin and chloramphenicol were used as specific inhibitors of mitochondrial protein synthesis. Erythromycin prevented the formation of mitochondrial respiratory systems but did not induce rho-mutants under the conditions used. However, repression of invertase was as strong as in the absence of the inhibitor. Chloramphenicol led only to a slight reduction of the respiratory systems and did not affect invertase levels. A combination of both

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

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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. Copyright © 2016 Elsevier Inc. All rights reserved.

The role of cholesterol in the association of endoplasmic reticulum membranes with mitochondria

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Fujimoto, Michiko [Cellular Stress Signaling Unit, Integrative Neuroscience Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, MD 21224 (United States); Hayashi, Teruo, E-mail: thayashi@mail.nih.gov [Cellular Stress Signaling Unit, Integrative Neuroscience Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, MD 21224 (United States); Su, Tsung-Ping, E-mail: tsu@intra.nida.nih.gov [Cellular Pathobiology Section, Integrative Neuroscience Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, MD 21224 (United States)

2012-01-06

Highlights: Black-Right-Pointing-Pointer The endoplasmic reticulum subdomain termed MAM associates with mitochondria. Black-Right-Pointing-Pointer The biophysical role of lipids in the MAM-mitochondria association is unknown. Black-Right-Pointing-Pointer The in vitro membrane association assay was used to examine the role of lipids. Black-Right-Pointing-Pointer Cholesterol was found to negatively regulate the association. -- Abstract: The unique endoplasmic reticulum (ER) subdomain termed the mitochondria-associated ER membrane (MAM) engages the physical connection between the ER and the mitochondrial outer membrane and plays a role in regulating IP{sub 3} receptor-mediated Ca{sup 2+} influx and the phospholipid transport between the two organelles. The MAM contains certain signaling and membrane-tethering proteins but also lipids including cholesterol. The biophysical role of lipids at the MAM, specifically in the physical interaction between the MAM of the ER and mitochondria, remains not totally clarified. Here we employed the in vitro membrane association assay to investigate the role of cholesterol in the association between MAMs and mitochondria. The purified MAMs and mitochondria were mixed in vitro in a test tube and then the physical association of the two subcellular organelles was quantified indirectly by measuring the presence of the MAM-specific protein sigma-1 receptors in the mitochondria fraction. Purified MAMs contained free cholesterol approximately 7 times higher than that in microsomes. We found that depletion of cholesterol in MAMs with methyl-{beta}-cyclodextrin (M{beta}C) significantly increases the association between MAMs and mitochondria, whereas M{beta}C saturated with cholesterol does not change the association. {sup 14}C-Serine pulse-labeling demonstrated that the treatment of living cells with M{beta}C decreases the level of de novo synthesized {sup 14}C-phosphatidylserine (PtSer) and concomitantly increases greatly the synthesis of

The role of cholesterol in the association of endoplasmic reticulum membranes with mitochondria

International Nuclear Information System (INIS)

Fujimoto, Michiko; Hayashi, Teruo; Su, Tsung-Ping

2012-01-01

Highlights: ► The endoplasmic reticulum subdomain termed MAM associates with mitochondria. ► The biophysical role of lipids in the MAM–mitochondria association is unknown. ► The in vitro membrane association assay was used to examine the role of lipids. ► Cholesterol was found to negatively regulate the association. -- Abstract: The unique endoplasmic reticulum (ER) subdomain termed the mitochondria-associated ER membrane (MAM) engages the physical connection between the ER and the mitochondrial outer membrane and plays a role in regulating IP 3 receptor-mediated Ca 2+ influx and the phospholipid transport between the two organelles. The MAM contains certain signaling and membrane-tethering proteins but also lipids including cholesterol. The biophysical role of lipids at the MAM, specifically in the physical interaction between the MAM of the ER and mitochondria, remains not totally clarified. Here we employed the in vitro membrane association assay to investigate the role of cholesterol in the association between MAMs and mitochondria. The purified MAMs and mitochondria were mixed in vitro in a test tube and then the physical association of the two subcellular organelles was quantified indirectly by measuring the presence of the MAM-specific protein sigma-1 receptors in the mitochondria fraction. Purified MAMs contained free cholesterol approximately 7 times higher than that in microsomes. We found that depletion of cholesterol in MAMs with methyl-β-cyclodextrin (MβC) significantly increases the association between MAMs and mitochondria, whereas MβC saturated with cholesterol does not change the association. 14 C-Serine pulse-labeling demonstrated that the treatment of living cells with MβC decreases the level of de novo synthesized 14 C-phosphatidylserine (PtSer) and concomitantly increases greatly the synthesis of 14 C-phosphatidylethanolamine (PtEt). Apparently, cholesterol depletion increased the PtSer transport from MAMs to mitochondria. Our

Penetrating cation/fatty acid anion pair as a mitochondria-targeted protonophore

Science.gov (United States)

Severin, Fedor F.; Severina, Inna I.; Antonenko, Yury N.; Rokitskaya, Tatiana I.; Cherepanov, Dmitry A.; Mokhova, Elena N.; Vyssokikh, Mikhail Yu.; Pustovidko, Antonina V.; Markova, Olga V.; Yaguzhinsky, Lev S.; Korshunova, Galina A.; Sumbatyan, Nataliya V.; Skulachev, Maxim V.; Skulachev, Vladimir P.

2010-01-01

A unique phenomenon of mitochondria-targeted protonophores is described. It consists in a transmembrane H+-conducting fatty acid cycling mediated by penetrating cations such as 10-(6’-plastoquinonyl)decyltriphenylphosphonium (SkQ1) or dodecyltriphenylphosphonium (C12TPP). The phenomenon has been modeled by molecular dynamics and directly proved by experiments on bilayer planar phospholipid membrane, liposomes, isolated mitochondria, and yeast cells. In bilayer planar phospholipid membrane, the concerted action of penetrating cations and fatty acids is found to result in conversion of a pH gradient (ΔpH) to a membrane potential (Δψ) of the Nernstian value (about 60 mV Δψ at ΔpH = 1). A hydrophobic cation with localized charge (cetyltrimethylammonium) failed to substitute for hydrophobic cations with delocalized charge. In isolated mitochondria, SkQ1 and C12TPP, but not cetyltrimethylammonium, potentiated fatty acid-induced (i) uncoupling of respiration and phosphorylation, and (ii) inhibition of H2O2 formation. In intact yeast cells, C12TPP stimulated respiration regardless of the extracellular pH value, whereas a nontargeted protonophorous uncoupler (trifluoromethoxycarbonylcyanide phenylhydrazone) stimulated respiration at pH 5 but not at pH 3. Hydrophobic penetrating cations might be promising to treat obesity, senescence, and some kinds of cancer that require mitochondrial hyperpolarization. PMID:20080732

Widespread Mitochondrial Depletion via Mitophagy Does Not Compromise Necroptosis

Directory of Open Access Journals (Sweden)

Stephen W.G. Tait

2013-11-01

Full Text Available Programmed necrosis (or necroptosis is a form of cell death triggered by the activation of receptor interacting protein kinase-3 (RIPK3. Several reports have implicated mitochondria and mitochondrial reactive oxygen species (ROS generation as effectors of RIPK3-dependent cell death. Here, we directly test this idea by employing a method for the specific removal of mitochondria via mitophagy. Mitochondria-deficient cells were resistant to the mitochondrial pathway of apoptosis, but efficiently died via tumor necrosis factor (TNF-induced, RIPK3-dependent programmed necrosis or as a result of direct oligomerization of RIPK3. Although the ROS scavenger butylated hydroxyanisole (BHA delayed TNF-induced necroptosis, it had no effect on necroptosis induced by RIPK3 oligomerization. Furthermore, although TNF-induced ROS production was dependent on mitochondria, the inhibition of TNF-induced necroptosis by BHA was observed in mitochondria-depleted cells. Our data indicate that mitochondrial ROS production accompanies, but does not cause, RIPK3-dependent necroptotic cell death.

Studies on the mechanism of pyrophosphate-mediated uptake of iron from transferrin by isolated rat-liver mitochondria

International Nuclear Information System (INIS)

Konopka, K.; Romslo, I.; Bergen Univ.

1981-01-01

1. Respiring rat liver mitochondria accumulate iron released from transferrin by pyrophosphate. The amount of iron accumulated is 1-1.5 nmol mg protein -1 h -1 , or approximately 60% of the amount of iron mobilized from transferrin. 2. The uptake declines if respiration is inhibited, substrate is depleted, or the experiments are run under anaerobic conditions. Substrate, depletion and respiratory inhibitors are less inhibitory under anaerobic conditions. 3. More than 80% of the amount of iron accumulated by aerobic, actively respiring mitochondria can be chelated by bathophenanthroline sulphonate, and with deuteroporphyrin included, up to 30% of the amount of iron accumulated is recovered as deuteroheme. Iron accumulated by respiration-inhibited mitochondria under aerobic conditions is not available for heme synthesis. 4. With time the uptake of iron increases eightfold relative to the uptake of pyrophosphate. 5. The results are compatible with a model in which ferric iron is mobilized from transferrin by pyrophosphate, ferric iron pyrophosphate is bound to the mitochondria, iron is reduced, dissociates from pyrophosphate and is taken up by the mitochondria. Ferrous irons thus formed is available for heme synthesis. (orig.) [de

A Powerful Mitochondria-Targeted Iron Chelator Affords High Photoprotection against Solar Ultraviolet A Radiation

OpenAIRE

Reelfs, Olivier; Abbate, Vincenzo; Hider, Robert C.; Pourzand, Charareh

2016-01-01

Mitochondria are the principal destination for labile iron, making these organelles particularly susceptible to oxidative damage on exposure to ultraviolet A (UVA, 320?400 nm), the oxidizing component of sunlight. The labile iron-mediated oxidative damage caused by UVA to mitochondria leads to necrotic cell death via adenosine triphosphate depletion. Therefore, targeted removal of mitochondrial labile iron via highly specific tools from these organelles may be an effective approach to protect...

Fe-S Cluster Biogenesis in Isolated Mammalian Mitochondria

Science.gov (United States)

Pandey, Alok; Pain, Jayashree; Ghosh, Arnab K.; Dancis, Andrew; Pain, Debkumar

2015-01-01

Iron-sulfur (Fe-S) clusters are essential cofactors, and mitochondria contain several Fe-S proteins, including the [4Fe-4S] protein aconitase and the [2Fe-2S] protein ferredoxin. Fe-S cluster assembly of these proteins occurs within mitochondria. Although considerable data exist for yeast mitochondria, this biosynthetic process has never been directly demonstrated in mammalian mitochondria. Using [35S]cysteine as the source of sulfur, here we show that mitochondria isolated from Cath.A-derived cells, a murine neuronal cell line, can synthesize and insert new Fe-35S clusters into aconitase and ferredoxins. The process requires GTP, NADH, ATP, and iron, and hydrolysis of both GTP and ATP is necessary. Importantly, we have identified the 35S-labeled persulfide on the NFS1 cysteine desulfurase as a genuine intermediate en route to Fe-S cluster synthesis. In physiological settings, the persulfide sulfur is released from NFS1 and transferred to a scaffold protein, where it combines with iron to form an Fe-S cluster intermediate. We found that the release of persulfide sulfur from NFS1 requires iron, showing that the use of iron and sulfur for the synthesis of Fe-S cluster intermediates is a highly coordinated process. The release of persulfide sulfur also requires GTP and NADH, probably mediated by a GTPase and a reductase, respectively. ATP, a cofactor for a multifunctional Hsp70 chaperone, is not required at this step. The experimental system described here may help to define the biochemical basis of diseases that are associated with impaired Fe-S cluster biogenesis in mitochondria, such as Friedreich ataxia. PMID:25398879

Apoptotic role of TGF-β mediated by Smad4 mitochondria translocation and cytochrome c oxidase subunit II interaction.

Science.gov (United States)

Pang, Lijuan; Qiu, Tao; Cao, Xu; Wan, Mei

2011-07-01

Smad4, originally isolated from the human chromosome 18q21, is a key factor in transducing the signals of the TGF-β superfamily of growth hormones and plays a pivotal role in mediating antimitogenic and proapoptotic effects of TGF-β, but the mechanisms by which Smad4 induces apoptosis are elusive. Here we report that Smad4 directly translocates to the mitochondria of apoptotic cells. Smad4 gene silencing by siRNA inhibits TGF-β-induced apoptosis in Hep3B cells and UV-induced apoptosis in PANC-1 cells. Cell fractionation assays demonstrated that a fraction of Smad4 translocates to mitochondria after long time TGF-β treatment or UV exposure, during which the cells were under apoptosis. Smad4 mitochondria translocation during apoptosis was also confirmed by fluorescence observation of Smad4 colocalization with MitoTracker Red. We searched for mitochondria proteins that have physical interactions with Smad4 using yeast two-hybrid screening approach. DNA sequence analysis identified 34 positive clones, five of which encoded subunits in mitochondria complex IV, i.e., one clone encoded cytochrome c oxidase COXII, three clones encoded COXIII and one clone encoded COXVb. Strong interaction between Smad4 with COXII, an important apoptosis regulator, was verified in yeast by β-gal activity assays and in mammalian cells by immunoprecipitation assays. Further, mitochondrial portion of cells was isolated and the interaction between COXII and Smad4 in mitochondria upon TGF-β treatment or UV exposure was confirmed. Importantly, targeting Smad4 to mitochondria using import leader fusions enhanced TGF-β-induced apoptosis. Collectively, the results suggest that Smad4 promote apoptosis of the cells through its mitochondrial translocation and association with mitochondria protein COXII. Copyright © 2011 Elsevier Inc. All rights reserved.

Mitochondrial localization of fission yeast manganese superoxide dismutase is required for its lysine acetylation and for cellular stress resistance and respiratory growth

International Nuclear Information System (INIS)

Takahashi, Hidekazu; Suzuki, Takehiro; Shirai, Atsuko; Matsuyama, Akihisa; Dohmae, Naoshi; Yoshida, Minoru

2011-01-01

Research highlights: → Fission yeast manganese superoxide dismutase (MnSOD) is acetylated. → The mitochondrial targeting sequence (MTS) is required for the acetylation of MnSOD. → The MTS is not crucial for MnSOD activity, but is important for respiratory growth. → Posttranslational regulation of MnSOD differs between budding and fission yeast. -- Abstract: Manganese-dependent superoxide dismutase (MnSOD) is localized in the mitochondria and is important for oxidative stress resistance. Although transcriptional regulation of MnSOD has been relatively well studied, much less is known about the protein's posttranslational regulation. In budding yeast, MnSOD is activated after mitochondrial import by manganese ion incorporation. Here we characterize posttranslational modification of MnSOD in the fission yeast Schizosaccharomyces pombe. Fission yeast MnSOD is acetylated at the 25th lysine residue. This acetylation was diminished by deletion of N-terminal mitochondrial targeting sequence, suggesting that MnSOD is acetylated after import into mitochondria. Mitochondrial localization of MnSOD is not essential for the enzyme activity, but is crucial for oxidative stress resistance and growth under respiratory conditions of fission yeast. These results suggest that, unlike the situation in budding yeast, S. pombe MnSOD is already active even before mitochondrial localization; nonetheless, mitochondrial localization is critical to allow the cell to cope with reactive oxygen species generated inside or outside of mitochondria.

Mitochondrial import of human and yeast fumarase in live mammalian cells: Retrograde translocation of the yeast enzyme is mainly caused by its poor targeting sequence

International Nuclear Information System (INIS)

Singh, Bhag; Gupta, Radhey S.

2006-01-01

Studies on yeast fumarase provide the main evidence for dual localization of a protein in mitochondria and cytosol by means of retrograde translocation. We have examined the subcellular targeting of yeast and human fumarase in live cells to identify factors responsible for this. The cDNAs for mature yeast or human fumarase were fused to the gene for enhanced green fluorescent protein (eGFP) and they contained, at their N-terminus, a mitochondrial targeting sequence (MTS) derived from either yeast fumarase, human fumarase, or cytochrome c oxidase subunit VIII (COX) protein. Two nuclear localization sequences (2x NLS) were also added to these constructs to facilitate detection of any cytosolic protein by its targeting to nucleus. In Cos-1 cells transfected with these constructs, human fumarase with either the native or COX MTSs was detected exclusively in mitochondria in >98% of the cells, while the remainder 1-2% of the cells showed varying amounts of nuclear labeling. In contrast, when human fumarase was fused to the yeast MTS, >50% of the cells showed nuclear labeling. Similar studies with yeast fumarase showed that with its native MTS, nuclear labeling was seen in 80-85% of the cells, but upon fusion to either human or COX MTS, nuclear labeling was observed in only 10-15% of the cells. These results provide evidence that extramitochondrial presence of yeast fumarase is mainly caused by the poor mitochondrial targeting characteristics of its MTS (but also affected by its primary sequence), and that the retrograde translocation mechanism does not play a significant role in the extramitochondrial presence of mammalian fumarase

Polyglutamine toxicity in yeast induces metabolic alterations and mitochondrial defects

KAUST Repository

Papsdorf, Katharina; Kaiser, Christoph J. O.; Drazic, Adrian; Grö tzinger, Stefan W.; Haeß ner, Carmen; Eisenreich, Wolfgang; Richter, Klaus

2015-01-01

formation. Indeed, we find that in vivo iron concentrations are misbalanced and observe a reduction in the activity of the prominent Fe-S cluster containing protein aconitase. Like in other yeast strains with impaired mitochondria, non-fermentative growth

Biogenesis of the yeast cytochrome bc1 complex.

Science.gov (United States)

Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L

2009-01-01

The mitochondrial respiratory chain is composed of four different protein complexes that cooperate in electron transfer and proton pumping across the inner mitochondrial membrane. The cytochrome bc1 complex, or complex III, is a component of the mitochondrial respiratory chain. This review will focus on the biogenesis of the bc1 complex in the mitochondria of the yeast Saccharomyces cerevisiae. In wild type yeast mitochondrial membranes the major part of the cytochrome bc1 complex was found in association with one or two copies of the cytochrome c oxidase complex. The analysis of several yeast mutant strains in which single genes or pairs of genes encoding bc1 subunits had been deleted revealed the presence of a common set of bc1 sub-complexes. These sub-complexes are represented by the central core of the bc1 complex, consisting of cytochrome b bound to subunit 7 and subunit 8, by the two core proteins associated with each other, by the Rieske protein associated with subunit 9, and by those deriving from the unexpected interaction of each of the two core proteins with cytochrome c1. Furthermore, a higher molecular mass sub-complex is that composed of cytochrome b, cytochrome c1, core protein 1 and 2, subunit 6, subunit 7 and subunit 8. The identification and characterization of all these sub-complexes may help in defining the steps and the molecular events leading to bc1 assembly in yeast mitochondria.

Live cell imaging of mitochondrial movement along actin cables in budding yeast.

Science.gov (United States)

Fehrenbacher, Kammy L; Yang, Hyeong-Cheol; Gay, Anna Card; Huckaba, Thomas M; Pon, Liza A

2004-11-23

Mitochondrial inheritance is essential for cell division. In budding yeast, mitochondrial movement from mother to daughter requires (1) actin cables, F-actin bundles that undergo retrograde movement during elongation from buds into mother cells; (2) the mitochore, a mitochondrial protein complex implicated in linking mitochondria to actin cables; and (3) Arp2/3 complex-mediated force generation on mitochondria. We observed three new classes of mitochondrial motility: anterograde movement at velocities of 0.2-0.33 microm/s, retrograde movement at velocities of 0.26-0.51 microm/s, and no net anterograde or retrograde movement. In all cases, motile mitochondria were associated with actin cables undergoing retrograde flow at velocities of 0.18-0.62 microm/s. Destabilization of actin cables or mutations of the mitochore blocked all mitochondrial movements. In contrast, mutations in the Arp2/3 complex affected anterograde but not retrograde mitochondrial movements. Actin cables are required for movement of mitochondria, secretory vesicles, mRNA, and spindle alignment elements in yeast. We provide the first direct evidence that one of the proposed cargos use actin cables as tracks. In the case of mitochondrial inheritance, anterograde movement drives transfer of the organelle from mothers to buds, while retrograde movement contributes to retention of the organelle in mother cells. Interaction of mitochondria with actin cables is required for anterograde and retrograde movement. In contrast, force generation on mitochondria is required only for anterograde movement. Finally, we propose a novel mechanism in which actin cables serve as "conveyor belts" that drive retrograde organelle movement.

Screening the yeast genome for energetic metabolism pathways involved in a phenotypic response to the anti-cancer agent 3-bromopyruvate.

Science.gov (United States)

Lis, Paweł; Jurkiewicz, Paweł; Cal-Bąkowska, Magdalena; Ko, Young H; Pedersen, Peter L; Goffeau, Andre; Ułaszewski, Stanisław

2016-03-01

In this study the detailed characteristic of the anti-cancer agent 3-bromopyruvate (3-BP) activity in the yeast Saccharomyces cerevisiae model is described, with the emphasis on its influence on energetic metabolism of the cell. It shows that 3-BP toxicity in yeast is strain-dependent and influenced by the glucose-repression system. Its toxic effect is mainly due to the rapid depletion of intracellular ATP. Moreover, lack of the Whi2p phosphatase results in strongly increased sensitivity of yeast cells to 3-BP, possibly due to the non-functional system of mitophagy of damaged mitochondria through the Ras-cAMP-PKA pathway. Single deletions of genes encoding glycolytic enzymes, the TCA cycle enzymes and mitochondrial carriers result in multiple effects after 3-BP treatment. However, it can be concluded that activity of the pentose phosphate pathway is necessary to prevent the toxicity of 3-BP, probably due to the fact that large amounts of NADPH are produced by this pathway, ensuring the reducing force needed for glutathione reduction, crucial to cope with the oxidative stress. Moreover, single deletions of genes encoding the TCA cycle enzymes and mitochondrial carriers generally cause sensitivity to 3-BP, while totally inactive mitochondrial respiration in the rho0 mutant resulted in increased resistance to 3-BP.

Radioprotective effects of Asparagus racemosus extracts against free radical damage in rat liver mitochondria

International Nuclear Information System (INIS)

Boloor, K.K.; Kamat, J.P.; Devasagayam, T.P.A.; Venkatachalam, S.R.

2000-01-01

The possible antioxidant effect of the extracts of Asparagus racemosus against membrane damage induced by free radicals generated during γ-radiation was examined in rat liver/brain mitochondria. These extracts displayed significant antioxidant properties in mitochondria against oxidation of both lipids and proteins as assessed by lipid peroxidation, protein oxidation and depletion of thiols. The inhibitory effect of the extracts, rich in polysaccharides like galactose, was more than that of the established antioxidants glutathione and ascorbic acid. (author)

Yeast as a Tool to Study Signaling Pathways in Mitochondrial Stress Response and Cytoprotection

Directory of Open Access Journals (Sweden)

Maša Ždralević

2012-01-01

Full Text Available Cell homeostasis results from the balance between cell capability to adapt or succumb to environmental stress. Mitochondria, in addition to supplying cellular energy, are involved in a range of processes deciding about cellular life or death. The crucial role of mitochondria in cell death is well recognized. Mitochondrial dysfunction has been associated with the death process and the onset of numerous diseases. Yet, mitochondrial involvement in cellular adaptation to stress is still largely unexplored. Strong interest exists in pharmacological manipulation of mitochondrial metabolism and signaling. The yeast Saccharomyces cerevisiae has proven a valuable model organism in which several intracellular processes have been characterized in great detail, including the retrograde response to mitochondrial dysfunction and, more recently, programmed cell death. In this paper we review experimental evidences of mitochondrial involvement in cytoprotection and propose yeast as a model system to investigate the role of mitochondria in the cross-talk between prosurvival and prodeath pathways.

[Genetic system for maintaining the mitochondrial human genome in yeast Yarrowia lipolytica].

Science.gov (United States)

Isakova, E P; Deryabina, Yu I; Velyakova, A V; Biryukova, J K; Teplova, V V; Shevelev, A B

2016-01-01

For the first time, the possibility of maintaining an intact human mitochondrial genome in a heterologous system in the mitochondria of yeast Yarrowia lipolytica is shown. A method for introducing directional changes into the structure of the mitochondrial human genome replicating in Y. lipolytica by an artificially induced ability of yeast mitochondria for homologous recombination is proposed. A method of introducing and using phenotypic selection markers for the presence or absence of defects in genes tRNA-Lys and tRNA-Leu of the mitochondrial genome is developed. The proposed system can be used to correct harmful mutations of the human mitochondrial genome associated with mitochondrial diseases and for preparative amplification of intact mitochondrial DNA with an adjusted sequence in yeast cells. The applicability of the new system for the correction of mutations in the genes of Lys- and Leu-specific tRNAs of the human mitochondrial genome associated with serious and widespread human mitochondrial diseases such as myoclonic epilepsy with lactic acidosis (MELAS) and myoclonic epilepsy with ragged-red fibers (MERRF) is shown.

Yeast as a system for modeling mitochondrial disease mechanisms and discovering therapies

Directory of Open Access Journals (Sweden)

Jean-Paul Lasserre

2015-06-01

Full Text Available Mitochondrial diseases are severe and largely untreatable. Owing to the many essential processes carried out by mitochondria and the complex cellular systems that support these processes, these diseases are diverse, pleiotropic, and challenging to study. Much of our current understanding of mitochondrial function and dysfunction comes from studies in the baker's yeast Saccharomyces cerevisiae. Because of its good fermenting capacity, S. cerevisiae can survive mutations that inactivate oxidative phosphorylation, has the ability to tolerate the complete loss of mitochondrial DNA (a property referred to as ‘petite-positivity’, and is amenable to mitochondrial and nuclear genome manipulation. These attributes make it an excellent model system for studying and resolving the molecular basis of numerous mitochondrial diseases. Here, we review the invaluable insights this model organism has yielded about diseases caused by mitochondrial dysfunction, which ranges from primary defects in oxidative phosphorylation to metabolic disorders, as well as dysfunctions in maintaining the genome or in the dynamics of mitochondria. Owing to the high level of functional conservation between yeast and human mitochondrial genes, several yeast species have been instrumental in revealing the molecular mechanisms of pathogenic human mitochondrial gene mutations. Importantly, such insights have pointed to potential therapeutic targets, as have genetic and chemical screens using yeast.

A survey of the interaction of calcium ions with mitochondria from different tissues and species

Science.gov (United States)

Carafoli, Ernesto; Lehninger, Albert L.

1971-01-01

A survey was made of the capacity of mitochondria isolated from a number of different tissues and species to accumulate Ca2+ from the suspending medium during electron transport. The species examined included the rat, mouse, rabbit, hamster, guinea pig, cow, chicken, turtle, blowfly, yeast and Neurospora crassa. The tissues examined included vertebrate liver, kidney, brain, heart, spleen, thyroid and adrenal cortex, and the flight muscle of the blowfly. The mitochondria from all vertebrate tissues examined showed: (a) stimulation of State 4 respiration by added Ca2+ (Ca2+/~ activation ratio about 2.0), accompanied by accumulation of Ca2+ and ejection of H+, with a H+/Ca2+ ratio about 1.0; (b) a requirement of phosphate for accumulation of large amounts of Ca2+; (c) respiration-independent high-affinity binding sites for Ca2+; (d) endogenous Ca2+, which is largely released by uncoupling agents. However, mitochondria from yeast and blowfly flight muscle are unable to accumulate Ca2+ in a respiration-dependent process and possess no high-affinity Ca2+-binding sites. These findings support the view that the high-affinity sites represent the ligand-binding sites of a specific Ca2+ `permease' or transport system in the membrane. The relatively high affinity for Ca2+, which equals or exceeds the affinity for ADP, and the generally uniform characteristics of Ca2+ transport in all the vertebrate mitochondria tested strongly suggest that respiration-linked Ca2+ accumulation plays a general and fundamental role in vertebrate cell physiology. PMID:5129264

Interaction Between Yeasts and Zinc

Science.gov (United States)

Nicola, Raffaele De; Walker, Graeme

Zinc is an essential trace element in biological systems. For example, it acts as a cellular membrane stabiliser, plays a critical role in gene expression and genome modification and activates nearly 300 enzymes, including alcohol dehydrogenase. The present chapter will be focused on the influence of zinc on cell physiology of industrial yeast strains of Saccharomyces cerevisiae, with special regard to the uptake and subsequent utilisation of this metal. Zinc uptake by yeast is metabolism-dependent, with most of the available zinc translocated very quickly into the vacuole. At cell division, zinc is distributed from mother to daughter cells and this effectively lowers the individual cellular zinc concentration, which may become zinc depleted at the onset of the fermentation. Zinc influences yeast fermentative performance and examples will be provided relating to brewing and wine fermentations. Industrial yeasts are subjected to several stresses that may impair fermentation performance. Such stresses may also impact on yeast cell zinc homeostasis. This chapter will discuss the practical implications for the correct management of zinc bioavailability for yeast-based biotechnologies aimed at improving yeast growth, viability, fermentation performance and resistance to environmental stresses

Electron transport chain in a thermotolerant yeast.

Science.gov (United States)

Mejía-Barajas, Jorge A; Martínez-Mora, José A; Salgado-Garciglia, Rafael; Noriega-Cisneros, Ruth; Ortiz-Avila, Omar; Cortés-Rojo, Christian; Saavedra-Molina, Alfredo

2017-04-01

Yeasts capable of growing and surviving at high temperatures are regarded as thermotolerant. For appropriate functioning of cellular processes and cell survival, the maintenance of an optimal redox state is critical of reducing and oxidizing species. We studied mitochondrial functions of the thermotolerant Kluyveromyces marxianus SLP1 and the mesophilic OFF1 yeasts, through the evaluation of its mitochondrial membrane potential (ΔΨ m ), ATPase activity, electron transport chain (ETC) activities, alternative oxidase activity, lipid peroxidation. Mitochondrial membrane potential and the cytoplasmic free Ca 2+ ions (Ca 2+ cyt) increased in the SLP1 yeast when exposed to high temperature, compared with the mesophilic yeast OFF1. ATPase activity in the mesophilic yeast diminished 80% when exposed to 40° while the thermotolerant SLP1 showed no change, despite an increase in the mitochondrial lipid peroxidation. The SLP1 thermotolerant yeast exposed to high temperature showed a diminution of 33% of the oxygen consumption in state 4. The uncoupled state 3 of oxygen consumption did not change in the mesophilic yeast when it had an increase of temperature, whereas in the thermotolerant SLP1 yeast resulted in an increase of 2.5 times when yeast were grown at 30 o , while a decrease of 51% was observed when it was exposed to high temperature. The activities of the ETC complexes were diminished in the SLP1 when exposed to high temperature, but also it was distinguished an alternative oxidase activity. Our results suggest that the mitochondria state, particularly ETC state, is an important characteristic of the thermotolerance of the SLP1 yeast strain.

Promotion of mitochondrial membrane complex assembly by a proteolytically inactive yeast Lon

NARCIS (Netherlands)

Rep, M; van Dijl, J M; Suda, K; Schatz, G; Grivell, L A; Suzuki, C K

1996-01-01

Afg3p and Rca1p are adenosine triphosphate (ATP)-dependent metalloproteases in yeast mitochondria. Cells lacking both proteins exhibit defects in respiration-dependent growth, degradation of mitochondrially synthesized proteins, and assembly of inner-membrane complexes. Defects in growth and protein

Mitochondria as determinant of nucleotide pools and chromosomal stability

DEFF Research Database (Denmark)

Madsen, Claus Desler; Munch-Petersen, Birgitte; Stevnsner, Tinna

2007-01-01

Mitochondrial function plays an important role in multiple human diseases and mutations in the mitochondrial genome have been detected in nearly every type of cancer investigated to date. However, the mechanism underlying the interrelation is unknown. We used human cell lines depleted of mitochon...... mitochondrial activity. Our results suggest that mitochondria are central players in maintaining genomic stability and in controlling essential nuclear processes such as upholding a balanced supply of nucleotides....

Supplementary Material for: Polyglutamine toxicity in yeast induces metabolic alterations and mitochondrial defects

KAUST Repository

Papsdorf, Katharina; Kaiser, Christoph; Drazic, Adrian; Grö tzinger, Stefan W.; Haeß ner, Carmen; Eisenreich, Wolfgang; Richter, Klaus

2015-01-01

-S cluster formation. Indeed, we find that in vivo iron concentrations are misbalanced and observe a reduction in the activity of the prominent Fe-S cluster containing protein aconitase. Like in other yeast strains with impaired mitochondria, non-fermentative

Derivatives of Rhodamine 19 as Mild Mitochondria-targeted Cationic Uncouplers*

Science.gov (United States)

Antonenko, Yuri N.; Avetisyan, Armine V.; Cherepanov, Dmitry A.; Knorre, Dmitry A.; Korshunova, Galina A.; Markova, Olga V.; Ojovan, Silvia M.; Perevoshchikova, Irina V.; Pustovidko, Antonina V.; Rokitskaya, Tatyana I.; Severina, Inna I.; Simonyan, Ruben A.; Smirnova, Ekaterina A.; Sobko, Alexander A.; Sumbatyan, Natalia V.; Severin, Fedor F.; Skulachev, Vladimir P.

2011-01-01

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C12R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H+ ions was generated in the presence of C12R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C12R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C12R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C12R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease. PMID:21454507

Role of mitochondria-associated hexokinase II in cancer cell death induced by 3-Bromopyruvate

Science.gov (United States)

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

2009-01-01

Summary 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 ρ0 cells that highly express HKII. Interestingly, the dissociation of HKII itself did no 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. PMID:19285479

Mitochondrion-mediated cell death: dissecting yeast apoptosis for a better understanding of neurodegeneration

Energy Technology Data Exchange (ETDEWEB)

Braun, Ralf J., E-mail: ralf.braun@uni-bayreuth.de [Institut für Zellbiologie, Universität Bayreuth, Bayreuth (Germany)

2012-11-28

Mitochondrial damage and dysfunction are common hallmarks for neurodegenerative disorders, including Alzheimer, Parkinson, Huntington diseases, and the motor neuron disorder amyotrophic lateral sclerosis. Damaged mitochondria pivotally contribute to neurotoxicity and neuronal cell death in these disorders, e.g., due to their inability to provide the high energy requirements for neurons, their generation of reactive oxygen species (ROS), and their induction of mitochondrion-mediated cell death pathways. Therefore, in-depth analyses of the underlying molecular pathways, including cellular mechanisms controlling the maintenance of mitochondrial function, is a prerequisite for a better understanding of neurodegenerative disorders. The yeast Saccharomyces cerevisiae is an established model for deciphering mitochondrial quality control mechanisms and the distinct mitochondrial roles during apoptosis and programmed cell death. Cell death upon expression of various human neurotoxic proteins has been characterized in yeast, revealing neurotoxic protein-specific differences. This review summarizes how mitochondria are affected in these neurotoxic yeast models, and how they are involved in the execution and prevention of cell death. I will discuss to which extent this mimics the situation in other neurotoxic model systems, and how this may contribute to a better understanding of the mitochondrial roles in the human disorders.

Mitochondrion-mediated cell death: dissecting yeast apoptosis for a better understanding of neurodegeneration

International Nuclear Information System (INIS)

Braun, Ralf J.

2012-01-01

Mitochondrial damage and dysfunction are common hallmarks for neurodegenerative disorders, including Alzheimer, Parkinson, Huntington diseases, and the motor neuron disorder amyotrophic lateral sclerosis. Damaged mitochondria pivotally contribute to neurotoxicity and neuronal cell death in these disorders, e.g., due to their inability to provide the high energy requirements for neurons, their generation of reactive oxygen species (ROS), and their induction of mitochondrion-mediated cell death pathways. Therefore, in-depth analyses of the underlying molecular pathways, including cellular mechanisms controlling the maintenance of mitochondrial function, is a prerequisite for a better understanding of neurodegenerative disorders. The yeast Saccharomyces cerevisiae is an established model for deciphering mitochondrial quality control mechanisms and the distinct mitochondrial roles during apoptosis and programmed cell death. Cell death upon expression of various human neurotoxic proteins has been characterized in yeast, revealing neurotoxic protein-specific differences. This review summarizes how mitochondria are affected in these neurotoxic yeast models, and how they are involved in the execution and prevention of cell death. I will discuss to which extent this mimics the situation in other neurotoxic model systems, and how this may contribute to a better understanding of the mitochondrial roles in the human disorders.

Long-term inhibition of cyclophilin D results in intracellular translocation of calcein AM from mitochondria to lysosomes.

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Shinohe, Daisuke; Kobayashi, Asuka; Gotoh, Marina; Tanaka, Kotaro; Ohta, Yoshihiro

2017-01-01

Cyclophilin D is a peptidyl-prolyl cis-trans isomerase localized in the mitochondrial matrix. Although its effects on mitochondrial characteristics have been well studied, its relation to the uptake of molecules by mitochondria remains unknown. Here, we demonstrated the effects of cyclophilin D on the intracellular translocation of calcein AM. Following addition of calcein AM to control cells or cells overexpressing wild-type cyclophilin D, calcein fluorescence was observed in mitochondria. However, long-term inhibition of cyclophilin D in these cells altered the localization of calcein fluorescence from mitochondria to lysosomes without changing mitochondrial esterase activity. In addition, depletion of glucose from the medium recovered calcein localization from lysosomes to mitochondria. This is the first demonstration of the effects of cyclophilin D on the intracellular translocation of molecules other than proteins and suggests that cyclophilin D may modify mitochondrial features by inducing the translocation of molecules to the mitochondria through the mechanism associated with cellular energy metabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

Adenine nucleotide translocator transports haem precursors into mitochondria.

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Motoki Azuma

2008-08-01

Full Text Available Haem is a prosthetic group for haem proteins, which play an essential role in oxygen transport, respiration, signal transduction, and detoxification. In haem biosynthesis, the haem precursor protoporphyrin IX (PP IX must be accumulated into the mitochondrial matrix across the inner membrane, but its mechanism is largely unclear. Here we show that adenine nucleotide translocator (ANT, the inner membrane transporter, contributes to haem biosynthesis by facilitating mitochondrial accumulation of its precursors. We identified that haem and PP IX specifically bind to ANT. Mitochondrial uptake of PP IX was inhibited by ADP, a known substrate of ANT. Conversely, ADP uptake into mitochondria was competitively inhibited by haem and its precursors, suggesting that haem-related porphyrins are accumulated into mitochondria via ANT. Furthermore, disruption of the ANT genes in yeast resulted in a reduction of haem biosynthesis by blocking the translocation of haem precursors into the matrix. Our results represent a new model that ANT plays a crucial role in haem biosynthesis by facilitating accumulation of its precursors into the mitochondrial matrix.

Structural Studies of the Yeast Mitochondrial Degradosome

DEFF Research Database (Denmark)

Feddersen, Ane; Jonstrup, Anette Thyssen; Brodersen, Ditlev Egeskov

The yeast mitochondrial degradosome/exosome (mtExo) is responsible for most RNA turnover in mitochondria and has been proposed to form a central part of a mitochondrial RNA surveillance system responsible for degradation of aberrant and unprocessed RNA ([1], [2]). In contrast to the cytoplasmic...... and nuclear exosome complexes, which consist of 10-12 different nuclease subunits, the mitochondrial degradosome is composed of only two large subunits - an RNase (Dss1p) and a helicase (Suv3p), belonging the Ski2 class of DExH box RNA helicases. Both subunits are encoded on the yeast nuclear genome...... and and Suv3p from the fission yeast, Schizosaccharomyces pombe, have been cloned for heterologous expression in E. coli. Of the two, we have succeeded in purifying the 73kDa Suv3p by Ni2+-affinity chromatography followed by cleavage of the N-terminal His-tag, cation exchange, and gel filtration. Crystals...

ISC1-dependent metabolic adaptation reveals an indispensable role for mitochondria in induction of nuclear genes during the diauxic shift in Saccharomyces cerevisiae.

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Kitagaki, Hiroshi; Cowart, L Ashley; Matmati, Nabil; Montefusco, David; Gandy, Jason; de Avalos, Silvia Vaena; Novgorodov, Sergei A; Zheng, Jim; Obeid, Lina M; Hannun, Yusuf A

2009-04-17

Growth of Saccharomyces cerevisiae following glucose depletion (the diauxic shift) depends on a profound metabolic adaptation accompanied by a global reprogramming of gene expression. In this study, we provide evidence for a heretofore unsuspected role for Isc1p in mediating this reprogramming. Initial studies revealed that yeast cells deleted in ISC1, the gene encoding inositol sphingolipid phospholipase C, which resides in mitochondria in the post-diauxic phase, showed defective aerobic respiration in the post-diauxic phase but retained normal intrinsic mitochondrial functions, including intact mitochondrial DNA, normal oxygen consumption, and normal mitochondrial polarization. Microarray analysis revealed that the Deltaisc1 strain failed to up-regulate genes required for nonfermentable carbon source metabolism during the diauxic shift, thus suggesting a mechanism for the defective supply of respiratory substrates into mitochondria in the post-diauxic phase. This defect in regulating nuclear gene induction in response to a defect in a mitochondrial enzyme raised the possibility that mitochondria may initiate diauxic shift-associated regulation of nucleus-encoded genes. This was established by demonstrating that in respiratory-deficient petite cells these genes failed to be up-regulated across the diauxic shift in a manner similar to the Deltaisc1 strain. Isc1p- and mitochondrial function-dependent genes significantly overlapped with Adr1p-, Snf1p-, and Cat8p-dependent genes, suggesting some functional link among these factors. However, the retrograde response was not activated in Deltaisc1, suggesting that the response of Deltaisc1 cannot be simply attributed to mitochondrial dysfunction. These results suggest a novel role for Isc1p in allowing the reprogramming of gene expression during the transition from anaerobic to aerobic metabolism.

rRNA maturation in yeast cells depleted of large ribosomal subunit proteins.

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Gisela Pöll

Full Text Available The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins. They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein-rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i how individual r-proteins control the productive processing of the major 5' end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events.

Heterologous expression of the Crassostrea gigas (Pacific oyster) alternative oxidase in the yeast Saccharomyces cerevisiae.

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Robertson, Aaron; Schaltz, Kyle; Neimanis, Karina; Staples, James F; McDonald, Allison E

2016-10-01

Alternative oxidase (AOX) is a terminal oxidase within the inner mitochondrial membrane (IMM) present in many organisms where it functions in the electron transport system (ETS). AOX directly accepts electrons from ubiquinol and is therefore capable of bypassing ETS Complexes III and IV. The human genome does not contain a gene coding for AOX, so AOX expression has been suggested as a gene therapy for a range of human mitochondrial diseases caused by genetic mutations that render Complex III and/or IV dysfunctional. An effective means of screening mutations amenable to AOX treatment remains to be devised. We have generated such a tool by heterologously expressing AOX from the Pacific oyster (Crassostrea gigas) in the yeast Saccharomyces cerevisiae under the control of a galactose promoter. Our results show that this animal AOX is monomeric and is correctly targeted to yeast mitochondria. Moreover, when expressed in yeast, Pacific oyster AOX is a functional quinol oxidase, conferring cyanide-resistant growth and myxothiazol-resistant oxygen consumption to yeast cells and isolated mitochondria. This system represents a high-throughput screening tool for determining which Complex III and IV genetic mutations in yeast will be amenable to AOX gene therapy. As many human genes are orthologous to those found in yeast, our invention represents an efficient and cost-effective way to evaluate viable research avenues. In addition, this system provides the opportunity to learn more about the localization, structure, and regulation of AOXs from animals that are not easily reared or manipulated in the lab.

Mitochondrial modification and respiratory deficiency in the yeast cell caused by cadmium poisoning

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Lindegren, C C; Lindegren, G

1973-01-01

Cells of Fleischmann bakers' yeast were grown in standard nutrient broth and in broth to which cobalt, or cadmium, or thallium, had been added. The cells were fixed by glutaraldehyde-permanganate and sectioned. Electron microscopy showed that (a) the endoplasmic reticulum was fixed well in cells grown in cobalt or cadmium, but the endoplasmic reticulum was not fixed in cells grown in normal or thallium broth; (b) the cristate mitochondria were normal in all cells except those grown in cadmium. No cristae were visible in the cristate mitochondria of cells grown in cadmium broth; (c) a large fraction of the cells recovered from cadmium broth were respiratory-deficient; (d) thallic oxide was present in the cristate mitochondria of cells recovered from thallium broth. 13 references, 3 figures.

Integrative analysis of the mitochondrial proteome in yeast.

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Holger Prokisch

2004-06-01

Full Text Available In this study yeast mitochondria were used as a model system to apply, evaluate, and integrate different genomic approaches to define the proteins of an organelle. Liquid chromatography mass spectrometry applied to purified mitochondria identified 546 proteins. By expression analysis and comparison to other proteome studies, we demonstrate that the proteomic approach identifies primarily highly abundant proteins. By expanding our evaluation to other types of genomic approaches, including systematic deletion phenotype screening, expression profiling, subcellular localization studies, protein interaction analyses, and computational predictions, we show that an integration of approaches moves beyond the limitations of any single approach. We report the success of each approach by benchmarking it against a reference set of known mitochondrial proteins, and predict approximately 700 proteins associated with the mitochondrial organelle from the integration of 22 datasets. We show that a combination of complementary approaches like deletion phenotype screening and mass spectrometry can identify over 75% of the known mitochondrial proteome. These findings have implications for choosing optimal genome-wide approaches for the study of other cellular systems, including organelles and pathways in various species. Furthermore, our systematic identification of genes involved in mitochondrial function and biogenesis in yeast expands the candidate genes available for mapping Mendelian and complex mitochondrial disorders in humans.

The Yeast Retrograde Response as a Model of Intracellular Signaling of Mitochondrial Dysfunction

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S. Michal eJazwinski

2012-05-01

Full Text Available Mitochondrial dysfunction activates intracellular signaling pathways that impact yeast longevity, and the best known of these pathways is the retrograde response. More recently, similar responses have been discerned in other systems, from invertebrates to human cells. However, the identity of the signal transducers is either unknown or apparently diverse, contrasting with the well-established signaling module of the yeast retrograde response. On the other hand, it has become equally clear that several other pathways and processes interact with the retrograde response, embedding it in a network responsive to a variety of cellular states. An examination of this network supports the notion that the master regulator NFkB aggregated a variety of mitochondria-related cellular responses at some point in evolution and has become the retrograde transcription factor. This has significant consequences for how we view some of the deficits associated with aging, such as inflammation. The support for NFkB as the retrograde response transcription factor is not only based on functional analyses. It is bolstered by the fact that NFkB can regulate Myc-Max, which is activated in human cells with dysfunctional mitochondria and impacts cellular metabolism. Myc-Max is homologous to the yeast retrograde response transcription factor Rtg1-Rtg3. Further research will be needed to disentangle the pro-aging from the anti-aging effects of NFkB. Interestingly, this is also a challenge for the complete understanding of the yeast retrograde response.

Light induces translocation of NF-κB p65 to the mitochondria and suppresses expression of cytochrome c oxidase subunit III (COX III) in the rat retina

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Tomita, Hiroshi, E-mail: htomita@iwate-u.ac.jp [Laboratory of Visual Neuroscience, Graduate Course in Biological Sciences, Iwate University Division of Science and Engineering, 4-3-5 Ueda, Morioka, Iwate 020-8551 (Japan); Soft-Path Engineering Research Center (SPERC), Faculty of Science and Engineering, Iwate University, Morioka 020-8551 (Japan); Clinical Research, Innovation and Education Center, Tohoku University Hospital, 1-1 Seiryo, Aoba, Sendai, Miyagi 980-8574 (Japan); Tabata, Kitako, E-mail: ktabata@iwate-u.ac.jp [Laboratory of Visual Neuroscience, Graduate Course in Biological Sciences, Iwate University Division of Science and Engineering, 4-3-5 Ueda, Morioka, Iwate 020-8551 (Japan); Takahashi, Maki, E-mail: mqdelta@iwate-u.ac.jp [Laboratory of Visual Neuroscience, Graduate Course in Biological Sciences, Iwate University Division of Science and Engineering, 4-3-5 Ueda, Morioka, Iwate 020-8551 (Japan); Nishiyama, Fumiaki, E-mail: t2114018@iwate-u.ac.jp [Laboratory of Visual Neuroscience, Graduate Course in Biological Sciences, Iwate University Division of Science and Engineering, 4-3-5 Ueda, Morioka, Iwate 020-8551 (Japan); Sugano, Eriko, E-mail: sseriko@iwate-u.ac.jp [Laboratory of Visual Neuroscience, Graduate Course in Biological Sciences, Iwate University Division of Science and Engineering, 4-3-5 Ueda, Morioka, Iwate 020-8551 (Japan); Soft-Path Engineering Research Center (SPERC), Faculty of Science and Engineering, Iwate University, Morioka 020-8551 (Japan)

2016-05-13

The transcription factor nuclear factor kappaB (NF-κB) plays various roles in cell survival, apoptosis, and inflammation. In the rat retina, NF-κB activity increases after exposure to damaging light, resulting in degeneration of photoreceptors. Here, we report that in dark-adapted rats exposed for 6 h to bright white light, the p65 subunit of retinal NF-κB translocates to the mitochondria, an event associated with a decrease in expression of cytochrome c oxidase subunit III (COX III). However, sustained exposure for 12 h depleted p65 from the mitochondria, and enhanced COX III expression. Treatment with the protective antioxidant PBN prior to light exposure prevents p65 depletion in the mitochondria and COX III upregulation during prolonged exposure, and apoptosis in photoreceptor cells. These results indicate that COX III expression is sensitive to the abundance of NF-κB p65 in the mitochondria, which, in turn, is affected by exposure to damaging light. - Highlights: • Damaging light exposure of the retina induces NF-κB p65 mitochondrial translocation. • NF-κB p65 mitochondrial translocation is associated with the decrease of COX III expression. • Prolonged light exposure depletes mitochondrial p65 resulting in the increase in COX III expression. • NF-κB p65 and COX III expression play an important role in the light-induced photoreceptor degeneration.

Light induces translocation of NF-κB p65 to the mitochondria and suppresses expression of cytochrome c oxidase subunit III (COX III) in the rat retina

International Nuclear Information System (INIS)

Tomita, Hiroshi; Tabata, Kitako; Takahashi, Maki; Nishiyama, Fumiaki; Sugano, Eriko

2016-01-01

The transcription factor nuclear factor kappaB (NF-κB) plays various roles in cell survival, apoptosis, and inflammation. In the rat retina, NF-κB activity increases after exposure to damaging light, resulting in degeneration of photoreceptors. Here, we report that in dark-adapted rats exposed for 6 h to bright white light, the p65 subunit of retinal NF-κB translocates to the mitochondria, an event associated with a decrease in expression of cytochrome c oxidase subunit III (COX III). However, sustained exposure for 12 h depleted p65 from the mitochondria, and enhanced COX III expression. Treatment with the protective antioxidant PBN prior to light exposure prevents p65 depletion in the mitochondria and COX III upregulation during prolonged exposure, and apoptosis in photoreceptor cells. These results indicate that COX III expression is sensitive to the abundance of NF-κB p65 in the mitochondria, which, in turn, is affected by exposure to damaging light. - Highlights: • Damaging light exposure of the retina induces NF-κB p65 mitochondrial translocation. • NF-κB p65 mitochondrial translocation is associated with the decrease of COX III expression. • Prolonged light exposure depletes mitochondrial p65 resulting in the increase in COX III expression. • NF-κB p65 and COX III expression play an important role in the light-induced photoreceptor degeneration.

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

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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

2017-06-01

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. © 2017 The Authors.

A Mitochondria-Dependent Pathway Mediates the Apoptosis of GSE-Induced Yeast

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Cao, Sishuo; Xu, Wentao; Zhang, Nan; Wang, Yan; Luo, YunBo; He, Xiaoyun; Huang, Kunlun

2012-01-01

Grapefruit seed extract (GSE), which has powerful anti-fungal activity, can induce apoptosis in S. cerevisiae. The yeast cells underwent apoptosis as determined by testing for apoptotic markers of DNA cleavage and typical chromatin condensation by Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) and 4,6'-diaminidino-2-phenylindole (DAPI) staining and electron microscopy. The changes of ΔΨmt (mitochondrial transmembrane potential) and ROS (reactive oxygen species) ...

Mitochondrial fission proteins regulate programmed cell death in yeast

OpenAIRE

Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J.; Qi, Bing; Pevsner, Jonathan; McCaffery, J. Michael; Hill, R. Blake; Basañez, Gorka; Hardwick, J. Marie

2004-01-01

The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we fo...

Flor Yeast: New Perspectives Beyond Wine Aging

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Legras, Jean-Luc; Moreno-Garcia, Jaime; Zara, Severino; Zara, Giacomo; Garcia-Martinez, Teresa; Mauricio, Juan C.; Mannazzu, Ilaria; Coi, Anna L.; Bou Zeidan, Marc; Dequin, Sylvie; Moreno, Juan; Budroni, Marilena

2016-01-01

The most important dogma in white-wine production is the preservation of the wine aroma and the limitation of the oxidative action of oxygen. In contrast, the aging of Sherry and Sherry-like wines is an aerobic process that depends on the oxidative activity of flor strains of Saccharomyces cerevisiae. Under depletion of nitrogen and fermentable carbon sources, these yeast produce aggregates of floating cells and form an air–liquid biofilm on the wine surface, which is also known as velum or flor. This behavior is due to genetic and metabolic peculiarities that differentiate flor yeast from other wine yeast. This review will focus first on the most updated data obtained through the analysis of flor yeast with -omic tools. Comparative genomics, proteomics, and metabolomics of flor and wine yeast strains are shedding new light on several features of these special yeast, and in particular, they have revealed the extent of proteome remodeling imposed by the biofilm life-style. Finally, new insights in terms of promotion and inhibition of biofilm formation through small molecules, amino acids, and di/tri-peptides, and novel possibilities for the exploitation of biofilm immobilization within a fungal hyphae framework, will be discussed. PMID:27148192

Mitochondrial fission proteins regulate programmed cell death in yeast.

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Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J; Qi, Bing; Pevsner, Jonathan; McCaffery, J Michael; Hill, R Blake; Basañez, Gorka; Hardwick, J Marie

2004-11-15

The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we found that the Saccharomyces cerevisiae homolog of human Drp1, Dnm1, promotes mitochondrial fragmentation/degradation and cell death following treatment with several death stimuli. Two Dnm1-interacting factors also regulate yeast cell death. The WD40 repeat protein Mdv1/Net2 promotes cell death, consistent with its role in mitochondrial fission. In contrast to its fission function in healthy cells, Fis1 unexpectedly inhibits Dnm1-mediated mitochondrial fission and cysteine protease-dependent cell death in yeast. Furthermore, the ability of yeast Fis1 to inhibit mitochondrial fission and cell death can be functionally replaced by human Bcl-2 and Bcl-xL. Together, these findings indicate that yeast and mammalian cells have a conserved programmed death pathway regulated by a common molecular component, Drp1/Dnm1, that is inhibited by a Bcl-2-like function.

Inhibition of free radical scavenging enzymes affects mitochondrial membrane permeability transition during growth and aging of yeast cells.

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Deryabina, Yulia; Isakova, Elena; Sekova, Varvara; Antipov, Alexey; Saris, Nils-Erik L

2014-12-01

In this study, we investigated the change in the antioxidant enzymes activity, cell respiration, reactive oxygen species (ROS), and impairment of membrane mitochondria permeability in the Endomyces magnusii yeasts during culture growth and aging. We showed that the transition into stationary phase is the key tool to understanding interaction of these processes. This growth stage is distinguished by two-fold increase in ROS production and respiration rate as compared to those in the logarithmic phase. It results in induction of alternative oxidase (AO) in the stationary phase, decline of the main antioxidant enzymes activities, ROS-production, and mitochondria membrane permeability. Significant increase in the share of mitochondrial isoform of superoxide dismutase (SOD2) occurred in the stationary phase from 51.8% (24 h of cultivation) to 68.6% (48 h of cultivation). Upon blocking the essential ROS-scavenging enzymes, SODs and catalases (CATs) some heterogeneity of cell population was observed: 80-90% of cells displayed evident signs of early apoptosis (such as disorientation of mitochondria cristae, mitochondrial fragmentation and deformation of nuclear chromatine). However, 10-20% of the population were definitely healthy. It allowed to draw the conclusion that a complete system of cell antioxidant protection underlies normal mitochondria functioning while the E. magnusii yeasts grow and age. Moreover, this system provides unimpaired cell physiology under oxidative stress during culture aging in the stationary phase. Failures in mitochondria functions due to inhibition of ROS-scavenging enzymes of CATs and SODs could lead to damage of the cells and some signs of early apoptosis.

Amino acid codes in mitochondria as possible clues to primitive codes

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Jukes, T. H.

1981-01-01

Differences between mitochondrial codes and the universal code indicate that an evolutionary simplification has taken place, rather than a return to a more primitive code. However, these differences make it evident that the universal code is not the only code possible, and therefore earlier codes may have differed markedly from the previous code. The present universal code is probably a 'frozen accident.' The change in CUN codons from leucine to threonine (Neurospora vs. yeast mitochondria) indicates that neutral or near-neutral changes occurred in the corresponding proteins when this code change took place, caused presumably by a mutation in a tRNA gene.

Bcl-xL stimulates Bax relocation to mitochondria and primes cells to ABT-737.

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Renault, Thibaud T; Teijido, Oscar; Missire, Florent; Ganesan, Yogesh Tengarai; Velours, Gisèle; Arokium, Hubert; Beaumatin, Florian; Llanos, Raul; Athané, Axel; Camougrand, Nadine; Priault, Muriel; Antonsson, Bruno; Dejean, Laurent M; Manon, Stéphen

2015-07-01

Bax cytosol-to-mitochondria translocation is a central event of the intrinsic pathway of apoptosis. Bcl-xL is an important regulator of this event and was recently shown to promote the retrotranslocation of mitochondrial Bax to the cytosol. The present study identifies a new aspect of the regulation of Bax localization by Bcl-xL: in addition to its role in Bax inhibition and retrotranslocation, we found that, like with Bcl-2, an increase of Bcl-xL expression levels led to an increase of Bax mitochondrial content. This finding was substantiated both in pro-lymphocytic FL5.12 cells and a yeast reporting system. Bcl-xL-dependent increase of mitochondrial Bax is counterbalanced by retrotranslocation, as we observed that Bcl-xLΔC, which is unable to promote Bax retrotranslocation, was more efficient than the full-length protein in stimulating Bax relocation to mitochondria. Interestingly, cells overexpressing Bcl-xL were more sensitive to apoptosis upon treatment with the BH3-mimetic ABT-737, suggesting that despite its role in Bax inhibition, Bcl-xL also primes mitochondria to permeabilization and cytochrome c release. Copyright © 2015 Elsevier Ltd. All rights reserved.

Electromagnetic probes of molecular motors in the electron transport chains of mitochondria and chloroplasts

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Miller, J. H., Jr.; Nawarathna, D.; Vajrala, V.; Gardner, J.; Widger, W. R.

2005-12-01

We report on measurements of harmonics generated by whole cells, mitochondria, and chloroplasts in response to applied sinusoidal electric fields. The frequency- and amplitude-dependence of the induced harmonics exhibit features that correlate with physiological processes. Budding yeast (S. cerevisiae) cells produce numerous harmonics, the amplitudes of which depend strongly on frequency. When the second or third harmonic amplitude is plotted vs. applied frequency, we observe two peaks, around 3 kHz and 12 kHz, which are suppressed by respiratory inhibitors. We observe similar peaks when measuring the harmonic response of B. indicas, a relative of the mitochondrial ancestor. In uncoupled mitochondria, in which most of the electron transport chain is active but the ATP-synthase molecular turbine is inactive, only one (lower frequency) of the two peaks is present. Finally, we find that harmonics generated by chloroplasts depend dramatically on incident light, and vanish in the absence of light.

Interaction between repressor Opi1p and ER membrane protein Scs2p facilitates transit of phosphatidic acid from the ER to mitochondria and is essential for INO1 gene expression in the presence of choline.

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Gaspar, Maria L; Chang, Yu-Fang; Jesch, Stephen A; Aregullin, Manuel; Henry, Susan A

2017-11-10

In the yeast Saccharomyces cerevisiae , the Opi1p repressor controls the expression of INO1 via the Opi1p/Ino2p-Ino4p regulatory circuit. Inositol depletion favors Opi1p interaction with both Scs2p and phosphatidic acid at the endoplasmic reticulum (ER) membrane. Inositol supplementation, however, favors the translocation of Opi1p from the ER into the nucleus, where it interacts with the Ino2p-Ino4p complex, attenuating transcription of INO1 A strain devoid of Scs2p ( scs2 Δ) and a mutant, OPI1FFAT , lacking the ability to interact with Scs2p were utilized to examine the specific role(s) of the Opi1p-Scs2p interaction in the regulation of INO1 expression and overall lipid metabolism. Loss of the Opi1p-Scs2p interaction reduced INO1 expression and conferred inositol auxotrophy. Moreover, inositol depletion in strains lacking this interaction resulted in Opi1p being localized to sites of lipid droplet formation, coincident with increased synthesis of triacylglycerol. Supplementation of choline to inositol-depleted growth medium led to decreased TAG synthesis in all three strains. However, in strains lacking the Opi1p-Scs2p interaction, Opi1p remained in the nucleus, preventing expression of INO1 These data support the conclusion that a specific pool of phosphatidic acid, associated with lipid droplet formation in the perinuclear ER, is responsible for the initial rapid exit of Opi1p from the nucleus to the ER and is required for INO1 expression in the presence of choline. Moreover, the mitochondria-specific phospholipid, cardiolipin, was significantly reduced in both strains compromised for Opi1p-Scs2p interaction, indicating that this interaction is required for the transfer of phosphatidic acid from the ER to the mitochondria for cardiolipin synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Mitochondrial depolarization in yeast zygotes inhibits clonal expansion of selfish mtDNA.

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Karavaeva, Iuliia E; Golyshev, Sergey A; Smirnova, Ekaterina A; Sokolov, Svyatoslav S; Severin, Fedor F; Knorre, Dmitry A

2017-04-01

Non-identical copies of mitochondrial DNA (mtDNA) compete with each other within a cell and the ultimate variant of mtDNA present depends on their relative replication rates. Using yeast Saccharomyces cerevisiae cells as a model, we studied the effects of mitochondrial inhibitors on the competition between wild-type mtDNA and mutant selfish mtDNA in heteroplasmic zygotes. We found that decreasing mitochondrial transmembrane potential by adding uncouplers or valinomycin changes the competition outcomes in favor of the wild-type mtDNA. This effect was significantly lower in cells with disrupted mitochondria fission or repression of the autophagy-related genes ATG8 , ATG32 or ATG33 , implying that heteroplasmic zygotes activate mitochondrial degradation in response to the depolarization. Moreover, the rate of mitochondrially targeted GFP turnover was higher in zygotes treated with uncoupler than in haploid cells or untreated zygotes. Finally, we showed that vacuoles of zygotes with uncoupler-activated autophagy contained DNA. Taken together, our data demonstrate that mitochondrial depolarization inhibits clonal expansion of selfish mtDNA and this effect depends on mitochondrial fission and autophagy. These observations suggest an activation of mitochondria quality control mechanisms in heteroplasmic yeast zygotes. © 2017. Published by The Company of Biologists Ltd.

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

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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

2015-01-01

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. PMID:26432643

VDAC electronics: 4. Novel electrical mechanism and thermodynamic estimations of glucose repression of yeast respiration.

Science.gov (United States)

Lemeshko, Victor V

2017-11-01

Inhibition of cell respiration by high concentrations of glucose (glucose repression), known as "Crabtree effect", has been demonstrated for various cancerous strains, highly proliferating cells and yeast lines. Although significant progress in understanding metabolic events associated with the glucose repression of cell respiration has been achieved, it is not yet clear whether the Crabtree effect is the result of a limited activity of the respiratory chain, or of some glucose-mediated regulation of mitochondrial metabolic state. In this work we propose an electrical mechanism of glucose repression of the yeast S. cerevisiae, resulting from generation of the mitochondrial outer membrane potential (OMP) coupled to the direct oxidation of cytosolic NADH in mitochondria. This yeast-type mechanism of OMP generation is different from the earlier proposed VDAC-hexokinase-mediated voltage generation of cancer-type, associated with the mitochondrial outer membrane. The model was developed assuming that VDAC is more permeable to NADH than to NAD + . Thermodynamic estimations of OMP, generated as a result of NADH(2-)/NAD + (1-) turnover through the outer membrane, demonstrated that the values of calculated negative OMP match the known range of VDAC voltage sensitivity, thus suggesting a possibility of OMP-dependent VDAC-mediated regulation of cell energy metabolism. According to the proposed mechanism, we suggest that the yeast-type Crabtree effect is the result of a fast VDAC-mediated electrical repression of mitochondria due to a decrease in the outer membrane permeability to charged metabolites and owing their redistribution between the mitochondrial intermembrane space and the cytosol, both controlled by metabolically-derived OMP. Copyright © 2017 Elsevier B.V. All rights reserved.

ATAD3 proteins: brokers of a mitochondria-endoplasmic reticulum connection in mammalian cells.

Science.gov (United States)

Baudier, Jacques

2018-05-01

In yeast, a sequence of physical and genetic interactions termed the endoplasmic reticulum (ER)-mitochondria organizing network (ERMIONE) controls mitochondria-ER interactions and mitochondrial biogenesis. Several functions that characterize ERMIONE complexes are conserved in mammalian cells, suggesting that a similar tethering complex must exist in metazoans. Recent studies have identified a new family of nuclear-encoded ATPases associated with diverse cellular activities (AAA+-ATPase) mitochondrial membrane proteins specific to multicellular eukaryotes, called the ATPase family AAA domain-containing protein 3 (ATAD3) proteins (ATAD3A and ATAD3B). These proteins are crucial for normal mitochondrial-ER interactions and lie at the heart of processes underlying mitochondrial biogenesis. ATAD3A orthologues have been studied in flies, worms, and mammals, highlighting the widespread importance of this gene during embryonic development and in adulthood. ATAD3A is a downstream effector of target of rapamycin (TOR) signalling in Drosophila and exhibits typical features of proteins from the ERMIONE-like complex in metazoans. In humans, mutations in the ATAD3A gene represent a new link between altered mitochondrial-ER interaction and recognizable neurological syndromes. The primate-specific ATAD3B protein is a biomarker of pluripotent embryonic stem cells. Through negative regulation of ATAD3A function, ATAD3B supports mitochondrial stemness properties. © 2017 Cambridge Philosophical Society.

Thymidine kinase 2 deficiency-induced mtDNA depletion in mouse liver leads to defect β-oxidation.

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Xiaoshan Zhou

Full Text Available Thymidine kinase 2 (TK2 deficiency in humans causes mitochondrial DNA (mtDNA depletion syndrome. To study the molecular mechanisms underlying the disease and search for treatment options, we previously generated and described a TK2 deficient mouse strain (TK2(-/- that progressively loses its mtDNA. The TK2(-/- mouse model displays symptoms similar to humans harboring TK2 deficient infantile fatal encephalomyopathy. Here, we have studied the TK2(-/- mouse model to clarify the pathological role of progressive mtDNA depletion in liver for the severe outcome of TK2 deficiency. We observed that a gradual depletion of mtDNA in the liver of the TK2(-/- mice was accompanied by increasingly hypertrophic mitochondria and accumulation of fat vesicles in the liver cells. The levels of cholesterol and nonesterified fatty acids were elevated and there was accumulation of long chain acylcarnitines in plasma of the TK2(-/- mice. In mice with hepatic mtDNA levels below 20%, the blood sugar and the ketone levels dropped. These mice also exhibited reduced mitochondrial β-oxidation due to decreased transport of long chain acylcarnitines into the mitochondria. The gradual loss of mtDNA in the liver of the TK2(-/- mice causes impaired mitochondrial function that leads to defect β-oxidation and, as a result, insufficient production of ketone bodies and glucose. This study provides insight into the mechanism of encephalomyopathy caused by TK2 deficiency-induced mtDNA depletion that may be used to explore novel therapeutic strategies.

Thymidine kinase 2 deficiency-induced mtDNA depletion in mouse liver leads to defect β-oxidation.

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Zhou, Xiaoshan; Kannisto, Kristina; Curbo, Sophie; von Döbeln, Ulrika; Hultenby, Kjell; Isetun, Sindra; Gåfvels, Mats; Karlsson, Anna

2013-01-01

Thymidine kinase 2 (TK2) deficiency in humans causes mitochondrial DNA (mtDNA) depletion syndrome. To study the molecular mechanisms underlying the disease and search for treatment options, we previously generated and described a TK2 deficient mouse strain (TK2(-/-)) that progressively loses its mtDNA. The TK2(-/-) mouse model displays symptoms similar to humans harboring TK2 deficient infantile fatal encephalomyopathy. Here, we have studied the TK2(-/-) mouse model to clarify the pathological role of progressive mtDNA depletion in liver for the severe outcome of TK2 deficiency. We observed that a gradual depletion of mtDNA in the liver of the TK2(-/-) mice was accompanied by increasingly hypertrophic mitochondria and accumulation of fat vesicles in the liver cells. The levels of cholesterol and nonesterified fatty acids were elevated and there was accumulation of long chain acylcarnitines in plasma of the TK2(-/-) mice. In mice with hepatic mtDNA levels below 20%, the blood sugar and the ketone levels dropped. These mice also exhibited reduced mitochondrial β-oxidation due to decreased transport of long chain acylcarnitines into the mitochondria. The gradual loss of mtDNA in the liver of the TK2(-/-) mice causes impaired mitochondrial function that leads to defect β-oxidation and, as a result, insufficient production of ketone bodies and glucose. This study provides insight into the mechanism of encephalomyopathy caused by TK2 deficiency-induced mtDNA depletion that may be used to explore novel therapeutic strategies.

A Yeast Mutant Deleted of GPH1 Bears Defects in Lipid Metabolism.

Directory of Open Access Journals (Sweden)

Martina Gsell

Full Text Available In a previous study we demonstrated up-regulation of the yeast GPH1 gene under conditions of phosphatidylethanolamine (PE depletion caused by deletion of the mitochondrial (M phosphatidylserine decarboxylase 1 (PSD1 (Gsell et al., 2013, PLoS One. 8(10:e77380. doi: 10.1371/journal.pone.0077380. Gph1p has originally been identified as a glycogen phosphorylase catalyzing degradation of glycogen to glucose in the stationary growth phase of the yeast. Here we show that deletion of this gene also causes decreased levels of phosphatidylcholine (PC, triacylglycerols and steryl esters. Depletion of the two non-polar lipids in a Δgph1 strain leads to lack of lipid droplets, and decrease of the PC level results in instability of the plasma membrane. In vivo labeling experiments revealed that formation of PC via both pathways of biosynthesis, the cytidine diphosphate (CDP-choline and the methylation route, is negatively affected by a Δgph1 mutation, although expression of genes involved is not down regulated. Altogether, Gph1p besides its function as a glycogen mobilizing enzyme appears to play a regulatory role in yeast lipid metabolism.

Toxicity of thallium on isolated rat liver mitochondria: the role of oxidative stress and MPT pore opening.

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Eskandari, M R; Mashayekhi, Vida; Aslani, Majid; Hosseini, Mir-Jamal

2015-02-01

Thallium(I) is a highly toxic heavy metal; however, up to now, its mechanisms are poorly understood. The authors' previous studies showed that this compound could induce reactive oxygen species (ROS) formation, reduced glutathione (GSH) oxidation, membrane lipid peroxidation, and mitochondrial membrane potential (MMP) collapse in isolated rat hepatocyte. Because the liver is the storage site of thallium, it seems that the liver mitochondria are one of the important targets for hepatotoxicity. In this investigation, the effects of thallium on mitochondria were studied to investigate its mechanisms of toxicity. Mitochondria were isolated from rat liver and incubated with different concentrations of thallium (25-200 µM). Thallium(I)-treated mitochondria showed a marked elevation in oxidative stress parameters accompanied by MMP collapse when compared with the control group. These results showed that different concentrations of thallium (25-200 µM) induced a significant (P thallium(I)-induced liver toxicity is a result of the disruptive effect of this metal on the mitochondrial respiratory complexes (I, II, and IV), which are the obvious causes of metal-induced ROS formation and ATP depletion. The latter two events, in turn, trigger cell death signaling via opening of mitochondrial permeability transition pore and cytochrome c expulsion. © 2013 Wiley Periodicals, Inc.

Isolation of rat adrenocortical mitochondria

Energy Technology Data Exchange (ETDEWEB)

Solinas, Paola [Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Department of Medicine, Center for Mitochondrial Disease, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Fujioka, Hisashi [Electron Microscopy Facility, Department of Pharmacology, Center for Mitochondrial Disease, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Tandler, Bernard [Department of Biological Sciences, School of Dental Medicine, Center for Mitochondrial Disease, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Hoppel, Charles L., E-mail: charles.hoppel@case.edu [Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Department of Medicine, Center for Mitochondrial Disease, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States)

2012-10-12

Highlights: Black-Right-Pointing-Pointer A method for isolation of adrenocortical mitochondria from the adrenal gland of rats is described. Black-Right-Pointing-Pointer The purified isolated mitochondria show excellent morphological integrity. Black-Right-Pointing-Pointer The properties of oxidative phosphorylation are excellent. Black-Right-Pointing-Pointer The method increases the opportunity of direct analysis of adrenal mitochondria from small animals. -- Abstract: This report describes a relatively simple and reliable method for isolating adrenocortical mitochondria from rats in good, reasonably pure yield. These organelles, which heretofore have been unobtainable in isolated form from small laboratory animals, are now readily accessible. A high degree of mitochondrial purity is shown by the electron micrographs, as well as the structural integrity of each mitochondrion. That these organelles have retained their functional integrity is shown by their high respiratory control ratios. In general, the biochemical performance of these adrenal cortical mitochondria closely mirrors that of typical hepatic or cardiac mitochondria.

The proline metabolism intermediate Δ1-pyrroline-5-carboxylate directly inhibits the mitochondrial respiration in budding yeast.

Science.gov (United States)

Nishimura, Akira; Nasuno, Ryo; Takagi, Hiroshi

2012-07-30

The proline metabolism intermediate Δ(1)-pyrroline-5-carboxylate (P5C) induces cell death in animals, plants and yeasts. To elucidate how P5C triggers cell death, we analyzed P5C metabolism, mitochondrial respiration and superoxide anion generation in the yeast Saccharomyces cerevisiae. Gene disruption analysis revealed that P5C-mediated cell death was not due to P5C metabolism. Interestingly, deficiency in mitochondrial respiration suppressed the sensitivity of yeast cells to P5C. In addition, we found that P5C inhibits the mitochondrial respiration and induces a burst of superoxide anions from the mitochondria. We propose that P5C regulates cell death via the inhibition of mitochondrial respiration. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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

Science.gov (United States)

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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Mitochondria in Lung Diseases

Science.gov (United States)

Aravamudan, Bharathi; Thompson, Michael A.; Pabelick, Christina M.; Prakash, Y. S.

2014-01-01

Summary Mitochondria are autonomous cellular organelles that oversee a variety of functions such as metabolism, energy production, calcium buffering, and cell fate determination. Regulation of their morphology and diverse activities beyond energy production are being recognized as playing major roles in cellular health and dysfunction. This review is aimed at summarizing what is known regarding mitochondrial contributions to pathogenesis of lung diseases. Emphasis is given to understanding the importance of structural and functional aspects of mitochondria in both normal cellular function (based on knowledge from other cell types) and in development and modulation of lung diseases such as asthma, COPD, cystic fibrosis and cancer. Emerging techniques that allow examination of mitochondria, and potential strategies to target mitochondria in the treatment of lung diseases are also discussed. PMID:23978003

Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae

OpenAIRE

Gadir, Noga; Haim-Vilmovsky, Liora; Kraut-Cohen, Judith; Gerst, Jeffrey E.

2011-01-01

Targeted mRNA localization is a likely determinant of localized protein synthesis. To investigate whether mRNAs encoding mitochondrial proteins (mMPs) localize to mitochondria and, thus, might confer localized protein synthesis and import, we visualized endogenously expressed mMPs in vivo for the first time. We determined the localization of 24 yeast mMPs encoding proteins of the mitochondrial matrix, outer and inner membrane, and intermembrane space and found that many mMPs colocalize with m...

Acrolein-Induced Oxidative Stress and Cell Death Exhibiting Features of Apoptosis in the Yeast Saccharomyces cerevisiae Deficient in SOD1.

Science.gov (United States)

Kwolek-Mirek, Magdalena; Zadrąg-Tęcza, Renata; Bednarska, Sabina; Bartosz, Grzegorz

2015-04-01

The yeast Saccharomyces cerevisiae is a useful eukaryotic model to study the toxicity of acrolein, an important environmental toxin and endogenous product of lipid peroxidation. The study was aimed at elucidation of the cytotoxic effect of acrolein on the yeast deficient in SOD1, Cu, Zn-superoxide dismutase which is hypersensitive to aldehydes. Acrolein generated within the cell from its precursor allyl alcohol caused growth arrest and cell death of the yeast cells. The growth inhibition involved an increase in production of reactive oxygen species and high level of protein carbonylation. DNA condensation and fragmentation, exposition of phosphatidylserine at the cell surface as well as decreased dynamic of actin microfilaments and mitochondria disintegration point to the induction of apoptotic-type cell death besides necrotic cell death.

The Aging Mitochondria

Directory of Open Access Journals (Sweden)

Pierre Theurey

2018-01-01

Full Text Available Mitochondrial dysfunction is a central event in many pathologies and contributes as well to age-related processes. However, distinguishing between primary mitochondrial dysfunction driving aging and a secondary mitochondrial impairment resulting from other cell alterations remains challenging. Indeed, even though mitochondria undeniably play a crucial role in aging pathways at the cellular and organismal level, the original hypothesis in which mitochondrial dysfunction and production of free radicals represent the main driving force of cell degeneration has been strongly challenged. In this review, we will first describe mitochondrial dysfunctions observed in aged tissue, and how these features have been linked to mitochondrial reactive oxygen species (ROS–mediated cell damage and mitochondrial DNA (mtDNA mutations. We will also discuss the clues that led to consider mitochondria as the starting point in the aging process, and how recent research has showed that the mitochondria aging axis represents instead a more complex and multifactorial signaling pathway. New working hypothesis will be also presented in which mitochondria are considered at the center of a complex web of cell dysfunctions that eventually leads to cell senescence and death.

Co-precipitation of phosphate and iron limits mitochondrial phosphate availability in Saccharomyces cerevisiae lacking the yeast frataxin homologue (YFH1).

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Seguin, Alexandra; Santos, Renata; Pain, Debkumar; Dancis, Andrew; Camadro, Jean-Michel; Lesuisse, Emmanuel

2011-02-25

Saccharomyces cerevisiae cells lacking the yeast frataxin homologue (Δyfh1) accumulate iron in the mitochondria in the form of nanoparticles of ferric phosphate. The phosphate content of Δyfh1 mitochondria was higher than that of wild-type mitochondria, but the proportion of mitochondrial phosphate that was soluble was much lower in Δyfh1 cells. The rates of phosphate and iron uptake in vitro by isolated mitochondria were higher for Δyfh1 than wild-type mitochondria, and a significant proportion of the phosphate and iron rapidly became insoluble in the mitochondrial matrix, suggesting co-precipitation of these species after oxidation of iron by oxygen. Increasing the amount of phosphate in the medium decreased the amount of iron accumulated by Δyfh1 cells and improved their growth in an iron-dependent manner, and this effect was mostly transcriptional. Overexpressing the major mitochondrial phosphate carrier, MIR1, slightly increased the concentration of soluble mitochondrial phosphate and significantly improved various mitochondrial functions (cytochromes, [Fe-S] clusters, and respiration) in Δyfh1 cells. We conclude that in Δyfh1 cells, soluble phosphate is limiting, due to its co-precipitation with iron.

Chemical genetic screen identifies lithocholic acid as an anti-aging compound that extends yeast chronological life span in a TOR-independent manner, by modulating housekeeping longevity assurance processes.

Science.gov (United States)

Goldberg, Alexander A; Richard, Vincent R; Kyryakov, Pavlo; Bourque, Simon D; Beach, Adam; Burstein, Michelle T; Glebov, Anastasia; Koupaki, Olivia; Boukh-Viner, Tatiana; Gregg, Christopher; Juneau, Mylène; English, Ann M; Thomas, David Y; Titorenko, Vladimir I

2010-07-01

In chronologically aging yeast, longevity can be extended by administering a caloric restriction (CR) diet or some small molecules. These life-extending interventions target the adaptable target of rapamycin (TOR) and cAMP/protein kinase A (cAMP/PKA) signaling pathways that are under the stringent control of calorie availability. We designed a chemical genetic screen for small molecules that increase the chronological life span of yeast under CR by targeting lipid metabolism and modulating housekeeping longevity pathways that regulate longevity irrespective of the number of available calories. Our screen identifies lithocholic acid (LCA) as one of such molecules. We reveal two mechanisms underlying the life-extending effect of LCA in chronologically aging yeast. One mechanism operates in a calorie availability-independent fashion and involves the LCA-governed modulation of housekeeping longevity assurance pathways that do not overlap with the adaptable TOR and cAMP/PKA pathways. The other mechanism extends yeast longevity under non-CR conditions and consists in LCA-driven unmasking of the previously unknown anti-aging potential of PKA. We provide evidence that LCA modulates housekeeping longevity assurance pathways by suppressing lipid-induced necrosis, attenuating mitochondrial fragmentation, altering oxidation-reduction processes in mitochondria, enhancing resistance to oxidative and thermal stresses, suppressing mitochondria-controlled apoptosis, and enhancing stability of nuclear and mitochondrial DNA.

Electron transport chain dysfunction by H(2)O (2) is linked to increased reactive oxygen species production and iron mobilization by lipoperoxidation: studies using Saccharomyces cerevisiae mitochondria.

Science.gov (United States)

Cortés-Rojo, Christian; Estrada-Villagómez, Mirella; Calderón-Cortés, Elizabeth; Clemente-Guerrero, Mónica; Mejía-Zepeda, Ricardo; Boldogh, Istvan; Saavedra-Molina, Alfredo

2011-04-01

The mitochondrial electron transport chain (ETC) contains thiol groups (-SH) which are reversibly oxidized to modulate ETC function during H(2)O(2) overproduction. Since deleterious effects of H(2)O(2) are not limited to -SH oxidation, due to the formation of other H(2)O(2)-derived species, some processes like lipoperoxidation could enhance the effects of H(2)O(2) over ETC enzymes, disrupt their modulation by -SH oxidation and increase superoxide production. To verify this hypothesis, we tested the effects of H(2)O(2) on ETC activities, superoxide production and iron mobilization in mitochondria from lipoperoxidation-resistant native yeast and lipoperoxidation-sensitized yeast. Only complex III activity from lipoperoxidation-sensitive mitochondria exhibited a higher susceptibility to H(2)O(2) and increased superoxide production. The recovery of ETC activity by the thiol reductanct β-mercaptoethanol (BME) was also altered at complex III, and a role was attributed to lipoperoxidation, the latter being also responsible for iron release. A hypothetical model linking lipoperoxidation, increased complex III damage, superoxide production and iron release is given.

Cooperation of TOM and TIM23 complexes during translocation of proteins into mitochondria.

Science.gov (United States)

Waegemann, Karin; Popov-Čeleketić, Dušan; Neupert, Walter; Azem, Abdussalam; Mokranjac, Dejana

2015-03-13

Translocation of the majority of mitochondrial proteins from the cytosol into mitochondria requires the cooperation of TOM and TIM23 complexes in the outer and inner mitochondrial membranes. The molecular mechanisms underlying this cooperation remain largely unknown. Here, we present biochemical and genetic evidence that at least two contacts from the side of the TIM23 complex play an important role in TOM-TIM23 cooperation in vivo. Tim50, likely through its very C-terminal segment, interacts with Tom22. This interaction is stimulated by translocating proteins and is independent of any other TOM-TIM23 contact known so far. Furthermore, the exposure of Tim23 on the mitochondrial surface depends not only on its interaction with Tim50 but also on the dynamics of the TOM complex. Destabilization of the individual contacts reduces the efficiency of import of proteins into mitochondria and destabilization of both contacts simultaneously is not tolerated by yeast cells. We conclude that an intricate and coordinated network of protein-protein interactions involving primarily Tim50 and also Tim23 is required for efficient translocation of proteins across both mitochondrial membranes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Exogenous ether lipids predominantly target mitochondria

DEFF Research Database (Denmark)

Kuerschner, Lars; Richter, Doris; Hannibal-Bach, Hans Kristian

2012-01-01

Ether lipids are ubiquitous constituents of cellular membranes with no discrete cell biological function assigned yet. Using fluorescent polyene-ether lipids we analyzed their intracellular distribution in living cells by microscopy. Mitochondria and the endoplasmic reticulum accumulated high......, accumulated to mitochondria and induced morphological changes and cellular apoptosis. These data indicate that edelfosine could exert its pro-apoptotic power by targeting and damaging mitochondria and thereby inducing cellular apoptosis. In general, this study implies an important role of mitochondria...

Hyperthyroidism causes cardiac dysfunction by mitochondrial impairment and energy depletion.

Science.gov (United States)

Maity, Sangeeta; Kar, Dipak; De, Kakali; Chander, Vivek; Bandyopadhyay, Arun

2013-05-01

This study elucidates the role of metabolic remodeling in cardiac dysfunction induced by hyperthyroidism. Cardiac hypertrophy, structural remodeling, and expression of the genes associated with fatty acid metabolism were examined in rats treated with triiodothyronine (T3) alone (8 μg/100 g body weight (BW), i.p.) for 15 days or along with a peroxisome proliferator-activated receptor alpha agonist bezafibrate (Bzf; 30 μg/100 g BW, oral) and were found to improve in the Bzf co-treated condition. Ultrastructure of mitochondria was damaged in T3-treated rat heart, which was prevented by Bzf co-administration. Hyperthyroidism-induced oxidative stress, reduction in cytochrome c oxidase activity, and myocardial ATP concentration were also significantly checked by Bzf. Heart function studied at different time points during the course of T3 treatment shows an initial improvement and then a gradual but progressive decline with time, which is prevented by Bzf co-treatment. In summary, the results demonstrate that hyperthyroidism inflicts structural and functional damage to mitochondria, leading to energy depletion and cardiac dysfunction.

Deoxyribonucleoside kinases in mitochondrial DNA depletion.

Science.gov (United States)

Saada-Reisch, Ann

2004-10-01

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are a heterogeneous group of mitochondrial disorders, manifested by a decreased mtDNA copy number and respiratory chain dysfunction. Primary MDS are inherited autosomally and may affect a single organ or multiple tissues. Mutated mitochondrial deoxyribonucleoside kinases; deoxyguanosine kinase (dGK) and thymidine kinase 2 (TK2), were associated with the hepatocerebral and myopathic forms of MDS respectively. dGK and TK2 are key enzymes in the mitochondrial nucleotide salvage pathway, providing the mitochondria with deoxyribonucleotides (dNP) essential for mtDNA synthesis. Although the mitochondrial dNP pool is physically separated from the cytosolic one, dNP's may still be imported through specific transport. Non-replicating tissues, where cytosolic dNP supply is down regulated, are thus particularly vulnerable to dGK and TK2 deficiency. The overlapping substrate specificity of deoxycytidine kinase (dCK) may explain the relative sparing of muscle in dGK deficiency, while low basal TK2 activity render this tissue susceptible to TK2 deficiency. The precise pathophysiological mechanisms of mtDNA depletion due to dGK and TK2 deficiencies remain to be determined, though recent findings confirm that it is attributed to imbalanced dNTP pools.

Depletion of cellular poly (A) binding protein prevents protein synthesis and leads to apoptosis in HeLa cells

Energy Technology Data Exchange (ETDEWEB)

Thangima Zannat, Mst.; Bhattacharjee, Rumpa B. [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada); Bag, Jnanankur, E-mail: jbag@uoguelph.ca [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada)

2011-05-13

Highlights: {yields} Depletion of cellular PABP level arrests mRNA translation in HeLa cells. {yields} PABP knock down leads to apoptotic cell death. {yields} PABP depletion does not affect transcription. {yields} PABP depletion does not lead to nuclear accumulation of mRNA. -- Abstract: The cytoplasmic poly (A) binding protein (PABP) is important in mRNA translation and stability. In yeast, depletion of PABP leads to translation arrest. Similarly, the PABP gene in Drosophila is important for proper development. It is however uncertain, whether mammalian PABP is essential for mRNA translation. Here we showed the effect of PABP depletion on mRNA metabolism in HeLa cells by using a small interfering RNA. Our results suggest that depletion of PABP prevents protein synthesis and consequently leads to cell death through apoptosis. Interestingly, no detectable effect of PABP depletion on transcription, transport and stability of mRNA was observed.

Depletion of cellular poly (A) binding protein prevents protein synthesis and leads to apoptosis in HeLa cells

International Nuclear Information System (INIS)

Thangima Zannat, Mst.; Bhattacharjee, Rumpa B.; Bag, Jnanankur

2011-01-01

Highlights: → Depletion of cellular PABP level arrests mRNA translation in HeLa cells. → PABP knock down leads to apoptotic cell death. → PABP depletion does not affect transcription. → PABP depletion does not lead to nuclear accumulation of mRNA. -- Abstract: The cytoplasmic poly (A) binding protein (PABP) is important in mRNA translation and stability. In yeast, depletion of PABP leads to translation arrest. Similarly, the PABP gene in Drosophila is important for proper development. It is however uncertain, whether mammalian PABP is essential for mRNA translation. Here we showed the effect of PABP depletion on mRNA metabolism in HeLa cells by using a small interfering RNA. Our results suggest that depletion of PABP prevents protein synthesis and consequently leads to cell death through apoptosis. Interestingly, no detectable effect of PABP depletion on transcription, transport and stability of mRNA was observed.

Mitochondria in neutrophil apoptosis

NARCIS (Netherlands)

van Raam, B. J.; Verhoeven, A. J.; Kuijpers, T. W.

2006-01-01

Central in the regulation of the short life span of neutrophils are their mitochondria. These organelles hardly contribute to the energy status of neutrophils but play a vital role in the apoptotic process. Not only do the mitochondria contain cytotoxic proteins that are released during apoptosis

A non-toxic fluorogenic dye for mitochondria labeling.

Science.gov (United States)

Han, Junyan; Han, Myung Shin; Tung, Ching-Hsuan

2013-11-01

Mitochondria, powerhouses of cells, are responsible for many critical cellular functions, such as cell energy metabolism, reactive oxygen species production, and apoptosis regulation. Monitoring mitochondria morphology in live cells temporally and spatially could help with the understanding of the mechanisms of mitochondrial functional regulation and the pathogenesis of mitochondria-related diseases. A novel non-cytotoxic fluorogenic compound, AcQCy7, was developed as a mitochondria-specific dye. AcQCy7 emitted no fluorescent signal outside of cells, but it became fluorescent after intracellular hydrolysis of the acetyl group. The hydrolyzed fluorescent product was well retained in mitochondria, enabling long-lasting fluorescence imaging of mitochondria without cell washing. A 2-day culture study using AcQCy7 showed no sign of cytotoxicity, whereas a commonly used mitochondria-staining probe, Mitochondria Tracker Green, caused significant cell death even at a much lower concentration. Apoptosis-causing mitochondria fission was monitored clearly in real time by AcQCy7. A simple add-and-read mitochondria specific dye AcQCy7 has been validated in various cell models. Bright mitochondria specific fluorescent signal in treated cells lasted several days without noticeable toxicity. The probe AcQCy7 has been proofed to be a non-toxic agent for long-term mitochondria imaging. © 2013.

The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria

Energy Technology Data Exchange (ETDEWEB)

Bhattacharjee, Ashima; Yang, Haojun; Duffy, Megan; Robinson, Emily; Conrad-Antoville, Arianrhod; Lu, Ya-Wen; Capps, Tony; Braiterman, Lelita; Wolfgang, Michael; Murphy, Michael P.; Yi, Ling; Kaler, Stephen G.; Lutsenko, Svetlana; Ralle, Martina

2016-05-16

Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).

Single molecule tracking fluorescence microscopy in mitochondria reveals highly dynamic but confined movement of Tom40

Science.gov (United States)

Kuzmenko, Anton; Tankov, Stoyan; English, Brian P.; Tarassov, Ivan; Tenson, Tanel; Kamenski, Piotr; Elf, Johan; Hauryliuk, Vasili

2011-12-01

Tom40 is an integral protein of the mitochondrial outer membrane, which as the central component of the Translocase of the Outer Membrane (TOM) complex forms a channel for protein import. We characterize the diffusion properties of individual Tom40 molecules fused to the photoconvertable fluorescent protein Dendra2 with millisecond temporal resolution. By imaging individual Tom40 molecules in intact isolated yeast mitochondria using photoactivated localization microscopy with sub-diffraction limited spatial precision, we demonstrate that Tom40 movement in the outer mitochondrial membrane is highly dynamic but confined in nature, suggesting anchoring of the TOM complex as a whole.

Dynamic survey of mitochondria by ubiquitin

Science.gov (United States)

Escobar-Henriques, Mafalda; Langer, Thomas

2014-01-01

Ubiquitin is a post-translational modifier with proteolytic and non-proteolytic roles in many biological processes. At mitochondria, it performs regulatory homeostatic functions and contributes to mitochondrial quality control. Ubiquitin is essential for mitochondrial fusion, regulates mitochondria-ER contacts, and participates in maternal mtDNA inheritance. Under stress, mitochondrial dysfunction induces ubiquitin-dependent responses that involve mitochondrial proteome remodeling and culminate in organelle removal by mitophagy. In addition, many ubiquitin-dependent mechanisms have been shown to regulate innate immune responses and xenophagy. Here, we review the emerging roles of ubiquitin at mitochondria. PMID:24569520

Identifying Cis-Regulatory Changes Involved in the Evolution of Aerobic Fermentation in Yeasts

Science.gov (United States)

Lin, Zhenguo; Wang, Tzi-Yuan; Tsai, Bing-Shi; Wu, Fang-Ting; Yu, Fu-Jung; Tseng, Yu-Jung; Sung, Huang-Mo; Li, Wen-Hsiung

2013-01-01

Gene regulation change has long been recognized as an important mechanism for phenotypic evolution. We used the evolution of yeast aerobic fermentation as a model to explore how gene regulation has evolved and how this process has contributed to phenotypic evolution and adaptation. Most eukaryotes fully oxidize glucose to CO2 and H2O in mitochondria to maximize energy yield, whereas some yeasts, such as Saccharomyces cerevisiae and its relatives, predominantly ferment glucose into ethanol even in the presence of oxygen, a phenomenon known as aerobic fermentation. We examined the genome-wide gene expression levels among 12 different yeasts and found that a group of genes involved in the mitochondrial respiration process showed the largest reduction in gene expression level during the evolution of aerobic fermentation. Our analysis revealed that the downregulation of these genes was significantly associated with massive loss of binding motifs of Cbf1p in the fermentative yeasts. Our experimental assays confirmed the binding of Cbf1p to the predicted motif and the activator role of Cbf1p. In summary, our study laid a foundation to unravel the long-time mystery about the genetic basis of evolution of aerobic fermentation, providing new insights into understanding the role of cis-regulatory changes in phenotypic evolution. PMID:23650209

Early manifestations of replicative aging in the yeast Saccharomyces cerevisiae

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Maksim I. Sorokin

2014-01-01

Full Text Available The yeast Saccharomyces cerevisiae is successfully used as a model organism to find genes responsible for lifespan control of higher organisms. As functional decline of higher eukaryotes can start as early as one quarter of the average lifespan, we asked whether S. cerevisiae can be used to model this manifestation of aging. While the average replicative lifespan of S. cerevisiae mother cells ranges between 15 and 30 division cycles, we found that resistances to certain stresses start to decrease much earlier. Looking into the mechanism, we found that knockouts of genes responsible for mitochondriato-nucleus (retrograde signaling, RTG1 or RTG3, significantly decrease the resistance of cells that generated more than four daughters, but not of the younger ones. We also found that even young mother cells frequently contain mitochondria with heterogeneous transmembrane potential and that the percentage of such cells correlates with replicative age. Together, these facts suggest that retrograde signaling starts to malfunction in relatively young cells, leading to accumulation of heterogeneous mitochondria within one cell. The latter may further contribute to a decline in stress resistances.

Mitochondria, Cybrids, Aging, and Alzheimer’s Disease

Science.gov (United States)

Swerdlow, Russell H.; Koppel, Scott; Weidling, Ian; Hayley, Clay; Ji, Yan; Wilkins, Heather M.

2018-01-01

Mitochondrial and bioenergetic function change with advancing age and may drive aging phenotypes. Mitochondrial and bioenergetic changes are also documented in various age-related neurodegenerative diseases, including Alzheimer’s disease (AD). In some instances AD mitochondrial and bioenergetic changes are reminiscent of those observed with advancing age, but are greater in magnitude. Mitochondrial and bioenergetic dysfunction could, therefore, link neurodegeneration to brain aging. Interestingly, mitochondrial defects in AD patients are not brain-limited, and mitochondrial function can be linked to classic AD histologic changes including amyloid precursor protein processing to beta amyloid. Also, transferring mitochondria from AD subjects to cell lines depleted of endogenous mitochondrial DNA (mtDNA) creates cytoplasmic hybrid (cybrid) cell lines that recapitulate specific biochemical, molecular, and histologic AD features. Such findings have led to the formulation of a “mitochondrial cascade hypothesis” that places mitochondrial dysfunction at the apex of the AD pathology pyramid. Data pertinent to this premise are reviewed. PMID:28253988

Functional Mitochondria in Health and Disease.

Science.gov (United States)

Herst, Patries M; Rowe, Matthew R; Carson, Georgia M; Berridge, Michael V

2017-01-01

The ability to rapidly adapt cellular bioenergetic capabilities to meet rapidly changing environmental conditions is mandatory for normal cellular function and for cancer progression. Any loss of this adaptive response has the potential to compromise cellular function and render the cell more susceptible to external stressors such as oxidative stress, radiation, chemotherapeutic drugs, and hypoxia. Mitochondria play a vital role in bioenergetic and biosynthetic pathways and can rapidly adjust to meet the metabolic needs of the cell. Increased demand is met by mitochondrial biogenesis and fusion of individual mitochondria into dynamic networks, whereas a decrease in demand results in the removal of superfluous mitochondria through fission and mitophagy. Effective communication between nucleus and mitochondria (mito-nuclear cross talk), involving the generation of different mitochondrial stress signals as well as the nuclear stress response pathways to deal with these stressors, maintains bioenergetic homeostasis under most conditions. However, when mitochondrial DNA (mtDNA) mutations accumulate and mito-nuclear cross talk falters, mitochondria fail to deliver critical functional outputs. Mutations in mtDNA have been implicated in neuromuscular and neurodegenerative mitochondriopathies and complex diseases such as diabetes, cardiovascular diseases, gastrointestinal disorders, skin disorders, aging, and cancer. In some cases, drastic measures such as acquisition of new mitochondria from donor cells occurs to ensure cell survival. This review starts with a brief discussion of the evolutionary origin of mitochondria and summarizes how mutations in mtDNA lead to mitochondriopathies and other degenerative diseases. Mito-nuclear cross talk, including various stress signals generated by mitochondria and corresponding stress response pathways activated by the nucleus are summarized. We also introduce and discuss a small family of recently discovered hormone-like mitopeptides

Photothermal imaging of skeletal muscle mitochondria.

Science.gov (United States)

Tomimatsu, Toru; Miyazaki, Jun; Kano, Yutaka; Kobayashi, Takayoshi

2017-06-01

The morphology and topology of mitochondria provide useful information about the physiological function of skeletal muscle. Previous studies of skeletal muscle mitochondria are based on observation with transmission, scanning electron microscopy or fluorescence microscopy. In contrast, photothermal (PT) microscopy has advantages over the above commonly used microscopic techniques because of no requirement for complex sample preparation by fixation or fluorescent-dye staining. Here, we employed the PT technique using a simple diode laser to visualize skeletal muscle mitochondria in unstained and stained tissues. The fine mitochondrial network structures in muscle fibers could be imaged with the PT imaging system, even in unstained tissues. PT imaging of tissues stained with toluidine blue revealed the structures of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria and the swelling behavior of mitochondria in damaged muscle fibers with sufficient image quality. PT image analyses based on fast Fourier transform (FFT) and Grey-level co-occurrence matrix (GLCM) were performed to derive the characteristic size of mitochondria and to discriminate the image patterns of normal and damaged fibers.

Peroxisome-mitochondria interplay and disease.

Science.gov (United States)

Schrader, Michael; Costello, Joseph; Godinho, Luis F; Islinger, Markus

2015-07-01

Peroxisomes and mitochondria are ubiquitous, highly dynamic organelles with an oxidative type of metabolism in eukaryotic cells. Over the years, substantial evidence has been provided that peroxisomes and mitochondria exhibit a close functional interplay which impacts on human health and development. The so-called "peroxisome-mitochondria connection" includes metabolic cooperation in the degradation of fatty acids, a redox-sensitive relationship, an overlap in key components of the membrane fission machineries and cooperation in anti-viral signalling and defence. Furthermore, combined peroxisome-mitochondria disorders with defects in organelle division have been revealed. In this review, we present the latest progress in the emerging field of peroxisomal and mitochondrial interplay in mammals with a particular emphasis on cooperative fatty acid β-oxidation, redox interplay, organelle dynamics, cooperation in anti-viral signalling and the resulting implications for disease.

Repression of mitochondrial translation, respiration and a metabolic cycle-regulated gene, SLF1, by the yeast Pumilio-family protein Puf3p.

Directory of Open Access Journals (Sweden)

Marc Chatenay-Lapointe

Full Text Available Synthesis and assembly of the mitochondrial oxidative phosphorylation (OXPHOS system requires genes located both in the nuclear and mitochondrial genomes, but how gene expression is coordinated between these two compartments is not fully understood. One level of control is through regulated expression mitochondrial ribosomal proteins and other factors required for mitochondrial translation and OXPHOS assembly, which are all products of nuclear genes that are subsequently imported into mitochondria. Interestingly, this cadre of genes in budding yeast has in common a 3'-UTR element that is bound by the Pumilio family protein, Puf3p, and is coordinately regulated under many conditions, including during the yeast metabolic cycle. Multiple functions have been assigned to Puf3p, including promoting mRNA degradation, localizing nucleus-encoded mitochondrial transcripts to the outer mitochondrial membrane, and facilitating mitochondria-cytoskeletal interactions and motility. Here we show that Puf3p has a general repressive effect on mitochondrial OXPHOS abundance, translation, and respiration that does not involve changes in overall mitochondrial biogenesis and largely independent of TORC1-mitochondrial signaling. We also identified the cytoplasmic translation factor Slf1p as yeast metabolic cycle-regulated gene that is repressed by Puf3p at the post-transcriptional level and promotes respiration and extension of yeast chronological life span when over-expressed. Altogether, these results should facilitate future studies on which of the many functions of Puf3p is most relevant for regulating mitochondrial gene expression and the role of nuclear-mitochondrial communication in aging and longevity.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine inhibits proton motive force in energized liver mitochondria

International Nuclear Information System (INIS)

Singh, Y.; Bhatnagar, R.; Sidhu, G.S.; Batra, J.K.; Krishna, G.

1989-01-01

It is known that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which induces Parkinson's-like disease in primates and humans, depletes hepatocytes of ATP and subsequently causes cell death. Incubation of rat liver mitochondria with MPTP and 1-methyl-4-phenyl pyridinium ion (MPP+) significantly inhibited incorporation of 32 Pi into ATP. MPTP and MPP+ inhibited the development of membrane potential and pH gradient in energized rat liver mitochondria, suggesting that reduction of the proton motive force may have reduced ATP synthesis. Since deprenyl, an inhibitor of monoamine oxidase, prevented the formation of MPP+ and inhibited the decrease in membrane potential caused by MPTP, but not that caused by MPP+, these effects of MPTP, as well as cell death, probably were mediated by MPP+. This mechanism may play a role in the specific loss of dopaminergic neurons resulting in MPTP-induced Parkinson's disease

Mam33 promotes cytochrome c oxidase subunit I translation in Saccharomyces cerevisiae mitochondria.

Science.gov (United States)

Roloff, Gabrielle A; Henry, Michael F

2015-08-15

Three mitochondrial DNA-encoded proteins, Cox1, Cox2, and Cox3, comprise the core of the cytochrome c oxidase complex. Gene-specific translational activators ensure that these respiratory chain subunits are synthesized at the correct location and in stoichiometric ratios to prevent unassembled protein products from generating free oxygen radicals. In the yeast Saccharomyces cerevisiae, the nuclear-encoded proteins Mss51 and Pet309 specifically activate mitochondrial translation of the largest subunit, Cox1. Here we report that Mam33 is a third COX1 translational activator in yeast mitochondria. Mam33 is required for cells to adapt efficiently from fermentation to respiration. In the absence of Mam33, Cox1 translation is impaired, and cells poorly adapt to respiratory conditions because they lack basal fermentative levels of Cox1. © 2015 Roloff and Henry. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Regulated release of Ca2+ from respiring mitochondria by Ca2+/2H+ antiport.

Science.gov (United States)

Fiskum, G; Lehninger, A L

1979-07-25

Simultaneous measurements of oxygen consumption and transmembrane transport of Ca2+, H+, and phosphate show that the efflux of Ca2+ from respiring tightly coupled rat liver mitochondria takes place by an electroneutral Ca2+/2H+ antiport process that is ruthenium red-insensitive and that is regulated by the oxidation-reduction state of the mitochondrial pyridine nucleotides. When mitochondrial pyridine nucleotides are kept in a reduced steady state, the efflux of Ca2+ is inhibited; when they are in an oxidized state, Ca2+ efflux is activated. These processes were demonstrated by allowing phosphate-depleted mitochondria respiring on succinate in the presence of rotenone to take up Ca2+ from the medium. Upon subsequent addition of ruthenium red to block Ca2+ transport via the electrophoretic influx pathway, and acetoacetate, to bring mitochondrial pyridine nucleotides into the oxidized state, Ca2+ efflux and H+ influx ensued. The observed H+ influx/Ca2+ efflux ratio was close to the value 2.0 predicted for the operation of an electrically neutral Ca2+/2H+ antiport process.

Interaction theory of mammalian mitochondria.

Science.gov (United States)

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. Copyright 2001 Academic Press.

Functional Mitochondria in Health and Disease

Directory of Open Access Journals (Sweden)

Patries M. Herst

2017-11-01

Full Text Available The ability to rapidly adapt cellular bioenergetic capabilities to meet rapidly changing environmental conditions is mandatory for normal cellular function and for cancer progression. Any loss of this adaptive response has the potential to compromise cellular function and render the cell more susceptible to external stressors such as oxidative stress, radiation, chemotherapeutic drugs, and hypoxia. Mitochondria play a vital role in bioenergetic and biosynthetic pathways and can rapidly adjust to meet the metabolic needs of the cell. Increased demand is met by mitochondrial biogenesis and fusion of individual mitochondria into dynamic networks, whereas a decrease in demand results in the removal of superfluous mitochondria through fission and mitophagy. Effective communication between nucleus and mitochondria (mito-nuclear cross talk, involving the generation of different mitochondrial stress signals as well as the nuclear stress response pathways to deal with these stressors, maintains bioenergetic homeostasis under most conditions. However, when mitochondrial DNA (mtDNA mutations accumulate and mito-nuclear cross talk falters, mitochondria fail to deliver critical functional outputs. Mutations in mtDNA have been implicated in neuromuscular and neurodegenerative mitochondriopathies and complex diseases such as diabetes, cardiovascular diseases, gastrointestinal disorders, skin disorders, aging, and cancer. In some cases, drastic measures such as acquisition of new mitochondria from donor cells occurs to ensure cell survival. This review starts with a brief discussion of the evolutionary origin of mitochondria and summarizes how mutations in mtDNA lead to mitochondriopathies and other degenerative diseases. Mito-nuclear cross talk, including various stress signals generated by mitochondria and corresponding stress response pathways activated by the nucleus are summarized. We also introduce and discuss a small family of recently discovered hormone

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

Energy Technology Data Exchange (ETDEWEB)

Mailloux, Ryan J.; Yumvihoze, Emmanuel; Chan, Hing Man, E-mail: laurie.chan@uottawa.ca

2015-12-15

The mechanism of intracellular metabolism of methylmercury (MeHg) is not fully known. It has been shown that superoxide (O{sub 2}·{sup −}), 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 O{sub 2}·{sup −} mediated degradation of MeHg in human neuroblastoma cells SH-K-SN. We also utilized paraquat (PQ) which generates O{sub 2}·{sup −} in the mitochondrial matrix. We found that the cleavage of the carbon-metal bond in MeHg was highly dependent on the topology of O{sub 2}·{sup −} production by mitochondria. Both rotenone and PQ, which increase O{sub 2}·{sup −} 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 O{sub 2}·{sup −} into the intermembrane space, did not have the same effect even though antimycin A induced a dose dependent increase in O{sub 2}·{sup −} 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 O{sub 2}·{sup −}, specifically within the matrix of mitochondria when O{sub 2}·{sup −} is in adequate supply. Our results also show that O{sub 2}·{sup −} 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. - Highlights: • Superoxide produced in the matrix of mitochondria degrades MeHg. • Superoxide produced in intermembrane space does not degrade MeHg. • Matrix-generated superoxide enhances Hg toxicity by converting MeHg to iHg.

Mitochondria in health and disease

DEFF Research Database (Denmark)

Durhuus, Jon Ambæk; Madsen, Claus Desler; Rasmussen, Lene Juel

2015-01-01

The primary role of mitochondria was long considered to be production of cellular energy. However, as the understanding of mitochondria in disease is ever expanding, so is their additional function for a healthy organism. Mitochondrial dysfunction is linked to a range of pathologies, including...... (SMRM) was titled "Mitochondria in Health and Disease". The conference was organized by Gayathri N, K Thangaraj, and KK Singh and was held at the National Institute of Mental Health & Neuro Sciences (NIMHANS) in Bangalore, India, from the 19th to 20th of December 2013. The meeting featured...

Assembly of the membrane domain of ATP synthase in human mitochondria.

Science.gov (United States)

He, Jiuya; Ford, Holly C; Carroll, Joe; Douglas, Corsten; Gonzales, Evvia; Ding, Shujing; Fearnley, Ian M; Walker, John E

2018-03-20

The ATP synthase in human mitochondria is a membrane-bound assembly of 29 proteins of 18 kinds. All but two membrane components are encoded in nuclear genes, synthesized on cytoplasmic ribosomes, and imported into the matrix of the organelle, where they are assembled into the complex with ATP6 and ATP8, the products of overlapping genes in mitochondrial DNA. Disruption of individual human genes for the nuclear-encoded subunits in the membrane portion of the enzyme leads to the formation of intermediate vestigial ATPase complexes that provide a description of the pathway of assembly of the membrane domain. The key intermediate complex consists of the F 1 -c 8 complex inhibited by the ATPase inhibitor protein IF 1 and attached to the peripheral stalk, with subunits e, f, and g associated with the membrane domain of the peripheral stalk. This intermediate provides the template for insertion of ATP6 and ATP8, which are synthesized on mitochondrial ribosomes. Their association with the complex is stabilized by addition of the 6.8 proteolipid, and the complex is coupled to ATP synthesis at this point. A structure of the dimeric yeast F o membrane domain is consistent with this model of assembly. The human 6.8 proteolipid (yeast j subunit) locks ATP6 and ATP8 into the membrane assembly, and the monomeric complexes then dimerize via interactions between ATP6 subunits and between 6.8 proteolipids (j subunits). The dimers are linked together back-to-face by DAPIT (diabetes-associated protein in insulin-sensitive tissue; yeast subunit k), forming long oligomers along the edges of the cristae.

Yeasts from Different Habitats and Their Potential as Biocontrol Agents

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Julia Pretscher

2018-04-01

Full Text Available Ever since plant diseases began causing losses in viticulture, the control of phytopathogenic fungi has become of vital interest for winemakers. The occurrence of novel pests, fungicide resistance, and changed consumer expectations have led to an enormous demand for novel plant protection strategies. As part of integrated protection measures, antagonistic microorganisms have been investigated to a large extent. Such microorganisms can be applied not only in conventional, but also in organic farming as biological control agents (BCA. Particularly, yeasts were found to be interesting candidates for the development of BCA. Many of these eukaryotic microorganisms are found as part of the phylloplane microflora. In this study, we assessed a set of 38 yeast isolates from different habitats, including the guts of termites, for inhibitory effects against some phytopathogenic fungi that have received less attention in earlier studies. The majority of yeasts were found to interfere with fungi infecting grapevine (Eutypa lata, Botrytis cinerea, and Roesleria subterranea, stone fruits (Monilinia fructicola, or rice (Magnaporte oryzae, as well in vitro and in model experiment on fruits. Although most yeast strains secreted glycoside hydrolases and proteases, attempts to demonstrate direct antagonistic activities of lytic enzymes failed. However, in culture filtrates of the termite yeast Papiliotrema odontotermitis OO5, a low molecular thermostable antagonistic factor was detected. Iron depletion as a BCA mechanism was confirmed for strains of Metschnikowia pulcherrima but not for other yeasts.

Molecular cloning of amphioxus uncoupling protein and assessment of its uncoupling activity using a yeast heterologous expression system

Energy Technology Data Exchange (ETDEWEB)

Chen, Kun [Jiangsu Diabetes Research Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu (China); Sun, Guoxun [Department of Hematology, Fourth Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001 (China); Lv, Zhiyuan; Wang, Chen [Jiangsu Diabetes Research Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu (China); Jiang, Xueyuan, E-mail: xueyuanjiang@yahoo.com.cn [Jiangsu Diabetes Research Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu (China); Li, Donghai, E-mail: lidonghai@gmail.com [Jiangsu Diabetes Research Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu (China); Zhang, Chenyu, E-mail: cyzhang@nju.edu.cn [Jiangsu Diabetes Research Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu (China)

2010-10-01

Research highlights: {yields} Invertebrates, for example amphioxus, do express uncoupling proteins. {yields} Both the sequence and the uncoupling activity of amphioxus UCP resemble UCP2. {yields} UCP1 is the only UCP that can form dimer on yeast mitochondria. -- Abstract: The present study describes the molecular cloning of a novel cDNA fragment from amphioxus (Branchiostoma belcheri) encoding a 343-amino acid protein that is highly homologous to human uncoupling proteins (UCP), this protein is therefore named amphioxus UCP. This amphioxus UCP shares more homology with and is phylogenetically more related to mammalian UCP2 as compared with UCP1. To further assess the functional similarity of amphioxus UCP to mammalian UCP1 and -2, the amphioxus UCP, rat UCP1, and human UCP2 were separately expressed in Saccharomyces cerevisiae, and the recombinant yeast mitochondria were isolated and assayed for the state 4 respiration rate and proton leak, using pYES2 empty vector as the control. UCP1 increased the state 4 respiration rate by 2.8-fold, and the uncoupling activity was strongly inhibited by GDP, while UCP2 and amphioxus UCP only increased the state 4 respiration rate by 1.5-fold and 1.7-fold in a GDP-insensitive manner, moreover, the proton leak kinetics of amphioxus UCP was very similar to UCP2, but much different from UCP1. In conclusion, the amphioxus UCP has a mild, unregulated uncoupling activity in the yeast system, which resembles mammalian UCP2, but not UCP1.

Molecular cloning of amphioxus uncoupling protein and assessment of its uncoupling activity using a yeast heterologous expression system

International Nuclear Information System (INIS)

Chen, Kun; Sun, Guoxun; Lv, Zhiyuan; Wang, Chen; Jiang, Xueyuan; Li, Donghai; Zhang, Chenyu

2010-01-01

Research highlights: → Invertebrates, for example amphioxus, do express uncoupling proteins. → Both the sequence and the uncoupling activity of amphioxus UCP resemble UCP2. → UCP1 is the only UCP that can form dimer on yeast mitochondria. -- Abstract: The present study describes the molecular cloning of a novel cDNA fragment from amphioxus (Branchiostoma belcheri) encoding a 343-amino acid protein that is highly homologous to human uncoupling proteins (UCP), this protein is therefore named amphioxus UCP. This amphioxus UCP shares more homology with and is phylogenetically more related to mammalian UCP2 as compared with UCP1. To further assess the functional similarity of amphioxus UCP to mammalian UCP1 and -2, the amphioxus UCP, rat UCP1, and human UCP2 were separately expressed in Saccharomyces cerevisiae, and the recombinant yeast mitochondria were isolated and assayed for the state 4 respiration rate and proton leak, using pYES2 empty vector as the control. UCP1 increased the state 4 respiration rate by 2.8-fold, and the uncoupling activity was strongly inhibited by GDP, while UCP2 and amphioxus UCP only increased the state 4 respiration rate by 1.5-fold and 1.7-fold in a GDP-insensitive manner, moreover, the proton leak kinetics of amphioxus UCP was very similar to UCP2, but much different from UCP1. In conclusion, the amphioxus UCP has a mild, unregulated uncoupling activity in the yeast system, which resembles mammalian UCP2, but not UCP1.

Your mitochondria are what you eat

DEFF Research Database (Denmark)

Jørgensen, Wenche; Rud, Kasper Abildgaard; Mortensen, Ole Hartvig

2017-01-01

of the mitochondria. Here, we report that rat muscle mitochondria does show the normal Randle‐type fat‐carbohydrate interaction seen in vivo. The mechanism behind this metabolic flexibility at the level of the isolated mitochondria is a regulation of the flux‐ratio: pyruvate dehydrogenase (PDH)/β‐oxidation to suit...... the actual substrate availability, with the PDH flux as the major point of regulation. We further report that this regulatory mechanism of carbohydrate‐fat metabolic interaction surprisingly is lost in mitochondria obtained from animals exposed for 12 weeks to a HF‐ or a HS diet as compared to rats given...... a normal chow diet. The mechanism seems to be a loss of the PDH flux decrease seen in controls, when fatty acid is supplied as substrate in addition to pyruvate, and vice versa for the supply of pyruvate as substrate to mitochondria oxidizing fatty acid. Finally, we report that the calculated TCA flux...

Mitochondria and Neurotransmission: Evacuating the Synapse

OpenAIRE

Hollenbeck, Peter J.

2005-01-01

An abundance of mitochondria has been the hallmark of synapses since their first ultrastructural description 50 years ago. Mitochondria have been shown to be essential for synaptic form and function in many systems, but until recently it has not been clear exactly what role(s) they play in neurotransmission. Now, evidence from the nervous system of Drosophila identifies the specific subcellular events that are most dependent upon nearby mitochondria.

Lipid droplets interact with mitochondria using SNAP23

DEFF Research Database (Denmark)

Jägerström, Sara; Polesie, Sam; Wickström, Ylva

2009-01-01

peroxisomes and the endoplasmic reticulum. We have used electron and confocal microscopy to demonstrate that LD form complexes with mitochondria in NIH 3T3 fibroblasts. Using an in vitro system of purified LD and mitochondria, we also show the formation of the LD-mitochondria complex, in which cytosolic...... factors are involved. Moreover, the presence of LD markers in mitochondria isolated by subcellular fractionations is demonstrated. Finally, ablation of SNAP23 using siRNA reduced complex formation and beta oxidation, which suggests that the LD-mitochondria complex is functional in the cell....

Calcium regulates cell death in cancer: Roles of the mitochondria and mitochondria-associated membranes (MAMs).

Science.gov (United States)

Danese, Alberto; Patergnani, Simone; Bonora, Massimo; Wieckowski, Mariusz R; Previati, Maurizio; Giorgi, Carlotta; Pinton, Paolo

2017-08-01

Until 1972, the term 'apoptosis' was used to differentiate the programmed cell death that naturally occurs in organismal development from the acute tissue death referred to as necrosis. Many studies on cell death and programmed cell death have been published and most are, at least to some degree, related to cancer. Some key proteins and molecular pathways implicated in cell death have been analyzed, whereas others are still being actively researched; therefore, an increasing number of cellular compartments and organelles are being implicated in cell death and cancer. Here, we discuss the mitochondria and subdomains of the endoplasmic reticulum (ER) that interact with mitochondria, the mitochondria-associated membranes (MAMs), which have been identified as critical hubs in the regulation of cell death and tumor growth. MAMs-dependent calcium (Ca 2+ ) release from the ER allows selective Ca 2+ uptake by the mitochondria. The perturbation of Ca 2+ homeostasis in cancer cells is correlated with sustained cell proliferation and the inhibition of cell death through the modulation of Ca 2+ signaling. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux. Copyright © 2017 Elsevier B.V. All rights reserved.

Depletion of a Drosophila homolog of yeast Sup35p disrupts spindle assembly, chromosome segregation, and cytokinesis during male meiosis.

Science.gov (United States)

Basu, J; Williams, B C; Li, Z; Williams, E V; Goldberg, M L

1998-01-01

In the course of a genetic screen for male-sterile mutations in Drosophila affecting chromosome segregation during the meiotic divisions in spermatocytes, we identified the mutation dsup35(63D). Examination of mutant testes showed that chromosome misbehavior was a consequence of major disruptions in meiotic spindle assembly. These perturbations included problems in aster formation, separation, and migration around the nuclear envelope; aberrations in spindle organization and integrity; and disappearance of the ana/telophase central spindle, which in turn disrupts cytokinesis. The dsup35(63D) mutation is caused by a P element insertion that affects, specifically in the testis, the expression of a gene (dsup35) encoding the Drosophila homolog of the yeast Sup35p and Xenopus eRF3 proteins. These proteins are involved in the termination of polypeptide synthesis on ribosomes, but previous studies have suggested that Sup35p and closely related proteins of the same family also interact directly with microtubules. An affinity-purified antibody directed against the product of the dsup35 gene was prepared; interestingly, this antibody specifically labels primary spermatocytes in one or two discrete foci of unknown structure within the nucleoplasm. We discuss how depletion of the dsup35 gene product in spermatocytes might lead to the global disruptions in meiotic spindle assembly seen in mutant spermatocytes.

Protonmotive force in muscle mitochondria

International Nuclear Information System (INIS)

Stumpf, D.A.; Haas, R.; Eguren, L.A.; Parks, J.K.; Eilert, R.E.

1982-01-01

The protonmotive force (delta p) of muscle mitochondria was measured by estimating the distribution of 14C-labeled TPMP (trimethylphenylphosphonium iodide) and 14C-labeled acetate across the inner membrane of muscle mitochondria. The matrix volume was simultaneously determined using 3H-labeled H2O and 3H-labeled mannitol and repeated drying to distinguish the label in these 2 compounds. Rapid separation of mitochondria from the incubation medium by centrifugation through silicone oil avoids the problems of potential anaerobic conditions associated with conventional centrifugation and large volumes of trapped media associated with filtration. The value for delta p (mean +/- SD) was 192+/- 26 mV in 30 determinations with rat muscle mitochondria during state 4. Measurement of oxygen consumption allowed calculation of membrane conductance (Cm,H+) which was 0.49 +/- 0.18 nmol of H+/min/mg protein/mV. The values for delta p and Cm,H+ are reported for a variety of experimental conditions and are consistent with Mitchell's chemiosmotic theory. Biopsy specimens obtained from human muscle gave state-4 delta p values of 197+/- 30 mV (n .5) and Cm,H+ values of 0.52 +/- 0.12 nmol of H+/min/mg/mV (n . 4). This delta p assay is the first described for coupled mammalian muscle mitochondria and will be useful in assessing membrane function

A chemical genetic screen for modulators of asymmetrical 2,2'-dimeric naphthoquinones cytotoxicity in yeast.

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Ashkan Emadi

Full Text Available BACKGROUND: Dimeric naphthoquinones (BiQ were originally synthesized as a new class of HIV integrase inhibitors but have shown integrase-independent cytotoxicity in acute lymphoblastic leukemia cell lines suggesting their use as potential anti-neoplastic agents. The mechanism of this cytotoxicity is unknown. In order to gain insight into the mode of action of binaphthoquinones we performed a systematic high-throughput screen in a yeast isogenic deletion mutant array for enhanced or suppressed growth in the presence of binaphthoquinones. METHODOLOGY/PRINCIPAL FINDINGS: Exposure of wild type yeast strains to various BiQs demonstrated inhibition of yeast growth with IC(50s in the microM range. Drug sensitivity and resistance screens were performed by exposing arrays of a haploid yeast deletion mutant library to BiQs at concentrations near their IC(50. Sensitivity screens identified yeast with deletions affecting mitochondrial function and cellular respiration as having increased sensitivity to BiQs. Corresponding to this, wild type yeast grown in the absence of a fermentable carbon source were particularly sensitive to BiQs, and treatment with BiQs was shown to disrupt the mitochondrial membrane potential and lead to the generation of reactive oxygen species (ROS. Furthermore, baseline ROS production in BiQ sensitive mutant strains was increased compared to wild type and could be further augmented by the presence of BiQ. Screens for resistance to BiQ action identified the mitochondrial external NAD(PH dehydrogenase, NDE1, as critical to BiQ toxicity and over-expression of this gene resulted in increased ROS production and increased sensitivity of wild type yeast to BiQ. CONCLUSIONS/SIGNIFICANCE: In yeast, binaphthoquinone cytotoxicity is likely mediated through NAD(PH:quonine oxidoreductases leading to ROS production and dysfunctional mitochondria. Further studies are required to validate this mechanism in mammalian cells.

Exogenous ether lipids predominantly target mitochondria.

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Lars Kuerschner

Full Text Available Ether lipids are ubiquitous constituents of cellular membranes with no discrete cell biological function assigned yet. Using fluorescent polyene-ether lipids we analyzed their intracellular distribution in living cells by microscopy. Mitochondria and the endoplasmic reticulum accumulated high amounts of ether-phosphatidylcholine and ether-phosphatidylethanolamine. Both lipids were specifically labeled using the corresponding lyso-ether lipids, which we established as supreme precursors for lipid tagging. Polyfosine, a fluorescent analogue of the anti-neoplastic ether lipid edelfosine, accumulated to mitochondria and induced morphological changes and cellular apoptosis. These data indicate that edelfosine could exert its pro-apoptotic power by targeting and damaging mitochondria and thereby inducing cellular apoptosis. In general, this study implies an important role of mitochondria in ether lipid metabolism and intracellular ether lipid trafficking.

Residual mitochondrial transmembrane potential decreases unsaturated fatty acid level in sake yeast during alcoholic fermentation

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Kazutaka Sawada

2016-01-01

Full Text Available Oxygen, a key nutrient in alcoholic fermentation, is rapidly depleted during this process. Several pathways of oxygen utilization have been reported in the yeast Saccharomyces cerevisiae during alcoholic fermentation, namely synthesis of unsaturated fatty acid, sterols and heme, and the mitochondrial electron transport chain. However, the interaction between these pathways has not been investigated. In this study, we showed that the major proportion of unsaturated fatty acids of ester-linked lipids in sake fermentation mash is derived from the sake yeast rather than from rice or koji (rice fermented with Aspergillus. Additionally, during alcoholic fermentation, inhibition of the residual mitochondrial activity of sake yeast increases the levels of unsaturated fatty acids of ester-linked lipids. These findings indicate that the residual activity of the mitochondrial electron transport chain reduces molecular oxygen levels and decreases the synthesis of unsaturated fatty acids, thereby increasing the synthesis of estery flavors by sake yeast. This is the first report of a novel link between residual mitochondrial transmembrane potential and the synthesis of unsaturated fatty acids by the brewery yeast during alcoholic fermentation.

ARTD1/PARP1 Negatively Regulates Glycolysis by Inhibiting Hexokinase 1 Independent of NAD+ Depletion

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Elise Fouquerel

2014-09-01

Full Text Available ARTD1 (PARP1 is a key enzyme involved in DNA repair through the synthesis of poly(ADP-ribose (PAR in response to strand breaks, and it plays an important role in cell death following excessive DNA damage. ARTD1-induced cell death is associated with NAD+ depletion and ATP loss; however, the molecular mechanism of ARTD1-mediated energy collapse remains elusive. Using real-time metabolic measurements, we compared the effects of ARTD1 activation and direct NAD+ depletion. We found that ARTD1-mediated PAR synthesis, but not direct NAD+ depletion, resulted in a block to glycolysis and ATP loss. We then established a proteomics-based PAR interactome after DNA damage and identified hexokinase 1 (HK1 as a PAR binding protein. HK1 activity is suppressed following nuclear ARTD1 activation and binding by PAR. These findings help explain how prolonged activation of ARTD1 triggers energy collapse and cell death, revealing insight into the importance of nucleus-to-mitochondria communication via ARTD1 activation.

Direct concentration and viability measurement of yeast in corn mash using a novel imaging cytometry method.

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Chan, Leo L; Lyettefi, Emily J; Pirani, Alnoor; Smith, Tim; Qiu, Jean; Lin, Bo

2011-08-01

Worldwide awareness of fossil-fuel depletion and global warming has been increasing over the last 30 years. Numerous countries, including the USA and Brazil, have introduced large-scale industrial fermentation facilities for bioethanol, biobutanol, or biodiesel production. Most of these biofuel facilities perform fermentation using standard baker's yeasts that ferment sugar present in corn mash, sugar cane, or other glucose media. In research and development in the biofuel industry, selection of yeast strains (for higher ethanol tolerance) and fermentation conditions (yeast concentration, temperature, pH, nutrients, etc.) can be studied to optimize fermentation performance. Yeast viability measurement is needed to identify higher ethanol-tolerant yeast strains, which may prolong the fermentation cycle and increase biofuel output. In addition, yeast concentration may be optimized to improve fermentation performance. Therefore, it is important to develop a simple method for concentration and viability measurement of fermenting yeast. In this work, we demonstrate an imaging cytometry method for concentration and viability measurements of yeast in corn mash directly from operating fermenters. It employs an automated cell counter, a dilution buffer, and staining solution from Nexcelom Bioscience to perform enumeration. The proposed method enables specific fluorescence detection of viable and nonviable yeasts, which can generate precise results for concentration and viability of yeast in corn mash. This method can provide an essential tool for research and development in the biofuel industry and may be incorporated into manufacturing to monitor yeast concentration and viability efficiently during the fermentation process.

Mitochondria-targeted vitamin E analogs inhibit breast cancer cell energy metabolism and promote cell death

International Nuclear Information System (INIS)

Cheng, Gang; Zielonka, Jacek; McAllister, Donna M; Mackinnon, A Craig Jr; Joseph, Joy; Dwinell, Michael B; Kalyanaraman, Balaraman

2013-01-01

Recent research has revealed that targeting mitochondrial bioenergetic metabolism is a promising chemotherapeutic strategy. Key to successful implementation of this chemotherapeutic strategy is the use of new and improved mitochondria-targeted cationic agents that selectively inhibit energy metabolism in breast cancer cells, while exerting little or no long-term cytotoxic effect in normal cells. In this study, we investigated the cytotoxicity and alterations in bioenergetic metabolism induced by mitochondria-targeted vitamin E analog (Mito-chromanol, Mito-ChM) and its acetylated ester analog (Mito-ChMAc). Assays of cell death, colony formation, mitochondrial bioenergetic function, intracellular ATP levels, intracellular and tissue concentrations of tested compounds, and in vivo tumor growth were performed. Both Mito-ChM and Mito-ChMAc selectively depleted intracellular ATP and caused prolonged inhibition of ATP-linked oxygen consumption rate in breast cancer cells, but not in non-cancerous cells. These effects were significantly augmented by inhibition of glycolysis. Mito-ChM and Mito-ChMAc exhibited anti-proliferative effects and cytotoxicity in several breast cancer cells with different genetic background. Furthermore, Mito-ChM selectively accumulated in tumor tissue and inhibited tumor growth in a xenograft model of human breast cancer. We conclude that mitochondria-targeted small molecular weight chromanols exhibit selective anti-proliferative effects and cytotoxicity in multiple breast cancer cells, and that esterification of the hydroxyl group in mito-chromanols is not a critical requirement for its anti-proliferative and cytotoxic effect

Isolation of Mitochondria from Potato Tubers

DEFF Research Database (Denmark)

Havelund, Jesper F.; Salvato, Fernanda; Chen, Mingjie

2014-01-01

One way to study the function of plant mitochondria is to extract them from plant tissues in an uncontaminated, intact and functional form. The reductionist assumption is that the components present in such a preparation and the in vitro measurable functions or activities reliably reflect...... the in vivo properties of the organelle inside the plant cell. Here, we describe a method to isolate mitochondria from a relatively homogeneous plant tissue, the dormant potato tuber. The homogenization is done using a juice extractor, which is a relatively gentle homogenization procedure where...... the mitochondria are only exposed to strong shearing forces once. After removal of starch and large tissue pieces by filtration, differential centrifugation is used to remove residual starch as well as larger organelles. The crude mitochondria are then first purified by using a step Percoll gradient...

Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System

Science.gov (United States)

Verghese, Jacob; Abrams, Jennifer; Wang, Yanyu

2012-01-01

Summary: The eukaryotic heat shock response is an ancient and highly conserved transcriptional program that results in the immediate synthesis of a battery of cytoprotective genes in the presence of thermal and other environmental stresses. Many of these genes encode molecular chaperones, powerful protein remodelers with the capacity to shield, fold, or unfold substrates in a context-dependent manner. The budding yeast Saccharomyces cerevisiae continues to be an invaluable model for driving the discovery of regulatory features of this fundamental stress response. In addition, budding yeast has been an outstanding model system to elucidate the cell biology of protein chaperones and their organization into functional networks. In this review, we evaluate our understanding of the multifaceted response to heat shock. In addition, the chaperone complement of the cytosol is compared to those of mitochondria and the endoplasmic reticulum, organelles with their own unique protein homeostasis milieus. Finally, we examine recent advances in the understanding of the roles of protein chaperones and the heat shock response in pathogenic fungi, which is being accelerated by the wealth of information gained for budding yeast. PMID:22688810

Bcs1p can rescue a large and productive cytochrome bc(1) complex assembly intermediate in the inner membrane of yeast mitochondria.

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Conte, Laura; Trumpower, Bernard L; Zara, Vincenzo

2011-01-01

The yeast cytochrome bc(1) complex, a component of the mitochondrial respiratory chain, is composed of ten distinct protein subunits. In the assembly of the bc(1) complex, some ancillary proteins, such as the chaperone Bcs1p, are actively involved. The deletion of the nuclear gene encoding this chaperone caused the arrest of the bc(1) assembly and the formation of a functionally inactive bc(1) core structure of about 500-kDa. This immature bc(1) core structure could represent, on the one hand, a true assembly intermediate or, on the other hand, a degradation product and/or an incorrect product of assembly. The experiments here reported show that the gradual expression of Bcs1p in the yeast strain lacking this protein was progressively able to rescue the bc(1) core structure leading to the formation of the functional homodimeric bc(1) complex. Following Bcs1p expression, the mature bc(1) complex was also progressively converted into two supercomplexes with the cytochrome c oxidase complex. The capability of restoring the bc(1) complex and the supercomplexes was also possessed by the mutated yeast R81C Bcsp1. Notably, in the human ortholog BCS1L, the corresponding point mutation (R45C) was instead the cause of a severe bc(1) complex deficiency. Differently from the yeast R81C Bcs1p, two other mutated Bcs1p's (K192P and F401I) were unable to recover the bc(1) core structure in yeast. This study identifies for the first time a productive assembly intermediate of the yeast bc(1) complex and gives new insights into the molecular mechanisms involved in the last steps of bc(1) assembly. Copyright © 2010 Elsevier B.V. All rights reserved.

Mitochondria and Mitochondrial ROS in Cancer: Novel Targets for Anticancer Therapy.

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Yang, Yuhui; Karakhanova, Svetlana; Hartwig, Werner; D'Haese, Jan G; Philippov, Pavel P; Werner, Jens; Bazhin, Alexandr V

2016-12-01

Mitochondria are indispensable for energy metabolism, apoptosis regulation, and cell signaling. Mitochondria in malignant cells differ structurally and functionally from those in normal cells and participate actively in metabolic reprogramming. Mitochondria in cancer cells are characterized by reactive oxygen species (ROS) overproduction, which promotes cancer development by inducing genomic instability, modifying gene expression, and participating in signaling pathways. Mitochondrial and nuclear DNA mutations caused by oxidative damage that impair the oxidative phosphorylation process will result in further mitochondrial ROS production, completing the "vicious cycle" between mitochondria, ROS, genomic instability, and cancer development. The multiple essential roles of mitochondria have been utilized for designing novel mitochondria-targeted anticancer agents. Selective drug delivery to mitochondria helps to increase specificity and reduce toxicity of these agents. In order to reduce mitochondrial ROS production, mitochondria-targeted antioxidants can specifically accumulate in mitochondria by affiliating to a lipophilic penetrating cation and prevent mitochondria from oxidative damage. In consistence with the oncogenic role of ROS, mitochondria-targeted antioxidants are found to be effective in cancer prevention and anticancer therapy. A better understanding of the role played by mitochondria in cancer development will help to reveal more therapeutic targets, and will help to increase the activity and selectivity of mitochondria-targeted anticancer drugs. In this review we summarized the impact of mitochondria on cancer and gave summary about the possibilities to target mitochondria for anticancer therapies. J. Cell. Physiol. 231: 2570-2581, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Role of the mitochondria in immune-mediated apoptotic death of the human pancreatic β cell line βLox5.

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Yaíma L Lightfoot

Full Text Available 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 ρ(0 cells. βLox5 ρ(0 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 ρ(0 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.

Polyglutamine toxicity in yeast induces metabolic alterations and mitochondrial defects

KAUST Repository

Papsdorf, Katharina

2015-09-03

Background Protein aggregation and its pathological effects are the major cause of several neurodegenerative diseases. In Huntington’s disease an elongated stretch of polyglutamines within the protein Huntingtin leads to increased aggregation propensity. This induces cellular defects, culminating in neuronal loss, but the connection between aggregation and toxicity remains to be established. Results To uncover cellular pathways relevant for intoxication we used genome-wide analyses in a yeast model system and identify fourteen genes that, if deleted, result in higher polyglutamine toxicity. Several of these genes, like UGO1, ATP15 and NFU1 encode mitochondrial proteins, implying that a challenged mitochondrial system may become dysfunctional during polyglutamine intoxication. We further employed microarrays to decipher the transcriptional response upon polyglutamine intoxication, which exposes an upregulation of genes involved in sulfur and iron metabolism and mitochondrial Fe-S cluster formation. Indeed, we find that in vivo iron concentrations are misbalanced and observe a reduction in the activity of the prominent Fe-S cluster containing protein aconitase. Like in other yeast strains with impaired mitochondria, non-fermentative growth is impossible after intoxication with the polyglutamine protein. NMR-based metabolic analyses reveal that mitochondrial metabolism is reduced, leading to accumulation of metabolic intermediates in polyglutamine-intoxicated cells. Conclusion These data show that damages to the mitochondrial system occur in polyglutamine intoxicated yeast cells and suggest an intricate connection between polyglutamine-induced toxicity, mitochondrial functionality and iron homeostasis in this model system.

Deoxynucleoside salvage enzymes and tissue specific mitochondrial DNA depletion.

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Wang, L

2010-06-01

Adequate mitochondrial DNA (mtDNA) copies are required for normal mitochondria function and reductions in mtDNA copy number due to genetic alterations cause tissue-specific mtDNA depletion syndrome (MDS). There are eight nuclear genes, directly or indirectly involved in mtDNA replication and mtDNA precursor synthesis, which have been identified as the cause of MDS. However, the tissue specific pathology of these nuclear gene mutations is not well understood. Here, mtDNA synthesis, mtDNA copy number control, and mtDNA turnover, as well as the synthesis of mtDNA precursors in relation to the levels of salvage enzymes are discussed. The question why MDS caused by TK2 and p53R2 mutations are predominantly muscle specific while dGK deficiency affected mainly liver will be addressed.

Intra- and Intercellular Quality Control Mechanisms of Mitochondria

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Yoshimitsu Kiriyama

2017-12-01

Full Text Available Mitochondria function to generate ATP and also play important roles in cellular homeostasis, signaling, apoptosis, autophagy, and metabolism. The loss of mitochondrial function results in cell death and various types of diseases. Therefore, quality control of mitochondria via intra- and intercellular pathways is crucial. Intracellular quality control consists of biogenesis, fusion and fission, and degradation of mitochondria in the cell, whereas intercellular quality control involves tunneling nanotubes and extracellular vesicles. In this review, we outline the current knowledge on the intra- and intercellular quality control mechanisms of mitochondria.

On Cellular Darwinism: Mitochondria.

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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.

X-ray irradiation of yeast cells

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Masini, Alessandra; Batani, Dimitri; Previdi, Fabio; Conti, Aldo; Pisani, Francesca; Botto, Cesare; Bortolotto, Fulvia; Torsiello, Flavia; Turcu, I. C. Edmond; Allott, Ric M.; Lisi, Nicola; Milani, Marziale; Costato, Michele; Pozzi, Achille; Koenig, Michel

1997-10-01

Saccharomyces Cerevisiae yeast cells were irradiated using the soft X-ray laser-plasma source at Rutherford Laboratory. The aim was to produce a selective damage of enzyme metabolic activity at the wall and membrane level (responsible for fermentation) without interfering with respiration (taking place in mitochondria) and with nuclear and DNA activity. The source was calibrated by PIN diodes and X-ray spectrometers. Teflon stripes were chosen as targets for the UV laser, emitting X-rays at about 0.9 keV, characterized by a very large decay exponent in biological matter. X-ray doses to the different cell compartments were calculated following a Lambert-Bouguet-Beer law. After irradiation, the selective damage to metabolic activity at the membrane level was measured by monitoring CO2 production with pressure silicon detectors. Preliminary results gave evidence of pressure reduction for irradiated samples and non-linear response to doses. Also metabolic oscillations were evidenced in cell suspensions and it was shown that X-ray irradiation changed the oscillation frequency.

Population FBA predicts metabolic phenotypes in yeast.

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Piyush Labhsetwar

2017-09-01

Full Text Available Using protein counts sampled from single cell proteomics distributions to constrain fluxes through a genome-scale model of metabolism, Population flux balance analysis (Population FBA successfully described metabolic heterogeneity in a population of independent Escherichia coli cells growing in a defined medium. We extend the methodology to account for correlations in protein expression arising from the co-regulation of genes and apply it to study the growth of independent Saccharomyces cerevisiae cells in two different growth media. We find the partitioning of flux between fermentation and respiration predicted by our model agrees with recent 13C fluxomics experiments, and that our model largely recovers the Crabtree effect (the experimentally known bias among certain yeast species toward fermentation with the production of ethanol even in the presence of oxygen, while FBA without proteomics constraints predicts respirative metabolism almost exclusively. The comparisons to the 13C study showed improvement upon inclusion of the correlations and motivated a technique to systematically identify inconsistent kinetic parameters in the literature. The minor secretion fluxes for glycerol and acetate are underestimated by our method, which indicate a need for further refinements to the metabolic model. For yeast cells grown in synthetic defined (SD medium, the calculated broad distribution of growth rates matches experimental observations from single cell studies, and we characterize several metabolic phenotypes within our modeled populations that make use of diverse pathways. Fast growing yeast cells are predicted to perform significant amount of respiration, use serine-glycine cycle and produce ethanol in mitochondria as opposed to slow growing cells. We use a genetic algorithm to determine the proteomics constraints necessary to reproduce the growth rate distributions seen experimentally. We find that a core set of 51 constraints are essential but

A Comparison of Two Yeast MnSODs: Mitochondrial Saccharomyces cerevisiae versus Cytosolic Candida albicans

International Nuclear Information System (INIS)

Sheng, Y.; Cabelli, D.; Stich, T.A.; Barnese, K.; Gralla, E.B.; Cascio, D.; Britt, R.D.; Valentine, J.S.

2011-01-01

Human MnSOD is significantly more product-inhibited than bacterial MnSODs at high concentrations of superoxide (O 2 - ). This behavior limits the amount of H 2 O 2 produced at high [O 2 - ]; its desirability can be explained by the multiple roles of H 2 O 2 in mammalian cells, particularly its role in signaling. To investigate the mechanism of product inhibition in MnSOD, two yeast MnSODs, one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), were isolated and characterized. ScMnSOD and CaMnSODc are similar in catalytic kinetics, spectroscopy, and redox chemistry, and they both rest predominantly in the reduced state (unlike most other MnSODs). At high [O 2 - ], the dismutation efficiencies of the yeast MnSODs surpass those of human and bacterial MnSODs, due to very low level of product inhibition. Optical and parallel-mode electron paramagnetic resonance (EPR) spectra suggest the presence of two Mn 3+ species in yeast Mn 3+ SODs, including the well-characterized 5-coordinate Mn 3+ species and a 6-coordinate L-Mn 3+ species with hydroxide as the putative sixth ligand (L). The first and second coordination spheres of ScMnSOD are more similar to bacterial than to human MnSOD. Gln154, an H-bond donor to the Mn-coordinated solvent molecule, is slightly further away from Mn in yeast MnSODs, which may result in their unusual resting state. Mechanistically, the high efficiency of yeast MnSODs could be ascribed to putative translocation of an outer-sphere solvent molecule, which could destabilize the inhibited complex and enhance proton transfer from protein to peroxide. Our studies on yeast MnSODs indicate the unique nature of human MnSOD in that it predominantly undergoes the inhibited pathway at high [O 2 - ].

Mitochondria and Endothelial Function

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Kluge, Matthew A.; Fetterman, Jessica L.; Vita, Joseph A.

2013-01-01

In contrast to their role in other cell types with higher energy demands, mitochondria in endothelial cells primarily function in signaling cellular responses to environmental cues. This article provides an overview of key aspects of mitochondrial biology in endothelial cells, including subcellular location, biogenesis, dynamics, autophagy, ROS production and signaling, calcium homeostasis, regulated cell death, and heme biosynthesis. In each section, we introduce key concepts and then review studies showing the importance of that mechanism to endothelial control of vasomotor tone, angiogenesis, and inflammatory activation. We particularly highlight the small number of clinical and translational studies that have investigated each mechanism in human subjects. Finally, we review interventions that target different aspects of mitochondrial function and their effects on endothelial function. The ultimate goal of such research is the identification of new approaches for therapy. The reviewed studies make it clear that mitochondria are important in endothelial physiology and pathophysiology. A great deal of work will be needed, however, before mitochondria-directed therapies are available for the prevention and treatment of cardiovascular disease. PMID:23580773

Immobilised Sarawak Malaysia yeast cells for production of bioethanol.

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Zain, Masniroszaime Mohd; Kofli, Noorhisham Tan; Rozaimah, Siti; Abdullah, Sheikh

2011-05-01

Bioethanol production using yeast has become a popular topic due to worrying depleting worldwide fuel reserve. The aim of the study was to investigate the capability of Malaysia yeast strains isolated from starter culture used in traditional fermented food and alcoholic beverages in producing Bioethanol using alginate beads entrapment method. The starter yeast consists of groups of microbes, thus the yeasts were grown in Sabouraud agar to obtain single colony called ST1 (tuak) and ST3 (tapai). The growth in Yeast Potatoes Dextrose (YPD) resulted in specific growth of ST1 at micro = 0.396 h-1 and ST3 at micro = 0.38 h-1, with maximum ethanol production of 7.36 g L-1 observed using ST1 strain. The two strains were then immobilized using calcium alginate entrapment method producing average alginate beads size of 0.51 cm and were grown in different substrates; YPD medium and Local Brown Sugar (LBS) for 8 h in flask. The maximum ethanol concentration measured after 7 h were at 6.63 and 6.59 g L-1 in YPD media and 1.54 and 1.39 g L-1in LBS media for ST1 and ST3, respectively. The use of LBS as carbon source showed higher yield of product (Yp/s), 0.59 g g-1 compared to YPD, 0.25 g g-1 in ST1 and (Yp/s), 0.54 g g-1 compared to YPD, 0.24 g g-1 in ST3 . This study indicated the possibility of using local strains (STI and ST3) to produce bioethanol via immobilization technique with local materials as substrate.

Mechanisms of communication between mitochondria and lysosomes.

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Raimundo, Nuno; Fernández-Mosquera, Lorena; Yambire, King Faisal; Diogo, Cátia V

2016-10-01

Mitochondria and lysosomes have long been studied in the context of their classic functions: energy factory and recycle bin, respectively. In the last twenty years, it became evident that these organelles are much more than simple industrial units, and are indeed in charge of many of cellular processes. Both mitochondria and lysosomes are now recognized as far-reaching signaling platforms, regulating many key aspects of cell and tissue physiology. It has furthermore become clear that mitochondria and lysosomes impact each other. The mechanisms underlying the cross-talk between these organelles are only now starting to be addressed. In this review, we briefly summarize how mitochondria, lysosomes and the lysosome-related process of autophagy affect each other in physiology and pathology. Copyright © 2016 Elsevier Ltd. All rights reserved.

AoAtg26, a putative sterol glucosyltransferase, is required for autophagic degradation of peroxisomes, mitochondria, and nuclei in the filamentous fungus Aspergillus oryzae.

Science.gov (United States)

Kikuma, Takashi; Tadokoro, Takayuki; Maruyama, Jun-Ichi; Kitamoto, Katsuhiko

2017-02-01

Autophagy is a conserved process in eukaryotic cells for degradation of cellular proteins and organelles. In filamentous fungi, autophagic degradation of organelles such as peroxisomes, mitochondria, and nuclei occurs in basal cells after the prolonged culture, but its mechanism is not well understood. Here, we functionally analyzed the filamentous fungus Aspergillus oryzae AoAtg26, an ortholog of the sterol glucosyltransferase PpAtg26 involved in pexophagy in the yeast Pichia pastoris. Deletion of Aoatg26 caused a severe decrease in conidiation and aerial hyphae formation, which is typically observed in the autophagy-deficient A. oryzae strains. In addition, cup-shaped AoAtg8-positive membrane structures were accumulated in the Aoatg26 deletion strain, indicating that autophagic process is impaired. Indeed, the Aoatg26 deletion strain was defective in the degradation of peroxisomes, mitochondria, and nuclei. Taken together, AoAtg26 plays an important role for autophagic degradation of organelles in A. oryzae, which may physiologically contribute to the differentiation in filamentous fungi.

Redox interplay between mitochondria and peroxisomes

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Celien eLismont

2015-05-01

Full Text Available Reduction-oxidation or ‘redox’ reactions are an integral part of a broad range of cellular processes such as gene expression, energy metabolism, protein import and folding, and autophagy. As many of these processes are intimately linked with cell fate decisions, transient or chronic changes in cellular redox equilibrium are likely to contribute to the initiation and progression of a plethora of human diseases. Since a long time, it is known that mitochondria are major players in redox regulation and signaling. More recently, it has become clear that also peroxisomes have the capacity to impact redox-linked physiological processes. To serve this function, peroxisomes cooperate with other organelles, including mitochondria. This review provides a comprehensive picture of what is currently known about the redox interplay between mitochondria and peroxisomes in mammals. We first outline the pro- and antioxidant systems of both organelles and how they may function as redox signaling nodes. Next, we critically review and discuss emerging evidence that peroxisomes and mitochondria share an intricate redox-sensitive relationship and cooperate in cell fate decisions. Key issues include possible physiological roles, messengers, and mechanisms. We also provide examples of how data mining of publicly-available datasets from ‘omics’ technologies can be a powerful means to gain additional insights into potential redox signaling pathways between peroxisomes and mitochondria. Finally, we highlight the need for more studies that seek to clarify the mechanisms of how mitochondria may act as dynamic receivers, integrators, and transmitters of peroxisome-derived mediators of oxidative stress. The outcome of such studies may open up exciting new avenues for the community of researchers working on cellular responses to organelle-derived oxidative stress, a research field in which the role of peroxisomes is currently highly underestimated and an issue of

The phytopathogenic virulent effector protein RipI induces apoptosis in budding yeast Saccharomyces cerevisiae.

Science.gov (United States)

Deng, Meng-Ying; Sun, Yun-Hao; Li, Pai; Fu, Bei; Shen, Dong; Lu, Yong-Jun

2016-10-01

Virulent protein toxins secreted by the bacterial pathogens can cause cytotoxicity by various molecular mechanisms to combat host cell defense. On the other hand, these proteins can also be used as probes to investigate the defense pathway of host innate immunity. Ralstonia solanacearum, one of the most virulent bacterial phytopathogens, translocates more than 70 effector proteins via type III secretion system during infection. Here, we characterized the cytotoxicity of effector RipI in budding yeast Saccharomyce scerevisiae, an alternative host model. We found that over-expression of RipI resulted in severe growth defect and arginine (R) 117 within the predicted integrase motif was required for inhibition of yeast growth. The phenotype of death manifested the hallmarks of apoptosis. Our data also revealed that RipI-induced apoptosis was independent of Yca1 and mitochondria-mediated apoptotic pathways because Δyca1 and Δaif1 were both sensitive to RipI as compared with the wild type. We further demonstrated that RipI was localized in the yeast nucleus and the N-terminal 1-174aa was required for the localization. High-throughput RNA sequencing analysis showed that upon RipI over-expression, 101 unigenes of yeast ribosome presented lower expression level, and 42 GO classes related to the nucleus or recombination were enriched with differential expression levels. Taken together, our data showed that a nuclear-targeting effector RipI triggers yeast apoptosis, potentially dependent on its integrase function. Our results also provided an alternative strategy to dissect the signaling pathway of cytotoxicity induced by the protein toxins. Copyright © 2016 Elsevier Ltd. All rights reserved.

Visible light alters yeast metabolic rhythms by inhibiting respiration.

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Robertson, James Brian; Davis, Chris R; Johnson, Carl Hirschie

2013-12-24

Exposure of cells to visible light in nature or in fluorescence microscopy often is considered to be relatively innocuous. However, using the yeast respiratory oscillation (YRO) as a sensitive measurement of metabolism, we find that non-UV visible light has a significant impact on yeast metabolism. Blue/green wavelengths of visible light shorten the period and dampen the amplitude of the YRO, which is an ultradian rhythm of cell metabolism and transcription. The wavelengths of light that have the greatest effect coincide with the peak absorption regions of cytochromes. Moreover, treating yeast with the electron transport inhibitor sodium azide has similar effects on the YRO as visible light. Because impairment of respiration by light would change several state variables believed to play vital roles in the YRO (e.g., oxygen tension and ATP levels), we tested oxygen's role in YRO stability and found that externally induced oxygen depletion can reset the phase of the oscillation, demonstrating that respiratory capacity plays a role in the oscillation's period and phase. Light-induced damage to the cytochromes also produces reactive oxygen species that up-regulate the oxidative stress response gene TRX2 that is involved in pathways that enable sustained growth in bright visible light. Therefore, visible light can modulate cellular rhythmicity and metabolism through unexpectedly photosensitive pathways.

Mitochondria: more than just a powerhouse.

Science.gov (United States)

McBride, Heidi M; Neuspiel, Margaret; Wasiak, Sylwia

2006-07-25

Pioneering biochemical studies have long forged the concept that the mitochondria are the 'energy powerhouse of the cell'. These studies, combined with the unique evolutionary origin of the mitochondria, led the way to decades of research focusing on the organelle as an essential, yet independent, functional component of the cell. Recently, however, our conceptual view of this isolated organelle has been profoundly altered with the discovery that mitochondria function within an integrated reticulum that is continually remodeled by both fusion and fission events. The identification of a number of proteins that regulate these activities is beginning to provide mechanistic details of mitochondrial membrane remodeling. However, the broader question remains regarding the underlying purpose of mitochondrial dynamics and the translation of these morphological transitions into altered functional output. One hypothesis has been that mitochondrial respiration and metabolism may be spatially and temporally regulated by the architecture and positioning of the organelle. Recent evidence supports and expands this idea by demonstrating that mitochondria are an integral part of multiple cell signaling cascades. Interestingly, proteins such as GTPases, kinases and phosphatases are involved in bi-directional communication between the mitochondrial reticulum and the rest of the cell. These proteins link mitochondrial function and dynamics to the regulation of metabolism, cell-cycle control, development, antiviral responses and cell death. In this review we will highlight the emerging evidence that provides molecular definition to mitochondria as a central platform in the execution of diverse cellular events.

CA V is present in rat kidney mitochondria

International Nuclear Information System (INIS)

Dodgson, S.J.; Contino, L.C.

1987-01-01

Guinea pig liver mitochondria contain the unique carbonic anhydrase isozyme, CA V. Prior to sacrifice, 15 rats and 15 guinea pigs were either fed normal lab chow (group 1), starved 48 hours (group 2) or fed normal lab chow and given to drink only water with added HCl, pH 2.5 (group 3). Mitochondria were prepared from excised livers and kidneys. CA V activity of disrupted mitochondria was measured by 18 O-mass spectrometric technique at pH 7.4, 37 0 C, 25 mM NaHCO 3 . Mass spectrometric CA assays with intact kidney mitochondria localize CA V activity to the matrix, as was found for liver mitochondria. It has been shown in hepatocytes prepared from starved guinea pigs and rats that inhibition of CA V results in decreased rate of gluconeogenesis from pyruvate. These present results are in line with the published observation that rat kidneys are much more gluconeogenic than guinea pig, and that this is increased by starvation and acidosis

Localization of mitochondria in living cells with rhodamine 123.

Science.gov (United States)

Johnson, L V; Walsh, M L; Chen, L B

1980-01-01

The laser dye rhodamine 123 is shown to be a specific probe for the localization of mitochondria in living cells. By virtue of its selectivity for mitochondria and its fluorescent properties, the detectability of mitochondria stained with rhodamine 123 is significantly improved over that provided by conventional light microscopic techniques. With the use of rhodamine 123, it is possible to detect alterations in mitochondrial distribution following transformation by Rous sarcoma virus and changes in the shape and organization of mitochondria induced by colchicine treatment. Images PMID:6965798

SLC25 Family Member Genetic Interactions Identify a Role for HEM25 in Yeast Electron Transport Chain Stability.

Science.gov (United States)

Dufay, J Noelia; Fernández-Murray, J Pedro; McMaster, Christopher R

2017-06-07

The SLC25 family member SLC25A38 (Hem25 in yeast) was recently identified as a mitochondrial glycine transporter that provides substrate to initiate heme/hemoglobin synthesis. Mutations in the human SLC25A38 gene cause congenital sideroblastic anemia. The full extent to which SLC25 family members coregulate heme synthesis with other mitochondrial functions is not clear. In this study, we surveyed 29 nonessential SLC25 family members in Saccharomyces cerevisiae for their ability to support growth in the presence and absence of HEM25 Six SLC25 family members were identified that were required for growth or for heme synthesis in cells lacking Hem25 function. Importantly, we determined that loss of function of the SLC25 family member Flx1, which imports FAD into mitochondria, together with loss of function of Hem25, resulted in inability to grow on media that required yeast cells to supply energy using mitochondrial respiration. We report that specific components of complexes of the electron transport chain are decreased in the absence of Flx1 and Hem25 function. In addition, we show that mitochondria from flx1 Δ hem25 Δ cells contain uncharacterized Cox2-containing high molecular weight aggregates. The functions of Flx1 and Hem25 provide a facile explanation for the decrease in heme level, and in specific electron transport chain complex components. Copyright © 2017 Dufay et al.

SLC25 Family Member Genetic Interactions Identify a Role for HEM25 in Yeast Electron Transport Chain Stability

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J. Noelia Dufay

2017-06-01

Full Text Available The SLC25 family member SLC25A38 (Hem25 in yeast was recently identified as a mitochondrial glycine transporter that provides substrate to initiate heme/hemoglobin synthesis. Mutations in the human SLC25A38 gene cause congenital sideroblastic anemia. The full extent to which SLC25 family members coregulate heme synthesis with other mitochondrial functions is not clear. In this study, we surveyed 29 nonessential SLC25 family members in Saccharomyces cerevisiae for their ability to support growth in the presence and absence of HEM25. Six SLC25 family members were identified that were required for growth or for heme synthesis in cells lacking Hem25 function. Importantly, we determined that loss of function of the SLC25 family member Flx1, which imports FAD into mitochondria, together with loss of function of Hem25, resulted in inability to grow on media that required yeast cells to supply energy using mitochondrial respiration. We report that specific components of complexes of the electron transport chain are decreased in the absence of Flx1 and Hem25 function. In addition, we show that mitochondria from flx1Δ hem25Δ cells contain uncharacterized Cox2-containing high molecular weight aggregates. The functions of Flx1 and Hem25 provide a facile explanation for the decrease in heme level, and in specific electron transport chain complex components.

Gis1 and Rph1 regulate glycerol and acetate metabolism in glucose depleted yeast cells.

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Jakub Orzechowski Westholm

Full Text Available Aging in organisms as diverse as yeast, nematodes, and mammals is delayed by caloric restriction, an effect mediated by the nutrient sensing TOR, RAS/cAMP, and AKT/Sch9 pathways. The transcription factor Gis1 functions downstream of these pathways in extending the lifespan of nutrient restricted yeast cells, but the mechanisms involved are still poorly understood. We have used gene expression microarrays to study the targets of Gis1 and the related protein Rph1 in different growth phases. Our results show that Gis1 and Rph1 act both as repressors and activators, on overlapping sets of genes as well as on distinct targets. Interestingly, both the activities and the target specificities of Gis1 and Rph1 depend on the growth phase. Thus, both proteins are associated with repression during exponential growth, targeting genes with STRE or PDS motifs in their promoters. After the diauxic shift, both become involved in activation, with Gis1 acting primarily on genes with PDS motifs, and Rph1 on genes with STRE motifs. Significantly, Gis1 and Rph1 control a number of genes involved in acetate and glycerol formation, metabolites that have been implicated in aging. Furthermore, several genes involved in acetyl-CoA metabolism are downregulated by Gis1.

Interorganelle Communication between Mitochondria and the Endolysosomal System

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Gonzalo Soto-Heredero

2017-11-01

Full Text Available The function of mitochondria and lysosomes has classically been studied separately. However, evidence has now emerged of intense crosstalk between these two organelles, such that the activity or stress status of one organelle may affect the other. Direct physical contacts between mitochondria and the endolysosomal compartment have been reported as a rapid means of interorganelle communication, mediating lipid or other metabolite exchange. Moreover, mitochondrial derived vesicles can traffic obsolete mitochondrial proteins into the endolysosomal system for their degradation or secretion to the extracellular milieu as exosomes, representing an additional mitochondrial quality control mechanism that connects mitochondria and lysosomes independently of autophagosome formation. Here, we present what is currently known about the functional and physical communication between mitochondria and lysosomes or lysosome-related organelles, and their role in sustaining cellular homeostasis.

Pathogenic lysosomal depletion in Parkinson's disease.

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Dehay, Benjamin; Bové, Jordi; Rodríguez-Muela, Natalia; Perier, Celine; Recasens, Ariadna; Boya, Patricia; Vila, Miquel

2010-09-15

Mounting evidence suggests a role for autophagy dysregulation in Parkinson's disease (PD). The bulk degradation of cytoplasmic proteins (including α-synuclein) and organelles (such as mitochondria) is mediated by macroautophagy, which involves the sequestration of cytosolic components into autophagosomes (AP) and its delivery to lysosomes. Accumulation of AP occurs in postmortem brain samples from PD patients, which has been widely attributed to an induction of autophagy. However, the cause and pathogenic significance of these changes remain unknown. Here we found in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of PD that AP accumulation and dopaminergic cell death are preceded by a marked decrease in the amount of lysosomes within dopaminergic neurons. Lysosomal depletion was secondary to the abnormal permeabilization of lysosomal membranes induced by increased mitochondrial-derived reactive oxygen species. Lysosomal permeabilization resulted in a defective clearance and subsequent accumulation of undegraded AP and contributed directly to neurodegeneration by the ectopic release of lysosomal proteases into the cytosol. Lysosomal breakdown and AP accumulation also occurred in PD brain samples, where Lewy bodies were strongly immunoreactive for AP markers. Induction of lysosomal biogenesis by genetic or pharmacological activation of lysosomal transcription factor EB restored lysosomal levels, increased AP clearance and attenuated 1-methyl-4-phenylpyridinium-induced cell death. Similarly, the autophagy-enhancer compound rapamycin attenuated PD-related dopaminergic neurodegeneration, both in vitro and in vivo, by restoring lysosomal levels. Our results indicate that AP accumulation in PD results from defective lysosomal-mediated AP clearance secondary to lysosomal depletion. Restoration of lysosomal levels and function may thus represent a novel neuroprotective strategy in PD.

Yeast Cells Exposed to Exogenous Palmitoleic Acid Either Adapt to Stress and Survive or Commit to Regulated Liponecrosis and Die

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Karamat Mohammad

2018-01-01

Full Text Available A disturbed homeostasis of cellular lipids and the resulting lipotoxicity are considered to be key contributors to many human pathologies, including obesity, metabolic syndrome, type 2 diabetes, cardiovascular diseases, and cancer. The yeast Saccharomyces cerevisiae has been successfully used for uncovering molecular mechanisms through which impaired lipid metabolism causes lipotoxicity and elicits different forms of regulated cell death. Here, we discuss mechanisms of the “liponecrotic” mode of regulated cell death in S. cerevisiae. This mode of regulated cell death can be initiated in response to a brief treatment of yeast with exogenous palmitoleic acid. Such treatment prompts the incorporation of exogenously added palmitoleic acid into phospholipids and neutral lipids. This orchestrates a global remodeling of lipid metabolism and transfer in the endoplasmic reticulum, mitochondria, lipid droplets, and the plasma membrane. Certain features of such remodeling play essential roles either in committing yeast to liponecrosis or in executing this mode of regulated cell death. We also outline four processes through which yeast cells actively resist liponecrosis by adapting to the cellular stress imposed by palmitoleic acid and maintaining viability. These prosurvival cellular processes are confined in the endoplasmic reticulum, lipid droplets, peroxisomes, autophagosomes, vacuoles, and the cytosol.

Oxidation and reduction of pyridine nucleotides in alamethicin-permeabilized plant mitochondria

DEFF Research Database (Denmark)

Johansson, F.I.; Michalecka, A.M.; Møller, I.M.

2004-01-01

method to permearbilize mitochondria and study the highly branched electron-transport chain in potato tuber (Solanum tuberosum) and pea leaf (Pisum sativum) mitochondria. We show that AlaM permeabilized the inner membrane of plant mitochondria to NAD(P)H, allowing the quantification of internal NAD......M-treated mitochondria was much higher than what has been previously measured by other techniques. Our results also show a difference in substrate specificities for complex I in mitochondria as compared with inside-out submitochondrial particles. AlaM facilitated the passage of cofactors to and from the mitochondrial...... environment not only in plant mitochondria but also in other membrane-enclosed compartments, such as intact cells, chloroplasts and peroxisomes....

Arsenate uncoupling of oxidative phosphorylation in isolated plant mitochondria

Energy Technology Data Exchange (ETDEWEB)

Wickes, W A; Wiskich, J T

1976-01-01

The uncoupling by arsenate of beetroot and cauliflower bud mitochondria showed the following characteristics: arsenate stimulation of respiration above the rate found with phosphate; inhibition of arsenate-stimulated respiration by phosphate; enhancement of arsenate-stimulated respiration by ADP; only partial prevention of this ADP-enhanced respiration by atractyloside; inhibition by oligomycin of the arsenate-stimulated respiration back to the phosphate rate; and the absence of any stimulatory effect of ADP in the presence of oligomycin. These results are qualitatively analogous to those reported for arsenate uncoupling in rat liver mitochondria. Arsenate stimulated malate oxidation, presumably by stimulating malate entry, in both beetroot and cauliflower bud mitochondria; however, high rates of oxidation, and presumably entry, were only sustained with arsenate in beetroot mitochondria. NADH was oxidized rapidly in cauliflower bud mitochondria in the presence of arsenate, showing that arsenate did not inhibit electron transfer processes.

Oxygen response of the wine yeast Saccharomyces cerevisiae EC1118 grown under carbon-sufficient, nitrogen-limited enological conditions.

Science.gov (United States)

Aceituno, Felipe F; Orellana, Marcelo; Torres, Jorge; Mendoza, Sebastián; Slater, Alex W; Melo, Francisco; Agosin, Eduardo

2012-12-01

Discrete additions of oxygen play a critical role in alcoholic fermentation. However, few studies have quantitated the fate of dissolved oxygen and its impact on wine yeast cell physiology under enological conditions. We simulated the range of dissolved oxygen concentrations that occur after a pump-over during the winemaking process by sparging nitrogen-limited continuous cultures with oxygen-nitrogen gaseous mixtures. When the dissolved oxygen concentration increased from 1.2 to 2.7 μM, yeast cells changed from a fully fermentative to a mixed respirofermentative metabolism. This transition is characterized by a switch in the operation of the tricarboxylic acid cycle (TCA) and an activation of NADH shuttling from the cytosol to mitochondria. Nevertheless, fermentative ethanol production remained the major cytosolic NADH sink under all oxygen conditions, suggesting that the limitation of mitochondrial NADH reoxidation is the major cause of the Crabtree effect. This is reinforced by the induction of several key respiratory genes by oxygen, despite the high sugar concentration, indicating that oxygen overrides glucose repression. Genes associated with other processes, such as proline uptake, cell wall remodeling, and oxidative stress, were also significantly affected by oxygen. The results of this study indicate that respiration is responsible for a substantial part of the oxygen response in yeast cells during alcoholic fermentation. This information will facilitate the development of temporal oxygen addition strategies to optimize yeast performance in industrial fermentations.

Phosphatidylserine transport by Ups2-Mdm35 in respiration-active mitochondria.

Science.gov (United States)

Miyata, Non; Watanabe, Yasunori; Tamura, Yasushi; Endo, Toshiya; Kuge, Osamu

2016-07-04

Phosphatidylethanolamine (PE) is an essential phospholipid for mitochondrial functions and is synthesized mainly by phosphatidylserine (PS) decarboxylase at the mitochondrial inner membrane. In Saccharomyces cerevisiae, PS is synthesized in the endoplasmic reticulum (ER), such that mitochondrial PE synthesis requires PS transport from the ER to the mitochondrial inner membrane. Here, we provide evidence that Ups2-Mdm35, a protein complex localized at the mitochondrial intermembrane space, mediates PS transport for PE synthesis in respiration-active mitochondria. UPS2- and MDM35-null mutations greatly attenuated conversion of PS to PE in yeast cells growing logarithmically under nonfermentable conditions, but not fermentable conditions. A recombinant Ups2-Mdm35 fusion protein exhibited phospholipid-transfer activity between liposomes in vitro. Furthermore, UPS2 expression was elevated under nonfermentable conditions and at the diauxic shift, the metabolic transition from glycolysis to oxidative phosphorylation. These results demonstrate that Ups2-Mdm35 functions as a PS transfer protein and enhances mitochondrial PE synthesis in response to the cellular metabolic state. © 2016 Miyata et al.

The depletion of nuclear glutathione impairs cell proliferation in 3t3 fibroblasts.

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Jelena Markovic

2009-07-01

Full Text Available Glutathione is considered essential for survival in mammalian cells and yeast but not in prokaryotic cells. The presence of a nuclear pool of glutathione has been demonstrated but its role in cellular proliferation and differentiation is still a matter of debate.We have studied proliferation of 3T3 fibroblasts for a period of 5 days. Cells were treated with two well known depleting agents, diethyl maleate (DEM and buthionine sulfoximine (BSO, and the cellular and nuclear glutathione levels were assessed by analytical and confocal microscopic techniques, respectively. Both agents decreased total cellular glutathione although depletion by BSO was more sustained. However, the nuclear glutathione pool resisted depletion by BSO but not with DEM. Interestingly, cell proliferation was impaired by DEM, but not by BSO. Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation.Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle.

Identification of Potential Calorie Restriction-Mimicking Yeast Mutants with Increased Mitochondrial Respiratory Chain and Nitric Oxide Levels

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Bin Li

2011-01-01

Full Text Available Calorie restriction (CR induces a metabolic shift towards mitochondrial respiration; however, molecular mechanisms underlying CR remain unclear. Recent studies suggest that CR-induced mitochondrial activity is associated with nitric oxide (NO production. To understand the role of mitochondria in CR, we identify and study Saccharomyces cerevisiae mutants with increased NO levels as potential CR mimics. Analysis of the top 17 mutants demonstrates a correlation between increased NO, mitochondrial respiration, and longevity. Interestingly, treating yeast with NO donors such as GSNO (S-nitrosoglutathione is sufficient to partially mimic CR to extend lifespan. CR-increased NO is largely dependent on mitochondrial electron transport and cytochrome c oxidase (COX. Although COX normally produces NO under hypoxic conditions, CR-treated yeast cells are able to produce NO under normoxic conditions. Our results suggest that CR may derepress some hypoxic genes for mitochondrial proteins that function to promote the production of NO and the extension of lifespan.

Mutations that Allow SIR2 Orthologs to Function in a NAD+-Depleted Environment.

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Ondracek, Caitlin R; Frappier, Vincent; Ringel, Alison E; Wolberger, Cynthia; Guarente, Leonard

2017-03-07

Sirtuin enzymes depend on NAD + to catalyze protein deacetylation. Therefore, the lowering of NAD + during aging leads to decreased sirtuin activity and may speed up aging processes in laboratory animals and humans. In this study, we used a genetic screen to identify two mutations in the catalytic domain of yeast Sir2 that allow the enzyme to function in an NAD + -depleted environment. These mutant enzymes give rise to a significant increase of yeast replicative lifespan and increase deacetylation by the Sir2 ortholog, SIRT1, in mammalian cells. Our data suggest that these mutations increase the stability of the conserved catalytic sirtuin domain, thereby increasing the catalytic efficiency of the mutant enzymes. Our approach to identifying sirtuin mutants that permit function in NAD + -limited environments may inform the design of small molecules that can maintain sirtuin activity in aging organisms. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

SAGA complex and Gcn5 are necessary for respiration in budding yeast.

Science.gov (United States)

Canzonetta, Claudia; Leo, Manuela; Guarino, Salvatore Rocco; Montanari, Arianna; Francisci, Silvia; Filetici, Patrizia

2016-12-01

In budding yeast, growth through fermentation and/or respiration is dependent on the type of carbon source present in the medium. SAGA complex is the main acetylation complex and is required, together with Rtg factors, for nucleus-mitochondria communication and transcriptional activation of specific nuclear genes. Even though acetylation is necessary for mitochondria activity and respiratory pathways the direct role of histone acetyltransferases and SAGA complex has never been investigated directly. In this study we demonstrate, for the first time, that Gcn5 and SAGA are needed for respiratory metabolism and oxygen consumption. According to a central role for acetylation in respiration we find that the Gcn5 inhibitor CPTH2 had higher efficacy on cells grown in glycerol containing media. We also demonstrated that the opposing activities of Gcn5 and Hda1 modify selectively H3-AcK18 and are essential for respiration. Taken together our results suggest a novel paradigm coupling acetyltransferase activity to respiratory metabolism. Correspondingly we propose the selective utilization of KAT inhibitor CPTH2, combined to the modulation of the respiratory metabolism of the cell, as a promising novel tool of intervention in cancer cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Depleted uranium

International Nuclear Information System (INIS)

Huffer, E.; Nifenecker, H.

2001-02-01

This document deals with the physical, chemical and radiological properties of the depleted uranium. What is the depleted uranium? Why do the military use depleted uranium and what are the risk for the health? (A.L.B.)

Human COQ9 Rescues a coq9 Yeast Mutant by Enhancing Coenzyme Q Biosynthesis from 4-Hydroxybenzoic Acid and Stabilizing the CoQ-Synthome

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Cuiwen H. He

2017-07-01

Full Text Available Coq9 is required for the stability of a mitochondrial multi-subunit complex, termed the CoQ-synthome, and the deamination step of Q intermediates that derive from para-aminobenzoic acid (pABA in yeast. In human, mutations in the COQ9 gene cause neonatal-onset primary Q10 deficiency. In this study, we determined whether expression of human COQ9 could complement yeast coq9 point or null mutants. We found that expression of human COQ9 rescues the growth of the temperature-sensitive yeast mutant, coq9-ts19, on a non-fermentable carbon source and increases the content of Q6, by enhancing Q biosynthesis from 4-hydroxybenzoic acid (4HB. To study the mechanism for the rescue by human COQ9, we determined the steady-state levels of yeast Coq polypeptides in the mitochondria of the temperature-sensitive yeast coq9 mutant expressing human COQ9. We show that the expression of human COQ9 significantly increased steady-state levels of yeast Coq4, Coq6, Coq7, and Coq9 at permissive temperature. Human COQ9 polypeptide levels persisted at non-permissive temperature. A small amount of the human COQ9 co-purified with tagged Coq6, Coq6-CNAP, indicating that human COQ9 interacts with the yeast Q-biosynthetic complex. These findings suggest that human COQ9 rescues the yeast coq9 temperature-sensitive mutant by stabilizing the CoQ-synthome and increasing Q biosynthesis from 4HB. This finding provides a powerful approach to studying the function of human COQ9 using yeast as a model.

Endurance training increases the efficiency of rat skeletal muscle mitochondria.

Science.gov (United States)

Zoladz, Jerzy A; Koziel, Agnieszka; Woyda-Ploszczyca, Andrzej; Celichowski, Jan; Jarmuszkiewicz, Wieslawa

2016-10-01

Endurance training enhances mitochondrial oxidative capacity, but its effect on mitochondria functioning is poorly understood. In the present study, the influence of an 8-week endurance training on the bioenergetic functioning of rat skeletal muscle mitochondria under different assay temperatures (25, 35, and 42 °C) was investigated. The study was performed on 24 adult 4-month-old male Wistar rats, which were randomly assigned to either a treadmill training group (n = 12) or a sedentary control group (n = 12). In skeletal muscles, endurance training stimulated mitochondrial biogenesis and oxidative capacity. In isolated mitochondria, endurance training increased the phosphorylation rate and elevated levels of coenzyme Q. Moreover, a decrease in mitochondrial uncoupling, including uncoupling protein-mediated proton leak, was observed after training, which could explain the increased reactive oxygen species production (in nonphosphorylating mitochondria) and enhanced oxidative phosphorylation efficiency. At all studied temperatures, endurance training significantly augmented H2O2 production (and coenzyme Q reduction level) in nonphosphorylating mitochondria and decreased H2O2 production (and coenzyme Q reduction level) in phosphorylating mitochondria. Endurance training magnified the hyperthermia-induced increase in oxidative capacity and attenuated the hyperthermia-induced decline in oxidative phosphorylation efficiency and reactive oxygen species formation of nonphosphorylating mitochondria via proton leak enhancement. Thus, endurance training induces both quantitative and qualitative changes in muscle mitochondria that are important for cell signaling as well as for maintaining muscle energy homeostasis, especially at high temperatures.

Nitric oxide and DOPAC-induced cell death: from GSH depletion to mitochondrial energy crisis.

Science.gov (United States)

Nunes, Carla; Barbosa, Rui M; Almeida, Leonor; Laranjinha, João

2011-09-01

The molecular mechanisms inherent to cell death associated with Parkinson's disease are not clearly understood. Diverse pathways, sequence of events and models have been explored in several studies. Recently, we have proposed an integrative mechanism, encompassing the interaction of nitric oxide (•NO) and a major dopamine metabolite, dihydroxyphenylacetic (DOPAC), leading to a synergistic mitochondrial dysfunction and cell death that may be operative in PD. In this study, we have studied the sequence of events underlying the mechanisms of cell death in PC12 cells exposed to •NO and DOPAC in terms of: a) free radical production; b) modulation by glutathione (GSH); c) energetic status and d) outer membrane mitochondria permeability. Using Electron Paramagnetic Resonance (EPR) it is shown the early production of oxygen free radicals followed by a depletion of GSH reflected by an increase of GSSG/GSH ratio in the cells treated with the mixture of •NO/DOPAC, as compared with the cells individually exposed to each of the stimulus. Glutathione ethyl ester (GSH-EE) and N-acetylcysteine (NAC) may rescue cells from death, increasing GSH content and preventing ATP loss in cells treated with the mixture DOPAC/•NO but failed to exert similar effects in the cells challenged only with •NO. The depletion of GSH is accompanied by a decreased activity of mitochondrial complex I. At a later stage, the concerted action of DOPAC and •NO include a rise in the ratio Bax/Bcl-2, an observation not evident when cells were exposed only to •NO. The results support a free radical-induced pathway leading to cell death involving the concerted action of DOPAC and •NO and the critical role of GSH in maintaining a functional mitochondria. Copyright © 2011 Elsevier Inc. All rights reserved.

Transport of N-acetylglutamate in rat-liver mitochondria

NARCIS (Netherlands)

Meijer, A. J.; van Woerkom, G. M.; Wanders, R. J.; Lof, C.

1982-01-01

The permeability properties of the rat-liver mitochondrial membrane for N-acetylglutamate, the activator of carbamoyl-phosphate synthetase (ammonia), were studied. 1. Transport of N-acetylglutamate into the mitochondria was only observed in partially or fully de-energized mitochondria and when the

Kex1 protease is involved in yeast cell death induced by defective N-glycosylation, acetic acid, and chronological aging.

Science.gov (United States)

Hauptmann, Peter; Lehle, Ludwig

2008-07-04

N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to humans. The key step of this pathway is the transfer of the lipid-linked core oligosaccharide to the nascent polypeptide chain, catalyzed by the oligosaccharyltransferase complex. Temperature-sensitive oligosaccharyltransferase mutants of Saccharomyces cerevisiae at the restrictive temperature, such as wbp1-1, as well as wild-type cells in the presence of the N-glycosylation inhibitor tunicamycin display typical apoptotic phenotypes like nuclear condensation, DNA fragmentation, phosphatidylserine translocation, caspase-like activity, and reactive oxygen species accumulation. Since deletion of the yeast metacaspase YCA1 did not abrogate this death pathway, we postulated a different proteolytic process to be responsible. Here, we show that Kex1 protease is involved in the programmed cell death caused by defective N-glycosylation. Its disruption decreases caspase-like activity, production of reactive oxygen species, and fragmentation of mitochondria and, conversely, improves growth and survival of cells. Moreover, we demonstrate that Kex1 contributes also to the active cell death program induced by acetic acid stress or during chronological aging, suggesting that Kex1 plays a more general role in cellular suicide of yeast.

Redox conditions and protein oxidation in plant mitochondria

DEFF Research Database (Denmark)

Møller, Ian Max; Kasimova, Marina R.; Krab, Klaas

2005-01-01

Redox conditions and protein oxidation in plant mitochondria NAD(P)H has a central position in respiratory metabolism. It is produced by a large number of enzymes, e.g. the Krebs cycle dehydrogenases, in the mitochondrial matrix and is oxidised by, amongst others, the respiratory chain. Most...... of this NAD(P)H appears to be bound to proteins, in fact free NAD(P)H – an important parameter in metabolic regulation - has never been observed in mitochondria. We have estimated free and bound NAD(P)H in isolated plant mitochondria under different metabolic conditions. The fluorescence spectra of free...... and bound NADH was determined and used to deconvolute fluorescence spectra of actively respiring mitochondria. Most of the mitochondrial NADH is bound in states 2 and 4. The amount of free NADH is lower but relatively constant even increasing a little in state 3 where it is about equal to bound NADH...

Supplementary Material for: Polyglutamine toxicity in yeast induces metabolic alterations and mitochondrial defects

KAUST Repository

Papsdorf, Katharina

2015-01-01

Abstract Background Protein aggregation and its pathological effects are the major cause of several neurodegenerative diseases. In Huntingtonâ s disease an elongated stretch of polyglutamines within the protein Huntingtin leads to increased aggregation propensity. This induces cellular defects, culminating in neuronal loss, but the connection between aggregation and toxicity remains to be established. Results To uncover cellular pathways relevant for intoxication we used genome-wide analyses in a yeast model system and identify fourteen genes that, if deleted, result in higher polyglutamine toxicity. Several of these genes, like UGO1, ATP15 and NFU1 encode mitochondrial proteins, implying that a challenged mitochondrial system may become dysfunctional during polyglutamine intoxication. We further employed microarrays to decipher the transcriptional response upon polyglutamine intoxication, which exposes an upregulation of genes involved in sulfur and iron metabolism and mitochondrial Fe-S cluster formation. Indeed, we find that in vivo iron concentrations are misbalanced and observe a reduction in the activity of the prominent Fe-S cluster containing protein aconitase. Like in other yeast strains with impaired mitochondria, non-fermentative growth is impossible after intoxication with the polyglutamine protein. NMR-based metabolic analyses reveal that mitochondrial metabolism is reduced, leading to accumulation of metabolic intermediates in polyglutamine-intoxicated cells. Conclusion These data show that damages to the mitochondrial system occur in polyglutamine intoxicated yeast cells and suggest an intricate connection between polyglutamine-induced toxicity, mitochondrial functionality and iron homeostasis in this model system.

Mitochondria As the Target for the Modulatory Effect of Curcumin in Oxaliplatin-induced Toxicity in Isolated Rat Liver Mitochondria.

Science.gov (United States)

Waseem, Mohammad; Parvez, Suhel; Tabassum, Heena

2017-01-01

To explore hepatoprotective action of curcumin (CMN, a bioflavonoid) on oxaliplatin (Oxa)-triggered mitochondrial oxidative stress and respiratory chain complexes in liver of rats. Oxa is a ubiquitously utilized platinum-based chemotherapeutic agent commonly used for the treatment of colorectal cancer. Mitochondria have recently emerged as targets for anticancer drugs in several kinds of toxicity including hepatotoxicity that can lead to neoplastic disease. There is a dearth of evidence involving the role of mitochondria in mediating Oxa-evoked hepatotoxicity and its underlying mechanism is still debatable. The study was performed in mitochondria isolated from liver of Wistar rats. Oxa (200 μg/mL) and CMN (5 μmol) were incubated under in vitro conditions. Oxa evoked a significant increase in the membrane lipid peroxidation (LPO) levels, protein carbonyl (PC) contents, decrease in reduced glutathione (GSH) and nonprotein thiol (NP-SH) levels. Oxa also caused a marked decline in the activities of enzymatic antioxidants and respiratory chain enzymes (I, II, III and V) in liver mitochondria. CMN pre-treatment significantly prevented the activities of enzymatic antioxidants and mitochondrial respiratory chain enzymes. CMN also restored the LPO and PC contents, GSH and NP-SH levels in liver mitochondria. CMN intake might be effective in regulation of Oxa-evoked mitotoxicity during chemotherapy. Moreover, it is included in the armamentarium for anticancer agent-induced oxidative stress. Copyright © 2017 IMSS. Published by Elsevier Inc. All rights reserved.

Toxicity of polyhydroxylated fullerene to mitochondria

Energy Technology Data Exchange (ETDEWEB)

Yang, Li-Yun [State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry for Biology and Medicine (MOE), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072 (China); Gao, Jia-Ling [Department of Chemistry, College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou 434023 (China); Gao, Tian; Dong, Ping; Ma, Long; Jiang, Feng-Lei [State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry for Biology and Medicine (MOE), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072 (China); Liu, Yi, E-mail: yiliuchem@whu.edu.cn [State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry for Biology and Medicine (MOE), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072 (China)

2016-01-15

Highlights: • Fullerenol-induced mitochondrial dysfunction was investigated at mitochondrial level. • Fullerenol disturbed mitochondrial inner membrane in polar protein regions. • Fullerenol affected the inner membrane and respiration chain of mitochondria. - Abstract: Mitochondrial dysfunction is considered as a crucial mechanism of nanomaterial toxicity. Herein, we investigated the effects of polyhydroxylated fullerene (C{sub 60}(OH){sub 44}, fullerenol), a model carbon-based nanomaterial with high water solubility, on isolated mitochondria. Our study demonstrated that fullerenol enhanced the permeabilization of mitochondrial inner membrane to H{sup +} and K{sup +} and induced mitochondrial permeability transition (MPT). The fullerenol-induced swelling was dose-dependent and could be effectively inhibited by MPT inhibitors such as cyclosporin A (CsA), adenosine diphosphate (ADP), ruthenium red (RR) and ethylenediaminetetraacetic acid (EDTA). After treating the mitochondria with fullerenol, the mitochondrial membrane potential (MMP) was found collapsed in a concentration-independent manner. The fluorescence anisotropy of hematoporphyrin (HP) changed significantly with the addition of fullerenol, while that of 1,6-diphenyl-hexatriene (DPH) changed slightly. Moreover, a decrease of respiration state 3 and increase of respiration state 4 were observed when mitochondria were energized with complex II substrate succinate. The results of transmission electron microscopy (TEM) provided direct evidence that fullerenol damaged the mitochondrial ultrastructure. The investigations can provide comprehensive information to elucidate the possible toxic mechanism of fullerenols at subcellular level.

Avocado oil induces long-term alleviation of oxidative damage in kidney mitochondria from type 2 diabetic rats by improving glutathione status.

Science.gov (United States)

Ortiz-Avila, Omar; Figueroa-García, María Del Consuelo; García-Berumen, Claudia Isabel; Calderón-Cortés, Elizabeth; Mejía-Barajas, Jorge A; Rodriguez-Orozco, Alain R; Mejía-Zepeda, Ricardo; Saavedra-Molina, Alfredo; Cortés-Rojo, Christian

2017-04-01

Hyperglycemia and mitochondrial ROS overproduction have been identified as key factors involved in the development of diabetic nephropathy. This has encouraged the search for strategies decreasing glucose levels and long-term improvement of redox status of glutathione, the main antioxidant counteracting mitochondrial damage. Previously, we have shown that avocado oil improves redox status of glutathione in liver and brain mitochondria from streptozotocin-induced diabetic rats; however, the long-term effects of avocado oil and its hypoglycemic effect cannot be evaluated because this model displays low survival and insulin depletion. Therefore, we tested during 1 year the effects of avocado oil on glycemia, ROS levels, lipid peroxidation and glutathione status in kidney mitochondria from type 2 diabetic Goto-Kakizaki rats. Diabetic rats exhibited glycemia of 120-186 mg/dL the first 9 months with a further increase to 250-300 mg/dL. Avocado oil decreased hyperglycemia at intermediate levels between diabetic and control rats. Diabetic rats displayed augmented lipid peroxidation and depletion of reduced glutathione throughout the study, while increased ROS generation was observed at the 3rd and 12th months along with diminished content of total glutathione at the 6th and 12th months. Avocado oil ameliorated all these defects and augmented the mitochondrial content of oleic acid. The beneficial effects of avocado oil are discussed in terms of the hypoglycemic effect of oleic acid and the probable dependence of glutathione transport on lipid peroxidation and thiol oxidation of mitochondrial carriers.

The affinity purification and characterization of ATP synthase complexes from mitochondria.

Science.gov (United States)

Runswick, Michael J; Bason, John V; Montgomery, Martin G; Robinson, Graham C; Fearnley, Ian M; Walker, John E

2013-02-13

The mitochondrial F₁-ATPase inhibitor protein, IF₁, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the α-helical inhibitory region of the bound IF₁ occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the γ-subunit in the enzyme's rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF₁ with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1-60 of bovine IF₁ with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione-S-transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme's stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region.

Functional characterization of mitochondria in neutrophils: a role restricted to apoptosis

NARCIS (Netherlands)

Maianski, N. A.; Geissler, J.; Srinivasula, S. M.; Alnemri, E. S.; Roos, D.; Kuijpers, T. W.

2004-01-01

Mitochondria are known to combine life-supporting functions with participation in apoptosis by controlling caspase activity. Here, we report that in human blood neutrophils the mitochondria are different, because they preserve mainly death-mediating abilities. Neutrophil mitochondria hardly

Ubiquinone accumulates in the mitochondria of yeast mutated in the ubiquinone binding protein, Qcr8p

International Nuclear Information System (INIS)

Hagerman, Ruth A.; Waring, Natashya J.; Willis, Richard A.; Hagerman, Ann E.

2006-01-01

In Saccharomyces cerevisiae, the trans-membrane helix of Qcr8p, the ubiquinone binding protein of complex III, contributes to the Q binding site. In wild-type cells, residue 62 of the helix is non-polar (proline). Substitution of proline 62 with a polar, uncharged residue does not impair the ability of the cells to respire, complex III assembly is unaffected, ubiquinone occupancy of the Q binding site is unchanged, and mitochondrial ubiquinone levels are in the wild-type range. Substitution with a +1 charged residue is associated with partial respiratory competence, impaired complex III assembly, and loss of cytochrome b. Although ubiquinone occupancy of the Q binding site is similar to wild-type, total mitochondrial ubiquinone doubled in these mutants. Mutants with a +2 charged substitution at position 62 are unable to respire. These results suggest that the accumulation of ubiquinone in the mitochondria may be a compensatory mechanism for impaired electron transport at cytochrome b

Characterization of a Plasmodium falciparum Orthologue of the Yeast Ubiquinone-Binding Protein, Coq10p.

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Bethany J Jenkins

Full Text Available Coenzyme Q (CoQ, ubiquinone is a central electron carrier in mitochondrial respiration. CoQ is synthesized through multiple steps involving a number of different enzymes. The prevailing view that the CoQ used in respiration exists as a free pool that diffuses throughout the mitochondrial inner membrane bilayer has recently been challenged. In the yeast Saccharomyces cerevisiae, deletion of the gene encoding Coq10p results in respiration deficiency without inhibiting the synthesis of CoQ, suggesting that the Coq10 protein is critical for the delivery of CoQ to the site(s of respiration. The precise mechanism by which this is achieved remains unknown at present. We have identified a Plasmodium orthologue of Coq10 (PfCoq10, which is predominantly expressed in trophozoite-stage parasites, and localizes to the parasite mitochondrion. Expression of PfCoq10 in the S. cerevisiae coq10 deletion strain restored the capability of the yeast to grow on respiratory substrates, suggesting a remarkable functional conservation of this protein over a vast evolutionary distance, and despite a relatively low level of amino acid sequence identity. As the antimalarial drug atovaquone acts as a competitive inhibitor of CoQ, we assessed whether over-expression of PfCoq10 altered the atovaquone sensitivity in parasites and in yeast mitochondria, but found no alteration of its activity.

Distinct Domestication Trajectories in Top-Fermenting Beer Yeasts and Wine Yeasts.

Science.gov (United States)

Gonçalves, Margarida; Pontes, Ana; Almeida, Pedro; Barbosa, Raquel; Serra, Marta; Libkind, Diego; Hutzler, Mathias; Gonçalves, Paula; Sampaio, José Paulo

2016-10-24

Beer is one of the oldest alcoholic beverages and is produced by the fermentation of sugars derived from starches present in cereal grains. Contrary to lager beers, made by bottom-fermenting strains of Saccharomyces pastorianus, a hybrid yeast, ale beers are closer to the ancient beer type and are fermented by S. cerevisiae, a top-fermenting yeast. Here, we use population genomics to investigate (1) the closest relatives of top-fermenting beer yeasts; (2) whether top-fermenting yeasts represent an independent domestication event separate from those already described; (3) whether single or multiple beer yeast domestication events can be inferred; and (4) whether top-fermenting yeasts represent non-recombinant or recombinant lineages. Our results revealed that top-fermenting beer yeasts are polyphyletic, with a main clade composed of at least three subgroups, dominantly represented by the German, British, and wheat beer strains. Other beer strains were phylogenetically close to sake, wine, or bread yeasts. We detected genetic signatures of beer yeast domestication by investigating genes previously linked to brewing and using genome-wide scans. We propose that the emergence of the main clade of beer yeasts is related with a domestication event distinct from the previously known cases of wine and sake yeast domestication. The nucleotide diversity of the main beer clade more than doubled that of wine yeasts, which might be a consequence of fundamental differences in the modes of beer and wine yeast domestication. The higher diversity of beer strains could be due to the more intense and different selection regimes associated to brewing. Copyright © 2016 Elsevier Ltd. All rights reserved.

THE ISLAMIC ETHICS OF MITOCHONDRIA TRANSPLANTATION

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Anke Iman Bouzenita

2017-12-01

Full Text Available ABSTRACT: Biotechnology has opened a new chapter with the advent of mitochondria transplantation for cell-based therapy. Mitochondrial transplantation was successfully led to birth; however, cytoplasmic transplantation has caused apprehension, since the mixing of human ooplasm from two different maternal sources may generate mitochondrial DNA (mtDNA heteroplasmy in the offspring. Islamic legal verdicts on human cloning and somatic cell transfer have been overweighing explicit as to its prohibition, due to the change of creation, mixing of lineage and other evaluations. Is mitochondria transplantation equivalent to human cloning in that genetic information is proliferated and does it, therefore, take the same legal rule? Are there possible benefits (masalih for medical treatment that may render mitochondria transplantation permissible, or are possible harms (mafasid overweighing? Or is it a completely different procedure, taking a different rule? The paper will investigate into these questions and discuss the dimensions of Islamic ethics on the issue.

Prions in yeast

OpenAIRE

Bezdíčka, Martin

2013-01-01

The thesis describes yeast prions and their biological effects on yeast in general. It defines the basic characteristics of yeast prions, that distinguish prions from other proteins. The thesis introduces various possibilities of prion formation, and propagation as well as specific types of yeast prions, including various functions of most studied types of prions. The thesis also focuses on chaperones that affect the state of yeast prions in cells. Lastly, the thesis indicates similarities be...

The destiny of Ca(2+) released by mitochondria.

Science.gov (United States)

Takeuchi, Ayako; Kim, Bongju; Matsuoka, Satoshi

2015-01-01

Mitochondrial Ca(2+) is known to regulate diverse cellular functions, for example energy production and cell death, by modulating mitochondrial dehydrogenases, inducing production of reactive oxygen species, and opening mitochondrial permeability transition pores. In addition to the action of Ca(2+) within mitochondria, Ca(2+) released from mitochondria is also important in a variety of cellular functions. In the last 5 years, the molecules responsible for mitochondrial Ca(2+) dynamics have been identified: a mitochondrial Ca(2+) uniporter (MCU), a mitochondrial Na(+)-Ca(2+) exchanger (NCLX), and a candidate for a mitochondrial H(+)-Ca(2+) exchanger (Letm1). In this review, we focus on the mitochondrial Ca(2+) release system, and discuss its physiological and pathophysiological significance. Accumulating evidence suggests that the mitochondrial Ca(2+) release system is not only crucial in maintaining mitochondrial Ca(2+) homeostasis but also participates in the Ca(2+) crosstalk between mitochondria and the plasma membrane and between mitochondria and the endoplasmic/sarcoplasmic reticulum.

Improving oocyte quality by transfer of autologous mitochondria from fully grown oocytes

DEFF Research Database (Denmark)

Kristensen, Stine Gry; Pors, Susanne Elisabeth; Andersen, Claus Yding

2017-01-01

options using autologous mitochondria to potentially augment pregnancy potential in ART. Autologous transfer of mitochondria from the patient's own germline cells has attracted much attention as a possible new treatment to revitalize deficient oocytes. IVF births have been reported after transfer...... of oogonial precursor cell-derived mitochondria; however, the source and quality of the mitochondria are still unclear. In contrast, fully grown oocytes are loaded with mitochondria which have passed the genetic bottleneck and are likely to be of high quality. An increased supply of such oocytes could...... with high quality mitochondria can be obtained from natural or stimulated ovaries and potentially be used to improve both quality and quantity of oocytes available for fertility treatment....

Kidney outer medulla mitochondria are more efficient compared to cortex mitochondria as a strategy to sustain ATP production in a suboptimal environment.

Science.gov (United States)

Schiffer, Tomas A; Gustafsson, Håkan; Palm, Fredrik

2018-05-30

The kidneys receive approximately 25% of cardiac output, which is a prerequisite in order to maintain sufficient glomerular filtration rate. However, both intrarenal regional renal blood flow and tissue oxygen levels are heterogeneous with decreasing levels in the inner part of the medulla. These differences in combination with the heterogeneous metabolic activity of the different nephron segment located in the different parts of the kidney may constitute a functional problem when challenged. The proximal tubule and the medullary thick ascending limb of Henle are considered to have the highest metabolic rate, which is relating to the high mitochondria content needed to sustain sufficient ATP production from oxidative phosphorylation in order to support high electrolyte transport activity in these nephron segments. Interestingly, the cells located in kidney medulla functions at the verge of hypoxia and the mitochondria may have adapted to the surrounding environment. However, little is known about intrarenal differences in mitochondria function. We therefore investigated functional differences between mitochondria isolated from kidney cortex and medulla of healthy normoglycemic rats were estimated using high-resolution respirometry. The results demonstrate that medullary mitochondria had a higher degree of coupling, are more efficient and have higher oxygen affinity, which would make them more suitable to function in an environment with limited oxygen supply. Furthermore, these results support the hypothesis that mitochondria of medullary cells have adapted to the normal hypoxic in vivo situation as a strategy of sustaining ATP production in a suboptimal environment.

Yeast petites and small colony variants: for everything there is a season.

Science.gov (United States)

Day, Martin

2013-01-01

The yeast petite mutant was first found in the yeast Saccharomyces cerevisiae. The colony is small because of a block in the aerobic respiratory chain pathway, which generates ATP. The petite yeasts are thus unable to grow on nonfermentable carbon sources (such as glycerol or ethanol), and form small anaerobic-sized colonies when grown in the presence of fermentable carbon sources (such as glucose). The petite phenotype results from mutations in the mitochondrial genome, loss of mitochondria, or mutations in the host cell genome. The latter mutations affect nuclear-encoded genes involved in oxidative phosphorylation and these mutants are termed neutral petites. They all produce wild-type progeny when crossed with a wild-type strain. The staphylococcal small colony variant (SCV) is a slow-growing mutant that typically exhibits the loss of many phenotypic characteristics and pathogenic traits. SCVs are mostly small, nonpigmented, and nonhaemolytic. Their small size is often due to an inability to synthesize electron transport chain components and so cannot generate ATP by oxidative phosphorylation. Evidence suggests that they are responsible for persistent and/or recurrent infections. This chapter compares the physiological and genetic basis of the petite mutants and SCVs. The review focuses principally on two representatives, the eukaryote S. cerevisiae and the prokaryote Staphylococcus aureus. There is, clearly, commonality in the physiological response. Interestingly, the similarity, based on their physiological states, has not been commented on previously. The finding of an overlapping physiological response that occurs across a taxonomic divide is novel. © 2013 Elsevier Inc. All rights reserved.

Ca(2+-dependent regulation of the Ca(2+ concentration in the myometrium mitochondria. II. Ca(2+ effects on mitochondria membranes polarization and [Ca(2+](m

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L. G. Babich

2017-06-01

Full Text Available It is known that Ca2+ accumulation in the mitochondria undergoes complex regulation by Ca2+ itself. But the mechanisms of such regulation are still discussed. In this paper we have shown that Ca ions directly or indirectly regulate the level of myometrium mitochondria membranes polarization. The additions of 100 µM Ca2+ were accompanied by depolarization of the mitochondria membranes. The following experiments were designed to study the impact of Ca2+ on the myometrium mitochondria [Ca2+]m. Isolated myometrium mitochondria were preincubated without or with 10 μM Са2+ followed by 100 μM Са2+ addition. Experiments were conducted in three mediums: without ATP and Mg2+ (0-medium, in the presence of 3 mM Mg2+ (Mg-medium and 3 mM Mg2+ + 3 mM ATP (Mg,ATP-medium. It was shown that the effects of 10 μM Са2+ addition were different in different mediums, namely in 0- and Mg-medium the [Ca2+]m values increased, whereas in Mg,ATP-medium statistically reliable changes were not registered. Preincubation of mitochondria with 10 μM Са2+ did not affect the [Ca2+]m value after the addition of 100 μM Са2+. The [Ca2+]m values after 100 μM Са2+ addition were the same in 0- and Mg,ATP-mediums and somewhat lower in Mg-medium. Preliminary incubation of mitochondria with 10 μM Са2+ in 0- and Mg-mediums reduced changes of Fluo 4 normalized fluorescence values that were induced by 100 μM Са2+ additions, but in Mg,ATP-medium such differences were not recorded. It is concluded that Са2+ exchange in myometrium mitochondria is regulated by the concentration of Ca ions as in the external medium, so in the matrix of mitochondria. The medium composition had a significant impact on the [Са2+]m values in the absence of exogenous cation. It is suggested that light increase of [Са2+]m before the addition of 100 μM Са2+ may have a positive effect on the functional activity of the mitochondria.

Oxygen Response of the Wine Yeast Saccharomyces cerevisiae EC1118 Grown under Carbon-Sufficient, Nitrogen-Limited Enological Conditions

Science.gov (United States)

Aceituno, Felipe F.; Orellana, Marcelo; Torres, Jorge; Mendoza, Sebastián; Slater, Alex W.; Melo, Francisco

2012-01-01

Discrete additions of oxygen play a critical role in alcoholic fermentation. However, few studies have quantitated the fate of dissolved oxygen and its impact on wine yeast cell physiology under enological conditions. We simulated the range of dissolved oxygen concentrations that occur after a pump-over during the winemaking process by sparging nitrogen-limited continuous cultures with oxygen-nitrogen gaseous mixtures. When the dissolved oxygen concentration increased from 1.2 to 2.7 μM, yeast cells changed from a fully fermentative to a mixed respirofermentative metabolism. This transition is characterized by a switch in the operation of the tricarboxylic acid cycle (TCA) and an activation of NADH shuttling from the cytosol to mitochondria. Nevertheless, fermentative ethanol production remained the major cytosolic NADH sink under all oxygen conditions, suggesting that the limitation of mitochondrial NADH reoxidation is the major cause of the Crabtree effect. This is reinforced by the induction of several key respiratory genes by oxygen, despite the high sugar concentration, indicating that oxygen overrides glucose repression. Genes associated with other processes, such as proline uptake, cell wall remodeling, and oxidative stress, were also significantly affected by oxygen. The results of this study indicate that respiration is responsible for a substantial part of the oxygen response in yeast cells during alcoholic fermentation. This information will facilitate the development of temporal oxygen addition strategies to optimize yeast performance in industrial fermentations. PMID:23001663

Bacterial infection increases risk of carcinogenesis by targeting mitochondria

DEFF Research Database (Denmark)

Strickertsson, Jesper A.B.; Desler, Claus; Rasmussen, Lene Juel

2017-01-01

pathways, and compares the impact of the bacterial alteration of mitochondrial function to that of cancer. Bacterial virulence factors have been demonstrated to induce mutations of mitochondrial DNA (mtDNA) and to modulate DNA repair pathways of the mitochondria. Furthermore, virulence factors can induce...... or impair the intrinsic apoptotic pathway. The effect of bacterial targeting of mitochondria is analogous to behavior of mitochondria in a wide array of tumours, and this strongly suggests that mitochondrial targeting of bacteria is a risk factor for carcinogenesis....

The rapid mode of calcium uptake into heart mitochondria (RaM): comparison to RaM in liver mitochondria.

Science.gov (United States)

Buntinas, L; Gunter, K K; Sparagna, G C; Gunter, T E

2001-04-02

A mechanism of Ca(2+) uptake, capable of sequestering significant amounts of Ca(2+) from cytosolic Ca(2+) pulses, has previously been identified in liver mitochondria. This mechanism, the Rapid Mode of Ca(2+) uptake (RaM), was shown to sequester Ca(2+) very rapidly at the beginning of each pulse in a sequence [Sparagna et al. (1995) J. Biol. Chem. 270, 27510-27515]. The existence and properties of RaM in heart mitochondria, however, are unknown and are the basis for this study. We show that RaM functions in heart mitochondria with some of the characteristics of RaM in liver, but its activation and inhibition are quite different. It is feasible that these differences represent different physiological adaptations in these two tissues. In both tissues, RaM is highly conductive at the beginning of a Ca(2+) pulse, but is inhibited by the rising [Ca(2+)] of the pulse itself. In heart mitochondria, the time required at low [Ca(2+)] to reestablish high Ca(2+) conductivity via RaM i.e. the 'resetting time' of RaM is much longer than in liver. RaM in liver mitochondria is strongly activated by spermine, activated by ATP or GTP and unaffected by ADP and AMP. In heart, RaM is activated much less strongly by spermine and unaffected by ATP or GTP. RaM in heart is strongly inhibited by AMP and has a biphasic response to ADP; it is activated at low concentrations and inhibited at high concentrations. Finally, an hypothesis consistent with the data and characteristics of liver and heart is presented to explain how RaM may function to control the rate of oxidative phosphorylation in each tissue. Under this hypothesis, RaM functions to create a brief, high free Ca(2+) concentration inside mitochondria which may activate intramitochondrial metabolic reactions with relatively small amounts of Ca(2+) uptake. This hypothesis is consistent with the view that intramitochondrial [Ca(2+)] may be used to control the rate of ADP phosphorylation in such a way as to minimize the probability of

Do the mitochondria of malaria parasites behave like the phoenix after return in the mosquito? Regeneration of degenerated mitochondria is required for successful Plasmodium infection.

Science.gov (United States)

Bongaerts, Ger

2005-01-01

Mitochondria are energy generators in eukaryotic organisms like man and the pathogenic malaria parasites, the Plasmodium spp. From the moment a mosquito-mediated malaria infection occurs in man the parasite multiplies profusely, but eventually the oxygen supply becomes the limiting factor in this process. Consequently, the parasite will increasingly generate energy (and lactic acid) from sugar fermentation. Simultaneously, the cristate structure of Plasmodium mitochondria degenerates and becomes acristate. The degenerated acristate mitochondria of mammalian Plasmodium parasites seem to be able to revitalise by transforming to cristate mitochondria inside the oxygen-rich mosquito, like the rebirth of the old phoenix. In this way the infectivity of the parasite is revitalised.

Effect of Myricetin, Pyrogallol, and Phloroglucinol on Yeast Resistance to Oxidative Stress

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Vanda Mendes

2015-01-01

Full Text Available The health beneficial effects of dietary polyphenols have been attributed to their intrinsic antioxidant activity, which depends on the structure of the compound and number of hydroxyl groups. In this study, the protective effects of pyrogallol, phloroglucinol, and myricetin on the yeast Saccharomyces cerevisiae were investigated. Pyrogallol and myricetin, which have a pyrogallol structure in the B ring, increased H2O2 resistance associated with a reduction in intracellular oxidation and protein carbonylation, whereas phloroglucinol did not exert protective effects. The acquisition of oxidative stress resistance in cells pretreated with pyrogallol and myricetin was not associated with an induction of endogenous antioxidant defences as assessed by the analysis of superoxide dismutase and catalase activities. However, myricetin, which provided greater stress resistance, prevented H2O2-induced glutathione oxidation. Moreover, myricetin increased the chronological lifespan of yeast lacking the mitochondrial superoxide dismutase (Sod2p, which exhibited a premature aging phenotype and oxidative stress sensitivity. These findings show that the presence of hydroxyl groups in the ortho position of the B ring in pyrogallol and myricetin contributes to the antioxidant protection afforded by these compounds. In addition, myricetin may alleviate aging-induced oxidative stress, particularly when redox homeostasis is compromised due to downregulation of endogenous defences present in mitochondria.

Melatonin as a mitochondria-targeted antioxidant: one of evolution's best ideas.

Science.gov (United States)

Reiter, Russel J; Rosales-Corral, Sergio; Tan, Dun Xian; Jou, Mei Jie; Galano, Annia; Xu, Bing

2017-11-01

Melatonin is an ancient antioxidant. After its initial development in bacteria, it has been retained throughout evolution such that it may be or may have been present in every species that have existed. Even though it has been maintained throughout evolution during the diversification of species, melatonin's chemical structure has never changed; thus, the melatonin present in currently living humans is identical to that present in cyanobacteria that have existed on Earth for billions of years. Melatonin in the systemic circulation of mammals quickly disappears from the blood presumably due to its uptake by cells, particularly when they are under high oxidative stress conditions. The measurement of the subcellular distribution of melatonin has shown that the concentration of this indole in the mitochondria greatly exceeds that in the blood. Melatonin presumably enters mitochondria through oligopeptide transporters, PEPT1, and PEPT2. Thus, melatonin is specifically targeted to the mitochondria where it seems to function as an apex antioxidant. In addition to being taken up from the circulation, melatonin may be produced in the mitochondria as well. During evolution, mitochondria likely originated when melatonin-forming bacteria were engulfed as food by ancestral prokaryotes. Over time, engulfed bacteria evolved into mitochondria; this is known as the endosymbiotic theory of the origin of mitochondria. When they did so, the mitochondria retained the ability to synthesize melatonin. Thus, melatonin is not only taken up by mitochondria but these organelles, in addition to many other functions, also probably produce melatonin as well. Melatonin's high concentrations and multiple actions as an antioxidant provide potent antioxidant protection to these organelles which are exposed to abundant free radicals.

Mitochondria-targeted nutraceuticals in sports medicine: a new perspective.

Science.gov (United States)

Ostojic, Sergej M

2017-01-01

Since mitochondria have been recognized as the cells' key organelles involved in the energy utilization during exercise, targeting the organelle with specifically designed compounds (mitochondria-targeted nutraceuticals, MTNs) may have a great promise in the prevention and treatment of heavy exercise-related mitochondrial dysfunction. In vitro studies suggested that MTNs have antioxidant effects at the molecular level, and might boost mitochondrial biogenesis and organelle bioenergetics, with both processes are known to positively affect exercise performance and recovery. However, while there are a number of different MTNs evaluated for a potential benefit as a therapy for mitochondria-related diseases and conditions, only few human studies evaluated the possible impact of novel MTNs in the field of sports medicine. This mini review summarizes recent research findings regarding the efficacy of different mitochondria-targeted nutritional agents, emphasizing their roles in sports medicine.

The fate of paternal mitochondria in marmoset pre-implantation embryos.

Science.gov (United States)

Luetjens, C M; Wesselmann, R

2008-06-01

Sperm-derived mitochondria are integrated into the oocyte at fertilization but seem to vanish during the early cleavage phase. The developmental potential of pre-implantation embryos seems to be closely related to their ability to induce degeneration of these mitochondria, but the mechanisms underlying their loss of function are not yet understood. This study focuses on the fate of paternal mitochondria in pre-implantation embryos. Stimulation, collection and in vitro culture of oocytes from Callithrix jacchus, allows the study of the destiny of paternal mitochondria by utilizing immunostaining of pre-implantation embryos, fluorescence and laserscanning microscopy. Live pre-implantation embryos were stained with a fluorescence indicator reflecting mitochondrial membrane potential. Evidence indicating the loss of mitochondrial function was not found nor that apoptosis pathways were involved in the disappearance of paternally derived mitochondria. These findings may have implications for mitochondrially inherited diseases and could lead to new strategies for improving assisted reproduction.

Mitochondria in anthropology and forensic medicine.

Science.gov (United States)

Grzybowski, Tomasz; Rogalla, Urszula

2012-01-01

Mitochondria's role in crucial metabolic pathways is probably the first answer which comes to our minds for the question: what do these tiny organelles serve for? However, specific features of their DNA made them extremely useful also in the field of anthropology and forensics. MtDNA analyses became a milestone in the complex task of unraveling earliest human migrations. Evidence provided by these experiments left no doubts on modern humans origins pointing to Africa being our cradle. It also contributed to interpretation of putative ways of our dispersal around Asia and Americas thousands years ago. On the other hand, analysis of mtDNA is well established and valuable tool in forensic genetics. When other definitely more popular markers give no answer on identity, it is the time to employ information carried by mitochondria. This chapter summarizes not only current reports on the role of mitochondria in forensics and reconstruction of modern humans phylogeny, but also calls one's attention to a broad range of difficulties and constraints associated with mtDNA analyses.

Role of mitochondria in parvovirus pathology.

Directory of Open Access Journals (Sweden)

Jonna Nykky

Full Text Available 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.

Radiation stimulation of yeast crops for increasing output of alcohol and baker yeasts

International Nuclear Information System (INIS)

Vlad, E.; Marsheu, P.

1974-01-01

The purpose of this study was to stimulate by gamma radiation the existing commercial types of yeast so as to obtain yeasts that would better reflect the substrate and have improved reproductive capacity. The experiments were conducted under ordinary conditions using commercial yeasts received from one factory producing alcohol and bakery yeasts and isolated as pure cultures. Irradiating yeast cultures with small doses (up to 10 krad) was found to stimulate the reproduction and fermenting activity of yeast cells as manifested in increased accumulation of yeast biomass and greater yield of ethyl alcohol. (E.T.)

mtDNA depletion myopathy: elucidation of the tissue specificity in the mitochondrial thymidine kinase (TK2) deficiency.

Science.gov (United States)

Saada, Ann; Shaag, Avraham; Elpeleg, Orly

2003-05-01

Decreased mitochondrial thymidine kinase (TK2) activity is associated with mitochondrial DNA (mtDNA) depletion and respiratory chain dysfunction and is manifested by isolated, fatal skeletal myopathy. Other tissues such as liver, brain, heart, and skin remain unaffected throughout the patients' life. In order to elucidate the mechanism of tissue specificity in the disease we have investigated the expression of the mitochondrial deoxynucleotide carrier, the mtDNA content and the activity of TK2 in mitochondria of various tissues. Our results suggest that low basal TK2 activity combined with a high requirement for mitochondrial encoded proteins in muscle predispose this tissue to the devastating effect of TK2 deficiency.

Huperzine A protects isolated rat brain mitochondria against beta-amyloid peptide.

Science.gov (United States)

Gao, Xin; Zheng, Chun Yan; Yang, Ling; Tang, Xi Can; Zhang, Hai Yan

2009-06-01

Our previous work in cells and animals showed that mitochondria are involved in the neuroprotective effect of huperzine A (HupA). In this study, the effects of HupA on isolated rat brain mitochondria were investigated. In addition to inhibiting the Abeta(25-35) (40 microM)-induced decrease in mitochondrial respiration, adenosine 5'-triphosphate (ATP) synthesis, enzyme activity, and transmembrane potential, HupA (0.01 or 0.1 microM) effectively prevented Abeta-induced mitochondrial swelling, reactive oxygen species increase, and cytochrome c release. More interestingly, administration of HupA to isolated mitochondria promoted the rate of ATP production and blocked mitochondrial swelling caused by normal osmosis. These results indicate that HupA protects mitochondria against Abeta at least in part by preserving membrane integrity and improving energy metabolism. These direct effects on mitochondria further extend the noncholinergic functions of HupA.

Fluoxetine and the mitochondria: A review of the toxicological aspects.

Science.gov (United States)

de Oliveira, Marcos Roberto

2016-09-06

Fluoxetine (a selective serotonin reuptake inhibitor (SSRI)) is used as an antidepressant by modulating the levels of serotonin in the synaptic cleft. Nevertheless, fluoxetine also induces undesirable effects, such as anxiety, sexual dysfunction, sleep disturbances, and gastrointestinal impairments. Fluoxetine has been viewed as an agent that may interfere with cell fate by triggering apoptosis. On the other hand, fluoxetine intake has been associated with increased cancer risk. Nonetheless, data remain contradictory and no conclusions were taken. Several studies demonstrated that fluoxetine interacts with mitochondria triggering apoptosis and/or altering mitochondrial function by modulating the activity of respiratory chain components and enzymes of the Krebs cycle. Furthermore, fluoxetine affects mitochondria-related redox parameters in different experimental models. In this review, data demonstrating the effects of fluoxetine upon mammalian mitochondria are described and discussed, as well as several unsolved questions in this field of research are addressed. A separate section deals with future needs regarding the research involving the impact of fluoxetine treatment upon mitochondria and mitochondria-related signaling. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Sensibility analysis of fuel depletion using different nuclear fuel depletion codes

Energy Technology Data Exchange (ETDEWEB)

Martins, F.; Velasquez, C.E.; Castro, V.F.; Pereira, C.; Silva, C. A. Mello da, E-mail: felipmartins94@gmail.com, E-mail: carlosvelcab@hotmail.com, E-mail: victorfariascastro@gmail.com, E-mail: claubia@nuclear.ufmg.br, E-mail: clarysson@nuclear.ufmg.br [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Departamento de Engenharia Nuclear

2017-07-01

Nowadays, the utilization of different nuclear codes to perform the depletion and criticality calculations has been used to simulated nuclear reactors problems. Therefore, the goal is to analyze the sensibility of the fuel depletion of a PWR assembly using three different nuclear fuel depletion codes. The burnup calculations are performed using the codes MCNP5/ORIGEN2.1 (MONTEBURNS), KENO-VI/ORIGEN-S (TRITONSCALE6.0) and MCNPX (MCNPX/CINDER90). Each nuclear code performs the burnup using different depletion codes. Each depletion code works with collapsed energies from a master library in 1, 3 and 63 groups, respectively. Besides, each code uses different ways to obtain neutron flux that influences the depletions calculation. The results present a comparison of the neutronic parameters and isotopes composition such as criticality and nuclides build-up, the deviation in results are going to be assigned to features of the depletion code in use, such as the different radioactive decay internal libraries and the numerical method involved in solving the coupled differential depletion equations. It is also seen that the longer the period is and the more time steps are chosen, the larger the deviation become. (author)

Sensibility analysis of fuel depletion using different nuclear fuel depletion codes

International Nuclear Information System (INIS)

Martins, F.; Velasquez, C.E.; Castro, V.F.; Pereira, C.; Silva, C. A. Mello da

2017-01-01

Nowadays, the utilization of different nuclear codes to perform the depletion and criticality calculations has been used to simulated nuclear reactors problems. Therefore, the goal is to analyze the sensibility of the fuel depletion of a PWR assembly using three different nuclear fuel depletion codes. The burnup calculations are performed using the codes MCNP5/ORIGEN2.1 (MONTEBURNS), KENO-VI/ORIGEN-S (TRITONSCALE6.0) and MCNPX (MCNPX/CINDER90). Each nuclear code performs the burnup using different depletion codes. Each depletion code works with collapsed energies from a master library in 1, 3 and 63 groups, respectively. Besides, each code uses different ways to obtain neutron flux that influences the depletions calculation. The results present a comparison of the neutronic parameters and isotopes composition such as criticality and nuclides build-up, the deviation in results are going to be assigned to features of the depletion code in use, such as the different radioactive decay internal libraries and the numerical method involved in solving the coupled differential depletion equations. It is also seen that the longer the period is and the more time steps are chosen, the larger the deviation become. (author)

BH3-only protein Bim inhibits activity of antiapoptotic members of Bcl-2 family when expressed in yeast.

Science.gov (United States)

Juhásová, Barbora; Mentel, Marek; Bhatia-Kiššová, Ingrid; Zeman, Igor; Kolarov, Jordan; Forte, Michael; Polčic, Peter

2011-09-02

Proteins of the Bcl-2 family regulate programmed cell death in mammals by promoting the release of cytochrome c from mitochondria in response to various proapoptotic stimuli. The mechanism by which BH3-only members of the family activate multidomain proapoptotic proteins Bax and Bak to form a pore in mitochondrial membranes remains under dispute. We report that cell death promoting activity of BH3-only protein Bim can be reconstituted in yeast when both Bax and antiapoptotic protein Bcl-X(L) are present, suggesting that Bim likely activates Bax indirectly by inhibiting antiapoptotic proteins. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Differential regulation of mitochondrial pyruvate carrier genes modulates respiratory capacity and stress tolerance in yeast.

Directory of Open Access Journals (Sweden)

Alba Timón-Gómez

Full Text Available Mpc proteins are highly conserved from yeast to humans and are necessary for the uptake of pyruvate at the inner mitochondrial membrane, which is used for leucine and valine biosynthesis and as a fuel for respiration. Our analysis of the yeast MPC gene family suggests that amino acid biosynthesis, respiration rate and oxidative stress tolerance are regulated by changes in the Mpc protein composition of the mitochondria. Mpc2 and Mpc3 are highly similar but functionally different: Mpc2 is most abundant under fermentative non stress conditions and important for amino acid biosynthesis, while Mpc3 is the most abundant family member upon salt stress or when high respiration rates are required. Accordingly, expression of the MPC3 gene is highly activated upon NaCl stress or during the transition from fermentation to respiration, both types of regulation depend on the Hog1 MAP kinase. Overexpression experiments show that gain of Mpc2 function leads to a severe respiration defect and ROS accumulation, while Mpc3 stimulates respiration and enhances tolerance to oxidative stress. Our results identify the regulated mitochondrial pyruvate uptake as an important determinant of respiration rate and stress resistance.

Differential regulation of mitochondrial pyruvate carrier genes modulates respiratory capacity and stress tolerance in yeast.

Science.gov (United States)

Timón-Gómez, Alba; Proft, Markus; Pascual-Ahuir, Amparo

2013-01-01

Mpc proteins are highly conserved from yeast to humans and are necessary for the uptake of pyruvate at the inner mitochondrial membrane, which is used for leucine and valine biosynthesis and as a fuel for respiration. Our analysis of the yeast MPC gene family suggests that amino acid biosynthesis, respiration rate and oxidative stress tolerance are regulated by changes in the Mpc protein composition of the mitochondria. Mpc2 and Mpc3 are highly similar but functionally different: Mpc2 is most abundant under fermentative non stress conditions and important for amino acid biosynthesis, while Mpc3 is the most abundant family member upon salt stress or when high respiration rates are required. Accordingly, expression of the MPC3 gene is highly activated upon NaCl stress or during the transition from fermentation to respiration, both types of regulation depend on the Hog1 MAP kinase. Overexpression experiments show that gain of Mpc2 function leads to a severe respiration defect and ROS accumulation, while Mpc3 stimulates respiration and enhances tolerance to oxidative stress. Our results identify the regulated mitochondrial pyruvate uptake as an important determinant of respiration rate and stress resistance.

Mitochondria-Associated Membranes As Networking Platforms and Regulators of Cancer Cell Fate

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Maria Livia Sassano

2017-08-01

Full Text Available The tight cross talk between two essential organelles of the cell, the endoplasmic reticulum (ER and mitochondria, is spatially and functionally regulated by specific microdomains known as the mitochondria-associated membranes (MAMs. MAMs are hot spots of Ca2+ transfer between the ER and mitochondria, and emerging data indicate their vital role in the regulation of fundamental physiological processes, chief among them mitochondria bioenergetics, proteostasis, cell death, and autophagy. Moreover, and perhaps not surprisingly, it has become clear that signaling events regulated at the ER–mitochondria intersection regulate key processes in oncogenesis and in the response of cancer cells to therapeutics. ER–mitochondria appositions have been shown to dynamically recruit oncogenes and tumor suppressors, modulating their activity and protein complex formation, adapt the bioenergetic demand of cancer cells and to regulate cell death pathways and redox signaling in cancer cells. In this review, we discuss some emerging players of the ER–mitochondria contact sites in mammalian cells, the key processes they regulate and recent evidence highlighting the role of MAMs in shaping cell-autonomous and non-autonomous signals that regulate cancer growth.

Mitochondrial alterations with mitochondrial DNA depletion in the nerves of AIDS patients with peripheral neuropathy induced by 2'3'-dideoxycytidine (ddC).

Science.gov (United States)

Dalakas, M C; Semino-Mora, C; Leon-Monzon, M

2001-11-01

The 2'3'-dideoxycytidine (ddC), a nonazylated dideoxynucleoside analog used for the treatment of AIDS, causes a dose-dependent, painful, sensorimotor axonal peripheral neuropathy in up to 30% of the patients. To investigate the cause of the neuropathy, we performed morphological and molecular studies on nerve biopsy specimens from well-selected patients with ddC-neuropathy and from control subjects with disease, including patients with AIDS-related neuropathy never treated with ddC. Because ddC, in vitro, inhibits the replication of mitochondrial DNA (mtDNA), we counted the number of normal and abnormal mitochondria in a 0.04 mm(2) cross-sectional area of the nerves and quantified the copy numbers of mtDNA by competitive PCR in all specimens. A varying degree of axonal degeneration was present in all nerves. Abnormal mitochondria with enlarged size, excessive vacuolization, electron-dense concentric inclusions and degenerative myelin structures were prominent in the ddC-neuropathy and accounted for 55% +/- 2.5% of all counted mitochondria in the axon and Schwann cells, compared with 9% +/- 0.7% of the controls (p ddC-treated patients compared with the controls. We conclude that ddC induces a mitochondrial neuropathy with depletion of the nerve's mtDNA. The findings are consistent with the ability of ddC to selectively inhibit the gamma-DNA polymerase in neuronal cell lines. Toxicity to mitochondria of the peripheral nerve is a new cause of acquired neuropathy induced by exogenous toxins and may be the cause of neuropathy associated with the other neurotoxic antiretroviral drugs or toxic-metabolic conditions.

Regulation of protein phosphorylation in oat mitochondria

International Nuclear Information System (INIS)

Pike, C.; Kopeck, K.; Sceppa, E.

1989-01-01

We sought to identify phosphorylated proteins in isolated oat mitocchondria and to characterize the enzymatic and regulatory properties of the protein kinase(s). Mitochondria from oats (Avena sativa L. cv. Garry) were purified on Percoll gradients. Mitochondria were incubated with 32 P-γ-ATP; proteins were separated by SDS-PAGE. A small number of bands was detected on autoradiograms, most prominently at 70 kD and 42 kD; the latter band has been tentatively identified as a subunit of the pyruvate dehydrogenase complex, a well-known phosphoprotein. The protein kinase(s) could also phosphorylate casein, but not histone. Spermine enhanced the phosphorylation of casein and inhibited the phosphorylation of the 42 kD band. These studies were carried out on both intact and burst mitochondria. Control by calcium and other ions was investigated. The question of the action of regulators on protein kinase or protein phosphatase was studied by the use of 35 S-adenosine thiotriphosphate

"When the going gets tough, who keeps going?" Depletion sensitivity moderates the ego-depletion effect.

Science.gov (United States)

Salmon, Stefanie J; Adriaanse, Marieke A; De Vet, Emely; Fennis, Bob M; De Ridder, Denise T D

2014-01-01

Self-control relies on a limited resource that can get depleted, a phenomenon that has been labeled ego-depletion. We argue that individuals may differ in their sensitivity to depleting tasks, and that consequently some people deplete their self-control resource at a faster rate than others. In three studies, we assessed individual differences in depletion sensitivity, and demonstrate that depletion sensitivity moderates ego-depletion effects. The Depletion Sensitivity Scale (DSS) was employed to assess depletion sensitivity. Study 1 employs the DSS to demonstrate that individual differences in sensitivity to ego-depletion exist. Study 2 shows moderate correlations of depletion sensitivity with related self-control concepts, indicating that these scales measure conceptually distinct constructs. Study 3 demonstrates that depletion sensitivity moderates the ego-depletion effect. Specifically, participants who are sensitive to depletion performed worse on a second self-control task, indicating a stronger ego-depletion effect, compared to participants less sensitive to depletion.

When the Going Gets Tough, Who Keeps Going? Depletion Sensitivity Moderates the Ego-Depletion Effect

Directory of Open Access Journals (Sweden)

Stefanie J. Salmon

2014-06-01

Full Text Available Self-control relies on a limited resource that can get depleted, a phenomenon that has been labeled ego-depletion. We argue that individuals may differ in their sensitivity to depleting tasks, and that consequently some people deplete their self-control resource at a faster rate than others. In three studies, we assessed individual differences in depletion sensitivity, and demonstrate that depletion sensitivity moderates ego-depletion effects. The Depletion Sensitivity Scale (DSS was employed to assess depletion sensitivity. Study 1 employs the DSS to demonstrate that individual differences in sensitivity to ego-depletion exist. Study 2 shows moderate correlations of depletion sensitivity with related self-control concepts, indicating that these scales measure conceptually distinct constructs. Study 3 demonstrates that depletion sensitivity moderates the ego-depletion effect. Specifically, participants who are sensitive to depletion performed worse on a second self-control task, indicating a stronger ego-depletion effect, compared to participants less sensitive to depletion.

Mitochondrial DNA repair and aging

Energy Technology Data Exchange (ETDEWEB)

Mandavilli, Bhaskar S.; Santos, Janine H.; Van Houten, Bennett

2002-11-30

The mitochondrial electron transport chain plays an important role in energy production in aerobic organisms and is also a significant source of reactive oxygen species that damage DNA, RNA and proteins in the cell. Oxidative damage to the mitochondrial DNA is implicated in various degenerative diseases, cancer and aging. The importance of mitochondrial ROS in age-related degenerative diseases is further strengthened by studies using animal models, Caenorhabditis elegans, Drosophila and yeast. Research in the last several years shows that mitochondrial DNA is more susceptible to various carcinogens and ROS when compared to nuclear DNA. DNA damage in mammalian mitochondria is repaired by base excision repair (BER). Studies have shown that mitochondria contain all the enzymes required for BER. Mitochondrial DNA damage, if not repaired, leads to disruption of electron transport chain and production of more ROS. This vicious cycle of ROS production and mtDNA damage ultimately leads to energy depletion in the cell and apoptosis.

Mitochondrial DNA repair and aging

International Nuclear Information System (INIS)

Mandavilli, Bhaskar S.; Santos, Janine H.; Van Houten, Bennett

2002-01-01

The mitochondrial electron transport chain plays an important role in energy production in aerobic organisms and is also a significant source of reactive oxygen species that damage DNA, RNA and proteins in the cell. Oxidative damage to the mitochondrial DNA is implicated in various degenerative diseases, cancer and aging. The importance of mitochondrial ROS in age-related degenerative diseases is further strengthened by studies using animal models, Caenorhabditis elegans, Drosophila and yeast. Research in the last several years shows that mitochondrial DNA is more susceptible to various carcinogens and ROS when compared to nuclear DNA. DNA damage in mammalian mitochondria is repaired by base excision repair (BER). Studies have shown that mitochondria contain all the enzymes required for BER. Mitochondrial DNA damage, if not repaired, leads to disruption of electron transport chain and production of more ROS. This vicious cycle of ROS production and mtDNA damage ultimately leads to energy depletion in the cell and apoptosis

Mitochondria, Energetics, Epigenetics, and Cellular Responses to Stress

Science.gov (United States)

McAllister, Kimberly; Worth, Leroy; Haugen, Astrid C.; Meyer, Joel N.; Domann, Frederick E.; Van Houten, Bennett; Mostoslavsky, Raul; Bultman, Scott J.; Baccarelli, Andrea A.; Begley, Thomas J.; Sobol, Robert W.; Hirschey, Matthew D.; Ideker, Trey; Santos, Janine H.; Copeland, William C.; Tice, Raymond R.; Balshaw, David M.; Tyson, Frederick L.

2014-01-01

Background: Cells respond to environmental stressors through several key pathways, including response to reactive oxygen species (ROS), nutrient and ATP sensing, DNA damage response (DDR), and epigenetic alterations. Mitochondria play a central role in these pathways not only through energetics and ATP production but also through metabolites generated in the tricarboxylic acid cycle, as well as mitochondria–nuclear signaling related to mitochondria morphology, biogenesis, fission/fusion, mitophagy, apoptosis, and epigenetic regulation. Objectives: We investigated the concept of bidirectional interactions between mitochondria and cellular pathways in response to environmental stress with a focus on epigenetic regulation, and we examined DNA repair and DDR pathways as examples of biological processes that respond to exogenous insults through changes in homeostasis and altered mitochondrial function. Methods: The National Institute of Environmental Health Sciences sponsored the Workshop on Mitochondria, Energetics, Epigenetics, Environment, and DNA Damage Response on 25–26 March 2013. Here, we summarize key points and ideas emerging from this meeting. Discussion: A more comprehensive understanding of signaling mechanisms (cross-talk) between the mitochondria and nucleus is central to elucidating the integration of mitochondrial functions with other cellular response pathways in modulating the effects of environmental agents. Recent studies have highlighted the importance of mitochondrial functions in epigenetic regulation and DDR with environmental stress. Development and application of novel technologies, enhanced experimental models, and a systems-type research approach will help to discern how environmentally induced mitochondrial dysfunction affects key mechanistic pathways. Conclusions: Understanding mitochondria–cell signaling will provide insight into individual responses to environmental hazards, improving prediction of hazard and susceptibility to

Protein oxidation in plant mitochondria as a stress indicator

DEFF Research Database (Denmark)

Møller, I.M.; Kristensen, B.K.

2004-01-01

oxidation of cysteine and methionine side chains is an important mechanism for regulating enzyme activity. Mitochondria from both mammalian and plant tissues contain a number of oxidised proteins, but the relative abundance of these post-translationally modified forms is as yet unknown......, as are the consequences of the modification for the properties and turnover time of the proteins. Specific proteins appear to be particularly vulnerable to oxidative carbonylation in the matrix of plant mitochondria; these include several enzymes of the Krebs cycle, glycine decarboxylase, superoxide dismutase and heat...... shock proteins. Plant mitochondria contain a number of different proteases, but their role in removing oxidatively damaged proteins is, as yet, unclear....

Pathology of mitochondria in MELAS syndrome: an ultrastructural study.

Science.gov (United States)

Felczak, Paulina; Lewandowska, Eliza; Stępniak, Iwona; Ołdak, Monika; Pollak, Agnieszka; Lechowicz, Urszula; Pasennik, Elżbieta; Stępień, Tomasz; Wierzba-Bobrowicz, Teresa

Ultrastructural changes in skeletal muscle biopsy in a 24-year-old female patient with clinically suspected mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS) syndrome are presented. We observed proliferation and/or pleomorphism of mitochondria in skeletal muscle and smooth muscle cells of arterioles, as well as in pericytes of capillaries. Paracrystalline inclusions were found only in damaged mitochondria of skeletal muscle. Genetic testing revealed a point mutation in A3243G tRNALeu(UUR) typical for MELAS syndrome. We conclude that differentiated pathological changes of mitochondria in the studied types of cells may be associated with the different energy requirements of these cells.

Fatty acids from oleaginous yeasts and yeast-like fungi and their potential applications.

Science.gov (United States)

Xue, Si-Jia; Chi, Zhe; Zhang, Yu; Li, Yan-Feng; Liu, Guang-Lei; Jiang, Hong; Hu, Zhong; Chi, Zhen-Ming

2018-02-01

Oleaginous yeasts, fatty acids biosynthesis and regulation in the oleaginous yeasts and the fatty acids from the oleaginous yeasts and their applications are reviewed in this article. Oleaginous yeasts such as Rhodosporidium toruloides, Yarrowia lipolytica, Rhodotorula mucilaginosa, and Aureobasidium melanogenum, which can accumulate over 50% lipid of their cell dry weight, have many advantages over other oleaginous microorganisms. The fatty acids from the oleaginous yeasts have many potential applications. Many oleaginous yeasts have now been genetically modified to over-produce fatty acids and their derivatives. The most important features of the oleaginous yeasts are that they have special enzymatic systems for enhanced biosynthesis and regulation of fatty acids in their lipid particles. Recently, some oleaginous yeasts such as R. toruloides have been found to have a unique fatty acids synthetase and other oleaginous yeasts such as A. melanogenum have a unique highly reducing polyketide synthase (HR-PKS) involved in the biosynthesis of hydroxyl fatty acids. It is necessary to further enhance lipid biosynthesis using metabolic engineering and explore new applications of fatty acids in biotechnology.

Chatty Mitochondria: Keeping Balance in Cellular Protein Homeostasis.

Science.gov (United States)

Topf, Ulrike; Wrobel, Lidia; Chacinska, Agnieszka

2016-08-01

Mitochondria are multifunctional cellular organelles that host many biochemical pathways including oxidative phosphorylation (OXPHOS). Defective mitochondria pose a threat to cellular homeostasis and compensatory responses exist to curtail the source of stress and/or its consequences. The mitochondrial proteome comprises proteins encoded by the nuclear and mitochondrial genomes. Disturbances in protein homeostasis may originate from mistargeting of nuclear encoded mitochondrial proteins. Defective protein import and accumulation of mistargeted proteins leads to stress that triggers translation alterations and proteasomal activation. These cytosolic pathways are complementary to the mitochondrial unfolded protein response (UPRmt) that aims to increase the capacity of protein quality control mechanisms inside mitochondria. They constitute putative targets for interventions aimed at increasing the fitness, stress resistance, and longevity of cells and organisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

Expression of Genes Related to Oxidative Stress in Yeast Treated with Ionizing Radiation and N-acetyl -L-cysteine

Energy Technology Data Exchange (ETDEWEB)

Park, Ji Young; Kim, Jin Kyu [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Nili, Mohammad [Dawnesh Radiation Research Institute, Barcelona (Spain)

2010-10-15

Ionizing radiation (IR) induces water radiolysis, which generates highly reactive hydroxyl radicals. Reactive oxygen species (ROS) cause apoptosis and cell damage including DNA strand breaks (DSBs), base damage, protein damage and lipid-hydroperoxide. Detoxifying enzymes are immediately triggered for ROS scavenging. Yeast contains two forms of superoxide dismutase (SOD). SOD1 as a cytosolic copper-zinc superoxide dismutase is located in the cytoplasm and cytosol. SOD2 as a manganese containing enzyme is act in mitochondria matrix and mitochondrion. These enzymes scavenge superoxide radicals by catalyzing the conversion of two of these radicals into hydrogen peroxide and molecular oxygen. The hydrogen peroxide formed by superoxide dismutase and by other processes is scavenged by catalase, a ubiquitous heme protein that catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen. Yeast contains two catalases. Catalase A (CTA1) and Cytosolic catalase T (CTT1) is located in peroxisome and cytoplasm, respectively. Yeast has two glutathione (GSH) peroxidases, which are GPX1 and GPX2. GPX1 and GPX2 are component of cellular component and cytoplasm, respectively. The biochemical function of GSH peroxidase is to reduce lipid-hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water. Otherwise, chemicals and materials help ROS detoxification against oxidative damage. N-acetyl-Lcysteine (NAC) having a thiol, a precursor for glutathione (GSH), is known as one of the antioxidants. In this study, we examined the effect of NAC through gene expressions related to protective enzyme against oxidative stress in yeast

Expression of Genes Related to Oxidative Stress in Yeast Treated with Ionizing Radiation and N-acetyl -L-cysteine

International Nuclear Information System (INIS)

Park, Ji Young; Kim, Jin Kyu; Nili, Mohammad

2010-01-01

Ionizing radiation (IR) induces water radiolysis, which generates highly reactive hydroxyl radicals. Reactive oxygen species (ROS) cause apoptosis and cell damage including DNA strand breaks (DSBs), base damage, protein damage and lipid-hydroperoxide. Detoxifying enzymes are immediately triggered for ROS scavenging. Yeast contains two forms of superoxide dismutase (SOD). SOD1 as a cytosolic copper-zinc superoxide dismutase is located in the cytoplasm and cytosol. SOD2 as a manganese containing enzyme is act in mitochondria matrix and mitochondrion. These enzymes scavenge superoxide radicals by catalyzing the conversion of two of these radicals into hydrogen peroxide and molecular oxygen. The hydrogen peroxide formed by superoxide dismutase and by other processes is scavenged by catalase, a ubiquitous heme protein that catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen. Yeast contains two catalases. Catalase A (CTA1) and Cytosolic catalase T (CTT1) is located in peroxisome and cytoplasm, respectively. Yeast has two glutathione (GSH) peroxidases, which are GPX1 and GPX2. GPX1 and GPX2 are component of cellular component and cytoplasm, respectively. The biochemical function of GSH peroxidase is to reduce lipid-hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water. Otherwise, chemicals and materials help ROS detoxification against oxidative damage. N-acetyl-Lcysteine (NAC) having a thiol, a precursor for glutathione (GSH), is known as one of the antioxidants. In this study, we examined the effect of NAC through gene expressions related to protective enzyme against oxidative stress in yeast

Lactate oxidation in human skeletal muscle mitochondria

DEFF Research Database (Denmark)

Jacobs, Robert A; Meinild, Anne-Kristine; Nordsborg, Nikolai B

2013-01-01

of four separate and specific substrate titration protocols, the respirometric analysis revealed that mitochondria were capable of oxidizing lactate in the absence of exogenous LDH. The titration of lactate and NAD(+) into the respiration medium stimulated respiration (P = 0.003). The addition...... of exogenous LDH failed to increase lactate-stimulated respiration (P = 1.0). The results further demonstrate that human skeletal muscle mitochondria cannot directly oxidize lactate within the mitochondrial matrix. Alternately, these data support previous claims that lactate is converted to pyruvate within...

Enhanced mitochondrial degradation of yeast cytochrome c with amphipathic structures.

Science.gov (United States)

Chen, Xi; Moerschell, Richard P; Pearce, David A; Ramanan, Durga D; Sherman, Fred

2005-02-01

The dispensable N-terminus of iso-1-cytochrome c (iso-1) in the yeast Saccharomyces cerevisiae was replaced by 11 different amphipathic structures. Rapid degradation of the corresponding iso-1 occurred, with the degree of degradation increasing with the amphipathic moments; and this amphipathic-dependent degradation was designated ADD. ADD occurred with the holo-forms in the mitochondria but not as the apo-forms in the cytosol. The extreme mutant type degraded with a half-life of approximately 12 min, whereas the normal iso-1 was stable over hours. ADD was influenced by the rho+/rho- state and by numerous chromosomal genes. Most importantly, ADD appeared to be specifically suppressed to various extents by deletions of any of the YME1, AFG3, or RCA1 genes encoding membrane-associated mitochondrial proteases, probably because the amphipathic structures caused a stronger association with the mitochondrial inner membrane and its associated proteases. The use of ADD assisted in the differentiation of substrates of different mitochondrial degradation pathways.

Air-liquid biofilm formation is dependent on ammonium depletion in a Saccharomyces cerevisiae flor strain.

Science.gov (United States)

Zara, Giacomo; Budroni, Marilena; Mannazzu, Ilaria; Zara, Severino

2011-12-01

Air-liquid biofilm formation appears to be an adaptive mechanism that promotes foraging of Saccharomyces cerevisiae flor strains in response to nutrient starvation. The FLO11 gene plays a central role in this phenotype as its expression allows yeast cells to rise to the liquid surface. Here, we investigated the role of ammonium depletion in air-liquid biofilm formation and FLO11 expression in a S. cerevisiae flor strain. The data obtained show that increasing ammonium concentrations from 0 to 450 m m reduce air-liquid biofilm in terms of biomass and velum formation and correlate with a reduction of FLO11 expression. Rapamycin inhibition of the TOR pathway and deletion of RAS2 gene significantly reduced biofilm formation and FLO11 expression. Taken together, these data suggest that ammonium depletion is a key factor in the induction of air-liquid biofilm formation and FLO11 expression in S. cerevisiae flor strains. Copyright © 2011 John Wiley & Sons, Ltd.

Specificity of DNA import into isolated mitochondria from plants and mammals

Directory of Open Access Journals (Sweden)

Koulintchenko M. V.

2014-01-01

Full Text Available Aim. Investigation of different features of DNA import into plant and human mitochondria, for a better understanding of mitochondrial genetics and generation of biotechnological tools. Methods. DNA up-take experiments with isolated plant mitochondria, using as substrates various sequences associated or not with the specific terminal inverted repeats (TIRs present at each end of the plant mitochondrial linear plasmids. Results. It was established that the DNA import efficiency has a non-linear dependence on DNA size. It was shown that import into plant mitochondria of DNA molecules of «medium» sizes, i. e. between 4 and 7 kb, barely has any sequence specificity: neither TIRs from the 11.6 kb Brassica plasmid, nor TIRs from the Zea mays S-plasmids influenced DNA import into Solanum tuberosum mitochondria. Conclusions. The data obtained support the hypothesis about species-specific import mechanism operating under the mitochondrial linear plasmids transfer into plant mitochondria.

Nanopreparations for mitochondria targeting drug delivery system: Current strategies and future prospective

Directory of Open Access Journals (Sweden)

Zhenjie Wang

2017-11-01

Full Text Available Mitochondria are a novel and promising therapeutic target for diagnosis, treatment and prevention of a lot of human diseases such as cancer, metabolic diseases and neurodegenerative disease. Owing to the mitochondrial special bilayer structure and highly negative potential nature, therapeutic molecules have multiple difficulties in reaching mitochondria. To overcome multiple barriers for targeting mitochondria, the researchers developed various pharmaceutical preparations such as liposomes, polymeric nanoparticles and inorganic nanoparticles modified by mitochondriotropic moieties like dequalinium (DQA, triphenylphosphonium (TPP, mitochondrial penetrating peptides (MPPs and mitochondrial protein import machinery that allow specific targeting. The targeted formulations exhibited enhanced pharmacological effect and better therapeutic effect than their untargeted counterpart both in vitro and in vivo. Nanocarriers may be used for bio-therapeutic delivery into specific mitochondria that possess a great potential treatment of mitochondria related diseases.

Yeast for virus research

Science.gov (United States)

Zhao, Richard Yuqi

2017-01-01

Budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are two popular model organisms for virus research. They are natural hosts for viruses as they carry their own indigenous viruses. Both yeasts have been used for studies of plant, animal and human viruses. Many positive sense (+) RNA viruses and some DNA viruses replicate with various levels in yeasts, thus allowing study of those viral activities during viral life cycle. Yeasts are single cell eukaryotic organisms. Hence, many of the fundamental cellular functions such as cell cycle regulation or programed cell death are highly conserved from yeasts to higher eukaryotes. Therefore, they are particularly suited to study the impact of those viral activities on related cellular activities during virus-host interactions. Yeasts present many unique advantages in virus research over high eukaryotes. Yeast cells are easy to maintain in the laboratory with relative short doubling time. They are non-biohazardous, genetically amendable with small genomes that permit genome-wide analysis of virologic and cellular functions. In this review, similarities and differences of these two yeasts are described. Studies of virologic activities such as viral translation, viral replication and genome-wide study of virus-cell interactions in yeasts are highlighted. Impacts of viral proteins on basic cellular functions such as cell cycle regulation and programed cell death are discussed. Potential applications of using yeasts as hosts to carry out functional analysis of small viral genome and to develop high throughput drug screening platform for the discovery of antiviral drugs are presented. PMID:29082230

Mitochondria from rat uterine smooth muscle possess ATP-sensitive potassium channel

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Olga B. Vadzyuk

2018-03-01

Full Text Available The objective of this study was to detect ATP-sensitive K+ uptake in rat uterine smooth muscle mitochondria and to determine possible effects of its activation on mitochondrial physiology. By means of fluorescent technique with usage of K+-sensitive fluorescent probe PBFI (potassium-binding benzofuran isophthalate we showed that accumulation of K ions in isolated mitochondria from rat myometrium is sensitive to effectors of KATP-channel (ATP-sensitive K+-channel – ATP, diazoxide, glibenclamide and 5HD (5-hydroxydecanoate. Our data demonstrates that K+ uptake in isolated myometrium mitochondria results in a slight decrease in membrane potential, enhancement of generation of ROS (reactive oxygen species and mitochondrial swelling. Particularly, the addition of ATP into incubation medium led to a decrease in mitochondrial swelling and ROS production, and an increase in membrane potential. These effects were eliminated by diazoxide. If blockers of KATP-channel were added along with diazoxide, the effects of diazoxide were removed. So, we postulate the existence of KATP-channels in rat uterus mitochondria and assume that their functioning may regulate physiological conditions of mitochondria, such as matrix volume, ROS generation and polarization of mitochondrial membrane. Keywords: ATP-sensitive potassium channel, Diazoxide, 5-hydroxydecanoate, Myometrium, Mitochondria, Mitochondrial swelling, Mitochondrial membrane potential, ROS

Structure of cells chloroplasts and mitochondria of cotton leaves following gamma irradiation

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Arslanova, S V [AN Uzbekskoj SSR, Tashkent. Inst. Ehksperimental' noj Biologii Rastenij

1975-01-01

The article investigates the structural changes in the plastides and mitochondria of cotton leaf cells after irradiation. Cotton seeds that had been moistened for 24 hours were irradiated by a gamma source with a dose of 10 kR (intensity: 19 R/s.). For the study of the plastides and mitochondria of the leaf cells samples were taken in the cotyledonous leaf and flowering phases of the cotton. The cells of the cotton leaf mesophillum in the standard consists of chloroplast with developed interior structures. Study of the ultrastructure of the cells of the mesophilic tissue of the cotyledonous leaf in irradiated cotton plants showed that the chloroplastide membranes are not damaged. A change in the form of the chloroplasts, an accumulation of starch and plastic substances in the chloroplasts, and a reduction in the number of inter-grain bonds were noted. It was discovered that gamma irradiation produces an excessive build-up of starch in the chloroplasts. The mitochondria are often located close to the plastides. The optical density is typical of the matrix of the mitochondria in non-irradiated plants. After cotton seeds that have sprouted are irradiated with a dose of 10 kR in the cotyledonous leaf phase, part of the mitochondria swells. The matrix becomes more transparent, and the number of chrysts decreases. Part of the mitochondria remains intact. The optical density and internal membranes of the mitochondria remain the same as in the control group. The disturbances of the chloroplast and the mitochondria are also observed in the budding and flowering phases (under conditions of a natural day). It was noted that a shortened day facilitated to some extent a normalization of metabolism, and this produced in turn a normal development of the chloroplasts, leaf mitochondria and ATF generation, which reduces the final biological effect of the radiation.

The effect of anthralin (dithranol) on mitochondria.

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Morlière, P; Dubertret, L; Sa e Melo, T; Salet, C; Fosse, M; Santus, R

1985-05-01

The short-term effect of topical application of anthralin (dithranol) on normal human skin was investigated by electron microscopy. Mitochondria appeared markedly damaged. By contrast other cellular structures, particularly the nuclear and cytoplasmic membranes were unchanged. In vitro experiments were therefore performed on isolated rat liver mitochondria and it was shown that anthralin acts as an uncoupler of oxidative phosphorylation. These results suggest that anthralin can inhibit the adenosine triphosphate supply in epidermal cells. This loss of energy supply in keratinocytes could explain, at least in part, the therapeutic efficiency of anthralin in psoriasis.

L-arabinose fermenting yeast

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Zhang, Min; Singh, Arjun; Suominen, Pirkko; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric

2013-02-12

An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. A yeast strain engineered to metabolize arabinose through a novel pathway is also disclosed. Methods of producing ethanol include utilizing these modified yeast strains.

Glutathione and Mitochondria

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Vicent eRibas

2014-07-01

Full Text Available Glutathione (GSH is the main nonprotein thiol in cells whose functions are dependent on the redox-active thiol of its cysteine moiety that serves as a cofactor for a number of antioxidant and detoxifying enzymes. While synthesized exclusively in the cytosol from its constituent amino acids, GSH is distributed in different compartments, including mitochondria where its concentration in the matrix equals that of the cytosol. This feature and its negative charge at physiological pH imply the existence of specific carriers to import GSH from the cytosol to the mitochondrial matrix, where it plays a key role in defense against respiration-induced reactive oxygen species and in the detoxification of lipid hydroperoxides and electrophiles. Moreover, as mitochondria play a central strategic role in the activation and mode of cell death, mitochondrial GSH has been shown to critically regulate the level of sensitization to secondary hits that induce mitochondrial membrane permeabilization and release of proteins confined in the intermembrane space that once in the cytosol engage the molecular machinery of cell death. In this review, we summarize recent data on the regulation of mitochondrial GSH and its role in cell death and prevalent human diseases, such as cancer, fatty liver disease and Alzheimer’s disease.

Effects of 60 Cobalt ionizing radiation in morphology and metabolism of yeasts and Chlamydospore of Candida albicans

International Nuclear Information System (INIS)

Grillo, Michel R.F.; Demicheli, Marina C.; Andrade Junior, Heitor F.; Galiesteo Junior, Andres A.J.

2015-01-01

Candida albicans is a fungus responsible for 80-90% of fungal infections, as the symptoms are similar to those of systemic bacterial infections there is a difficulty for immediate diagnosis. These difficulties can lead to delays of antifungal therapy, which contributes to the high mortality rates associated with this infection. Resistance structures referred to as chlamydospores are very common in the pathogen, representing different cell types that form in response to certain genetic or environmental conditions. Recently, various antifungal agents and new therapeutic strategies have come into use, allowing the fungus to acquire a resistance to the drugs. The use of ionizing radiation has been widely employed for the production of immunogens against various parasites. In this work, we evaluate the effects of gamma radiation ( 60 Co) in yeast and chlamydospore of C. albicans with doses ranging from 320 to 10.240 Gy with Cobalt 60. Subsequently the samples were plated and after seven days, the colony forming units (CFU) told. The viability of irradiated cells were evaluated using the Janus green dye. A dose of 6000 Gy was considered ideal for the mitigation of chlamydospore and yeast. The dimorphic change mechanisms of both fungal structures were not harmed. The viability of chlamydospores remained above 70% while the yeast viability remained above 85%. By transmission electron microscopy and fluorescence microscopy may be noted cytoplasmic changes, defects in the cell wall, mitochondria, and the presence of partially preserved vesicles of both morphological forms of C. albicans. Irradiation both chlamydospore as C. albicans yeast allows the suppression of their reproduction, opening the possibility of their use in future candidate immunogens. (author)

Effects of 60 Cobalt ionizing radiation in morphology and metabolism of yeasts and Chlamydospore of Candida albicans

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Grillo, Michel R.F.; Demicheli, Marina C.; Andrade Junior, Heitor F.; Galiesteo Junior, Andres A.J., E-mail: galisteo@usp.br [Universidade de Sao Paulo (IMTSP/USP), Sao Paulo, SP (Brazil). Instituto de Medicina Tropical. Lab. de Protozoologia; Takakura, Cleusa F.H. [Universidade de Sao Paulo (FM/USP), Sao Paulo, SP (Brazil). Departamento de Patologia de Molestias Transmissiveis. Lab. de Patologia; Negro, Gilda M.B. del [Universidade de Sao Paulo (HCFM/USP/IMTSP/LIM-53), Sao Paulo, SP (Brazil). Hospital das Clinicas. Lab. de Micologia; Nascimento, Nanci do [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

2015-07-01

Candida albicans is a fungus responsible for 80-90% of fungal infections, as the symptoms are similar to those of systemic bacterial infections there is a difficulty for immediate diagnosis. These difficulties can lead to delays of antifungal therapy, which contributes to the high mortality rates associated with this infection. Resistance structures referred to as chlamydospores are very common in the pathogen, representing different cell types that form in response to certain genetic or environmental conditions. Recently, various antifungal agents and new therapeutic strategies have come into use, allowing the fungus to acquire a resistance to the drugs. The use of ionizing radiation has been widely employed for the production of immunogens against various parasites. In this work, we evaluate the effects of gamma radiation ({sup 60}Co) in yeast and chlamydospore of C. albicans with doses ranging from 320 to 10.240 Gy with Cobalt 60. Subsequently the samples were plated and after seven days, the colony forming units (CFU) told. The viability of irradiated cells were evaluated using the Janus green dye. A dose of 6000 Gy was considered ideal for the mitigation of chlamydospore and yeast. The dimorphic change mechanisms of both fungal structures were not harmed. The viability of chlamydospores remained above 70% while the yeast viability remained above 85%. By transmission electron microscopy and fluorescence microscopy may be noted cytoplasmic changes, defects in the cell wall, mitochondria, and the presence of partially preserved vesicles of both morphological forms of C. albicans. Irradiation both chlamydospore as C. albicans yeast allows the suppression of their reproduction, opening the possibility of their use in future candidate immunogens. (author)

Qualitative and quantitative modifications of root mitochondria during senescence of above-ground parts of Arabidopis thaliana.

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Fanello, Diego Darío; Bartoli, Carlos Guillermo; Guiamet, Juan José

2017-05-01

This work studied modifications experienced by root mitochondria during whole plant senescence or under light deprivation, using Arabidopsis thaliana plants with YFP tagged to mitochondria. During post-bolting development, root respiratory activity started to decline after aboveground organs (i.e., rosette leaves) had senesced. This suggests that carbohydrate starvation may induce root senescence. Similarly, darkening the whole plant induced a decrease in respiration of roots. This was partially due to a decrease in the number of total mitochondria (YFP-labelled mitochondria) and most probably to a decrease in the quantity of mitochondria with a developed inner membrane potential (ΔΨm, i.e., Mitotracker red- labelled mitochondria). Also, the lower amount of mitochondria with ΔΨm compared to YFP-labelled mitochondria at 10d of whole darkened plant, suggests the presence of mitochondria in a "standby state". The experiments also suggest that small mitochondria made the main contribution to the respiratory activity that was lost during root senescence. Sugar supplementation partially restored the respiration of mitochondria after 10d of whole plant dark treatment. These results suggest that root senescence is triggered by carbohydrate starvation, with loss of ΔΨm mitochondria and changes in mitochondrial size distribution. Copyright © 2017 Elsevier B.V. All rights reserved.

Are mitochondria a permanent source of reactive oxygen species?

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Staniek, K; Nohl, H

2000-11-20

The observation that in isolated mitochondria electrons may leak out of the respiratory chain to form superoxide radicals (O(2)(radical-)) has prompted the assumption that O(2)(radical-) formation is a compulsory by-product of respiration. Since mitochondrial O(2)(radical-) formation under homeostatic conditions could not be demonstrated in situ so far, conclusions drawn from isolated mitochondria must be considered with precaution. The present study reveals a link between electron deviation from the respiratory chain to oxygen and the coupling state in the presence of antimycin A. Another important factor is the analytical system applied for the detection of activated oxygen species. Due to the presence of superoxide dismutase in mitochondria, O(2)(radical-) release cannot be realistically determined in intact mitochondria. We therefore followed the release of the stable dismutation product H(2)O(2) by comparing most frequently used H(2)O(2) detection methods. The possible interaction of the detection systems with the respiratory chain was avoided by a recently developed method, which was compared with conventional methods. Irrespective of the methods applied, the substrates used for respiration and the state of respiration established, intact mitochondria could not be made to release H(2)O(2) from dismutating O(2)(radical-). Although regular mitochondrial respiration is unlikely to supply single electrons for O(2)(radical-) formation our study does not exclude the possibility of the respiratory chain becoming a radical source under certain conditions.

Mature Erythrocytes of Iguana iguana (Squamata, Iguanidae) Possess Functional Mitochondria.

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Di Giacomo, Giuseppina; Campello, Silvia; Corrado, Mauro; Di Giambattista, Livia; Cirotti, Claudia; Filomeni, Giuseppe; Gentile, Gabriele

2015-01-01

Electron microscopy analyses of Iguana iguana blood preparations revealed the presence of mitochondria within erythrocytes with well-structured cristae. Fluorescence microscopy analyses upon incubation with phalloidin-FITC, Hoechst 33342 and mitochondrial transmembrane potential (Δψm)-sensitive probe MitoTracker Red indicated that mitochondria i) widely occur in erythrocytes, ii) are polarized, and iii) seem to be preferentially confined at a "perinuclear" region, as confirmed by electron microscopy. The analysis of NADH-dependent oxygen consumption showed that red blood cells retain the capability to consume oxygen, thereby providing compelling evidence that mitochondria of Iguana erythrocytes are functional and capable to perform oxidative phosphorylation.

Bcl-2 prevents loss of mitochondria in CCCP-induced apoptosis

International Nuclear Information System (INIS)

Graaf, Aniek O. de; Heuvel, Lambert P. van den; Dijkman, Henry B.P.M.; Abreu, Ronney A. de; Birkenkamp, Kim U.; Witte, Theo de; Reijden, Bert A. van der; Smeitink, Jan A.M.; Jansen, Joop H.

2004-01-01

Bcl-2 family proteins regulate apoptosis at the level of mitochondria. To examine the mechanism of Bcl-2 function, we investigated the effects of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) on two hematopoietic cell lines and Bcl-2 overexpressing transfectants. CCCP directly interferes with mitochondrial function and induces apoptosis. We show that Bcl-2 inhibits apoptosis and that the antiapoptotic effect of Bcl-2 takes place upstream of caspase activation and nuclear changes associated with apoptosis, since these were markedly inhibited in cells overexpressing Bcl-2. Bcl-2 does not prevent the decrease in mitochondrial membrane potential nor the alterations in cellular ATP content induced by CCCP in FL5.12 and Jurkat cells. A higher number of mitochondria was observed in untreated Bcl-2 transfected cells compared to parental cells, as shown by electron microscopy. Exposure to CCCP induced a dramatic decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, with apparent swelling and loss of cristae in parental cells. Bcl-2 clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. These data suggest that CCCP induces apoptosis by structural disruption of mitochondria and that Bcl-2 prevents apoptosis and mitochondrial degeneration by preserving mitochondrial integrity

Bcl-2 prevents loss of mitochondria in CCCP-induced apoptosis.

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de Graaf, Aniek O; van den Heuvel, Lambert P; Dijkman, Henry B P M; de Abreu, Ronney A; Birkenkamp, Kim U; de Witte, Theo; van der Reijden, Bert A; Smeitink, Jan A M; Jansen, Joop H

2004-10-01

Bcl-2 family proteins regulate apoptosis at the level of mitochondria. To examine the mechanism of Bcl-2 function, we investigated the effects of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) on two hematopoietic cell lines and Bcl-2 overexpressing transfectants. CCCP directly interferes with mitochondrial function and induces apoptosis. We show that Bcl-2 inhibits apoptosis and that the antiapoptotic effect of Bcl-2 takes place upstream of caspase activation and nuclear changes associated with apoptosis, since these were markedly inhibited in cells overexpressing Bcl-2. Bcl-2 does not prevent the decrease in mitochondrial membrane potential nor the alterations in cellular ATP content induced by CCCP in FL5.12 and Jurkat cells. A higher number of mitochondria was observed in untreated Bcl-2 transfected cells compared to parental cells, as shown by electron microscopy. Exposure to CCCP induced a dramatic decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, with apparent swelling and loss of cristae in parental cells. Bcl-2 clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. These data suggest that CCCP induces apoptosis by structural disruption of mitochondria and that Bcl-2 prevents apoptosis and mitochondrial degeneration by preserving mitochondrial integrity.

Deuterium-depleted water

International Nuclear Information System (INIS)

Stefanescu, Ion; Steflea, Dumitru; Saros-Rogobete, Irina; Titescu, Gheorghe; Tamaian, Radu

2001-01-01

Deuterium-depleted water represents water that has an isotopic content smaller than 145 ppm D/(D+H) which is the natural isotopic content of water. Deuterium depleted water is produced by vacuum distillation in columns equipped with structured packing made from phosphor bronze or stainless steel. Deuterium-depleted water, the production technique and structured packing are patents of National Institute of Research - Development for Cryogenics and Isotopic Technologies at Rm. Valcea. Researches made in the last few years showed the deuterium-depleted water is a biological active product that could have many applications in medicine and agriculture. (authors)

Psychological Stress and Mitochondria: A Systematic Review.

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Picard, Martin; McEwen, Bruce S

Mitochondria are multifunctional life-sustaining organelles that represent a potential intersection point between psychosocial experiences and biological stress responses. This article provides a systematic review of the effects of psychological stress on mitochondrial structure and function. A systematic review of the literature investigating the effects of psychological stress on mitochondrial function was conducted. The review focused on experimentally controlled studies allowing us to draw causal inference about the effect of induced psychological stress on mitochondria. A total of 23 studies met the inclusion criteria. All studies involved male laboratory animals, and most demonstrated that acute and chronic stressors influenced specific facets of mitochondrial function, particularly within the brain. Nineteen studies showed significant adverse effects of psychological stress on mitochondria and four found increases in function or size after stress. In humans, only six observational studies were available, none with experimental designs, and most only measured biological markers that do not directly reflect mitochondrial function, such as mitochondrial DNA copy number. Overall, evidence supports the notion that acute and chronic stressors influence various aspects of mitochondrial biology, and that chronic stress exposure can lead to molecular and functional recalibrations among mitochondria. Limitations of current animal and human studies are discussed. Maladaptive mitochondrial changes that characterize this subcellular state of stress are termed mitochondrial allostatic load. Prospective studies with sensitive measures of specific mitochondrial outcomes will be needed to establish the link between psychosocial stressors, emotional states, the resulting neuroendocrine and immune processes, and mitochondrial energetics relevant to mind-body research in humans.

Effects of Various Kynurenine Metabolites on Respiratory Parameters of Rat Brain, Liver and Heart Mitochondria

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Halina Baran*

2016-01-01

Full Text Available Previously, we demonstrated that the endogenous glutamate receptor antagonist kynurenic acid dose-dependently and significantly affected rat heart mitochondria. Now we have investigated the effects of L-tryptophan, L-kynurenine, 3-hydroxykynurenine and kynurenic, anthranilic, 3-hydroxyanthranilic, xanthurenic and quinolinic acids on respiratory parameters (ie, state 2, state 3, respiratory control index (RC and ADP/oxygen ratio in brain, liver and heart mitochondria of adult rats. Mitochondria were incubated with glutamate/malate (5 mM or succinate (10 mM and in the presence of L-tryptophan metabolites (1 mM or in the absence, as control. Kynurenic and anthranilic acids significantly reduced RC values of heart mitochondria in the presence of glutamate/malate. Xanthurenic acid significantly reduced RC values of brain mitochondria in the presence of glutamate/malate. Furthermore, 3-hydroxykynurenine and 3-hydroxyanthranilic acid decreased RC values of brain, liver and heart mitochondria using glutamate/malate. In the presence of succinate, 3-hydroxykynurenine and 3-hydroxyanthranilic acid affected RC values of brain mitochondria, whereas in liver and heart mitochondria only 3-hydroxykynurenine lowered RC values significantly. Furthermore, lowered ADP/oxygen ratios were observed in brain mitochondria in the presence of succinate with 3-hydroxykynurenine and 3-hydroxyanthranilic acid, and to a lesser extent with glutamate/malate. In addition, 3-hydroxyanthranilic acid significantly lowered the ADP/oxygen ratio in heart mitochondria exposed to glutamate/malate, while in the liver mitochondria only a mild reduction was found. Tests of the influence of L-tryptophan and its metabolites on complex I in liver mitochondria showed that only 3-hydroxykynurenine, 3-hydroxyanthranilic acid and L-kynurenine led to a significant acceleration of NADH-driven complex I activities. The data indicate that L-tryptophan metabolites had different effects on brain, liver

BioMEMS for mitochondria medicine

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Padmaraj, Divya

A BioMEMS device to study cell-mitochondrial physiological functionalities was developed. The pathogenesis of many diseases including obesity, diabetes and heart failure as well as aging has been linked to functional defects of mitochondria. The synthesis of Adenosine Tri Phosphate (ATP) is determined by the electrical potential across the inner mitochondrial membrane and by the pH difference due to proton flux across it. Therefore, electrical characterization by E-fields with complementary chemical testing was used here. The BioMEMS device was fabricated as an SU-8 based microfluidic system with gold electrodes on SiO2/Si wafers for electromagnetic interrogation. Ion Sensitive Field Effect Transistors (ISFETs) were incorporated for proton studies important in the electron transport chain, together with monitoring Na+, K+ and Ca++ ions for ion channel studies. ISFETs are chemically sensitive Metal Oxide Semiconductor Field Effect Transistor (MOSFET) devices and their threshold voltage is directly proportional to the electrolytic H+ ion variation. These ISFETs (sensitivity ˜55 mV/pH for H+) were further realized as specific ion sensitive Chemical Field Effect Transistors (CHEMFETs) by depositing a specific ion sensitive membrane on the gate. Electrodes for dielectric spectroscopy studies of mitochondria were designed as 2- and 4-probe structures for optimized operation over a wide frequency range. In addition, to limit polarization effects, a 4-electrode set-up with unique meshed pickup electrodes (7.5x7.5 mum2 loops with 4 mum wires) was fabricated. Sensitivity of impedance spectroscopy to membrane potential changes was confirmed by studying the influence of uncouplers and glucose on mitochondria. An electrical model was developed for the mitochondrial sample, and its frequency response correlated with impedance spectroscopy experiments of sarcolemmal mitochondria. Using the mesh electrode structure, we obtained a reduction of 83.28% in impedance at 200 Hz. COMSOL

Mitochondria morphologic changes and metabolic effects of rat hippocampus after microwave irradiation

International Nuclear Information System (INIS)

Zhao Li; Peng Ruiyun; Gao Yabing; Wang Shuiming; Wang Lifeng; Dong Qi; Xu Xinping; Ma Junjie

2007-01-01

Objective: To investigate the effect of microwave on mitochondria morphologic and metabolism of rat hippocampus. Methods: 30 male rats were exposed to microwave with the average power density of 30 mW/cm 2 . Rats were sacrificed at 6 h, 1 d, 3 d and 7 d after irradiation. Electron microscope, enzymatic activity staining and spectrophotometer were used to study ultrastructure change of hippocampus mitochondria and activity of ATPase, SDH and MAO. Mitochondrial ATP, ADP and AMP contents were measured by high performance liquid chromatography (HPLC). Results: At 6 h after microwave radiation, the sizes and shapes of hippocampus mitochondria were abnormal and the injury of mitochondria was aggravated at 1 and 3 d after radiation. The mitochondria presented swell, cavitation including disorder, shortness and decrease of crest. The activity of SDH and content of ATP were decreased at 6 h, most serious at 3 d(P<0.01), and recovered at 7 d after radiation. The activity of ATPase and MAO increased notably at 1 d and 3 d after radiation (P<0.01). Conclusions: Microwave can damage the structure and function of mitochondria in rat hippocampus, and cause the energy metabolism of enzyme disorder. (authors)

Doubly uniparental inheritance of mitochondria as a model system for studying germ line formation.

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Liliana Milani

Full Text Available BACKGROUND: Doubly Uniparental Inheritance (DUI of mitochondria occurs when both mothers and fathers are capable of transmitting mitochondria to their offspring, in contrast to the typical Strictly Maternal Inheritance (SMI. DUI was found in some bivalve molluscs, in which two mitochondrial genomes are inherited, one through eggs, the other through sperm. During male embryo development, spermatozoon mitochondria aggregate in proximity of the first cleavage furrow and end up in the primordial germ cells, while they are dispersed in female embryos. METHODOLOGY/PRINCIPAL FINDINGS: We used MitoTracker, microtubule staining and transmission electron microscopy to examine the mechanisms of this unusual distribution of sperm mitochondria in the DUI species Ruditapes philippinarum. Our results suggest that in male embryos the midbody deriving from the mitotic spindle of the first division concurs in positioning the aggregate of sperm mitochondria. Furthermore, an immunocytochemical analysis showed that the germ line determinant Vasa segregates close to the first cleavage furrow. CONCLUSIONS/SIGNIFICANCE: In DUI male embryos, spermatozoon mitochondria aggregate in a stable area on the animal-vegetal axis: in organisms with spiral segmentation this zone is not involved in cleavage, so the aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area in which also germ plasm is transferred. In 2-blastomere embryos, the segregation of sperm mitochondria in the same region with Vasa suggests their contribution in male germ line formation. In DUI male embryos, M-type mitochondria must be recognized by egg factors to be actively transferred in the germ line, where they become dominant replacing the Balbiani body mitochondria. The typical features of germ line assembly point to a common biological mechanism shared by DUI and SMI organisms. Although the molecular dynamics of the segregation of sperm mitochondria in DUI species are unknown

SEGMENTATION OF MITOCHONDRIA IN ELECTRON MICROSCOPY IMAGES USING ALGEBRAIC CURVES.

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Seyedhosseini, Mojtaba; Ellisman, Mark H; Tasdizen, Tolga

2013-01-01

High-resolution microscopy techniques have been used to generate large volumes of data with enough details for understanding the complex structure of the nervous system. However, automatic techniques are required to segment cells and intracellular structures in these multi-terabyte datasets and make anatomical analysis possible on a large scale. We propose a fully automated method that exploits both shape information and regional statistics to segment irregularly shaped intracellular structures such as mitochondria in electron microscopy (EM) images. The main idea is to use algebraic curves to extract shape features together with texture features from image patches. Then, these powerful features are used to learn a random forest classifier, which can predict mitochondria locations precisely. Finally, the algebraic curves together with regional information are used to segment the mitochondria at the predicted locations. We demonstrate that our method outperforms the state-of-the-art algorithms in segmentation of mitochondria in EM images.

Mitochondria mediate septin cage assembly to promote autophagy of Shigella.

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Sirianni, Andrea; Krokowski, Sina; Lobato-Márquez, Damián; Buranyi, Stephen; Pfanzelter, Julia; Galea, Dieter; Willis, Alexandra; Culley, Siân; Henriques, Ricardo; Larrouy-Maumus, Gerald; Hollinshead, Michael; Sancho-Shimizu, Vanessa; Way, Michael; Mostowy, Serge

2016-07-01

Septins, cytoskeletal proteins with well-characterised roles in cytokinesis, form cage-like structures around cytosolic Shigella flexneri and promote their targeting to autophagosomes. However, the processes underlying septin cage assembly, and whether they influence S. flexneri proliferation, remain to be established. Using single-cell analysis, we show that the septin cages inhibit S. flexneri proliferation. To study mechanisms of septin cage assembly, we used proteomics and found mitochondrial proteins associate with septins in S. flexneri-infected cells. Strikingly, mitochondria associated with S. flexneri promote septin assembly into cages that entrap bacteria for autophagy. We demonstrate that the cytosolic GTPase dynamin-related protein 1 (Drp1) interacts with septins to enhance mitochondrial fission. To avoid autophagy, actin-polymerising Shigella fragment mitochondria to escape from septin caging. Our results demonstrate a role for mitochondria in anti-Shigella autophagy and uncover a fundamental link between septin assembly and mitochondria. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

The Fab1/PIKfyve Phosphoinositide Phosphate Kinase Is Not Necessary to Maintain the pH of Lysosomes and of the Yeast Vacuole*

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Ho, Cheuk Y.; Choy, Christopher H.; Wattson, Christina A.; Johnson, Danielle E.; Botelho, Roberto J.

2015-01-01

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH lysosomes. PMID:25713145

Atorvastatin affects negatively respiratory function of isolated endothelial mitochondria.

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Broniarek, Izabela; Jarmuszkiewicz, Wieslawa

2018-01-01

The purpose of this research was to elucidate the direct effects of two popular blood cholesterol-lowering drugs used to treat cardiovascular diseases, atorvastatin and pravastatin, on respiratory function, membrane potential, and reactive oxygen species formation in mitochondria isolated from human umbilical vein endothelial cells (EA.hy926 cell line). Hydrophilic pravastatin did not significantly affect endothelial mitochondria function. In contrast, hydrophobic calcium-containing atorvastatin induced a loss of outer mitochondrial membrane integrity, an increase in hydrogen peroxide formation, and reductions in maximal (phosphorylating or uncoupled) respiratory rate, membrane potential and oxidative phosphorylation efficiency. The atorvastatin-induced changes indicate an impairment of mitochondrial function at the level of ATP synthesis and at the level of the respiratory chain, likely at complex I and complex III. The atorvastatin action on endothelial mitochondria was highly dependent on calcium ions and led to a disturbance in mitochondrial calcium homeostasis. Uptake of calcium ions included in atorvastatin molecule induced mitochondrial uncoupling that enhanced the inhibition of the mitochondrial respiratory chain by atorvastatin. Our results indicate that hydrophobic calcium-containing atorvastatin, widely used as anti-atherosclerotic agent, has a direct negative action on isolated endothelial mitochondria. Copyright © 2017. Published by Elsevier Inc.

Ego depletion in visual perception: Ego-depleted viewers experience less ambiguous figure reversal.

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Wimmer, Marina C; Stirk, Steven; Hancock, Peter J B

2017-10-01

This study examined the effects of ego depletion on ambiguous figure perception. Adults (N = 315) received an ego depletion task and were subsequently tested on their inhibitory control abilities that were indexed by the Stroop task (Experiment 1) and their ability to perceive both interpretations of ambiguous figures that was indexed by reversal (Experiment 2). Ego depletion had a very small effect on reducing inhibitory control (Cohen's d = .15) (Experiment 1). Ego-depleted participants had a tendency to take longer to respond in Stroop trials. In Experiment 2, ego depletion had small to medium effects on the experience of reversal. Ego-depleted viewers tended to take longer to reverse ambiguous figures (duration to first reversal) when naïve of the ambiguity and experienced less reversal both when naïve and informed of the ambiguity. Together, findings suggest that ego depletion has small effects on inhibitory control and small to medium effects on bottom-up and top-down perceptual processes. The depletion of cognitive resources can reduce our visual perceptual experience.

Yeast Flocculation—Sedimentation and Flotation

Directory of Open Access Journals (Sweden)

Graham G. Stewart

2018-04-01

Full Text Available Unlike most fermentation alcohol beverage production processes, brewers recycle their yeast. This is achieved by employing a yeast culture’s: flocculation, adhesion, sedimentation, flotation, and cropping characteristics. As a consequence of yeast recycling, the quality of the cropped yeast culture’s characteristics is critical. However, the other major function of brewer’s yeast is to metabolise wort into ethanol, carbon dioxide, glycerol, and other fermentation products, many of which contribute to beer’s overall flavour characteristics. This review will only focus on brewer’s yeast flocculation characteristics.

Yeast cell differentiation: Lessons from pathogenic and non-pathogenic yeasts.

Science.gov (United States)

Palková, Zdena; Váchová, Libuše

2016-09-01

Yeasts, historically considered to be single-cell organisms, are able to activate different differentiation processes. Individual yeast cells can change their life-styles by processes of phenotypic switching such as the switch from yeast-shaped cells to filamentous cells (pseudohyphae or true hyphae) and the transition among opaque, white and gray cell-types. Yeasts can also create organized multicellular structures such as colonies and biofilms, and the latter are often observed as contaminants on surfaces in industry and medical care and are formed during infections of the human body. Multicellular structures are formed mostly of stationary-phase or slow-growing cells that diversify into specific cell subpopulations that have unique metabolic properties and can fulfill specific tasks. In addition to the development of multiple protective mechanisms, processes of metabolic reprogramming that reflect a changed environment help differentiated individual cells and/or community cell constituents to survive harmful environmental attacks and/or to escape the host immune system. This review aims to provide an overview of differentiation processes so far identified in individual yeast cells as well as in multicellular communities of yeast pathogens of the Candida and Cryptococcus spp. and the Candida albicans close relative, Saccharomyces cerevisiae. Molecular mechanisms and extracellular signals potentially involved in differentiation processes are also briefly mentioned. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mature Erythrocytes of Iguana iguana (Squamata, Iguanidae Possess Functional Mitochondria.

Directory of Open Access Journals (Sweden)

Giuseppina Di Giacomo

Full Text Available Electron microscopy analyses of Iguana iguana blood preparations revealed the presence of mitochondria within erythrocytes with well-structured cristae. Fluorescence microscopy analyses upon incubation with phalloidin-FITC, Hoechst 33342 and mitochondrial transmembrane potential (Δψm-sensitive probe MitoTracker Red indicated that mitochondria i widely occur in erythrocytes, ii are polarized, and iii seem to be preferentially confined at a "perinuclear" region, as confirmed by electron microscopy. The analysis of NADH-dependent oxygen consumption showed that red blood cells retain the capability to consume oxygen, thereby providing compelling evidence that mitochondria of Iguana erythrocytes are functional and capable to perform oxidative phosphorylation.

L-arabinose fermenting yeast

Science.gov (United States)

Zhang, Min; Singh, Arjun; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric; Suominen, Pirkko

2010-12-07

An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. Methods of producing ethanol include utilizing these modified yeast strains. ##STR00001##

Gene introduction into the mitochondria of Arabidopsis thaliana via peptide-based carriers

Science.gov (United States)

Chuah, Jo-Ann; Yoshizumi, Takeshi; Kodama, Yutaka; Numata, Keiji

2015-01-01

Available methods in plant genetic transformation are nuclear and plastid transformations because similar procedures have not yet been established for the mitochondria. The double membrane and small size of the organelle, in addition to its large population in cells, are major obstacles in mitochondrial transfection. Here we report the intracellular delivery of exogenous DNA localized to the mitochondria of Arabidopsis thaliana using a combination of mitochondria-targeting peptide and cell-penetrating peptide. Low concentrations of peptides were sufficient to deliver DNA into the mitochondria and expression of imported DNA reached detectable levels within a short incubation period (12 h). We found that electrostatic interaction with the cell membrane is not a critical factor for complex internalization, instead, improved intracellular penetration of mitochondria-targeted complexes significantly enhanced gene transfer efficiency. Our results delineate a simple and effective peptide-based method, as a starting point for the development of more sophisticated plant mitochondrial transfection strategies.

A YEAST SPECIFIC INSERTION AMIDST OBG FOLD IS CRITICAL FOR THE MITOCHONDRIAL FUNCTION OF Mtg2p IN SACCHAROMYCES CEREVISIAE

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Upasana Mehra

2017-06-01

Full Text Available Protein expression in mitochondria is carried out by ribosomes that are distinct from their cytosolic counterpart. Mitochondrial ribosomes are made of individual proteins having distinct lineages: those with clear bacterial orthologues, those conserved in eukaryotes only and proteins that are species specific. MTG2 is the mitochondrial member of the universally conserved Obg family of GTPases in Saccharomyces cerevisiae which associates with and regulates mitochondrial large ribosomal subunit assembly. In this study we demonstrate that MTG2, in addition to the universally conserved OBG and GTPase domains, has an essential yeast specific insertion domain positioned within the N terminal OBG fold. Cells expressing mtg2∆201-294, deleted for the insertion domain are not able to support cellular respiration. In addition, we show that large stretches of amino acids can be inserted into MTG2 at the end of the yeast specific insertion domain and the OBG fold without perturbing its cellular functions, consistent with the insertion domain folding into a species specific protein binding platform.

Nitrile Metabolizing Yeasts

Science.gov (United States)

Bhalla, Tek Chand; Sharma, Monica; Sharma, Nitya Nand

Nitriles and amides are widely distributed in the biotic and abiotic components of our ecosystem. Nitrile form an important group of organic compounds which find their applications in the synthesis of a large number of compounds used as/in pharmaceutical, cosmetics, plastics, dyes, etc>. Nitriles are mainly hydro-lyzed to corresponding amide/acid in organic chemistry. Industrial and agricultural activities have also lead to release of nitriles and amides into the environment and some of them pose threat to human health. Biocatalysis and biotransformations are increasingly replacing chemical routes of synthesis in organic chemistry as a part of ‘green chemistry’. Nitrile metabolizing organisms or enzymes thus has assumed greater significance in all these years to convert nitriles to amides/ acids. The nitrile metabolizing enzymes are widely present in bacteria, fungi and yeasts. Yeasts metabolize nitriles through nitrilase and/or nitrile hydratase and amidase enzymes. Only few yeasts have been reported to possess aldoxime dehydratase. More than sixty nitrile metabolizing yeast strains have been hither to isolated from cyanide treatment bioreactor, fermented foods and soil. Most of the yeasts contain nitrile hydratase-amidase system for metabolizing nitriles. Transformations of nitriles to amides/acids have been carried out with free and immobilized yeast cells. The nitrilases of Torulopsis candida>and Exophiala oligosperma>R1 are enantioselec-tive and regiospecific respectively. Geotrichum>sp. JR1 grows in the presence of 2M acetonitrile and may have potential for application in bioremediation of nitrile contaminated soil/water. The nitrilase of E. oligosperma>R1 being active at low pH (3-6) has shown promise for the hydroxy acids. Immobilized yeast cells hydrolyze some additional nitriles in comparison to free cells. It is expected that more focus in future will be on purification, characterization, cloning, expression and immobilization of nitrile metabolizing

Defining the Architecture of the Core Machinery for the Assembly of Fe-S Clusters in Human Mitochondria.

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Gakh, Oleksandr; Ranatunga, Wasantha; Galeano, Belinda K; Smith, Douglas S; Thompson, James R; Isaya, Grazia

2017-01-01

Although Fe-S clusters may assemble spontaneously from elemental iron and sulfur in protein-free systems, the potential toxicity of free Fe 2+ , Fe 3+ , and S 2- ions in aerobic environments underscores the requirement for specialized proteins to oversee the safe assembly of Fe-S clusters in living cells. Prokaryotes first developed multiprotein systems for Fe-S cluster assembly, from which mitochondria later derived their own system and became the main Fe-S cluster suppliers for eukaryotic cells. Early studies in yeast and human mitochondria indicated that Fe-S cluster assembly in eukaryotes is centered around highly conserved Fe-S proteins (human ISCU) that serve as scaffolds upon which new Fe-S clusters are assembled from (i) elemental sulfur, provided by a pyridoxal phosphate-dependent cysteine desulfurase (human NFS1) and its stabilizing-binding partner (human ISD11), and (ii) elemental iron, provided by an iron-binding protein of the frataxin family (human FXN). Further studies revealed that all of these proteins could form stable complexes that could reach molecular masses of megadaltons. However, the protein-protein interaction surfaces, catalytic mechanisms, and overall architecture of these macromolecular machines remained undefined for quite some time. The delay was due to difficulties inherent in reconstituting these very large multiprotein complexes in vitro or isolating them from cells in sufficient quantities to enable biochemical and structural studies. Here, we describe approaches we developed to reconstitute the human Fe-S cluster assembly machinery in Escherichia coli and to define its remarkable architecture. © 2017 Elsevier Inc. All rights reserved.

Doxorubicin Action on Mitochondria: Relevance to Osteosarcoma Therapy?

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Armstrong, Jo; Dass, Crispin R

2018-01-01

The mitochondria may very well determine the final commitment of the cell to death, particularly in times of energy stress. Cancer chemotherapeutics such as the anthracycline doxorubicin perturb mitochondrial structure and function in tumour cells, as evidenced in osteosarcoma, for which doxorubicin is used clinically as frontline therapy. This same mechanism of cell inhibition is also pertinent to doxorubicin's primary cause of side-effects, that to the cardiac tissue, culminating in such dire events as congestive heart failure. Reactive oxygen species are partly to blame for this effect on the mitochondria, which impact the electron transport chain. As this review highlights that, there is much more to be learnt about the mitochondria and how it is affected by such effective but toxic drugs as doxorubicin. Such information will aid researchers who search for cancer treatment able to preserve mitochondrial number and function in normal cells. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

“When the going gets tough, who keeps going?” Depletion sensitivity moderates the ego-depletion effect

Science.gov (United States)

Salmon, Stefanie J.; Adriaanse, Marieke A.; De Vet, Emely; Fennis, Bob M.; De Ridder, Denise T. D.

2014-01-01

Self-control relies on a limited resource that can get depleted, a phenomenon that has been labeled ego-depletion. We argue that individuals may differ in their sensitivity to depleting tasks, and that consequently some people deplete their self-control resource at a faster rate than others. In three studies, we assessed individual differences in depletion sensitivity, and demonstrate that depletion sensitivity moderates ego-depletion effects. The Depletion Sensitivity Scale (DSS) was employed to assess depletion sensitivity. Study 1 employs the DSS to demonstrate that individual differences in sensitivity to ego-depletion exist. Study 2 shows moderate correlations of depletion sensitivity with related self-control concepts, indicating that these scales measure conceptually distinct constructs. Study 3 demonstrates that depletion sensitivity moderates the ego-depletion effect. Specifically, participants who are sensitive to depletion performed worse on a second self-control task, indicating a stronger ego-depletion effect, compared to participants less sensitive to depletion. PMID:25009523

PEGylated anticancer-carbon nanotubes complex targeting mitochondria of lung cancer cells

Science.gov (United States)

Kim, Sang-Woo; Lee, Yeon Kyung; Lee, Jong Yeon; Hong, Jeong Hee; Khang, Dongwoo

2017-11-01

Although activating apoptosis in cancer cells by targeting the mitochondria is an effective strategy for cancer therapy, insufficient targeting of the mitochondria in cancer cells restricts the availability in clinical treatment. Here, we report on a polyethylene glycol-coated carbon nanotube (CNT)-ABT737 nanodrug that improves the mitochondrial targeting of lung cancer cells. The polyethylene glycol-coated CNT-ABT737 nanodrug internalized into the early endosomes via macropinocytosis and clathrin-mediated endocytosis in advance of early endosomal escape and delivered into the mitochondria. Cytosol release of the nanodrug led to apoptosis of lung cancer cells by abruption of the mitochondrial membrane potential, inducing Bcl-2-mediated apoptosis and generating intracellular reactive oxygen species. As such, this study provides an effective strategy for increasing the anti-lung cancer efficacy by increasing mitochondria accumulation rate of cytosol released anticancer nanodrugs.

Response of skeletal muscle mitochondria to hypoxia.

Science.gov (United States)

Hoppeler, Hans; Vogt, Michael; Weibel, Ewald R; Flück, Martin

2003-01-01

This review explores the current concepts relating the structural and functional modifications of skeletal muscle mitochondria to the molecular mechanisms activated when organisms are exposed to a hypoxic environment. In contrast to earlier assumptions it is now established that permanent or long-term exposure to severe environmental hypoxia decreases the mitochondrial content of muscle fibres. Oxidative muscle metabolism is shifted towards a higher reliance on carbohydrates as a fuel, and intramyocellular lipid substrate stores are reduced. Moreover, in muscle cells of mountaineers returning from the Himalayas, we find accumulations of lipofuscin, believed to be a mitochondrial degradation product. Low mitochondrial contents are also observed in high-altitude natives such as Sherpas. In these subjects high-altitude performance seems to be improved by better coupling between ATP demand and supply pathways as well as better metabolite homeostasis. The hypoxia-inducible factor 1 (HIF-1) has been identified as a master regulator for the expression of genes involved in the hypoxia response, such as genes coding for glucose transporters, glycolytic enzymes and vascular endothelial growth factor (VEGF). HIF-1 achieves this by binding to hypoxia response elements in the promoter regions of these genes, whereby the increase of HIF-1 in hypoxia is the consequence of a reduced degradation of its dominant subunit HIF-1a. A further mechanism that seems implicated in the hypoxia response of muscle mitochondria is related to the formation of reactive oxygen species (ROS) in mitochondria during oxidative phosphorylation. How exactly ROS interfere with HIF-1a as well as MAP kinase and other signalling pathways is debated. The current evidence suggests that mitochondria themselves could be important players in oxygen sensing.

Drosophila Regulate Yeast Density and Increase Yeast Community Similarity in a Natural Substrate

OpenAIRE

Stamps, Judy A.; Yang, Louie H.; Morales, Vanessa M.; Boundy-Mills, Kyria L.

2012-01-01

Drosophila melanogaster adults and larvae, but especially larvae, had profound effects on the densities and community structure of yeasts that developed in banana fruits. Pieces of fruit exposed to adult female flies previously fed fly-conditioned bananas developed higher yeast densities than pieces of the same fruits that were not exposed to flies, supporting previous suggestions that adult Drosophila vector yeasts to new substrates. However, larvae alone had dramatic effects on yeast densit...

Mitochondria in aging cell differentiation

Czech Academy of Sciences Publication Activity Database

Palková, Zdena; Váchová, Libuše

2016-01-01

Roč. 8, č. 7 (2016), s. 1287-1288 ISSN 1945-4589 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : mitochondria * cell differentiation * retrograde signaling Subject RIV: EE - Microbiology, Virology Impact factor: 4.867, year: 2016

An epigrammatic (abridged) recounting of the myriad tales of astonishing deeds and dire consequences pertaining to nitric oxide and reactive oxygen species in mitochondria with an ancillary missive concerning the origins of apoptosis

International Nuclear Information System (INIS)

Heck, Diane E.; Kagan, Valerian E.; Shvedova, Anna A.; Laskin, Jeffrey D.

2005-01-01

Mitochondria play a central role in the life and death of cells. These organelles serve as the major energy-producing powerhouse, whereby the generation of ATP is associated with the utilization of molecular oxygen. A significant fraction (2-3%) of molecular oxygen consumed by mitochondria may be reduced in a one-electron fashion to yield a series of reactive oxygen species (ROS) such as superoxide anion radical, hydrogen peroxide, and hydroxyl radical. ROS are capable of damaging components of the electron transport apparatus and can, in turn, disrupt mitochondrial functioning, limiting cellular ATP levels and ultimately resulting in cell death. ROS-induced disruption of electron transport can perpetuate production of deleterious ROS and propagate mitochondrial damage. Consequently, mitochondria are highly enriched with water-soluble and lipid-soluble antioxidants (glutathione, ascorbate, Vitamin E, and coenzyme Q) and antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase, catalase, thioredoxins, and peroxiredoxin. Another important antioxidant acting as a very effective scavenger of reactive lipid (peroxyl, alkoxyl) radicals is nitric oxide (NO) generated by mitochondrial nitric oxide synthase. However, NO can also be very disruptive to mitochondria function, a process facilitated by its high reactivity with superoxide. This interaction results in the formation of peroxynitrite, an oxidant capable of causing oxidative/nitrosative stress, further aggravating mitochondrial dysfunction, causing ATP depletion and damage to cells. Thus, in the most general sense, the effects of NO in mitochondria may be either protective or deleterious depending on specific conditions of local redox environment (redox potential, ratio of oxidized to reduced glutathione, transition metals, and the presence of other oxygen- and nitrogen-centered radicals)

Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells.

Science.gov (United States)

Tomioku, Kan-Na; Shigekuni, Mikiko; Hayashi, Hiroki; Yoshida, Akane; Futagami, Taiki; Tamaki, Hisanori; Tanabe, Kenji; Fujita, Akikazu

2018-05-01

In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane. Copyright © 2018 Elsevier GmbH. All rights reserved.

[Organization of mitochondria in the growing hyphae of Neurospora crassa].

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Potapova, T V; Boĭtsova, L Iu; Golyshev, S A; Popinako, A V

2013-01-01

In vivo fluorescent labeling of mitochondria in Neurospora crassa showed the concentration of filamentous mitochondria within 30 μm of apex in growing hyphae. These mitochondrial assemblies propagated forward with the elongation of hyphae, split and segregated as the growing tip bifurcated and formed de novo when new branches formed farther away from the apex. The efficiency of the mitochondria concentration in the apical 30 μm zone is related to the growth rate and identical in hyphae cultivated in glucose- and sorbitol-containing media. The obtained data are discussed in connection with the behavior of microtubules in growing hyphae as well as with the electric heterogeneity of N. crassa hyphal apex described previously.

RECQL4 localizes to mitochondria and preserves mitochondrial DNA integrity

DEFF Research Database (Denmark)

Croteau, Deborah L; Rossi, Marie L; Canugovi, Chandrika

2012-01-01

in premature aging. There is no information about whether any of the RecQ helicases play roles in mitochondrial biogenesis, which is strongly implicated in the aging process. Here, we used microscopy to visualize RECQL4 in mitochondria. Fractionation of human and mouse cells also showed that RECQL4 was present...... in mitochondria. Q-PCR amplification of mitochondrial DNA demonstrated that mtDNA damage accumulated in RECQL4-deficient cells. Microarray analysis suggested that mitochondrial bioenergetic pathways might be affected in RTS. Measurements of mitochondrial bioenergetics showed a reduction in the mitochondrial......Q helicase to be found in both human and mouse mitochondria, and the loss of RECQL4 alters mitochondrial integrity....

A novel nuclear genetic code alteration in yeasts and the evolution of codon reassignment in eukaryotes.

Science.gov (United States)

Mühlhausen, Stefanie; Findeisen, Peggy; Plessmann, Uwe; Urlaub, Henning; Kollmar, Martin

2016-07-01

The genetic code is the cellular translation table for the conversion of nucleotide sequences into amino acid sequences. Changes to the meaning of sense codons would introduce errors into almost every translated message and are expected to be highly detrimental. However, reassignment of single or multiple codons in mitochondria and nuclear genomes, although extremely rare, demonstrates that the code can evolve. Several models for the mechanism of alteration of nuclear genetic codes have been proposed (including "codon capture," "genome streamlining," and "ambiguous intermediate" theories), but with little resolution. Here, we report a novel sense codon reassignment in Pachysolen tannophilus, a yeast related to the Pichiaceae. By generating proteomics data and using tRNA sequence comparisons, we show that Pachysolen translates CUG codons as alanine and not as the more usual leucine. The Pachysolen tRNACAG is an anticodon-mutated tRNA(Ala) containing all major alanine tRNA recognition sites. The polyphyly of the CUG-decoding tRNAs in yeasts is best explained by a tRNA loss driven codon reassignment mechanism. Loss of the CUG-tRNA in the ancient yeast is followed by gradual decrease of respective codons and subsequent codon capture by tRNAs whose anticodon is not part of the aminoacyl-tRNA synthetase recognition region. Our hypothesis applies to all nuclear genetic code alterations and provides several testable predictions. We anticipate more codon reassignments to be uncovered in existing and upcoming genome projects. © 2016 Mühlhausen et al.; Published by Cold Spring Harbor Laboratory Press.

Rejuvenating cellular respiration for optimizing respiratory function: targeting mitochondria.

Science.gov (United States)

Agrawal, Anurag; Mabalirajan, Ulaganathan

2016-01-15

Altered bioenergetics with increased mitochondrial reactive oxygen species production and degradation of epithelial function are key aspects of pathogenesis in asthma and chronic obstructive pulmonary disease (COPD). This motif is not unique to obstructive airway disease, reported in related airway diseases such as bronchopulmonary dysplasia and parenchymal diseases such as pulmonary fibrosis. Similarly, mitochondrial dysfunction in vascular endothelium or skeletal muscles contributes to the development of pulmonary hypertension and systemic manifestations of lung disease. In experimental models of COPD or asthma, the use of mitochondria-targeted antioxidants, such as MitoQ, has substantially improved mitochondrial health and restored respiratory function. Modulation of noncoding RNA or protein regulators of mitochondrial biogenesis, dynamics, or degradation has been found to be effective in models of fibrosis, emphysema, asthma, and pulmonary hypertension. Transfer of healthy mitochondria to epithelial cells has been associated with remarkable therapeutic efficacy in models of acute lung injury and asthma. Together, these form a 3R model--repair, reprogramming, and replacement--for mitochondria-targeted therapies in lung disease. This review highlights the key role of mitochondrial function in lung health and disease, with a focus on asthma and COPD, and provides an overview of mitochondria-targeted strategies for rejuvenating cellular respiration and optimizing respiratory function in lung diseases. Copyright © 2016 the American Physiological Society.

The Fab1/PIKfyve phosphoinositide phosphate kinase is not necessary to maintain the pH of lysosomes and of the yeast vacuole.

Science.gov (United States)

Ho, Cheuk Y; Choy, Christopher H; Wattson, Christina A; Johnson, Danielle E; Botelho, Roberto J

2015-04-10

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH lysosomes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

New yeasts-new brews: modern approaches to brewing yeast design and development.

Science.gov (United States)

Gibson, B; Geertman, J-M A; Hittinger, C T; Krogerus, K; Libkind, D; Louis, E J; Magalhães, F; Sampaio, J P

2017-06-01

The brewing industry is experiencing a period of change and experimentation largely driven by customer demand for product diversity. This has coincided with a greater appreciation of the role of yeast in determining the character of beer and the widespread availability of powerful tools for yeast research. Genome analysis in particular has helped clarify the processes leading to domestication of brewing yeast and has identified domestication signatures that may be exploited for further yeast development. The functional properties of non-conventional yeast (both Saccharomyces and non-Saccharomyces) are being assessed with a view to creating beers with new flavours as well as producing flavoursome non-alcoholic beers. The discovery of the psychrotolerant S. eubayanus has stimulated research on de novo S. cerevisiae × S. eubayanus hybrids for low-temperature lager brewing and has led to renewed interest in the functional importance of hybrid organisms and the mechanisms that determine hybrid genome function and stability. The greater diversity of yeast that can be applied in brewing, along with an improved understanding of yeasts' evolutionary history and biology, is expected to have a significant and direct impact on the brewing industry, with potential for improved brewing efficiency, product diversity and, above all, customer satisfaction. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Vaginal yeast infection

Science.gov (United States)

Yeast infection - vagina; Vaginal candidiasis; Monilial vaginitis ... Most women have a vaginal yeast infection at some time. Candida albicans is a common type of fungus. It is often found in small amounts ...

Human cultured cells are capable to incorporate isolated plant mitochondria loaded with exogenous DNA

Directory of Open Access Journals (Sweden)

Laktionov P. P.

2012-07-01

Full Text Available Aim. To investigate the possibility of human cultured cells to incorporate isolated mitochondria together with exogenous DNA introduced into organelles. Methods. Two approaches were used for this purpose, fluorescent labelling of mitochondria and/or DNA with subsequent analysis of the cells subjected to incubation by microscopy or by quantitative PCR. Results. We have shown that human cultured cells lines, HeLa and HUVEC, are capable to uptake isolated plant mitochondria and that this process depends on the incubation time and concentration of organelles present in medium. The incorporated mitochondria can serve as vehicles to deliver exogenous DNA into human cells, this DNA is then distributed in different cell compartments. Conclusions. These results are preliminary and need further investigations, including testing the possibility of human cells to incorporate the mitochondria of human or animal origin and creating genetic construction which could provide certain selectivity or stability of the transferred exogenous DNA upon cell uptake of the mitochondria as vectors.

Regulation of the Stress-Activated Degradation of Mitochondrial Respiratory Complexes in Yeast

Directory of Open Access Journals (Sweden)

Alba Timón-Gómez

2018-01-01

Full Text Available Repair and removal of damaged mitochondria is a key process for eukaryotic cell homeostasis. Here we investigate in the yeast model how different protein complexes of the mitochondrial electron transport chain are subject to specific degradation upon high respiration load and organelle damage. We find that the turnover of subunits of the electron transport complex I equivalent and complex III is preferentially stimulated upon high respiration rates. Particular mitochondrial proteases, but not mitophagy, are involved in this activated degradation. Further mitochondrial damage by valinomycin treatment of yeast cells triggers the mitophagic removal of the same respiratory complexes. This selective protein degradation depends on the mitochondrial fusion and fission apparatus and the autophagy adaptor protein Atg11, but not on the mitochondrial mitophagy receptor Atg32. Loss of autophagosomal protein function leads to valinomycin sensitivity and an overproduction of reactive oxygen species upon mitochondrial damage. A specific event in this selective turnover of electron transport chain complexes seems to be the association of Atg11 with the mitochondrial network, which can be achieved by overexpression of the Atg11 protein even in the absence of Atg32. Furthermore, the interaction of various Atg11 molecules via the C-terminal coil domain is specifically and rapidly stimulated upon mitochondrial damage and could therefore be an early trigger of selective mitophagy in response to the organelles dysfunction. Our work indicates that autophagic quality control upon mitochondrial damage operates in a selective manner.

Studying anti-oxidative properties of inclusion complexes of α-lipoic acid with γ-cyclodextrin in single living fission yeast by confocal Raman microspectroscopy

Science.gov (United States)

Noothalapati, Hemanth; Ikarashi, Ryo; Iwasaki, Keita; Nishida, Tatsuro; Kaino, Tomohiro; Yoshikiyo, Keisuke; Terao, Keiji; Nakata, Daisuke; Ikuta, Naoko; Ando, Masahiro; Hamaguchi, Hiro-o.; Kawamukai, Makoto; Yamamoto, Tatsuyuki

2018-05-01

α-lipoic acid (ALA) is an essential cofactor for many enzyme complexes in aerobic metabolism, especially in mitochondria of eukaryotic cells where respiration takes place. It also has excellent anti-oxidative properties. The acid has two stereo-isomers, R- and S- lipoic acid (R-LA and S-LA), but only the R-LA has biological significance and is exclusively produced in our body. A mutant strain of fission yeast, Δdps1, cannot synthesize coenzyme Q10, which is essential during yeast respiration, leading to oxidative stress. Therefore, it shows growth delay in the minimal medium. We studied anti-oxidant properties of ALA in its free form and their inclusion complexes with γ-cyclodextrin using this mutant yeast model. Both free forms R- and S-LA as well as 1:1 inclusion complexes with γ-cyclodextrin recovered growth of Δdps1 depending on the concentration and form. However, it has no effect on the growth of wild type fission yeast strain at all. Raman microspectroscopy was employed to understand the anti-oxidant property at the molecular level. A sensitive Raman band at 1602 cm-1 was monitored with and without addition of ALAs. It was found that 0.5 mM and 1.0 mM concentrations of ALAs had similar effect in both free and inclusion forms. At 2.5 mM ALAs, free forms inhibited the growth while inclusion complexes helped in recovered. 5.0 mM ALA showed inhibitory effect irrespective of form. Our results suggest that the Raman band at 1602 cm-1 is a good measure of oxidative stress in fission yeast.

Potentiality of Yeasts in the Direct Conversion of Starchy Materials to Ethanol and Its Relevance in the New Millennium

Science.gov (United States)

Reddy, L. V. A.; Reddy, O. V. S.; Basappa, S. C.

In recent years, the use of renewable and abundantly available starchy and cellulosic materials for industrial production of ethanol is gaining importance, in view of the fact, that ethanol is one of the most prospective future motor fuels, that can be expected to replace fossil fuels, which are fast depleting in the world scenario. Although, the starch and the starchy substrates could be converted successfully to ethanol on industrial scales by the use of commercial amylolytic enzymes and yeast fermentation, the cost of production is rather very high. This is mainly due to the non-enzymatic and enzymatic conversion (gelatinization, liquefaction and saccharification) of starch to sugars, which costs around 20 % of the cost of production of ethanol from starch. In this context, the use of amylolytic yeasts, that can directly convert starch to ethanol by a single step, are potentially suited to reduce the cost of production of ethanol from starch. Research advances made in this direction have shown encouraging results, both in terms of identifying the potentially suited yeasts for the purpose and also their economic ethanol yields. This chapter focuses on the types of starch and starchy substrates and their digestion to fermentable sugars, optimization of fermentation conditions to ethanol from starch, factors that affect starch fermentation, potential amylolytic yeasts which can directly convert starch to ethanol, genetic improvement of these yeasts for better conversion efficiency and their future economic prospects in the new millennium.

Calculation of ion currents across the inner membrane of functionally intact mitochondria

Science.gov (United States)

Kane, Daniel A; Pavlov, Evgeny V

2013-01-01

Mitochondrial ion transport systems play a central role in cell physiology. Rates of Ca2+ and K+ transport across the inner mitochondrial membrane have been derived from the measurement of ion accumulation over time within functional isolated mitochondria or mitochondria of cultured cells. Alternatively, the electrical currents generated by ionic flux have been directly measured in purified and swollen mitochondrial samples (mitoplasts) or reconstituted channels, and typically range from 1 pA to several 100s pA. However, the direct electrophysiological approach necessarily requires extensive processing of the mitochondria prior to measurement, which can only be performed on isolated mitoplasts. To compare rates of mitochondrial ion transport measured in electrophysiological experiments to those measured in intact mitochondria and cells, we converted published rates of mitochondrial ion uptake into units of ionic current. We estimate that for monovalent ions, uptake by intact mitochondria at the rate of 1 nmol ∙ mg−1 protein ∙ min−1 is equivalent to 0.2 fA of current per whole single mitochondrion (0.4 fA for divalent ions). In intact mitochondria, estimated rates of electrogenic cation uptake are limited to 1–100 fA of integral current per single mitochondrion. These estimates are orders of magnitude lower than the currents through mitochondrial channels directly measured via patch-clamp or artificial lipid bilayer approaches. PMID:24037064

Effect of sclerin on amino acid incorporation into mitochondria isolated from rat liver

International Nuclear Information System (INIS)

Yamaguchi, Masanori; Satomura, Yukio

1975-01-01

Though sclerin (SCL) stimulated amino acid incorporation into the protein fraction of post mitochondrial supernatant of rat liver homogenate, it had no effect on the incorporation into the isolated mitochondria at pH 7.2, despite of its stimulating effect on mitochondrial oxidative phosphorylation. SCL stimulated amino acid incorporation into the mitochondria at pH 6.1, and to some extent maintained the activity on that in mitochondria during aging in hypotonic Tris-HCl buffer (pH 7.2). Since SCL prevented leakage of amino acids from the mitochondria into these buffers, it was suggested that SCL may protect a structure of mitochondrial membrane which appeared to have a significance on transport of amino acids. In liver slices, SCL stimulated amino acid incorporation only into the extra-mitochondrial fraction for the first 3 min, but gradually turned to simulate incorporation into mitochondria within 30 min. (auth.)

Effect of sclerin on amino acid incorporation into mitochondria isolated from rat liver

Energy Technology Data Exchange (ETDEWEB)

Yamaguchi, M; Satomura, Y [Osaka City Univ. (Japan). Faculty of Science

1975-08-01

Though sclerin (SCL) stimulated amino acid incorporation into the protein fraction of post mitochondrial supernatant of rat liver homogenate, it had no effect on the incorporation into the isolated mitochondria at pH 7.2, despite of its stimulating effect on mitochondrial oxidative phosphorylation. SCL stimulated amino acid incorporation into the mitochondria at pH 6.1, and to some extent maintained the activity on that in mitochondria during aging in hypotonic Tris-HCl buffer (pH 7.2). Since SCL prevented leakage of amino acids from the mitochondria into these buffers, it was suggested that SCL may protect a structure of mitochondrial membrane which appeared to have a significance on transport of amino acids. In liver slices, SCL stimulated amino acid incorporation only into the extra-mitochondrial fraction for the first 3 min, but gradually turned to simulate incorporation into mitochondria within 30 min.

Schizosaccharomyces japonicus: the fission yeast is a fusion of yeast and hyphae.

Science.gov (United States)

Niki, Hironori

2014-03-01

The clade of Schizosaccharomyces includes 4 species: S. pombe, S. octosporus, S. cryophilus, and S. japonicus. Although all 4 species exhibit unicellular growth with a binary fission mode of cell division, S. japonicus alone is dimorphic yeast, which can transit from unicellular yeast to long filamentous hyphae. Recently it was found that the hyphal cells response to light and then synchronously activate cytokinesis of hyphae. In addition to hyphal growth, S. japonicas has many properties that aren't shared with other fission yeast. Mitosis of S. japonicas is referred to as semi-open mitosis because dynamics of nuclear membrane is an intermediate mode between open mitosis and closed mitosis. Novel genetic tools and the whole genomic sequencing of S. japonicas now provide us with an opportunity for revealing unique characters of the dimorphic yeast. © 2013 The Author. Yeast Published by John Wiley & Sons Ltd.

Fluoroacetylcarnitine: metabolism and metabolic effects in mitochondria

Energy Technology Data Exchange (ETDEWEB)

Bremer, J; Davis, E J

1973-01-01

The metabolism and metabolic effects of fluoroacetylcarnitine have been investigated. Carnitineacetyltransferase transfers the fluoro-acetyl group of fluoroacetylcarnitine nearly as rapidly to CoA as the acetyl group of acetylcarnitine. Fluorocitrate is then formed by citrate synthase, but this second reaction is relatively slow. The fluorocitrate formed intramitochondrially inhibits the metabolism of citrate. In heart and skeletal muscle mitochondria the accumulated citrate inhibits citrate synthesis and the ..beta..-oxidation of fatty acids. Free acetate is formed, presumably because accumulated acetyl-CoA is hydrolyzed. In liver mitochondria the accumulation of citrate leads to a relatively increased rate of ketogenesis. Increased ketogenesis is obtained also upon the addition of citrate to the reaction mixture.

ANGULAR LIGHT-SCATTERING STUDIES ON ISOLATED MITOCHONDRIA

Science.gov (United States)

Gotterer, Gerald S.; Thompson, Thomas E.; Lehninger, Albert L.

1961-01-01

Angular light-scattering studies have been carried out on suspensions of isolated rat liver mitochondria. The angular scatter pattern has a large forward component, typical of large particles. Changes in dissymmetry and in the intensity of light scattered at 90° have been correlated with changes in optical density during the course of mitochondrial swelling and contraction. Such changes can be measured at mitochondrial concentrations much below those required for optical density measurements. Changes in mitochondrial geometry caused by factors "leaking" from mitochondria, not detectable by optical density measurements, have been demonstrated by measuring changes in dissymmetry. Angular light-scattering measurements therefore offer the advantages of increased sensitivity and of added indices of changes in mitochondrial conformation. PMID:19866589

Obesity-exposed oocytes accumulate and transmit damaged mitochondria due to an inability to activate mitophagy.

Science.gov (United States)

Boudoures, Anna L; Saben, Jessica; Drury, Andrea; Scheaffer, Suzanne; Modi, Zeel; Zhang, Wendy; Moley, Kelle H

2017-06-01

Mitochondria are the most prominent organelle in the oocyte. Somatic cells maintain a healthy population of mitochondria by degrading damaged mitochondria via mitophagy, a specialized autophagy pathway. However, evidence from previous work investigating the more general macroautophagy pathway in oocytes suggests that mitophagy may not be active in the oocyte. This would leave the vast numbers of mitochondria - poised to be inherited by the offspring - vulnerable to damage. Here we test the hypothesis that inactive mitophagy in the oocyte underlies maternal transmission of dysfunctional mitochondria. To determine whether oocytes can complete mitophagy, we used either CCCP or AntimycinA to depolarize mitochondria and trigger mitophagy. After depolarization, we did not detect co-localization of mitochondria with autophagosomes and mitochondrial DNA copy number remained unchanged, indicating the non-functional mitochondrial population was not removed. To investigate the impact of an absence of mitophagy in oocytes with damaged mitochondria on offspring mitochondrial function, we utilized in vitro fertilization of high fat high sugar (HF/HS)-exposed oocytes, which have lower mitochondrial membrane potential and damaged mitochondria. Here, we demonstrate that blastocysts generated from HF/HS oocytes have decreased mitochondrial membrane potential, lower metabolites involved in ATP generation, and accumulation of PINK1, a mitophagy marker protein. This mitochondrial phenotype in the blastocyst mirrors the phenotype we show in HF/HS exposed oocytes. Taken together, these data suggest that the mechanisms governing oocyte mitophagy are fundamentally distinct from those governing somatic cell mitophagy and that the absence of mitophagy in the setting of HF/HS exposure contributes to the oocyte-to-blastocyst transmission of dysfunctional mitochondria. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Multi-domain CGFS-type glutaredoxin Grx4 regulates iron homeostasis via direct interaction with a repressor Fep1 in fission yeast

Energy Technology Data Exchange (ETDEWEB)

Kim, Kyoung-Dong; Kim, Hyo-Jin; Lee, Kyung-Chang [Laboratory of Molecular Microbiology, School of Biological Sciences and Institute of Microbiology, Seoul National University, Seoul 151-742 (Korea, Republic of); Roe, Jung-Hye, E-mail: jhroe@snu.ac.kr [Laboratory of Molecular Microbiology, School of Biological Sciences and Institute of Microbiology, Seoul National University, Seoul 151-742 (Korea, Republic of)

2011-05-20

Research highlights: {yields} Monothiol glutaredoxin Grx4 allows Fep1-mediated de-repression of iron uptake genes at low iron. {yields} Grx4 directly interacts with Fep1 in vivo and in vitro. {yields} The Cys172 in the CGFS motif of Grx4 is necessary for cell proliferation and iron regulation. {yields} The Cys172 of Grx4 is required for normal interaction with Fep1. -- Abstract: The fission yeast Schizosaccharomyces pombe contains two CGFS-type monothiol glutaredoxins, Grx4 and Grx5, which are localized primarily in the nucleus and mitochondria, respectively. We observed involvement of Grx4 in regulating iron-responsive gene expression, which is modulated by a repressor Fep1. Lack of Grx4 caused defects not only in growth but also in the expression of both iron-uptake and iron-utilizing genes regardless of iron availability. In order to unravel how Grx4 is involved in Fep1-mediated regulation, interaction between them was investigated. Co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) revealed that Grx4 physically interacts with Fep1 in vivo. BiFC revealed localized nuclear dots produced by interaction of Grx4 with Fep1. Mutation of cysteine-172 in the CGFS motif to serine (C172S) produced effects similarly observed under Grx4 depletion, such as the loss of iron-dependent gene regulation and the absence of nuclear dots in BiFC analysis. These results suggest that the ability of Grx4 to bind iron, most likely Fe-S cofactor, could be critical in interacting with and modulating the activity of Fep1.

Halo Star Lithium Depletion

International Nuclear Information System (INIS)

Pinsonneault, M. H.; Walker, T. P.; Steigman, G.; Narayanan, Vijay K.

1999-01-01

The depletion of lithium during the pre-main-sequence and main-sequence phases of stellar evolution plays a crucial role in the comparison of the predictions of big bang nucleosynthesis with the abundances observed in halo stars. Previous work has indicated a wide range of possible depletion factors, ranging from minimal in standard (nonrotating) stellar models to as much as an order of magnitude in models that include rotational mixing. Recent progress in the study of the angular momentum evolution of low-mass stars permits the construction of theoretical models capable of reproducing the angular momentum evolution of low-mass open cluster stars. The distribution of initial angular momenta can be inferred from stellar rotation data in young open clusters. In this paper we report on the application of these models to the study of lithium depletion in main-sequence halo stars. A range of initial angular momenta produces a range of lithium depletion factors on the main sequence. Using the distribution of initial conditions inferred from young open clusters leads to a well-defined halo lithium plateau with modest scatter and a small population of outliers. The mass-dependent angular momentum loss law inferred from open cluster studies produces a nearly flat plateau, unlike previous models that exhibited a downward curvature for hotter temperatures in the 7Li-Teff plane. The overall depletion factor for the plateau stars is sensitive primarily to the solar initial angular momentum used in the calibration for the mixing diffusion coefficients. Uncertainties remain in the treatment of the internal angular momentum transport in the models, and the potential impact of these uncertainties on our results is discussed. The 6Li/7Li depletion ratio is also examined. We find that the dispersion in the plateau and the 6Li/7Li depletion ratio scale with the absolute 7Li depletion in the plateau, and we use observational data to set bounds on the 7Li depletion in main-sequence halo

Connecting Mitochondria, Metabolism, and Stem Cell Fate

Science.gov (United States)

Wanet, Anaïs; Arnould, Thierry; Najimi, Mustapha

2015-01-01

As sites of cellular respiration and energy production, mitochondria play a central role in cell metabolism. Cell differentiation is associated with an increase in mitochondrial content and activity and with a metabolic shift toward increased oxidative phosphorylation activity. The opposite occurs during reprogramming of somatic cells into induced pluripotent stem cells. Studies have provided evidence of mitochondrial and metabolic changes during the differentiation of both embryonic and somatic (or adult) stem cells (SSCs), such as hematopoietic stem cells, mesenchymal stem cells, and tissue-specific progenitor cells. We thus propose to consider those mitochondrial and metabolic changes as hallmarks of differentiation processes. We review how mitochondrial biogenesis, dynamics, and function are directly involved in embryonic and SSC differentiation and how metabolic and sensing pathways connect mitochondria and metabolism with cell fate and pluripotency. Understanding the basis of the crosstalk between mitochondria and cell fate is of critical importance, given the promising application of stem cells in regenerative medicine. In addition to the development of novel strategies to improve the in vitro lineage-directed differentiation of stem cells, understanding the molecular basis of this interplay could lead to the identification of novel targets to improve the treatment of degenerative diseases. PMID:26134242

Yeast ecology of Kombucha fermentation.

Science.gov (United States)

Teoh, Ai Leng; Heard, Gillian; Cox, Julian

2004-09-01

Kombucha is a traditional fermentation of sweetened tea, involving a symbiosis of yeast species and acetic acid bacteria. Despite reports of different yeast species being associated with the fermentation, little is known of the quantitative ecology of yeasts in Kombucha. Using oxytetracycline-supplemented malt extract agar, yeasts were isolated from four commercially available Kombucha products and identified using conventional biochemical and physiological tests. During the fermentation of each of the four products, yeasts were enumerated from both the cellulosic pellicle and liquor of the Kombucha. The number and diversity of species varied between products, but included Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii and Zygosaccharomyces bailii. While these yeast species are known to occur in Kombucha, the enumeration of each species present throughout fermentation of each of the four Kombucha cultures demonstrated for the first time the dynamic nature of the yeast ecology. Kombucha fermentation is, in general, initiated by osmotolerant species, succeeded and ultimately dominated by acid-tolerant species.

Mitochondria: An Organelle of Bacterial Origin Controlling Inflammation

Directory of Open Access Journals (Sweden)

Alain Meyer

2018-04-01

Full Text Available Inflammation is a cellular and molecular response to infection and/or tissues injury. While a suited inflammatory response in intensity and time allows for killing pathogens, clearing necrotic tissue, and healing injury; an excessive inflammatory response drives various diseases in which inflammation and tissues damages/stress self-sustain each other. Microbes have been poorly implied in non-resolving inflammation, emphasizing the importance of endogenous regulation of inflammation. Mitochondria have been historically identified as the main source of cellular energy, by coupling the oxidation of fatty acids and pyruvate with the production of high amount of adenosine triphosphate by the electron transport chain. Mitochondria are also the main source of reactive oxygen species. Interestingly, research in the last decade has highlighted that since its integration in eukaryote cells, this organelle of bacterial origin has not only been tolerated by immunity, but has also been placed as a central regulator of cell defense. In intact cells, mitochondria regulate cell responses to critical innate immune receptors engagement. Downstream intracellular signaling pathways interact with mitochondrial proteins and are tuned by mitochondrial functioning. Moreover, upon cell stress or damages, mitochondrial components are released into the cytoplasm or the extra cellular milieu, where they act as danger signals when recognized by innate immune receptors. Finally, by regulating the energetic state of immunological synapse between dendritic cells and lymphocytes, mitochondria regulate the inflammation fate toward immunotolerance or immunogenicity. As dysregulations of these processes have been recently involved in various diseases, the identification of the underlying mechanisms might open new avenues to modulate inflammation.

Yeast genome sequencing:

DEFF Research Database (Denmark)

Piskur, Jure; Langkjær, Rikke Breinhold

2004-01-01

For decades, unicellular yeasts have been general models to help understand the eukaryotic cell and also our own biology. Recently, over a dozen yeast genomes have been sequenced, providing the basis to resolve several complex biological questions. Analysis of the novel sequence data has shown...... of closely related species helps in gene annotation and to answer how many genes there really are within the genomes. Analysis of non-coding regions among closely related species has provided an example of how to determine novel gene regulatory sequences, which were previously difficult to analyse because...... they are short and degenerate and occupy different positions. Comparative genomics helps to understand the origin of yeasts and points out crucial molecular events in yeast evolutionary history, such as whole-genome duplication and horizontal gene transfer(s). In addition, the accumulating sequence data provide...

Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast

International Nuclear Information System (INIS)

Kim, Kyoung-Dong; Chung, Woo-Hyun; Kim, Hyo-Jin; Lee, Kyung-Chang; Roe, Jung-Hye

2010-01-01

Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The Δgrx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1 + gene encoding a putative A-type Fe-S scaffold, in addition to mas5 + and hsc1 + genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2 + , but not isu1 + and ssc1 + , complemented the growth phenotype of Δgrx5 mutant as isa1 + did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.

Ca2+ Transport by Mitochondria from L1210 Mouse Ascites Tumor Cells

Science.gov (United States)

Reynafarje, Baltazar; Lehninger, Albert L.

1973-01-01

Mitochondria isolated from the ascites form of L1210 mouse leukemia cells readily accumulate Ca2+ from the suspending medium and eject H+ during oxidation of succinate in the presence of phosphate and Mg2+, with normal stoichiometry between Ca2+ uptake and electron transport. Ca2+ loads up to 1600 ng-atoms per mg of protein are attained. As is the case in mitochondria from normal tissues, Ca2+ uptake takes precedence over oxidative phosphorylation. However, Ca2+ 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 Ca2+ in stimulation of oxygen uptake is about 3-fold greater than in normal liver mitochondria; moreover, the maximal rate of Ca2+ transport is also considerably higher. Furthermore, when Ca2+ pulses are added to L1210 mitochondria in the absence of phosphate or other permeant anions, much larger amounts of Ca2+ are bound and H+ ejected per atom of oxygen consumed than in the presence of phosphate; up to 7 Ca2+ ions are bound per pair of electrons passing each energy-conserving site of the electron-transport chain. Such “superstoichiometry” of Ca2+ uptake can be accounted for by two distinct types of respiration-dependent interaction of Ca2+ with the L1210 mitochondria. One is the stimulation of oxygen consumption, which is achieved by relatively low concentrations of Ca2+ (Km ≅ 8 μM) and is accompanied by binding of Ca2+ up to 40 ng-atoms per mg of protein. The second process, also dependent on electron transport, is the binding of further Ca2+ from the medium in exchange with previously stored membrane-bound protons, in which the affinity for Ca2+ is much lower (Km ≅ 120 μM). PMID:4515933

"When the going gets tough, who keeps going?" Depletion sensitivity moderates the ego-depletion effect

NARCIS (Netherlands)

Salmon, Stefanie J.; Adriaanse, Marieke A.; De Vet, Emely; Fennis, Bob M.; De Ridder, Denise T D

2014-01-01

Self-control relies on a limited resource that can get depleted, a phenomenon that has been labeled ego-depletion. We argue that individuals may differ in their sensitivity to depleting tasks, and that consequently some people deplete their self-control resource at a faster rate than others. In

"When the going gets tough, who keeps going?" : Depletion sensitivity moderates the ego-depletion effect

NARCIS (Netherlands)

Salmon, Stefanie J.; Adriaanse, Marieke A.; De Vet, Emely; Fennis, Bob M.; De Ridder, Denise T. D.

2014-01-01

Self-control relies on a limited resource that can get depleted, a phenomenon that has been labeled ego-depletion. We argue that individuals may differ in their sensitivity to depleting tasks, and that consequently some people deplete their self-control resource at a faster rate than others. In

RNA Editing in Plant Mitochondria

Science.gov (United States)

Hiesel, Rudolf; Wissinger, Bernd; Schuster, Wolfgang; Brennicke, Axel

1989-12-01

Comparative sequence analysis of genomic and complementary DNA clones from several mitochondrial genes in the higher plant Oenothera revealed nucleotide sequence divergences between the genomic and the messenger RNA-derived sequences. These sequence alterations could be most easily explained by specific post-transcriptional nucleotide modifications. Most of the nucleotide exchanges in coding regions lead to altered codons in the mRNA that specify amino acids better conserved in evolution than those encoded by the genomic DNA. Several instances show that the genomic arginine codon CGG is edited in the mRNA to the tryptophan codon TGG in amino acid positions that are highly conserved as tryptophan in the homologous proteins of other species. This editing suggests that the standard genetic code is used in plant mitochondria and resolves the frequent coincidence of CGG codons and tryptophan in different plant species. The apparently frequent and non-species-specific equivalency of CGG and TGG codons in particular suggests that RNA editing is a common feature of all higher plant mitochondria.

The modality effect of ego depletion: Auditory task modality reduces ego depletion.

Science.gov (United States)

Li, Qiong; Wang, Zhenhong

2016-08-01

An initial act of self-control that impairs subsequent acts of self-control is called ego depletion. The ego depletion phenomenon has been observed consistently. The modality effect refers to the effect of the presentation modality on the processing of stimuli. The modality effect was also robustly found in a large body of research. However, no study to date has examined the modality effects of ego depletion. This issue was addressed in the current study. In Experiment 1, after all participants completed a handgrip task, one group's participants completed a visual attention regulation task and the other group's participants completed an auditory attention regulation task, and then all participants again completed a handgrip task. The ego depletion phenomenon was observed in both the visual and the auditory attention regulation task. Moreover, participants who completed the visual task performed worse on the handgrip task than participants who completed the auditory task, which indicated that there was high ego depletion in the visual task condition. In Experiment 2, participants completed an initial task that either did or did not deplete self-control resources, and then they completed a second visual or auditory attention control task. The results indicated that depleted participants performed better on the auditory attention control task than the visual attention control task. These findings suggest that altering task modality may reduce ego depletion. © 2016 Scandinavian Psychological Associations and John Wiley & Sons Ltd.

NetPhosYeast: prediction of protein phosphorylation sites in yeast

DEFF Research Database (Denmark)

Ingrell, C.R.; Miller, Martin Lee; Jensen, O.N.

2007-01-01

sites compared to those in humans, suggesting the need for an yeast-specific phosphorylation site predictor. NetPhosYeast achieves a correlation coefficient close to 0.75 with a sensitivity of 0.84 and specificity of 0.90 and outperforms existing predictors in the identification of phosphorylation sites...

The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Van Rossom, Sofie; Op de Beeck, Ken; Franssens, Vanessa; Swinnen, Erwin; Schepers, Anne; Ghillebert, Ruben; Caldara, Marina; Van Camp, Guy; Winderickx, Joris

2012-01-01

DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2–6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae

Energy Technology Data Exchange (ETDEWEB)

Van Rossom, Sofie [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium); Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Op de Beeck, Ken [Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Franssens, Vanessa; Swinnen, Erwin [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium); Schepers, Anne [Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Ghillebert, Ruben; Caldara, Marina [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium); Van Camp, Guy [Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Winderickx, Joris, E-mail: guy.vancamp@ua.ac.be, E-mail: joris.winderickx@bio.kuleuven.be [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium)

2012-07-25

DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2–6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

Mitochondria and ageing: role in heart, skeletal muscle and adipose tissue

Science.gov (United States)

Boengler, Kerstin; Kosiol, Maik; Mayr, Manuel; Schulz, Rainer

2017-01-01

Abstract Age is the most important risk factor for most diseases. Mitochondria play a central role in bioenergetics and metabolism. In addition, several lines of evidence indicate the impact of mitochondria in lifespan determination and ageing. The best‐known hypothesis to explain ageing is the free radical theory, which proposes that cells, organs, and organisms age because they accumulate reactive oxygen species (ROS) damage over time. Mitochondria play a central role as the principle source of intracellular ROS, which are mainly formed at the level of complex I and III of the respiratory chain. Dysfunctional mitochondria generating less ATP have been observed in various aged organs. Mitochondrial dysfunction comprises different features including reduced mitochondrial content, altered mitochondrial morphology, reduced activity of the complexes of the electron transport chain, opening of the mitochondrial permeability transition pore, and increased ROS formation. Furthermore, abnormalities in mitochondrial quality control or defects in mitochondrial dynamics have also been linked to senescence. Among the tissues affected by mitochondrial dysfunction are those with a high‐energy demand and thus high mitochondrial content. Therefore, the present review focuses on the impact of mitochondria in the ageing process of heart and skeletal muscle. In this article, we review different aspects of mitochondrial dysfunction and discuss potential therapeutic strategies to improve mitochondrial function. Finally, novel aspects of adipose tissue biology and their involvement in the ageing process are discussed. PMID:28432755

Dual peptide conjugation strategy for improved cellular uptake and mitochondria targeting.

Science.gov (United States)

Lin, Ran; Zhang, Pengcheng; Cheetham, Andrew G; Walston, Jeremy; Abadir, Peter; Cui, Honggang

2015-01-21

Mitochondria are critical regulators of cellular function and survival. Delivery of therapeutic and diagnostic agents into mitochondria is a challenging task in modern pharmacology because the molecule to be delivered needs to first overcome the cell membrane barrier and then be able to actively target the intracellular organelle. Current strategy of conjugating either a cell penetrating peptide (CPP) or a subcellular targeting sequence to the molecule of interest only has limited success. We report here a dual peptide conjugation strategy to achieve effective delivery of a non-membrane-penetrating dye 5-carboxyfluorescein (5-FAM) into mitochondria through the incorporation of both a mitochondrial targeting sequence (MTS) and a CPP into one conjugated molecule. Notably, circular dichroism studies reveal that the combined use of α-helix and PPII-like secondary structures has an unexpected, synergistic contribution to the internalization of the conjugate. Our results suggest that although the use of positively charged MTS peptide allows for improved targeting of mitochondria, with MTS alone it showed poor cellular uptake. With further covalent linkage of the MTS-5-FAM conjugate to a CPP sequence (R8), the dually conjugated molecule was found to show both improved cellular uptake and effective mitochondria targeting. We believe these results offer important insight into the rational design of peptide conjugates for intracellular delivery.

Up-regulation of ROS by mitochondria-dependent bystander signaling contributes to genotoxicity of bystander effects

International Nuclear Information System (INIS)

Chen Shaopeng; Zhao Ye; Zhao Guoping; Han Wei; Bao Lingzhi; Yu, K.N.; Wu Lijun

2009-01-01

Genomic instability can be observed in bystander cells. However, the underlying mechanism(s) is still relatively unclear. In a previous study, we found that irradiated cells released mitochondria-dependent intracellular factor(s) which could lead to bystander γ-H2AX induction. In this paper, we used normal (ρ + ) and mtDNA-depleted (ρ 0 ) human-hamster hybrid cells to investigate mitochondrial effects on the genotoxicity in bystander effect through medium transfer experiments. Through the detection of DNA double-strand breaks with γ-H2AX, we found that the fraction of γ-H2AX positive cells changed with time when irradiation conditioned cell medium (ICCM) were harvested. ICCM harvested from irradiated ρ + cells at 10 min post-irradiation (ρ + ICCM 10min ) caused larger increases of bystander γ-H2AX induction comparing to ρ 0 ICCM 10min , which only caused a slight increase of bystander γ-H2AX induction. The ρ + ICCM 10min could also result in the up-regulation of ROS production (increased by 35% at 10 min), while there was no significant increase in cells treated with ρ 0 ICCM 10min . We treated cells with dimethyl sulfoxide (DMSO), the scavenger of ROS, and quenched γ-H2AX induction by ρ + ICCM. Furthermore, after the medium had been transferred and the cells were continuously cultured for 7 days, we found significantly increased CD59 - gene loci mutation (increased by 45.9%) and delayed cell death in the progeny of ρ + ICCM-treated bystander cells. In conclusion, the work presented here suggested that up-regulation of the mitochondria-dependent ROS might be very important in mediating genotoxicity of bystander effects.

The Toxicity of Depleted Uranium

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Wayne Briner

2010-01-01

Full Text Available Depleted uranium (DU is an emerging environmental pollutant that is introduced into the environment primarily by military activity. While depleted uranium is less radioactive than natural uranium, it still retains all the chemical toxicity associated with the original element. In large doses the kidney is the target organ for the acute chemical toxicity of this metal, producing potentially lethal tubular necrosis. In contrast, chronic low dose exposure to depleted uranium may not produce a clear and defined set of symptoms. Chronic low-dose, or subacute, exposure to depleted uranium alters the appearance of milestones in developing organisms. Adult animals that were exposed to depleted uranium during development display persistent alterations in behavior, even after cessation of depleted uranium exposure. Adult animals exposed to depleted uranium demonstrate altered behaviors and a variety of alterations to brain chemistry. Despite its reduced level of radioactivity evidence continues to accumulate that depleted uranium, if ingested, may pose a radiologic hazard. The current state of knowledge concerning DU is discussed.

Rapid efflux of Ca2+ from heart mitochondria in the presence of inorganic pyrophosphate.

Science.gov (United States)

Vercesi, A; Lehninger, A L

1984-01-13

Inorganic pyrophosphate (PPi) in the intracellular concentration range causes rapid efflux of Ca2+ from rat heart mitochondria oxidizing pyruvate + malate in a low Na+ medium. Half-maximal rates of Ca2+ efflux were given by 20 microM PPi. During and after PPi-stimulated Ca2+ efflux the mitochondria retain their structural integrity and complete respiratory control. Carboxyatractyloside inhibits PPi-stimulated Ca2+ efflux, indicating PPi must enter the matrix in order to promote Ca2+ efflux. Heart mitochondria have a much higher affinity for PPi uptake and PPi-induced Ca2+ efflux than liver mitochondria.

Identification of nitric oxide in mitochondria of myometrium cell

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Danylovych Yu. V.

2015-06-01

Full Text Available Aim. To demonstrate the possibility of NO synthesis in intact myocytes of uterus. Methods. Confocal scanning microscopy method, NO-sensitive fluorescent probe DAF-FM, MitoTracker Orange CM-H2TMRos. Results. The basal production of NO in intact myocytes was shown using DAF-FM. Incubation of myocytes with NO donor – sodium nitroprusside (SNP – led to an increase of the DAF-FM-T fluorescent signal. On the contrary, the addition of NO-synthase inhibitor – N-nitro-L-arginine (NA – results in the reduction of fluorescent intensity. It was demonstrated colocalizition of specific probe for mitochondria MitoTracker Orange CM-H2TMRos and NO-sensitive dye DAF-FM. Conclusions. For the first time it has been demonstrated the presence of NO in smooth muscle cell mitochondria using laser confocal microscopy, NO-sensitive probe DAF-FM and specific marker of the functionally active mitochondria MitoTracker Orange CM-H2TMRos.

An origin-deficient yeast artificial chromosome triggers a cell cycle checkpoint.

Science.gov (United States)

van Brabant, A J; Buchanan, C D; Charboneau, E; Fangman, W L; Brewer, B J

2001-04-01

Checkpoint controls coordinate entry into mitosis with the completion of DNA replication. Depletion of nucleotide precursors by treatment with the drug hydroxyurea triggers such a checkpoint response. However, it is not clear whether the signal for this hydroxyurea-induced checkpoint pathway is the presence of unreplicated DNA, or rather the persistence of single-stranded or damaged DNA. In a yeast artificial chromosome (YAC) we have engineered an approximately 170 kb region lacking efficient replication origins that allows us to explore the specific effects of unreplicated DNA on cell cycle progression. Replication of this YAC extends the length of S phase and causes cells to engage an S/M checkpoint. In the absence of Rad9 the YAC becomes unstable, undergoing deletions within the origin-free region.

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

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Jesper Foged Havelund

2013-03-01

Full Text Available Mitochondria fulfill some basic roles in all plant cells. They supply the cell with energy in the form of ATP and reducing equivalents (NAD(PH 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.

Study of oxidative and phosphorylative activity in mitochondria from cereal seedlings

Energy Technology Data Exchange (ETDEWEB)

Plhak, F

1973-01-01

In the present paper the oxidative and phosphorylative activity of mitochondria isolated from rye, wheat, barley and corn seedlings are compared. Mitochondria from the shoots as well as from the roots of rye, wheat and corn oxidized succinate and in the presence of ATP or ADP exhibited the respiratory control which reached the values mostly of about 2. In the presence of ATP or ADP the decrease of inorganic phosphorus was contemporarily remarkable. The P/O ratio reached the values mostly of about 0.8 up to 1.0. The presence of ATP effected in some cases more favorable the respiratory control as well as the P/O ratio in comparison with ADP. With regard to the fact that a trapping hexokinase system was not added, the presence of endogenous hexokinase in mitochondria of experimental plants is presumed. The barley mitochondria exhibited the respiratory control as well, but instead of the decrease in inorganic phosphorus content in reaction mixture, an increase took place. It was caused by the presence of active ATP-ase which was not effectively inhibited by present NaF.

Differences between flocculating yeast and regular industrial yeast in transcription and metabolite profiling during ethanol fermentation

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Lili Li

2017-03-01

Full Text Available Objectives: To improve ethanolic fermentation performance of self-flocculating yeast, difference between a flocculating yeast strain and a regular industrial yeast strain was analyzed by transcriptional and metabolic approaches. Results: The number of down-regulated (industrial yeast YIC10 vs. flocculating yeast GIM2.71 and up-regulated genes were 4503 and 228, respectively. It is the economic regulation for YIC10 that non-essential genes were down-regulated, and cells put more “energy” into growth and ethanol production. Hexose transport and phosphorylation were not the limiting-steps in ethanol fermentation for GIM2.71 compared to YIC10, whereas the reaction of 1,3-disphosphoglycerate to 3-phosphoglycerate, the decarboxylation of pyruvate to acetaldehyde and its subsequent reduction to ethanol were the most limiting steps. GIM2.71 had stronger stress response than non-flocculating yeast and much more carbohydrate was distributed to other bypass, such as glycerol, acetate and trehalose synthesis. Conclusions: Differences between flocculating yeast and regular industrial yeast in transcription and metabolite profiling will provide clues for improving the fermentation performance of GIM2.71.

The construction of the zinc finger nucleases

Indian Academy of Sciences (India)

Michel Morange

in yeast mitochondria, and to provide evidence that, in contrast to nuclear introns, some ... the yeast genome, and to use them to obtain large fragments of the yeast .... and flanking exons in wild-type and box3 mutants of cytochrome b reveals ...

A Yeast/Drosophila Screen to Identify New Compounds Overcoming Frataxin Deficiency

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Alexandra Seguin

2015-01-01

Full Text Available Friedreich’s ataxia (FA is a rare neurodegenerative disease which is very debilitating for the patients who progressively lose their autonomy. The lack of efficient therapeutic treatment of the disease strongly argues for urgent need to search for new active compounds that may stop the progression of the disease or prevent the appearance of the symptoms when the genetic defect is diagnosed early enough. In the present study, we used a yeast strain with a deletion of the frataxin homologue gene as a model of FA cells in a primary screen of two chemical libraries, a fraction of the French National Chemical Library (5500 compounds and the Prestwick collection (880 compounds. We ran a secondary screen on Drosophila melanogaster flies expressing reduced levels of frataxin during larval development. Half of the compounds selected in yeast appeared to be active in flies in this developmental paradigm, and one of the two compounds with highest activities in this assay partially rescued the heart dilatation phenotype resulting from heart specific depletion of frataxin. The unique complementarity of these two frataxin-deficient models, unicellular and multicellular, appears to be very efficient to select new compounds with improved selectivity, bringing significant perspectives towards improvements in FA therapy.

One-year high fat diet affects muscle-but not brain mitochondria

DEFF Research Database (Denmark)

Joergensen, Tenna; Grunnet, Niels; Quistorff, Bjørn

2015-01-01

It is well known that few weeks of high fat (HF) diet may induce metabolic disturbances and mitochondrial dysfunction in skeletalmuscle. However, little is known about the effects of long-term HF exposure and effects on brain mitochondria are unknown. Wistarrats were fed either chow (13E% fat......) or HF diet (60E% fat) for 1 year. The HF animals developed obesity, dyslipidemia, insulinresistance, and dysfunction of isolated skeletal muscle mitochondria: state 3 and state 4 were 30% to 50% increased (P .... Adding also succinate in state 3 resulted in ahigher substrate control ratio (SCR) with PC, but a lower SCR with pyruvate (P mitochondria from the same animal showed no changes with the substrates relevant...

Management of depleted uranium

International Nuclear Information System (INIS)

2001-01-01

Large stocks of depleted uranium have arisen as a result of enrichment operations, especially in the United States and the Russian Federation. Countries with depleted uranium stocks are interested in assessing strategies for the use and management of depleted uranium. The choice of strategy depends on several factors, including government and business policy, alternative uses available, the economic value of the material, regulatory aspects and disposal options, and international market developments in the nuclear fuel cycle. This report presents the results of a depleted uranium study conducted by an expert group organised jointly by the OECD Nuclear Energy Agency and the International Atomic Energy Agency. It contains information on current inventories of depleted uranium, potential future arisings, long term management alternatives, peaceful use options and country programmes. In addition, it explores ideas for international collaboration and identifies key issues for governments and policy makers to consider. (authors)

Uncoupling and oxidative stress in liver mitochondria isolated from rats with acute iron overload

Energy Technology Data Exchange (ETDEWEB)

Pardo Andreu, G.L. [Centro de Quimica Farmaceutica, Departamento de Investigaciones Biomedicas, Ciudad de La Habana (Cuba); Inada, N.M.; Vercesi, A.E. [Universidade Estadual de Campinas, Departamento de Patologia Clinica, Faculdade de Ciencias Medicas, Campinas, SP (Brazil); Curti, C. [Universidade de Sao Paulo, Departamento de Fisica e Quimica, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, SP (Brazil)

2009-01-15

One hypothesis for the etiology of cell damage arising from iron overload is that its excess selectively affects mitochondria. Here we tested the effects of acute iron overload on liver mitochondria isolated from rats subjected to a single dose of i.p. 500 mg/kg iron-dextran. The treatment increased the levels of iron in mitochondria (from 21{+-}4 to 130{+-}7 nmol/mg protein) and caused both lipid peroxidation and glutathione oxidation. The mitochondria of iron-treated rats showed lower respiratory control ratio in association with higher resting respiration. The mitochondrial uncoupling elicited by iron-treatment did not affect the phosphorylation efficiency or the ATP levels, suggesting that uncoupling is a mitochondrial protective mechanism against acute iron overload. Therefore, the reactive oxygen species (ROS)/H{sup +} leak couple, functioning as a mitochondrial redox homeostatic mechanism could play a protective role in the acutely iron-loaded mitochondria. (orig.)

Mitochondria and the evolutionary roots of cancer

International Nuclear Information System (INIS)

Davila, Alfonso F; Zamorano, Pedro

2013-01-01

Cancer disease is inherent to, and widespread among, metazoans. Yet, some of the hallmarks of cancer such as uncontrolled cell proliferation, lack of apoptosis, hypoxia, fermentative metabolism and free cell motility (metastasis) are akin to a prokaryotic lifestyle, suggesting a link between cancer disease and evolution. In this hypothesis paper, we propose that cancer cells represent a phenotypic reversion to the earliest stage of eukaryotic evolution. This reversion is triggered by the dysregulation of the mitochondria due to cumulative oxidative damage to mitochondrial and nuclear DNA. As a result, the phenotype of normal, differentiated cells gradually reverts to the phenotype of a facultative anaerobic, heterotrophic cell optimized for survival and proliferation in hypoxic environments. This phenotype matches the phenotype of the last eukaryotic common ancestor (LECA) that resulted from the endosymbiosis between an α-proteobacteria (which later became the mitochondria) and an archaebacteria. As such, the evolution of cancer within one individual can be viewed as a recapitulation of the evolution of the eukaryotic cell from fully differentiated cells to LECA. This evolutionary model of cancer is compatible with the current understanding of the disease, and explains the evolutionary basis for most of the hallmarks of cancer, as well as the link between the disease and aging. It could also open new avenues for treatment directed at reestablishing the synergy between the mitochondria and the cancerous cell. (paper)

Comparative Analysis of VERA Depletion Problems

International Nuclear Information System (INIS)

Park, Jinsu; Kim, Wonkyeong; Choi, Sooyoung; Lee, Hyunsuk; Lee, Deokjung

2016-01-01

Each code has its own solver for depletion, which can produce different depletion calculation results. In order to produce reference solutions for depletion calculation comparison, sensitivity studies should be preceded for each depletion solver. The sensitivity tests for burnup interval, number of depletion zones, and recoverable energy per fission (Q-value) were performed in this paper. For the comparison of depletion calculation results, usually the multiplication factors are compared as a function of burnup. In this study, new comparison methods have been introduced by using the number density of isotope or element, and a cumulative flux instead of burnup. In this paper, optimum depletion calculation options are determined through the sensitivity study of the burnup intervals and the number of depletion intrazones. Because the depletion using CRAM solver performs well for large burnup intervals, smaller number of burnup steps can be used to produce converged solutions. It was noted that the depletion intra-zone sensitivity is only pin-type dependent. The 1 and 10 depletion intra-zones for the normal UO2 pin and gadolinia rod, respectively, are required to obtain the reference solutions. When the optimized depletion calculation options are used, the differences of Q-values are found to be a main cause of the differences of solutions. In this paper, new comparison methods were introduced for consistent code-to-code comparisons even when different kappa libraries were used in the depletion calculations

Yeasts and yeast-like organisms associated with fruits and blossoms of different fruit trees.

Science.gov (United States)

Vadkertiová, Renáta; Molnárová, Jana; Vránová, Dana; Sláviková, Elena

2012-12-01

Yeasts are common inhabitants of the phyllosphere, but our knowledge of their diversity in various plant organs is still limited. This study focused on the diversity of yeasts and yeast-like organisms associated with matured fruits and fully open blossoms of apple, plum, and pear trees, during 2 consecutive years at 3 localities in southwest Slovakia. The occurrence of yeasts and yeast-like organisms in fruit samples was 2½ times higher and the yeast community more diverse than that in blossom samples. Only 2 species (Aureobasidium pullulans and Metschnikowia pulcherrima) occurred regularly in the blossom samples, whereas Galactomyces candidus, Hanseniaspora guilliermondii, Hanseniaspora uvarum, M. pulcherrima, Pichia kluyveri, Pichia kudriavzevii, and Saccharomyces cerevisiae were the most frequently isolated species from the fruit samples. The ratio of the number of samples where only individual species were present to the number of samples where 2 or more species were found (consortium) was counted. The occurrence of individual species in comparison with consortia was much higher in blossom samples than in fruit samples. In the latter, consortia predominated. Aureobasidium pullulans, M. pulcherrima, and S. cerevisiae, isolated from both the fruits and blossoms, can be considered as resident yeast species of various fruit tree species cultivated in southwest Slovakia localities.

Squalene epoxidase as a target for manipulation of squalene levels in the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Garaiová, Martina; Zambojová, Veronika; Simová, Zuzana; Griač, Peter; Hapala, Ivan

2014-03-01

Squalene is a valuable natural substance with several biotechnological applications. In the yeast Saccharomyces cerevisiae, it is produced in the isoprenoid pathway as the first precursor dedicated to ergosterol biosynthesis. The aim of this study was to explore the potential of squalene epoxidase encoded by the ERG1 gene as the target for manipulating squalene levels in yeast. Highest squalene levels (over 1000 μg squalene per 10(9)  cells) were induced by specific point mutations in ERG1 gene that reduced activity of squalene epoxidase and caused hypersensitivity to terbinafine. This accumulation of squalene in erg1 mutants did not significantly disturb their growth. Treatment with squalene epoxidase inhibitor terbinafine revealed a limit in squalene accumulation at 700 μg squalene per 10(9)  cells which was associated with pronounced growth defects. Inhibition of squalene epoxidase activity by anaerobiosis or heme deficiency resulted in relatively low squalene levels. These levels were significantly increased by ergosterol depletion in anaerobic cells which indicated feedback inhibition of squalene production by ergosterol. Accumulation of squalene in erg1 mutants and terbinafine-treated cells were associated with increased cellular content and aggregation of lipid droplets. Our results prove that targeted genetic manipulation of the ERG1 gene is a promising tool for increasing squalene production in yeast. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd.

An F-actin-depleted zone is present at the hyphal tip of invasive hyphae of Neurospora crassa.

Science.gov (United States)

Suei, S; Garrill, A

2008-01-01

The distribution of filamentous actin (F-actin) in invasive and noninvasive hyphae of the ascomycete Neurospora crassa was investigated. Eighty six percent of noninvasive hyphae had F-actin in the tip region compared to only 9% of invasive hyphae. The remaining 91% of the invasive hyphae had no obvious tip high concentration of F-actin staining; instead they had an F-actin-depleted zone in this region, although some F-actin, possibly associated with the Spitzenkörper, remained at the tip. The size of the F-actin-depleted zone in invasive hyphae increased with an increase in agar concentration. The membrane stain FM 4-64 reveals a slightly larger accumulation of vesicles at the tips of invasive hyphae relative to noninvasive hyphae, although this difference is unlikely to be sufficient to account for the exclusion of F-actin from the depleted zone. Antibodies raised against the actin filament-severing protein cofilin from both yeast and human cells localize to the tips of invasive hyphae. The human cofilin antibody shows a more random distribution in noninvasive hyphae locating primarily at the hyphal periphery but with some diffuse cytoplasmic staining. This antibody also identifies a single band at 21 kDa in immunoblots of whole hyphal fractions. These data suggest that a protein with epitopic similarity to cofilin may function in F-actin dynamics that underlie invasive growth. The F-actin-depleted zone may play a role in the regulation of tip yielding to turgor pressure, thus increasing the protrusive force necessary for invasive growth.

Polyamine conjugation of curcumin analogues toward the discovery of mitochondria-directed neuroprotective agents.

Science.gov (United States)

Simoni, Elena; Bergamini, Christian; Fato, Romana; Tarozzi, Andrea; Bains, Sandip; Motterlini, Roberto; Cavalli, Andrea; Bolognesi, Maria Laura; Minarini, Anna; Hrelia, Patrizia; Lenaz, Giorgio; Rosini, Michela; Melchiorre, Carlo

2010-10-14

Mitochondria-directed antioxidants 2-5 were designed by conjugating curcumin congeners with different polyamine motifs as vehicle tools. The conjugates emerged as efficient antioxidants in mitochondria and fibroblasts and also exerted a protecting role through heme oxygenase-1 activation. Notably, the insertion of a polyamine function into the curcumin-like moiety allowed an efficient intracellular uptake and mitochondria targeting. It also resulted in a significant decrease in the cytotoxicity effects. 2-5 are therefore promising molecules for neuroprotectant lead discovery.

Erythroid precursors from patients with low-risk myelodysplasia demonstrate ultrastructural features of enhanced autophagy of mitochondria

NARCIS (Netherlands)

Houwerzijl, E. J.; Pol, H-W D.; Blom, N. R.; van der Want, J. J. L.; de Wolf, J. Thm; Vellenga, E.

Recent studies in erythroid cells have shown that autophagy is an important process for the physiological clearance of mitochondria during terminal differentiation. However, autophagy also plays an important role in removing damaged and dysfunctional mitochondria. Defective mitochondria and impaired

Functional expression of human adenine nucleotide translocase 4 in Saccharomyces cerevisiae.

Directory of Open Access Journals (Sweden)

Takashi Hamazaki

2011-04-01

Full Text Available The adenine nucleotide translocase (ANT mediates the exchange of ADP and ATP across the inner mitochondrial membrane. The human genome encodes multiple ANT isoforms that are expressed in a tissue-specific manner. Recently a novel germ cell-specific member of the ANT family, ANT4 (SLC25A31 was identified. Although it is known that targeted depletion of ANT4 in mice resulted in male infertility, the functional biochemical differences between ANT4 and other somatic ANT isoforms remain undetermined. To gain insight into ANT4, we expressed human ANT4 (hANT4 in yeast mitochondria. Unlike the somatic ANT proteins, expression of hANT4 failed to complement an AAC-deficient yeast strain for growth on media requiring mitochondrial respiration. Moreover, overexpression of hANT4 from a multi-copy plasmid interfered with optimal yeast growth. However, mutation of specific amino acids of hANT4 improved yeast mitochondrial expression and supported growth of the AAC-deficient yeast on non-fermentable carbon sources. The mutations affected amino acids predicted to interact with phospholipids, suggesting the importance of lipid interactions for function of this protein. Each mutant hANT4 and the somatic hANTs exhibited similar ADP/ATP exchange kinetics. These data define common and distinct biochemical characteristics of ANT4 in comparison to ANT1, 2 and 3 providing a basis for study of its unique adaptation to germ cells.

Amplification of arsenic genotoxicity by TiO2 nanoparticles in mammalian cells: new insights from physicochemical interactions and mitochondria.

Science.gov (United States)

Wang, Xinan; Liu, Yun; Wang, Juan; Nie, Yaguang; Chen, Shaopeng; Hei, Tom K; Deng, Zhaoxiang; Wu, Lijun; Zhao, Guoping; Xu, An

2017-10-01

Titanium dioxide nanoparticles (TiO 2 NPs) have shown great adsorption capacity for arsenic (As); however, the potential impact of TiO 2 NPs on the behavior and toxic responses of As remains largely unexplored. In the present study, we focused on the physicochemical interaction between TiO 2 NPs and As(III) to clarify the underlying mechanisms involved in their synergistic genotoxic effect on mammalian cells. Our data showed that As(III) mainly interacted with TiO 2 NPs by competitively occupying the sites of hydroxyl groups on the surface of TiO 2 NP aggregates, resulting in more aggregation of TiO 2 NPs. Although TiO 2 NPs at concentrations used here had no cytotoxic or genotoxic effects on cells, they efficiently increased the genotoxicity of As(III) in human-hamster hybrid (A L ) cells. The synergistic genotoxicity of TiO 2 NPs and As(III) was partially inhibited by various endocytosis pathway inhibitors while it was completely blocked by an As(III)-specific chelator. Using a mitochondrial membrane potential fluorescence probe, a reactive oxygen species (ROS) probe together with mitochondrial DNA-depleted ρ 0 A L cells, we discovered that mitochondria were essential for mediating the synergistic DNA-damaging effects of TiO 2 NPs and As(III). These data provide novel mechanistic proof that TiO 2 NPs enhanced the genotoxicity of As(III) via physicochemical interactions, which were mediated by mitochondria-dependent ROS.

Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondria-dependent apoptosis.

Science.gov (United States)

Tanaka, Toru; Hosoi, Fumihito; Yamaguchi-Iwai, Yuko; Nakamura, Hajime; Masutani, Hiroshi; Ueda, Shugo; Nishiyama, Akira; Takeda, Shunichi; Wada, Hiromi; Spyrou, Giannis; Yodoi, Junji

2002-04-02

Thioredoxin-2 (Trx-2) is a mitochondria-specific member of the thioredoxin superfamily. Mitochondria have a crucial role in the signal transduction for apoptosis. To investigate the biological significance of Trx-2, we cloned chicken TRX-2 cDNA and generated clones of the conditional Trx-2-deficient cells using chicken B-cell line, DT40. Here we show that TRX-2 is an essential gene and that Trx-2-deficient cells undergo apoptosis upon repression of the TRX-2 transgene, showing an accumulation of intracellular reactive oxygen species (ROS). Cytochrome c is released from mitochondria, while caspase-9 and caspase-3, but not caspase-8, are activated upon inhibition of the TRX-2 transgene. In addition, Trx-2 and cytochrome c are co-immunoprecipitated in an in vitro assay. These results suggest that mitochondrial Trx-2 is essential for cell viability, playing a crucial role in the scavenging ROS in mitochondria and regulating the mitochondrial apoptosis signaling pathway.

Effects of Ca2+ on oxidative phosphorylation in mitochondria from the thermogenic organ of marlin.

Science.gov (United States)

O'Brien, J; Block, B A

1996-12-01

Mitochondria from the muscle-derived thermogenic (heater) organ and oxidative red muscle of the blue marlin (Makaira nigricans) were studied in order to evaluate aspects of the mechanism of thermogenesis in heater tissue. We investigated whether short-term Ca(2+)-induced uncoupling of mitochondria contributes to the thermogenic cycle of the heater organ by enhancing the respiration rate. Specific electrodes were used to obtain simultaneous measurements of oxygen consumption and Ca2+ fluxes on isolated mitochondria, and the effects of various concentrations of Ca2+ on respiration rates and the ADP phosphorylated/atomic oxygen consumed (P/O) ratio were examined. Addition of Ca2+ in excess of 10 mumol l-1 to respiring heater organ or red muscle mitochondria partially inhibited state 3 respiration and reduced the P/O ratio, indicating that the mitochondria were partially uncoupled. These effects were blocked by 2 mumol l-1 Ruthenium Red. In heater organ mitochondria, state 3 respiration rate and the P/O ratio were not significantly reduced by 1 mumol l-1 free Ca2+, a concentration likely to be near the maximum achieved in a stimulated cell. This indicates that transient increases in cytosolic Ca2+ concentration may not significantly reduce the P/O ratio of heater organ mitochondria. The activity of 2-oxoglutarate dehydrogenase in heater organ mitochondria was stimulated by approximately 15% by Ca2+ concentrations between 0.2 and 1 mumol l-1. These results suggest that heater organ mitochondria are able to maintain a normal P/O ratio and should maintain ATP output during transient increases in Ca2+ concentration, supporting a model in which an ATP-consuming process drives thermogenesis. Activation of mitochondrial dehydrogenases by low levels of Ca2+ may also enhance respiration and contribute to thermogenesis.

Two-Photon Probes for Lysosomes and Mitochondria: Simultaneous Detection of Lysosomes and Mitochondria in Live Tissues by Dual-Color Two-Photon Microscopy Imaging.

Science.gov (United States)

Lim, Chang Su; Hong, Seung Taek; Ryu, Seong Shick; Kang, Dong Eun; Cho, Bong Rae

2015-10-01

Novel two-photon (TP) probes were developed for lysosomes (PLT-yellow) and mitochondria (BMT-blue and PMT-yellow). These probes emitted strong TP-excited fluorescence in cells at widely separated wavelength regions and displayed high organelle selectivity, good cell permeability, low cytotoxicity, and pH insensitivity. The BMT-blue and PLT-yellow probes could be utilized to detect lysosomes and mitochondria simultaneously in live tissues by using dual-color two-photon microscopy, with minimum interference from each other. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Entropy analysis in yeast DNA

International Nuclear Information System (INIS)

Kim, Jongkwang; Kim, Sowun; Lee, Kunsang; Kwon, Younghun

2009-01-01

In this article, we investigate the language structure in yeast 16 chromosomes. In order to find it, we use the entropy analysis for codons (or amino acids) of yeast 16 chromosomes, developed in analysis of natural language by Montemurro et al. From the analysis, we can see that there exists a language structure in codons (or amino acids) of yeast 16 chromosomes. Also we find that the grammar structure of amino acids of yeast 16 chromosomes has a deep relationship with secondary structure of protein.

Ovarian ageing: the role of mitochondria in oocytes and follicles.

Science.gov (United States)

May-Panloup, Pascale; Boucret, Lisa; Chao de la Barca, Juan-Manuel; Desquiret-Dumas, Valérie; Ferré-L'Hotellier, Véronique; Morinière, Catherine; Descamps, Philippe; Procaccio, Vincent; Reynier, Pascal

2016-11-01

There is a great inter-individual variability of ovarian ageing, and almost 20% of patients consulting for infertility show signs of premature ovarian ageing. This feature, taken together with delayed childbearing in modern society, leads to the emergence of age-related ovarian dysfunction concomitantly with the desire for pregnancy. Assisted reproductive technology is frequently inefficacious in cases of ovarian ageing, thus raising the economic, medical and societal costs of the procedures. Ovarian ageing is characterized by quantitative and qualitative alteration of the ovarian oocyte reserve. Mitochondria play a central role in follicular atresia and could be the main target of the ooplasmic factors determining oocyte quality adversely affected by ageing. Indeed, the oocyte is the richest cell of the body in mitochondria and depends largely on these organelles to acquire competence for fertilization and early embryonic development. Moreover, the oocyte ensures the uniparental transmission and stability of the mitochondrial genome across the generations. This review focuses on the role played by mitochondria in ovarian ageing and on the possible consequences over the generations. PubMed was used to search the MEDLINE database for peer-reviewed original articles and reviews concerning mitochondria and ovarian ageing, in animal and human species. Searches were performed using keywords belonging to three groups: 'mitochondria' or 'mitochondrial DNA'; 'ovarian reserve', 'oocyte', 'ovary' or 'cumulus cells'; and 'ageing' or 'ovarian ageing'. These keywords were combined with other search phrases relevant to the topic. References from these articles were used to obtain additional articles. There is a close relationship, in mammalian models and humans, between mitochondria and the decline of oocyte quality with ageing. Qualitatively, ageing-related mitochondrial (mt) DNA instability, which leads to the accumulation of mtDNA mutations in the oocyte, plays a key role in

Probing cytochrome c in living mitochondria with surface-enhanced Raman spectroscopy

DEFF Research Database (Denmark)

Brazhe, Nadezda A.; Evlyukhin, Andrey B.; Goodilin, Eugene A.

2015-01-01

Selective study of the electron transport chain components in living mitochondria is essential for fundamental biophysical research and for the development of new medical diagnostic methods. However, many important details of inter- and intramembrane mitochondrial processes have remained in shadow...... due to the lack of non-invasive techniques. Here we suggest a novel label-free approach based on the surface-enhanced Raman spectroscopy (SERS) to monitor the redox state and conformation of cytochrome c in the electron transport chain in living mitochondria. We demonstrate that SERS spectra of living...... mitochondria placed on hierarchically structured silver-ring substrates provide exclusive information about cytochrome c behavior under modulation of inner mitochondrial membrane potential, proton gradient and the activity of ATP-synthetase. Mathematical simulation explains the observed enhancement of Raman...

Post-transcriptional regulation of ribosome biogenesis in yeast

Directory of Open Access Journals (Sweden)

Isabelle C. Kos-Braun

2017-05-01

Full Text Available Most microorganisms are exposed to the constantly and often rapidly changing environment. As such they evolved mechanisms to balance their metabolism and energy expenditure with the resources available to them. When resources become scarce or conditions turn out to be unfavourable for growth, cells reduce their metabolism and energy usage to survive. One of the major energy consuming processes in the cell is ribosome biogenesis. Unsurprisingly, cells encountering adverse conditions immediately shut down production of new ribosomes. It is well established that nutrient depletion leads to a rapid repression of transcription of the genes encoding ribosomal proteins, ribosome biogenesis factors as well as ribosomal RNA (rRNA. However, if pre-rRNA processing and ribosome assembly are regulated post-transcriptionally remains largely unclear. We have recently uncovered that the yeast Saccharomyces cerevisiae rapidly switches between two alternative pre-rRNA processing pathways depending on the environmental conditions. Our findings reveal a new level of complexity in the regulation of ribosome biogenesis.

The yeast metacaspase is implicated in oxidative stress response in frataxin-deficient cells.

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Lefevre, Sophie; Sliwa, Dominika; Auchère, Françoise; Brossas, Caroline; Ruckenstuhl, Christoph; Boggetto, Nicole; Lesuisse, Emmanuel; Madeo, Frank; Camadro, Jean-Michel; Santos, Renata

2012-01-20

Friedreich ataxia is the most common recessive neurodegenerative disease and is caused by reduced expression of mitochondrial frataxin. Frataxin depletion causes impairment in iron-sulfur cluster and heme biosynthesis, disruption of iron homeostasis and hypersensitivity to oxidants. Currently no pharmacological treatment blocks disease progression, although antioxidant therapies proved to benefit patients. We show that sensitivity of yeast frataxin-deficient cells to hydrogen peroxide is partially mediated by the metacaspase. Metacaspase deletion in frataxin-deficient cells results in recovery of antioxidant capacity and heme synthesis. In addition, our results suggest that metacaspase is associated with mitochondrial respiration, intracellular redox control and genomic stability. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Yeast flocculation: New story in fuel ethanol production.

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Zhao, X Q; Bai, F W

2009-01-01

Yeast flocculation has been used in the brewing industry to facilitate biomass recovery for a long time, and thus its mechanism of yeast flocculation has been intensively studied. However, the application of flocculating yeast in ethanol production garnered attention mainly in the 1980s and 1990s. In this article, updated research progress in the molecular mechanism of yeast flocculation and the impact of environmental conditions on yeast flocculation are reviewed. Construction of flocculating yeast strains by genetic approach and utilization of yeast flocculation for ethanol production from various feedstocks were presented. The concept of self-immobilized yeast cells through their flocculation is revisited through a case study of continuous ethanol fermentation with the flocculating yeast SPSC01, and their technical and economic advantages are highlighted by comparing with yeast cells immobilized with supporting materials and regular free yeast cells as well. Taking the flocculating yeast SPSC01 as an example, the ethanol tolerance of the flocculating yeast was also discussed.

Erythroid cell mitochondria receive endosomal iron by a "kiss-and-run" mechanism.

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Hamdi, Amel; Roshan, Tariq M; Kahawita, Tanya M; Mason, Anne B; Sheftel, Alex D; Ponka, Prem

2016-12-01

In erythroid cells, more than 90% of transferrin-derived iron enters mitochondria where ferrochelatase inserts Fe 2+ into protoporphyrin IX. However, the path of iron from endosomes to mitochondrial ferrochelatase remains elusive. The prevailing opinion is that, after its export from endosomes, the redox-active metal spreads into the cytosol and mysteriously finds its way into mitochondria through passive diffusion. In contrast, this study supports the hypothesis that the highly efficient transport of iron toward ferrochelatase in erythroid cells requires a direct interaction between transferrin-endosomes and mitochondria (the "kiss-and-run" hypothesis). Using a novel method (flow sub-cytometry), we analyze lysates of reticulocytes after labeling these organelles with different fluorophores. We have identified a double-labeled population definitively representing endosomes interacting with mitochondria, as demonstrated by confocal microscopy. Moreover, we conclude that this endosome-mitochondrion association is reversible, since a "chase" with unlabeled holotransferrin causes a time-dependent decrease in the size of the double-labeled population. Importantly, the dissociation of endosomes from mitochondria does not occur in the absence of holotransferrin. Additionally, mutated recombinant holotransferrin, that cannot release iron, significantly decreases the uptake of 59 Fe by reticulocytes and diminishes 59 Fe incorporation into heme. This suggests that endosomes, which are unable to provide iron to mitochondria, cause a "traffic jam" leading to decreased endocytosis of holotransferrin. Altogether, our results suggest that a molecular mechanism exists to coordinate the iron status of endosomal transferrin with its trafficking. Besides its contribution to the field of iron metabolism, this study provides evidence for a new intracellular trafficking pathway of organelles. Copyright © 2016 Elsevier B.V. All rights reserved.

A conserved endoplasmic reticulum membrane protein complex (EMC facilitates phospholipid transfer from the ER to mitochondria.

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Sujoy Lahiri

2014-10-01

Full Text Available Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC has decreased phosphatidylserine (PS transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE. Cells lacking EMC proteins and the ER-mitochondria tethering complex called ERMES (the ER-mitochondria encounter structure are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER-mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.

EFFECT OF ACTIVE ACCUMULATION OF CALCIUM AND PHOSPHATE IONS ON THE STRUCTURE OF RAT LIVER MITOCHONDRIA

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Greenawalt, John W.; Rossi, Carlo S.; Lehninger, Albert L.

1964-01-01

Rat liver mitochondria allowed to accumulate maximal amounts of Ca++ and HPO4 = ions from the suspending medium in vitro during respiration have a considerably higher specific gravity than normal mitochondria and may be easily separated from the latter by isopycnic centrifugation in density gradients of sucrose or cesium chloride. When the mitochondria are allowed to accumulate less than maximal amounts of Ca++ and HPO4 = from the medium, they have intermediate specific gravities which are roughly proportional to their content of calcium phosphate. Maximally "loaded" mitochondria are relatively homogeneous with respect to specific gravity. Correlated biochemical and electron microscopic studies show that Ca++-loaded mitochondria contain numerous dense granules, of which some 85 per cent are over 500 A in diameter. These granules are electron-opaque not only following fixation and staining with heavy metal reagents, but also following fixation with formaldehyde, demonstrating that the characteristic granules in Ca++-loaded mitochondria have intrinsic electron-opacity. The dense granules are almost always located within the inner compartment of the mitochondria and not in the space between the inner and outer membranes. They are frequently located at or near the cristae and they often show electron-transparent "cores." Such granules appear to be made up of clusters of smaller dense particles, but preliminary x-ray diffraction analysis and electron diffraction studies have revealed no evidence of crystallinity in the deposits. The electron-opaque granules decrease in number when the Ca++-loaded mitochondria are incubated with 2,4-dinitrophenol; simultaneously there is discharge of Ca++ and phosphate from the mitochondria into the medium. PMID:14228516

Natural selection of mitochondria during somatic lifetime promotes healthy aging

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Rodell, Anders; Rasmussen, Lene J; Bergersen, Linda H

2013-01-01

Stimulation of mitochondrial biogenesis during life-time challenges both eliminates disadvantageous properties and drives adaptive selection of advantageous phenotypic variations. Intermittent fission and fusion of mitochondria provide specific targets for health promotion by brief temporal...... stressors, interspersed with periods of recovery and biogenesis. For mitochondria, the mechanisms of selection, variability, and heritability, are complicated by interaction of two independent genomes, including the multiple copies of DNA in each mitochondrion, as well as the shared nuclear genome of each...

Brewing characteristics of piezosensitive sake yeasts

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Nomura, Kazuki; Hoshino, Hirofumi; Igoshi, Kazuaki; Onozuka, Haruka; Tanaka, Erika; Hayashi, Mayumi; Yamazaki, Harutake; Takaku, Hiroaki; Iguchi, Akinori; Shigematsu, Toru

2018-04-01

Application of high hydrostatic pressure (HHP) treatment to food processing is expected as a non-thermal fermentation regulation technology that supresses over fermentation. However, the yeast Saccharomyces cerevisiae used for Japanese rice wine (sake) brewing shows high tolerance to HHP. Therefore, we aimed to generate pressure-sensitive (piezosensitive) sake yeast strains by mating sake with piezosensitive yeast strains to establish an HHP fermentation regulation technology and extend the shelf life of fermented foods. The results of phenotypic analyses showed that the generated yeast strains were piezosensitive and exhibited similar fermentation ability compared with the original sake yeast strain. In addition, primary properties of sake brewed using these strains, such as ethanol concentration, sake meter value and sake flavor compounds, were almost equivalent to those obtained using the sake yeast strain. These results suggest that the piezosensitive strains exhibit brewing characteristics essentially equivalent to those of the sake yeast strain.

Ozone-depleting Substances (ODS)

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U.S. Environmental Protection Agency — This site includes all of the ozone-depleting substances (ODS) recognized by the Montreal Protocol. The data include ozone depletion potentials (ODP), global warming...

The role of uncoupling protein 3 regulating calcium ion uptake into mitochondria during sarcopenia

Science.gov (United States)

Nikawa, Takeshi; Choi, Inho; Haruna, Marie; Hirasaka, Katsuya; Maita Ohno, Ayako; Kondo Teshima, Shigetada

Overloaded mitochondrial calcium concentration contributes to progression of mitochondrial dysfunction in aged muscle, leading to sarcopenia. Uncoupling protein 3 (UCP3) is primarily expressed in the inner membrane of skeletal muscle mitochondria. Recently, it has been reported that UCP3 is associated with calcium uptake into mitochondria. However, the mechanisms by which UCP3 regulates mitochondrial calcium uptake are not well understood. Here we report that UCP3 interacts with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that is localized in mitochondria, which is involved in cellular responses to calcium ion. The hydrophilic sequences within the loop 2, matrix-localized hydrophilic domain of mouse UCP3 are necessary for binding to Hax-1 of the C-terminal domain in adjacent to mitochondrial innermembrane. Interestingly, these proteins interaction occur the calcium-dependent manner. Indeed, overexpression of UCP3 significantly enhanced calcium uptake into mitochondria on Hax-1 endogenously expressing C2C12 myoblasts. In addition, Hax-1 knock-down enhanced calcium uptake into mitochondria on both UCP3 and Hax-1 endogenously expressing C2C12 myotubes, but not myoblasts. Finally, the dissociation of UCP3 and Hax-1 enhances calcium uptake into mitochondria in aged muscle. These studies identify a novel UCP3-Hax-1 complex regulates the influx of calcium ion into mitochondria in muscle. Thus, the efficacy of UCP3-Hax-1 in mitochondrial calcium regulation may provide a novel therapeutic approach against mitochondrial dysfunction-related disease containing sarcopenia.

Production of Food Grade Yeasts

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Argyro Bekatorou

2006-01-01

Full Text Available Yeasts have been known to humans for thousands of years as they have been used in traditional fermentation processes like wine, beer and bread making. Today, yeasts are also used as alternative sources of high nutritional value proteins, enzymes and vitamins, and have numerous applications in the health food industry as food additives, conditioners and flavouring agents, for the production of microbiology media and extracts, as well as livestock feeds. Modern scientific advances allow the isolation, construction and industrial production of new yeast strains to satisfy the specific demands of the food industry. Types of commercial food grade yeasts, industrial production processes and raw materials are highlighted. Aspects of yeast metabolism, with respect to carbohydrate utilization, nutritional aspects and recent research advances are also discussed.

Mitochondria in biology and medicine--2012

DEFF Research Database (Denmark)

Madsen, Claus Desler; Rasmussen, Lene Juel

2014-01-01

as biomarkers for the diseases and most important, it opens the possibility of a treatment or a cure for a disease. "Mitochondria in Biology and Medicine" was the title of the second annual conference of Society of Mitochondrial Research and Medicine-India. The conference was organized by Rana P. Singh, Keshav...

High Throughput Microplate Respiratory Measurements Using Minimal Quantities Of Isolated Mitochondria

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Rogers, George W.; Brand, Martin D.; Petrosyan, Susanna; Ashok, Deepthi; Elorza, Alvaro A.; Ferrick, David A.; Murphy, Anne N.

2011-01-01

Recently developed technologies have enabled multi-well measurement of O2 consumption, facilitating the rate of mitochondrial research, particularly regarding the mechanism of action of drugs and proteins that modulate metabolism. Among these technologies, the Seahorse XF24 Analyzer was designed for use with intact cells attached in a monolayer to a multi-well tissue culture plate. In order to have a high throughput assay system in which both energy demand and substrate availability can be tightly controlled, we have developed a protocol to expand the application of the XF24 Analyzer to include isolated mitochondria. Acquisition of optimal rates requires assay conditions that are unexpectedly distinct from those of conventional polarography. The optimized conditions, derived from experiments with isolated mouse liver mitochondria, allow multi-well assessment of rates of respiration and proton production by mitochondria attached to the bottom of the XF assay plate, and require extremely small quantities of material (1–10 µg of mitochondrial protein per well). Sequential measurement of basal, State 3, State 4, and uncoupler-stimulated respiration can be made in each well through additions of reagents from the injection ports. We describe optimization and validation of this technique using isolated mouse liver and rat heart mitochondria, and apply the approach to discover that inclusion of phosphatase inhibitors in the preparation of the heart mitochondria results in a specific decrease in rates of Complex I-dependent respiration. We believe this new technique will be particularly useful for drug screening and for generating previously unobtainable respiratory data on small mitochondrial samples. PMID:21799747

High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria.

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George W Rogers

Full Text Available Recently developed technologies have enabled multi-well measurement of O(2 consumption, facilitating the rate of mitochondrial research, particularly regarding the mechanism of action of drugs and proteins that modulate metabolism. Among these technologies, the Seahorse XF24 Analyzer was designed for use with intact cells attached in a monolayer to a multi-well tissue culture plate. In order to have a high throughput assay system in which both energy demand and substrate availability can be tightly controlled, we have developed a protocol to expand the application of the XF24 Analyzer to include isolated mitochondria. Acquisition of optimal rates requires assay conditions that are unexpectedly distinct from those of conventional polarography. The optimized conditions, derived from experiments with isolated mouse liver mitochondria, allow multi-well assessment of rates of respiration and proton production by mitochondria attached to the bottom of the XF assay plate, and require extremely small quantities of material (1-10 µg of mitochondrial protein per well. Sequential measurement of basal, State 3, State 4, and uncoupler-stimulated respiration can be made in each well through additions of reagents from the injection ports. We describe optimization and validation of this technique using isolated mouse liver and rat heart mitochondria, and apply the approach to discover that inclusion of phosphatase inhibitors in the preparation of the heart mitochondria results in a specific decrease in rates of Complex I-dependent respiration. We believe this new technique will be particularly useful for drug screening and for generating previously unobtainable respiratory data on small mitochondrial samples.

Biochemistry of Mitochondria

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Filiz Koc

2003-02-01

Full Text Available Mitochondria are energy source of cells. They have external and internal membranes, cristas and matrix. External membranes consist of specialized transport proteins. They have monoamine oxidase and citokrome-c reductase which both play role in KREBS cycle as catalyst and many enzymes which are necessary for phospholipid and phosphoric acid synthesis. Enzymes of electron transport chain and oxidative phosphorylation are located in the internal membranes. Also, here, there are transport systems for specific substances, such as ATP, ADP, P1, pyruvate, succinate, malate, citrate, and -ketoglutarate . Matrix; having gel-like consistency, contains a large number of enzymes. [Archives Medical Review Journal 2003; 12(0.100: 1-13

Viral degradasome hijacks mitochondria to suppress innate immunity

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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

Modeling population dynamics of mitochondria in mammalian cells

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Kornick, Kellianne; Das, Moumita

Mitochondria are organelles located inside eukaryotic cells and are essential for several key cellular processes such as energy (ATP) production, cell signaling, differentiation, and apoptosis. All organisms are believed to have low levels of variation in mitochondrial DNA (mtDNA), and alterations in mtDNA are connected to a range of human health conditions, including epilepsy, heart failure, Parkinsons disease, diabetes, and multiple sclerosis. Therefore, understanding how changes in mtDNA accumulate over time and are correlated to changes in mitochondrial function and cell properties can have a profound impact on our understanding of cell physiology and the origins of some diseases. Motivated by this, we develop and study a mathematical model to determine which cellular parameters have the largest impact on mtDNA population dynamics. The model consists of coupled ODEs to describe subpopulations of healthy and dysfunctional mitochondria subject to mitochondrial fission, fusion, autophagy, and mutation. We study the time evolution and stability of each sub-population under specific selection biases and pressures by tuning specific terms in our model. Our results may provide insights into how sub-populations of mitochondria survive and evolve under different selection pressures. This work was supported by a Grant from the Moore Foundation.

Paramagnetic properties of the low- and high-spin states of yeast cytochrome c peroxidase

International Nuclear Information System (INIS)

Vanwetswinkel, Sophie; Nuland, Nico A. J. van; Volkov, Alexander N.

2013-01-01

Here we describe paramagnetic NMR analysis of the low- and high-spin forms of yeast cytochrome c peroxidase (CcP), a 34 kDa heme enzyme involved in hydroperoxide reduction in mitochondria. Starting from the assigned NMR spectra of a low-spin CN-bound CcP and using a strategy based on paramagnetic pseudocontact shifts, we have obtained backbone resonance assignments for the diamagnetic, iron-free protein and the high-spin, resting-state enzyme. The derived chemical shifts were further used to determine low- and high-spin magnetic susceptibility tensors and the zero-field splitting constant (D) for the high-spin CcP. The D value indicates that the latter contains a hexacoordinate heme species with a weak field ligand, such as water, in the axial position. Being one of the very few high-spin heme proteins analyzed in this fashion, the resting state CcP expands our knowledge of the heme coordination chemistry in biological systems

Paramagnetic properties of the low- and high-spin states of yeast cytochrome c peroxidase

Energy Technology Data Exchange (ETDEWEB)

Vanwetswinkel, Sophie; Nuland, Nico A. J. van; Volkov, Alexander N., E-mail: ovolkov@vub.ac.be [Vrije Universiteit Brussel, Jean Jeener NMR Centre, Structural Biology Brussels (Belgium)

2013-09-15

Here we describe paramagnetic NMR analysis of the low- and high-spin forms of yeast cytochrome c peroxidase (CcP), a 34 kDa heme enzyme involved in hydroperoxide reduction in mitochondria. Starting from the assigned NMR spectra of a low-spin CN-bound CcP and using a strategy based on paramagnetic pseudocontact shifts, we have obtained backbone resonance assignments for the diamagnetic, iron-free protein and the high-spin, resting-state enzyme. The derived chemical shifts were further used to determine low- and high-spin magnetic susceptibility tensors and the zero-field splitting constant (D) for the high-spin CcP. The D value indicates that the latter contains a hexacoordinate heme species with a weak field ligand, such as water, in the axial position. Being one of the very few high-spin heme proteins analyzed in this fashion, the resting state CcP expands our knowledge of the heme coordination chemistry in biological systems.

Cardiovascular Disease, Mitochondria, and Traditional Chinese Medicine

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Jie Wang

2015-01-01

Full Text Available Recent studies demonstrated that mitochondria play an important role in the cardiovascular system and mutations of mitochondrial DNA affect coronary artery disease, resulting in hypertension, atherosclerosis, and cardiomyopathy. Traditional Chinese medicine (TCM has been used for thousands of years to treat cardiovascular disease, but it is not yet clear how TCM affects mitochondrial function. By reviewing the interactions between the cardiovascular system, mitochondrial DNA, and TCM, we show that cardiovascular disease is negatively affected by mutations in mitochondrial DNA and that TCM can be used to treat cardiovascular disease by regulating the structure and function of mitochondria via increases in mitochondrial electron transport and oxidative phosphorylation, modulation of mitochondrial-mediated apoptosis, and decreases in mitochondrial ROS. However further research is still required to identify the mechanism by which TCM affects CVD and modifies mitochondrial DNA.

Up-regulation of ROS by mitochondria-dependent bystander signaling contributes to genotoxicity of bystander effects

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Chen Shaopeng [Key Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031 (China); Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Zhao Ye; Zhao Guoping [Key Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031 (China); Han Wei [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Bao Lingzhi [Key Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031 (China); Yu, K.N., E-mail: peter.yu@cityu.edu.hk [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Wu Lijun, E-mail: ljw@ipp.ac.cn [Key Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031 (China)

2009-06-18

Genomic instability can be observed in bystander cells. However, the underlying mechanism(s) is still relatively unclear. In a previous study, we found that irradiated cells released mitochondria-dependent intracellular factor(s) which could lead to bystander {gamma}-H2AX induction. In this paper, we used normal ({rho}{sup +}) and mtDNA-depleted ({rho}{sup 0}) human-hamster hybrid cells to investigate mitochondrial effects on the genotoxicity in bystander effect through medium transfer experiments. Through the detection of DNA double-strand breaks with {gamma}-H2AX, we found that the fraction of {gamma}-H2AX positive cells changed with time when irradiation conditioned cell medium (ICCM) were harvested. ICCM harvested from irradiated {rho}{sup +} cells at 10 min post-irradiation ({rho}{sup +} ICCM{sub 10min}) caused larger increases of bystander {gamma}-H2AX induction comparing to {rho}{sup 0} ICCM{sub 10min}, which only caused a slight increase of bystander {gamma}-H2AX induction. The {rho}{sup +} ICCM{sub 10min} could also result in the up-regulation of ROS production (increased by 35% at 10 min), while there was no significant increase in cells treated with {rho}{sup 0} ICCM{sub 10min}. We treated cells with dimethyl sulfoxide (DMSO), the scavenger of ROS, and quenched {gamma}-H2AX induction by {rho}{sup +} ICCM. Furthermore, after the medium had been transferred and the cells were continuously cultured for 7 days, we found significantly increased CD59{sup -} gene loci mutation (increased by 45.9%) and delayed cell death in the progeny of {rho}{sup +} ICCM-treated bystander cells. In conclusion, the work presented here suggested that up-regulation of the mitochondria-dependent ROS might be very important in mediating genotoxicity of bystander effects.

New nanocomposites for SERS studies of living cells and mitochondria

DEFF Research Database (Denmark)

Sarycheva, A. S.; Brazhe, N. A.; Baizhumanov, A. A.

2016-01-01

A great enhancement in Raman scattering (SERS) from heme-containing submembrane biomolecules inside intact erythrocytes and functional mitochondria is demonstrated for the first time using silver–silica beads prepared using a new method involving aerosol pyrolysis with aqueous diamminesilver...... molecules. The SERS spectra of functional mitochondria are sensitive to the activity of the mitochondrial electron transport chain, thus making the method a novel label-free approach to monitor the redox state and conformation of cytochromes in their natural cell environment. The developed nanocomposites...

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

DEFF Research Database (Denmark)

Havelund, Jesper; Thelen, Jay J.; Møller, Ian Max

2013-01-01

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......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......, 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....

Not your ordinary yeast: non-Saccharomyces yeasts in wine production uncovered.

Science.gov (United States)

Jolly, Neil P; Varela, Cristian; Pretorius, Isak S

2014-03-01

Saccharomyces cerevisiae and grape juice are 'natural companions' and make a happy wine marriage. However, this relationship can be enriched by allowing 'wild' non-Saccharomyces yeast to participate in a sequential manner in the early phases of grape must fermentation. However, such a triangular relationship is complex and can only be taken to 'the next level' if there are no spoilage yeast present and if the 'wine yeast' - S. cerevisiae - is able to exert its dominance in time to successfully complete the alcoholic fermentation. Winemakers apply various 'matchmaking' strategies (e.g. cellar hygiene, pH, SO2 , temperature and nutrient management) to keep 'spoilers' (e.g. Dekkera bruxellensis) at bay, and allow 'compatible' wild yeast (e.g. Torulaspora delbrueckii, Pichia kluyveri, Lachancea thermotolerans and Candida/Metschnikowia pulcherrima) to harmonize with potent S. cerevisiae wine yeast and bring the best out in wine. Mismatching can lead to a 'two is company, three is a crowd' scenario. More than 40 of the 1500 known yeast species have been isolated from grape must. In this article, we review the specific flavour-active characteristics of those non-Saccharomyces species that might play a positive role in both spontaneous and inoculated wine ferments. We seek to present 'single-species' and 'multi-species' ferments in a new light and a new context, and we raise important questions about the direction of mixed-fermentation research to address market trends regarding so-called 'natural' wines. This review also highlights that, despite the fact that most frontier research and technological developments are often focussed primarily on S. cerevisiae, non-Saccharomyces research can benefit from the techniques and knowledge developed by research on the former. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Ergosterol is mainly located in the cytoplasmic leaflet of the yeast plasma membrane.

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Solanko, Lukasz M; Sullivan, David P; Sere, Yves Y; Szomek, Maria; Lunding, Anita; Solanko, Katarzyna A; Pizovic, Azra; Stanchev, Lyubomir D; Pomorski, Thomas Günther; Menon, Anant K; Wüstner, Daniel

2018-03-01

Transbilayer lipid asymmetry is a fundamental characteristic of the eukaryotic cell plasma membrane (PM). While PM phospholipid asymmetry is well documented, the transbilayer distribution of PM sterols such as mammalian cholesterol and yeast ergosterol is not reliably known. We now report that sterols are asymmetrically distributed across the yeast PM, with the majority (~80%) located in the cytoplasmic leaflet. By exploiting the sterol-auxotrophic hem1Δ yeast strain we obtained cells in which endogenous ergosterol was quantitatively replaced with dehydroergosterol (DHE), a closely related fluorescent sterol that functionally and accurately substitutes for ergosterol in vivo. Using fluorescence spectrophotometry and microscopy we found that membrane-impermeant collisional quenchers (spin-labeled phosphatidylcholine and trinitrobenzene sulfonic acid). Efficient quenching was seen only after the cells were disrupted by glass-bead lysis or repeated freeze-thaw to allow quenchers access to the cell interior. The extent of quenching was unaffected by treatments that deplete cellular ATP levels, collapse the PM electrochemical gradient or affect the actin cytoskeleton. However, alterations in PM phospholipid asymmetry in cells lacking phospholipid flippases resulted in a more symmetric transbilayer distribution of sterol. Similarly, an increase in the quenchable pool of DHE was observed when PM sphingolipid levels were reduced by treating cells with myriocin. We deduce that sterols comprise up to ~45% of all inner leaflet lipids in the PM, a result that necessitates revision of current models of the architecture of the PM lipid bilayer. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

DISC1 Modulates Neuronal Stress Responses by Gate-Keeping ER-Mitochondria Ca2+ Transfer through the MAM

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Sung Jin Park

2017-12-01

Full Text Available Summary: A wide range of Ca2+-mediated functions are enabled by the dynamic properties of Ca2+, all of which are dependent on the endoplasmic reticulum (ER and mitochondria. Disrupted-in-schizophrenia 1 (DISC1 is a scaffold protein that is involved in the function of intracellular organelles and is linked to cognitive and emotional deficits. Here, we demonstrate that DISC1 localizes to the mitochondria-associated ER membrane (MAM. At the MAM, DISC1 interacts with IP3R1 and downregulates its ligand binding, modulating ER-mitochondria Ca2+ transfer through the MAM. The disrupted regulation of Ca2+ transfer caused by DISC1 dysfunction leads to abnormal Ca2+ accumulation in mitochondria following oxidative stress, which impairs mitochondrial functions. DISC1 dysfunction alters corticosterone-induced mitochondrial Ca2+ accumulation in an oxidative stress-dependent manner. Together, these findings link stress-associated neural stimuli with intracellular ER-mitochondria Ca2+ crosstalk via DISC1, providing mechanistic insight into how environmental risk factors can be interpreted by intracellular pathways under the control of genetic components in neurons. : Park et al. show that DISC1 regulates ER-mitochondria Ca2+ transfer through mitochondria-associated ER membrane (MAM. DISC1 dysfunction at MAM increases ER-mitochondria Ca2+ transfer during oxidative stress and excessive amounts of corticosterone, which impairs mitochondrial function. Keywords: DISC1, MAM, mitochondria, Ca2+, IP3R1, oxidative stress

Comparative studies on mitochondria isolated from neuron-enriched and glia-enriched fractions of rabbit and beef brain.

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Hamberger, A; Blomstrand, C; Lehninger, A L

1970-05-01

Fractions enriched in neuronal and glial cells were obtained from dispersions of whole beef brain and rabbit cerebral cortex by large-scale density gradient centrifugation procedures. The fractions were characterized by appropriate microscopic observation. Mitochondria were then isolated from these fractions by differential centrifugation of their homogenates. The two different types of mitochondria were characterized with respect to certain enzyme activities, respiratory rate, rate of protein synthesis, and their buoyant density in sucrose gradients. The mitochondria from the neuron-enriched fraction were distinguished by a higher rate of incorporation of amino acids into protein, higher cytochrome oxidase activity, and a higher buoyant density in sucrose density gradients. Mitochondria from the glia-enriched fraction showed relatively high monoamine oxidase and Na(+)- and K(+)-stimulated ATPase activities. The rates of oxidation of various substrates and the acceptor control ratios did not differ appreciably between the two types of mitochondria. The difference in the buoyant density of mitochondria isolated from the neuron-enriched and glia-enriched cell fractions was utilized in attempts to separate neuronal and glial mitochondria from the mixed mitochondria obtained from whole brain homogenates in shallow sucrose gradients. The appearance of two peaks of cytochrome oxidase, monoamine oxidase, and protein concentration in such gradients shows the potential feasibility of such an approach.

Visual light effects on mitochondria: The potential implications in relation to glaucoma.

Science.gov (United States)

Osborne, Neville N; Núñez-Álvarez, Claudia; Del Olmo-Aguado, Susana; Merrayo-Lloves, Jesús

2017-09-01

Light of different wave-lengths have the potential to interact with four major mitochondrial protein complexes that are involved in the generation of ATP. Neurones of the central nervous system have an absolute dependence on mitochondrial generated ATP. Laboratory studies show that short-wave or blue light (400-480nm) that impinges on the retina affect flavin and cytochrome constituents associated with mitochondria to decrease the rate of ATP formation, stimulate ROS and results in cell death. This suggests that blue light could potentially have a negative influence on retinal ganglion cell (RGC) mitochondria that are abundant and not shielded by macular pigments as occurs for photoreceptor mitochondria. This might be of significance in glaucoma where it is likely that RGC mitochondria are already affected and therefore be more susceptible to blue light. Thus simply filtering out some natural blue light from entering the eye might be beneficial for the treatment of glaucoma. Long-wave or red light (650-800nm) affects mitochondrial complex IV or cytochrome oxidase to increase the rate of formation of ATP and ROS causing the generation of a number of beneficial factors. Significantly, laboratory studies show that increasing the normal amount of natural red light reaching rat RGC mitochondria in situ, subjected to ischemia, proved to be beneficial. A challenge now is to test whether extra red light delivered to the human retina can slow-down RGC loss in glaucoma. Such a methodology has also the advantage of being non-invasive. One very exciting possibility might be in the production of a lens where solar UV light is convertes to add to the amount of natural red light entering the eye. Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Aspirin Induces Apoptosis through Release of Cytochrome c from Mitochondria

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Katja C. Zimmermann

2000-01-01

Full Text Available Nonsteroidal anti-inflammatory drugs (NSAID reduce the risk for cancer, due to their anti proliferative and apoptosis-inducing effects. A critical pathway for apoptosis involves the release of cytochrome c from mitochondria, which then interacts with Apaf-1 to activate caspase proteases that orchestrate cell death. In this study we found that treatment of a human cancer cell line with aspirin induced caspase activation and the apoptotic cell morphology, which was blocked by the caspase inhibitor zVAD-fmk. Further analysis of the mechanism underlying this apoptotic event showed that aspirin induces translocation of Bax to the mitochondria and triggers release of cytochrome c into the cytosol. The release of cytochrome c from mitochondria was inhibited by overexpression of the antiapoptotic protein Bcl-2 and cells that lack Apaf-1 were resistant to aspirin-induced apoptosis. These data provide evidence that the release of cytochrome c is an important part of the apoptotic mechanism of aspirin.

Effect of yeast storage temperature and flour composition on fermentative activities of baker's yeast

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Pejin Dušanka J.

2009-01-01

Full Text Available Baker's yeast is a set of living cells of Saccharomyces cerevisiae. It contains around 70-72% of water, 42-45% of proteins, around 40% of carbohydrates, around 7.5% of lipids (based on dry matter, and vitamin B-complex. On the basis of yeast cell analysis it can be concluded that yeast is a complex biological system which changes in time. The intensity of the changes depends on temperature. Yeast sample was stored at 4°C i 24°C for 12 days. During storage at 4°C, the content of total carbohydrates decreased from 48.81% to 37.50% (dry matter, whereas carbohydrate loss ranged from 40.81% to 29.28% at 24°C. The content of trehalose was 12.33% in the yeast sample stored at 4°C and 0.24% at 24°C. Loss of fermentative activity was 81.76% in the sample stored at 24°C for 12 days. The composition of five samples of 1st category flour was investigated. It was found that flours containing more reducing sugars and maltose enable higher fermentation activities. The flours with higher ash content (in the range 0.5-0.94% had higher contents of phytic acid. Higher ash and phytic contents in flour increased the yeast fermentative efficiency. In bakery industry, a range of ingredients has been applied to improve the product's quality such as surface active substances (emulsifiers, enzymes, sugars and fats. In the paper, the effect of some ingredients added to dough (margarine, saccharose, sodium chloride and malted barley on the yeast fermentative activity was studied. The mentioned ingredients were added to dough at different doses: 0.5, 1.0, 1.5 and 2.0%, flour basis. It was found that the investigated ingredients affected the fermentative activity of yeast and improved the bread quality.

Lager Yeast Comes of Age

Science.gov (United States)

2014-01-01

Alcoholic fermentations have accompanied human civilizations throughout our history. Lager yeasts have a several-century-long tradition of providing fresh beer with clean taste. The yeast strains used for lager beer fermentation have long been recognized as hybrids between two Saccharomyces species. We summarize the initial findings on this hybrid nature, the genomics/transcriptomics of lager yeasts, and established targets of strain improvements. Next-generation sequencing has provided fast access to yeast genomes. Its use in population genomics has uncovered many more hybridization events within Saccharomyces species, so that lager yeast hybrids are no longer the exception from the rule. These findings have led us to propose network evolution within Saccharomyces species. This “web of life” recognizes the ability of closely related species to exchange DNA and thus drain from a combined gene pool rather than be limited to a gene pool restricted by speciation. Within the domesticated lager yeasts, two groups, the Saaz and Frohberg groups, can be distinguished based on fermentation characteristics. Recent evidence suggests that these groups share an evolutionary history. We thus propose to refer to the Saaz group as Saccharomyces carlsbergensis and to the Frohberg group as Saccharomyces pastorianus based on their distinct genomes. New insight into the hybrid nature of lager yeast will provide novel directions for future strain improvement. PMID:25084862

Respiration and substrate transport rates as well as reactive oxygen species production distinguish mitochondria from brain and liver.

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Gusdon, Aaron M; Fernandez-Bueno, Gabriel A; Wohlgemuth, Stephanie; Fernandez, Jenelle; Chen, Jing; Mathews, Clayton E

2015-09-10

Aberrant mitochondrial function, including excessive reactive oxygen species (ROS) production, has been implicated in the pathogenesis of human diseases. The use of mitochondrial inhibitors to ascertain the sites in the electron transport chain (ETC) resulting in altered ROS production can be an important tool. However, the response of mouse mitochondria to ETC inhibitors has not been thoroughly assessed. Here we set out to characterize the differences in phenotypic response to ETC inhibitors between the more energetically demanding brain mitochondria and less energetically demanding liver mitochondria in commonly utilized C57BL/6J mice. We show that in contrast to brain mitochondria, inhibiting distally within complex I or within complex III does not increase liver mitochondrial ROS production supported by complex I substrates, and liver mitochondrial ROS production supported by complex II substrates occurred primarily independent of membrane potential. Complex I, II, and III enzymatic activities and membrane potential were equivalent between liver and brain and responded to ETC. inhibitors similarly. Brain mitochondria exhibited an approximately two-fold increase in complex I and II supported respiration compared with liver mitochondria while exhibiting similar responses to inhibitors. Elevated NADH transport and heightened complex II-III coupled activity accounted for increased complex I and II supported respiration, respectively in brain mitochondria. We conclude that important mechanistic differences exist between mouse liver and brain mitochondria and that mouse mitochondria exhibit phenotypic differences compared with mitochondria from other species.

“When the going gets tough, who keeps going?” Depletion sensitivity moderates the ego-depletion effect

NARCIS (Netherlands)

Salmon, S.J.; Adriaanse, M.A.; Vet, de E.W.M.L.; Fennis, B.M.; Ridder, de D.T.D.

2014-01-01

Self-control relies on a limited resource that can get depleted, a phenomenon that has been labeled ego-depletion. We argue that individuals may differ in their sensitivity to depleting tasks, and that consequently some people deplete their self-control resource at a faster rate than others. In

Analysis of the behavior of mitochondria in the ovaries of the earthworm Dendrobaena veneta Rosa 1839.

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Justyna Faron

Full Text Available We examined six types of cells that form the ovary of the earthworm Dendrobena veneta ogonia, prooocytes, vitellogenic oocytes, trophocytes, fully grown postvitellogenic oocytes and somatic cells of the gonad. The quantitative stereological method revealed a much higher "volume density" of mitochondria in all of the types of germ-line cells except for the somatic cells. Fluorescent vital stain JC-1, however, showed a much higher oxidative activity of mitochondria in the somatic cells than in the germ-line cells. The distribution of active and inactive mitochondria within the studied cells was assessed using the computer program ImageJ. The analysis showed a higher luminosity of inactive mitochondria in all of the types of germ-line cells and a higher luminosity of active mitochondria in somatic cells. The OXPHOS activity was found in somatic cells mitochondria and in the peripheral mitochondria of the vitellogenic oocytes. The detection of reactive oxygen species (ROS revealed a differentiated distribution of ROS in the different cell types. The amount of ROS substances was lower in somatic cells than in younger germ-line cells. The ROS level was also low in the cytoplasm of fully grown postwitellogenic oocytes. The distribution of the MnSOD enzyme that protects mitochondria against destructive role of ROS substances was high in the oogonia and in prooocytes and it was very high in vitellogenic and postvitellogenic oocytes. However, a much lower level of this protective enzyme was observed in the trophocytes and the lowest level was found in the cytoplasm of somatic cells. The lower mitochondrial activity and higher level of MnSOD activity in germ-line cells when compared to somatic cells testifies to the necessity of the organisms to protect the mitochondria of oocytes against the destructive role of the ROS that are produced during oxidative phosphorylation. The protection of the mitochondria in oocytes is essential for the transfer of healthy

Yeast strains and methods of use thereof

OpenAIRE

Goddard, Matthew Robert; Gardner, Richard Clague; Anfang, Nicole

2013-01-01

The present invention relates to yeast strains and, in particular, to yeast stains for use in fermentation processes. The invention also relates to methods of fermentation using the yeast strains of the invention either alone or in combination with other yeast strains. The invention thither relates to methods for the selection of yeast strains suitable for fermentation cultures by screening for various metabolic products and the use of specific nutrient sources.

Swelling and functional disorders of isolated liver mitochondria induced by ultraviolet light exposure

International Nuclear Information System (INIS)

Sayanagi, Hideaki

1977-01-01

Biochemical and morphological disruption of liver mitochondria exposed to ultraviolet light were discussed. The mitochondria was prepared from rat liver, and the suspension was exposed to a broad spectrum ultraviolet light. The ultraviolet exposure of isolated rat liver mitochondria prepared from group 1 (regular laboratory chow), caused the great acceleration of swelling of mitochondria and the loss of the ability to couple the phosphorylation with respiration chain. The irradiated mitochondria produced an increase of lipid peroxide which was proportional to the dose of ultraviolet energy. By the use of a difference spectra technic, the absorption bands of cytochrome b, c (c 1 ), and flavoprotein were found to decrease in absorption after ultraviolet exposure. However, mitochondrial suspension prepared from rat in group 2 (regular chow supplemented with 3 mg% riboflavin free form), 3 (with 3 mg% riboflavin tetrabutyrate), 4 (with 5 mg% glutathione (GSH)), provided some degree of protection against the above deleterious effects of ultraviolet radiation. The irradiation effects could be reduced in the irradiated mitochondrial suspension which was incubated with riboflavin and GSH respectively after exposure. Riboflavin B 2 tetrabutyrate was found to show the significant effect of anti-oxidation. Riboflavin free-form was also active in this respect but to a lesser extent. (auth.)

Differentiating between apparent and actual rates of H2O2 metabolism by isolated rat muscle mitochondria to test a simple model of mitochondria as regulators of H2O2 concentration

Science.gov (United States)

Treberg, Jason R.; Munro, Daniel; Banh, Sheena; Zacharias, Pamela; Sotiri, Emianka

2015-01-01

Mitochondria are often regarded as a major source of reactive oxygen species (ROS) in animal cells, with H2O2 being the predominant ROS released from mitochondria; however, it has been recently demonstrated that energized brain mitochondria may act as stabilizers of H2O2 concentration (Starkov et al. [1]) based on the balance between production and the consumption of H2O2, the later of which is a function of [H2O2] and follows first order kinetics. Here we test the hypothesis that isolated skeletal muscle mitochondria, from the rat, are able to modulate [H2O2] based upon the interaction between the production of ROS, as superoxide/H2O2, and the H2O2 decomposition capacity. The compartmentalization of detection systems for H2O2 and the intramitochondrial metabolism of H2O2 leads to spacial separation between these two components of the assay system. This results in an underestimation of rates when relying solely on extramitochondrial H2O2 detection. We find that differentiating between these apparent rates found when using extramitochondrial H2O2 detection and the actual rates of metabolism is important to determining the rate constant for H2O2 consumption by mitochondria in kinetic experiments. Using the high rate of ROS production by mitochondria respiring on succinate, we demonstrate that net H2O2 metabolism by mitochondria can approach a stable steady-state of extramitochondrial [H2O2]. Importantly, the rate constant determined by extrapolation of kinetic experiments is similar to the rate constant determined as the [H2O2] approaches a steady state. PMID:26001520

The dynamic regulation of NAD metabolism in mitochondria

Science.gov (United States)

Stein, Liana Roberts; Imai, Shin-ichiro

2012-01-01

Mitochondria are intracellular powerhouses that produce ATP and carry out diverse functions for cellular energy metabolism. While the maintenance of an optimal NAD/NADH ratio is essential for mitochondrial function, it has recently become apparent that the maintenance of the mitochondrial NAD pool also has critical importance. The biosynthesis, transport, and catabolism of NAD and its key intermediates play an important role in the regulation of NAD-consuming mediators, such as sirtuins, poly-ADP-ribose polymerases, and CD38/157 ectoenzymes, in intra- and extracellular compartments. Mitochondrial NAD biosynthesis is also modulated in response to nutritional and environmental stimuli. In this article, we discuss this dynamic regulation of NAD metabolism in mitochondria to shed light on the intimate connection between NAD and mitochondrial function. PMID:22819213

The Rise of Mitochondria in Medicine

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Picard, Martin; Wallace, Douglas C; Burelle, Yan

2016-01-01

Once considered exclusively the cell's powerhouse, mitochondria are now recognized to perform multiple essential cellular functions beyond energy production, impacting most areas of cell biology and medicine. Since the emergence of molecular biology and the discovery of pathogenic mitochondrial DNA defects in the 1980's, research advances have revealed a number of common human diseases which share an underlying pathogenesis involving mitochondrial dysfunction. Mitochondria undergo function-defining dynamic shape changes, communicate with each other, regulate gene expression within the nucleus, modulate synaptic transmission within the brain, release molecules that contribute to oncogenic transformation and trigger inflammatory responses systemically, and influence the regulation of complex physiological systems. Novel “mitopathogenic” mechanisms are thus being uncovered across a number of medical disciplines including genetics, oncology, neurology, immunology, and critical care medicine. Increasing knowledge of the bioenergetic aspects of human disease has provided new opportunities for diagnosis, therapy, prevention, and in connecting various domains of medicine. In this article, we overview specific aspects of mitochondrial biology that have contributed to – and likely will continue to enhance the progress of modern medicine. PMID:27423788

Atherosclerotic lesions and mitochondria DNA deletions in brain microvessels: implication in the pathogenesis of Alzheimer's disease.

Science.gov (United States)

Aliev, Gjumrakch; Gasimov, Eldar; Obrenovich, Mark E; Fischbach, Kathryn; Shenk, Justin C; Smith, Mark A; Perry, George

2008-01-01

The pathogenesis that is primarily responsible for Alzheimer's disease (AD) and cerebrovascular accidents (CVA) appears to involve chronic hypoperfusion. We studied the ultrastructural features of vascular lesions and mitochondria in brain vascular wall cells from human AD biopsy samples and two transgenic mouse models of AD, yeast artificial chromosome (YAC) and C57B6/SJL Tg (+), which overexpress human amyloid beta precursor protein (AbetaPP). In situ hybridization using probes for normal and 5 kb deleted human and mouse mitochondrial DNA (mtDNA) was performed along with immunocytochemistry using antibodies against the Abeta peptide processed from AbetaPP, 8-hydroxy-2'-guanosine (8OHG), and cytochrome c oxidase (COX). More amyloid deposition, oxidative stress markers as well as mitochondrial DNA deletions and structural abnormalities were present in the vascular walls of the human AD samples and the AbetaPP-YAC and C57B6/SJL Tg (+) transgenic mice compared to age-matched controls. Ultrastructural damage in perivascular cells highly correlated with endothelial lesions in all samples. Therefore, pharmacological interventions, directed at correcting the chronic hypoperfusion state, may change the natural course of the development of dementing neurodegeneration.

Immobilization of yeast cells by radiation-induced polymerization

International Nuclear Information System (INIS)

Fujimura, T.; Kaetsu, I.

1982-01-01

Radiation-induced polymerization method was applied to the immobilization of yeast cells. The effects of irradiation, cooling and monomer, which are neccessary for polymerization, were recovered completely by subsequent aerobical incubation of yeast cells. The ethanol productive in immobilized yeast cells increased with the increase of aerobical incubation period. The growth of yeast cells in immobilized yeast cells was indicated. The maximum ethanol productivity in immobilized yeast cell system was around three times as much as that in free yeast cell system. (orig.)

Evolutionary History of Ascomyceteous Yeasts

Energy Technology Data Exchange (ETDEWEB)

Haridas, Sajeet; Riley, Robert; Salamov, Asaf; Goker, Markus; Klenk, Hans-Peter; Kurtzman, Cletus P.; Blackwell, Meredith; Grigoriev, Igor; Jeffries, Thomas W.

2014-06-06

Yeasts are important for many industrial and biotechnological processes and show remarkable diversity despite morphological similarities. We have sequenced the genomes of 16 ascomycete yeasts of taxonomic and industrial importance including members of Saccharomycotina and Taphrinomycotina. A comparison of these with several other previously published yeast genomes have added increased confidence to the phylogenetic positions of previously poorly placed species including Saitoella complicata, Babjeviella inositovora and Metschnikowia bicuspidata. Phylogenetic analysis also showed that yeasts with alternative nuclear codon usage where CUG encodes serine instead of leucine are monophyletic within the Saccharomycotina. Most of the yeasts have compact genomes with a large fraction of single exon genes with Lipomyces starkeyi and the previously published Pneumocystis jirovecii being notable exceptions. Intron analysis suggests that early diverging species have more introns. We also observed a large number of unclassified lineage specific non-simple repeats in these genomes.

In vivo imaging reveals mitophagy independence in the maintenance of axonal mitochondria during normal aging.

Science.gov (United States)

Cao, Xu; Wang, Haiqiong; Wang, Zhao; Wang, Qingyao; Zhang, Shuang; Deng, Yuanping; Fang, Yanshan

2017-10-01

Mitophagy is thought to be a critical mitochondrial quality control mechanism in neurons and has been extensively studied in neurological disorders such as Parkinson's disease. However, little is known about how mitochondria are maintained in the lengthy neuronal axons in the context of physiological aging. Here, we utilized the unique Drosophila wing nerve model and in vivo imaging to rigorously profile changes in axonal mitochondria during aging. We revealed that mitochondria became fragmented and accumulated in aged axons. However, lack of Pink1 or Parkin did not lead to the accumulation of axonal mitochondria or axonal degeneration. Further, unlike in in vitro cultured neurons, we found that mitophagy rarely occurred in intact axons in vivo, even in aged animals. Furthermore, blocking overall mitophagy by knockdown of the core autophagy genes Atg12 or Atg17 had little effect on the turnover of axonal mitochondria or axonal integrity, suggesting that mitophagy is not required for axonal maintenance; this is regardless of whether the mitophagy is PINK1-Parkin dependent or independent. In contrast, downregulation of mitochondrial fission-fusion genes caused age-dependent axonal degeneration. Moreover, Opa1 expression in the fly head was significantly decreased with age, which may underlie the accumulation of fragmented mitochondria in aged axons. Finally, we showed that adult-onset, neuronal downregulation of the fission-fusion, but not mitophagy genes, dramatically accelerated features of aging. We propose that axonal mitochondria are maintained independently of mitophagy and that mitophagy-independent mechanisms such as fission-fusion may be central to the maintenance of axonal mitochondria and neural integrity during normal aging. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Ego Depletion Impairs Implicit Learning

Science.gov (United States)

Thompson, Kelsey R.; Sanchez, Daniel J.; Wesley, Abigail H.; Reber, Paul J.

2014-01-01

Implicit skill learning occurs incidentally and without conscious awareness of what is learned. However, the rate and effectiveness of learning may still be affected by decreased availability of central processing resources. Dual-task experiments have generally found impairments in implicit learning, however, these studies have also shown that certain characteristics of the secondary task (e.g., timing) can complicate the interpretation of these results. To avoid this problem, the current experiments used a novel method to impose resource constraints prior to engaging in skill learning. Ego depletion theory states that humans possess a limited store of cognitive resources that, when depleted, results in deficits in self-regulation and cognitive control. In a first experiment, we used a standard ego depletion manipulation prior to performance of the Serial Interception Sequence Learning (SISL) task. Depleted participants exhibited poorer test performance than did non-depleted controls, indicating that reducing available executive resources may adversely affect implicit sequence learning, expression of sequence knowledge, or both. In a second experiment, depletion was administered either prior to or after training. Participants who reported higher levels of depletion before or after training again showed less sequence-specific knowledge on the post-training assessment. However, the results did not allow for clear separation of ego depletion effects on learning versus subsequent sequence-specific performance. These results indicate that performance on an implicitly learned sequence can be impaired by a reduction in executive resources, in spite of learning taking place outside of awareness and without conscious intent. PMID:25275517

Ego depletion impairs implicit learning.

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Kelsey R Thompson

Full Text Available Implicit skill learning occurs incidentally and without conscious awareness of what is learned. However, the rate and effectiveness of learning may still be affected by decreased availability of central processing resources. Dual-task experiments have generally found impairments in implicit learning, however, these studies have also shown that certain characteristics of the secondary task (e.g., timing can complicate the interpretation of these results. To avoid this problem, the current experiments used a novel method to impose resource constraints prior to engaging in skill learning. Ego depletion theory states that humans possess a limited store of cognitive resources that, when depleted, results in deficits in self-regulation and cognitive control. In a first experiment, we used a standard ego depletion manipulation prior to performance of the Serial Interception Sequence Learning (SISL task. Depleted participants exhibited poorer test performance than did non-depleted controls, indicating that reducing available executive resources may adversely affect implicit sequence learning, expression of sequence knowledge, or both. In a second experiment, depletion was administered either prior to or after training. Participants who reported higher levels of depletion before or after training again showed less sequence-specific knowledge on the post-training assessment. However, the results did not allow for clear separation of ego depletion effects on learning versus subsequent sequence-specific performance. These results indicate that performance on an implicitly learned sequence can be impaired by a reduction in executive resources, in spite of learning taking place outside of awareness and without conscious intent.

Ego depletion impairs implicit learning.

Science.gov (United States)

Thompson, Kelsey R; Sanchez, Daniel J; Wesley, Abigail H; Reber, Paul J

2014-01-01

Implicit skill learning occurs incidentally and without conscious awareness of what is learned. However, the rate and effectiveness of learning may still be affected by decreased availability of central processing resources. Dual-task experiments have generally found impairments in implicit learning, however, these studies have also shown that certain characteristics of the secondary task (e.g., timing) can complicate the interpretation of these results. To avoid this problem, the current experiments used a novel method to impose resource constraints prior to engaging in skill learning. Ego depletion theory states that humans possess a limited store of cognitive resources that, when depleted, results in deficits in self-regulation and cognitive control. In a first experiment, we used a standard ego depletion manipulation prior to performance of the Serial Interception Sequence Learning (SISL) task. Depleted participants exhibited poorer test performance than did non-depleted controls, indicating that reducing available executive resources may adversely affect implicit sequence learning, expression of sequence knowledge, or both. In a second experiment, depletion was administered either prior to or after training. Participants who reported higher levels of depletion before or after training again showed less sequence-specific knowledge on the post-training assessment. However, the results did not allow for clear separation of ego depletion effects on learning versus subsequent sequence-specific performance. These results indicate that performance on an implicitly learned sequence can be impaired by a reduction in executive resources, in spite of learning taking place outside of awareness and without conscious intent.

Melatonin, mitochondria, and the skin.

Science.gov (United States)

Slominski, Andrzej T; Zmijewski, Michal A; Semak, Igor; Kim, Tae-Kang; Janjetovic, Zorica; Slominski, Radomir M; Zmijewski, Jaroslaw W

2017-11-01

The skin being a protective barrier between external and internal (body) environments has the sensory and adaptive capacity to maintain local and global body homeostasis in response to noxious factors. An important part of the skin response to stress is its ability for melatonin synthesis and subsequent metabolism through the indolic and kynuric pathways. Indeed, melatonin and its metabolites have emerged as indispensable for physiological skin functions and for effective protection of a cutaneous homeostasis from hostile environmental factors. Moreover, they attenuate the pathological processes including carcinogenesis and other hyperproliferative/inflammatory conditions. Interestingly, mitochondria appear to be a central hub of melatonin metabolism in the skin cells. Furthermore, substantial evidence has accumulated on the protective role of the melatonin against ultraviolet radiation and the attendant mitochondrial dysfunction. Melatonin and its metabolites appear to have a modulatory impact on mitochondrion redox and bioenergetic homeostasis, as well as the anti-apoptotic effects. Of note, some metabolites exhibit even greater impact than melatonin alone. Herein, we emphasize that melatonin-mitochondria axis would control integumental functions designed to protect local and perhaps global homeostasis. Given the phylogenetic origin and primordial actions of melatonin, we propose that the melatonin-related mitochondrial functions represent an evolutionary conserved mechanism involved in cellular adaptive response to skin injury and repair.

Sexual differentiation in fission yeast

DEFF Research Database (Denmark)

Egel, R; Nielsen, O; Weilguny, D

1990-01-01

The regulation of sexual reproduction in yeast constitutes the highest level of differentiation observed in these unicellular organisms. The various ramifications of this system involve DNA rearrangement, transcriptional control, post-translational modification (such as protein phosphorylation) a......) and receptor/signal processing. A few basic similarities are common to both fission and budding yeasts. The wiring of the regulatory circuitry, however, varies considerably between these divergent yeast groups....

Exposure to nature counteracts aggression after depletion.

Science.gov (United States)

Wang, Yan; She, Yihan; Colarelli, Stephen M; Fang, Yuan; Meng, Hui; Chen, Qiuju; Zhang, Xin; Zhu, Hongwei

2018-01-01

Acts of self-control are more likely to fail after previous exertion of self-control, known as the ego depletion effect. Research has shown that depleted participants behave more aggressively than non-depleted participants, especially after being provoked. Although exposure to nature (e.g., a walk in the park) has been predicted to replenish resources common to executive functioning and self-control, the extent to which exposure to nature may counteract the depletion effect on aggression has yet to be determined. The present study investigated the effects of exposure to nature on aggression following depletion. Aggression was measured by the intensity of noise blasts participants delivered to an ostensible opponent in a competition reaction-time task. As predicted, an interaction occurred between depletion and environmental manipulations for provoked aggression. Specifically, depleted participants behaved more aggressively in response to provocation than non-depleted participants in the urban condition. However, provoked aggression did not differ between depleted and non-depleted participants in the natural condition. Moreover, within the depletion condition, participants in the natural condition had lower levels of provoked aggression than participants in the urban condition. This study suggests that a brief period of nature exposure may restore self-control and help depleted people regain control over aggressive urges. © 2017 Wiley Periodicals, Inc.

Formation and mobilization of neutral lipids in the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Wagner, A; Daum, G

2005-11-01

Since energy storage is a basic metabolic process, the synthesis of neutral lipids occurs in all kingdoms of life. The yeast Saccharomyces cerevisiae, widely accepted as a model eukaryotic cell, contains two classes of neutral lipids, namely STEs (steryl esters) and TAGs (triacylglycerols). TAGs are synthesized through two pathways governed by the acyl-CoA diacylglycerol acyltransferase Dga1p and the phospholipid diacylglycerol acyltransferase Lro1p. STEs are formed by two STE synthases Are1p and Are2p, two enzymes with overlapping function, which also catalyse TAG formation, although to a minor extent. Neutral lipids are stored in the so-called lipid particles and can be utilized for membrane formation under conditions of lipid depletion. For this purpose, storage lipids have to be mobilized by TAG lipases and STE hydrolases. A TAG lipase named Tgl3p was identified as a major yeast TAG hydrolytic enzyme in lipid particles. Recently, a new family of hydrolases was detected which is required for STE mobilization in S. cerevisiae. These enzymes, named Yeh1p, Yeh2p and Tgl1p, are paralogues of the mammalian acid lipase family. The role of these proteins in biosynthesis and mobilization of TAG and STE, and the regulation of these processes will be discussed in this minireview.

Phospholipase activity in rat liver mitochondria studied by the use of endogenous substrates.

Science.gov (United States)

Bjornstad, P

1966-09-01

The hydrolysis of endogenous phosphatidyl ethanolamine and lecithin in rat liver mitochondria has been studied by using mitochondria from rats injected with ethanolamine-1,2-(14)C or choline-1,2-(14)C. A phospholipase A-like enzyme has been demonstrated, which catalyzes the hydrolysis of one fatty acid ester linkage in phosphatidyl ethanolamine and lecithin. Phosphatidyl ethanolamine is hydrolyzed in preference to lecithin and the main reaction products are free fatty acids and lysophosphatidyl ethanolamine. The further breakdown of lysophospholipids appears to be limited in mitochondria, which indicates that lysophospholipase activity is mainly located extramitochondrially. The enzymic system is greatly stimulated by calcium ions, and also slightly by magnesium ions, while EDTA inhibits it almost completely. These findings are discussed in relation to previous observations on the effect of calcium and of EDTA on the functions of mitochondria. The possible function of the mitochondrial phospholipase for the formation of phospholipids with special fatty acids at the alpha- and -position is discussed.

Occurrence of Killer Yeast Strains in Fruit and Berry Wine Yeast Populations

Directory of Open Access Journals (Sweden)

Gintare Gulbiniene

2004-01-01

Full Text Available Apple, cranberry, chokeberry and Lithuanian red grape wine yeast populations were used for the determination of killer yeast occurrence. According to the tests of the killer characteristics and immunity the isolated strains were divided into seven groups. In this work the activity of killer toxins purified from some typical strains was evaluated. The analysed strains produced different amounts of active killer toxin and some of them possessed new industrially significant killer properties. Total dsRNA extractions in 11 killer strains of yeast isolated from spontaneous fermentations revealed that the molecular basis of the killer phenomenon was not only dsRNAs, but also unidentified genetic determinants.

Deoxynucleoside salvage in fission yeast allows rescue of ribonucleotide reductase deficiency but not Spd1-mediated inhibition of replication

DEFF Research Database (Denmark)

Fleck, Oliver; Fahnøe, Ulrik; Løvschal, Katrine Vyff

2017-01-01

In fission yeast, the small, intrinsically disordered protein S-phase delaying protein 1 (Spd1) blocks DNA replication and causes checkpoint activation at least in part, by inhibiting the enzyme ribonucleotide reductase, which is responsible for the synthesis of DNA. The CRL4(Cdt2) E3 ubiquitin...... ligase mediates degradation of Spd1 and the related protein Spd2 at S phase of the cell cycle. We have generated a conditional allele of CRL4(Cdt2), by expressing the highly unstable substrate-recruiting protein Cdt2 from a repressible promoter. Unlike Spd1, Spd2 does not regulate deoxynucleotide...... triphosphate (dNTP) pools; yet we find that Spd1 and Spd2 together inhibit DNA replication upon Cdt2 depletion. To directly test whether this block of replication was solely due to insufficient dNTP levels, we established a deoxy-nucleotide salvage pathway in fission yeast by expressing the human nucleoside...

Ca(2+)-dependent nonspecific permeability of the inner membrane of liver mitochondria in the guinea fowl (Numida meleagris).

Science.gov (United States)

Vedernikov, Aleksander A; Dubinin, Mikhail V; Zabiakin, Vladimir A; Samartsev, Victor N

2015-06-01

This comparative study presents the results of the induction of Ca(2+)-dependent nonspecific permeability of the inner membrane (pore opening) of rat and guinea fowl liver mitochondria by mechanisms that are both sensitive and insensitive to cyclosporin A (CsA). It was established that energized rat and guinea fowl liver mitochondria incubated with 1 mM of inorganic phosphate (Pi) are capable of swelling upon addition of at least 125 and 875 nmol of CaCl2 per 1 mg protein, respectively. Under these conditions, the Ca(2+) release from the mitochondria of these animals and a drop in Δψ are observed. All of these processes are inhibited by 1 μM of CsA. FCCP, causing organelle de-energization, induces pore opening in rat and guinea fowl liver mitochondria upon addition of 45 и 625 nmol of CaCl2 per 1 mg protein, respectively. These results suggest the existence of a CsA-sensitive mechanism for the induction of Ca(2+)-dependent pores in guinea fowl liver mitochondria, which has been reported in rat liver mitochondria. However, guinea fowl liver mitochondria have a significantly greater resistance to Ca(2+) as a pore inducer compared to rat liver mitochondria. It was found that the addition of α,ω-hexadecanedioic acid (HDA) to rat and guinea fowl liver mitochondria incubated with CsA and loaded with Ca(2+) causes organelle swelling and Ca(2+) release from the matrix. It is assumed that in contrast to the CsA-sensitive pore, the CsA-insensitive pore induced by HDA in the inner membrane of guinea fowl liver mitochondria, as well as in rat liver mitochondria, is lipid in nature.

Oxidative stress induces mitochondrial fragmentation in frataxin-deficient cells

Energy Technology Data Exchange (ETDEWEB)

Lefevre, Sophie [Mitochondria, Metals and Oxidative Stress Laboratory, Institut Jacques Monod, CNRS-Universite Paris-Diderot, Sorbonne Paris Cite, 15 rue Helene Brion, 75205 Paris cedex 13 (France); ED515 UPMC, 4 place Jussieu 75005 Paris (France); Sliwa, Dominika [Mitochondria, Metals and Oxidative Stress Laboratory, Institut Jacques Monod, CNRS-Universite Paris-Diderot, Sorbonne Paris Cite, 15 rue Helene Brion, 75205 Paris cedex 13 (France); Rustin, Pierre [Inserm, U676, Physiopathology and Therapy of Mitochondrial Disease Laboratory, 75019 Paris (France); Universite Paris-Diderot, Faculte de Medecine Denis Diderot, IFR02 Paris (France); Camadro, Jean-Michel [Mitochondria, Metals and Oxidative Stress Laboratory, Institut Jacques Monod, CNRS-Universite Paris-Diderot, Sorbonne Paris Cite, 15 rue Helene Brion, 75205 Paris cedex 13 (France); Santos, Renata, E-mail: santos.renata@ijm.univ-paris-diderot.fr [Mitochondria, Metals and Oxidative Stress Laboratory, Institut Jacques Monod, CNRS-Universite Paris-Diderot, Sorbonne Paris Cite, 15 rue Helene Brion, 75205 Paris cedex 13 (France)

2012-02-10

Highlights: Black-Right-Pointing-Pointer Yeast frataxin-deficiency leads to increased proportion of fragmented mitochondria. Black-Right-Pointing-Pointer Oxidative stress induces complete mitochondrial fragmentation in {Delta}yfh1 cells. Black-Right-Pointing-Pointer Oxidative stress increases mitochondrial fragmentation in patient fibroblasts. Black-Right-Pointing-Pointer Inhibition of mitochondrial fission in {Delta}yfh1 induces oxidative stress resistance. -- Abstract: Friedreich ataxia (FA) is the most common recessive neurodegenerative disease. It is caused by deficiency in mitochondrial frataxin, which participates in iron-sulfur cluster assembly. Yeast cells lacking frataxin ({Delta}yfh1 mutant) showed an increased proportion of fragmented mitochondria compared to wild-type. In addition, oxidative stress induced complete fragmentation of mitochondria in {Delta}yfh1 cells. Genetically controlled inhibition of mitochondrial fission in these cells led to increased resistance to oxidative stress. Here we present evidence that in yeast frataxin-deficiency interferes with mitochondrial dynamics, which might therefore be relevant for the pathophysiology of FA.

Biomedical applications of yeast- a patent view, part one: yeasts as workhorses for the production of therapeutics and vaccines.

Science.gov (United States)

Roohvand, Farzin; Shokri, Mehdi; Abdollahpour-Alitappeh, Meghdad; Ehsani, Parastoo

2017-08-01

Yeasts, as Eukaryotes, offer unique features for ease of growth and genetic manipulation possibilities, making it an exceptional microbial host. Areas covered: This review provides general and patent-oriented insights into production of biopharmaceuticals by yeasts. Patents, wherever possible, were correlated to the original or review articles. The review describes applications of major GRAS (generally regarded as safe) yeasts for the production of therapeutic proteins and subunit vaccines; additionally, immunomodulatory properties of yeast cell wall components were reviewed for use of whole yeast cells as a new vaccine platform. The second part of the review will discuss yeast- humanization strategies and innovative applications. Expert opinion: Biomedical applications of yeasts were initiated by utilization of Saccharomyces cerevisiae, for production of leavened (fermented) products, and advanced to serve to produce biopharmaceuticals. Higher biomass production and expression/secretion yields, more similarity of glycosylation patterns to mammals and possibility of host-improvement strategies through application of synthetic biology might enhance selection of Pichia pastoris (instead of S. cerevisiae) as a host for production of biopharmaceutical in future. Immunomodulatory properties of yeast cell wall β-glucans and possibility of intracellular expression of heterologous pathogen/tumor antigens in yeast cells have expanded their application as a new platform, 'Whole Yeast Vaccines'.

Mitochondrial recombination increases with age in Podospora anserina

NARCIS (Netherlands)

van Diepeningen, Anne D; Goedbloed, Daniël J; Slakhorst, S Marijke; Koopmanschap, A Bertha; Maas, Marc F P M; Hoekstra, Rolf F; Debets, Alfons J M

With uniparental inheritance of mitochondria, there seems little reason for homologous recombination in mitochondria, but the machinery for mitochondrial recombination is quite well-conserved in many eukaryote species. In fungi and yeasts heteroplasmons may be formed when strains fuse and transfer

Diabetes-Induced Dysfunction of Mitochondria and Stem Cells in Skeletal Muscle and the Nervous System

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Fujimaki, Shin; Kuwabara, Tomoko

2017-01-01

Diabetes mellitus is one of the most common metabolic diseases spread all over the world, which results in hyperglycemia caused by the breakdown of insulin secretion or insulin action or both. Diabetes has been reported to disrupt the functions and dynamics of mitochondria, which play a fundamental role in regulating metabolic pathways and are crucial to maintain appropriate energy balance. Similar to mitochondria, the functions and the abilities of stem cells are attenuated under diabetic condition in several tissues. In recent years, several studies have suggested that the regulation of mitochondria functions and dynamics is critical for the precise differentiation of stem cells. Importantly, physical exercise is very useful for preventing the diabetic alteration by improving the functions of both mitochondria and stem cells. In the present review, we provide an overview of the diabetic alterations of mitochondria and stem cells and the preventive effects of physical exercise on diabetes, focused on skeletal muscle and the nervous system. We propose physical exercise as a countermeasure for the dysfunction of mitochondria and stem cells in several target tissues under diabetes complication and to improve the physiological function of patients with diabetes, resulting in their quality of life being maintained. PMID:29036909

Virgin olive oil yeasts: A review.

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Ciafardini, Gino; Zullo, Biagi Angelo

2018-04-01

This review summarizes current knowledge on virgin olive oil yeasts. Newly produced olive oil contains solid particles and micro drops of vegetation water in which yeasts reproduce to become the typical microbiota of olive oil. To date, about seventeen yeast species have been isolated from different types of olive oils and their by-products, of which six species have been identified as new species. Certain yeast species contribute greatly to improving the sensorial characteristics of the newly produced olive oil, whereas other species are considered harmful as they can damage the oil quality through the production of unpleasant flavors and triacylglycerol hydrolysis. Studies carried out in certain yeast strains have demonstrated the presence of defects in olive oil treated with Candida adriatica, Nakazawaea wickerhamii and Candida diddensiae specific strains, while other olive oil samples treated with other Candida diddensiae strains were defect-free after four months of storage and categorized as extra virgin. A new acetic acid producing yeast species, namely, Brettanomyces acidodurans sp. nov., which was recently isolated from olive oil, could be implicated in the wine-vinegary defect of the product. Other aspects related to the activity of the lipase-producing yeasts and the survival of the yeast species in the flavored olive oils are also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Terroir of yeasts? – Application of FTIR spectroscopy and molecular methods for strain typing of yeasts

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Gerhards Daniel

2015-01-01

Full Text Available The site specific influence on wine (Terroir is an often by wine producers, consumers and scientists discussed topic in the world of wine. A study on grapes and (spontaneous fermentations from six different vineyards was done to investigate the biodiversity of yeasts and to answer the question if there is a terroir of yeast and how it could be influenced. Randomly isolated yeasts were identified by FTIR-spectroscopy and molecular methods on species and strain level. Vineyard specific yeast floras would be observed but they are not such important as expected. Only a few overlapping strain patterns would be identified during both vintages. The yeast flora of the winery had a huge impact on the spontaneous fermentations, but is not really constant and influenced by different factors from outside.

Xanthurenic acid translocates proapoptotic Bcl-2 family proteins into mitochondria and impairs mitochondrial function

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Hess Otto M

2004-04-01

Full Text Available Abstract Background Xanthurenic acid is an endogenous molecule produced by tryptophan degradation, produced in the cytoplasm and mitochondria. Its accumulation can be observed in aging-related diseases, e.g. senile cataract and infectious disease. We previously reported that xanthurenic acid provokes apoptosis, and now present a study of the response of mitochondria to xanthurenic acid. Results Xanthurenic acid at 10 or 20 μM in culture media of human aortic smooth muscle cells induces translocation of the proteins Bax, Bak, Bclxs, and Bad into mitochondria. In 20 μM xanthurenic acid, Bax is also translocated to the nucleus. In isolated mitochondria xanthurenic acid leads to Bax and Bclxs oligomerization, accumulation of Ca2+, and increased oxygen consumption. Conclusion Xanthurenic acid interacts directly with Bcl-2 family proteins, inducing mitochondrial pathways of apoptosis and impairing mitochondrial functions.

Mitochondria-meditated pathways of organ failure upon inflammation

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Andrey V. Kozlov

2017-10-01

Full Text Available Liver failure induced by systemic inflammatory response (SIRS is often associated with mitochondrial dysfunction but the mechanism linking SIRS and mitochondria-mediated liver failure is still a matter of discussion. Current hypotheses suggest that causative events could be a drop in ATP synthesis, opening of mitochondrial permeability transition pore, specific changes in mitochondrial morphology, impaired Ca2+ uptake, generation of mitochondrial reactive oxygen species (mtROS, turnover of mitochondria and imbalance in electron supply to the respiratory chain. The aim of this review is to critically analyze existing hypotheses, in order to highlight the most promising research lines helping to prevent liver failure induced by SIRS. Evaluation of the literature shows that there is no consistent support that impaired Ca++ metabolism, electron transport chain function and ultrastructure of mitochondria substantially contribute to liver failure. Moreover, our analysis suggests that the drop in ATP levels has protective rather than a deleterious character. Recent data suggest that the most critical mitochondrial event occurring upon SIRS is the release of mtROS in cytoplasm, which can activate two specific intracellular signaling cascades. The first is the mtROS-mediated activation of NADPH-oxidase in liver macrophages and endothelial cells; the second is the acceleration of the expression of inflammatory genes in hepatocytes. The signaling action of mtROS is strictly controlled in mitochondria at three points, (i at the site of ROS generation at complex I, (ii the site of mtROS release in cytoplasm via permeability transition pore, and (iii interaction with specific kinases in cytoplasm. The systems controlling mtROS-signaling include pro- and anti-inflammatory mediators, nitric oxide, Ca2+ and NADPH-oxidase. Analysis of the literature suggests that further research should be focused on the impact of mtROS on organ failure induced by inflammation

N-Acetyl Cysteine Depletes Reactive Oxygen Species and Prevents Dental Monomer-Induced Intrinsic Mitochondrial Apoptosis In Vitro in Human Dental Pulp Cells.

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Yang Jiao

Full Text Available To investigate the involvement of intrinsic mitochondrial apoptosis in dental monomer-induced cytotoxicity and the influences of N-acetyl cysteine (NAC on this process.Human dental pulp cells (hDPCs were exposed to several dental monomers in the absence or presence of NAC, and cell viability, intracellular redox balance, morphology and function of mitochondria and key indicators of intrinsic mitochondrial apoptosis were evaluated using various commercial kits.Dental monomers exerted dose-dependent cytotoxic effects on hDPCs. Concomitant to the over-production of reactive oxygen species (ROS and depletion of glutathione (GSH, differential changes in activities of superoxide dismutase, glutathione peroxidase, and catalase were detected. Apoptosis, as indicated by positive Annexin V/propidium iodide (PI staining and activation of caspase-3, was observed after dental monomer treatment. Dental monomers impaired the morphology and function of mitochondria, and induced intrinsic mitochondrial apoptosis in hDPCs via up-regulation of p53, Bax and cleaved caspase-3, and down-regulation of Bcl-2. NAC restored cell viability, relieved oxidative stress and blocked the apoptotic effects of dental monomers.Dental monomers induced oxidative stress and mitochondrial intrinsic apoptosis in hDPCs. NAC could reduce the oxidative stress and thus protect hDPCs against dental monomer-induced apoptosis.

21 CFR 184.1983 - Bakers yeast extract.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Bakers yeast extract. 184.1983 Section 184.1983... Listing of Specific Substances Affirmed as GRAS § 184.1983 Bakers yeast extract. (a) Bakers yeast extract... a selected strain of yeast, Saccharomyces cerevisiae. It may be concentrated or dried. (b) The...

Endoplasmic reticulum involvement in yeast cell death

International Nuclear Information System (INIS)

Nicanor Austriaco, O.

2012-01-01

Yeast cells undergo programed cell death (PCD) with characteristic markers associated with apoptosis in mammalian cells including chromatin breakage, nuclear fragmentation, reactive oxygen species generation, and metacaspase activation. Though significant research has focused on mitochondrial involvement in this phenomenon, more recent work with both Saccharomyces cerevisiae and Schizosaccharomyces pombe has also implicated the endoplasmic reticulum (ER) in yeast PCD. This minireview provides an overview of ER stress-associated cell death (ER-SAD) in yeast. It begins with a description of ER structure and function in yeast before moving to a discussion of ER-SAD in both mammalian and yeast cells. Three examples of yeast cell death associated with the ER will be highlighted here including inositol starvation, lipid toxicity, and the inhibition of N-glycosylation. It closes by suggesting ways to further examine the involvement of the ER in yeast cell death.

Gamma radiation effect on the mitochondria ultrastructure in different radiosensitive types of cotton plants

Energy Technology Data Exchange (ETDEWEB)

Arslanov, S V

1973-01-01

When germinated seeds are irradiated with a dose of 10 krad, the mitochondrial ultrastructure is disrupted in the early-ripening 1306-DV and the late-ripening S-1622 varieties of the cotton-plant. The mitochondria exhibited swelling, breakdown of internal structure and vacuolation. In the S-1622 variety the mitochondria shrink owing to their small number and larger size and to the smaller number of cristae. Changes in the ultrafine organization of mitochondria lead to inhibition of carbohydrate oxidation through the Krebs cycle and intensification of oxidation through the pentosophosphate cycle.

Tumor cell phenotype is sustained by selective MAPK oxidation in mitochondria.

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Soledad Galli

2008-06-01

Full Text Available Mitochondria are major cellular sources of hydrogen peroxide (H(2O(2, the production of which is modulated by oxygen availability and the mitochondrial energy state. An increase of steady-state cell H(2O(2 concentration is able to control the transition from proliferating to quiescent phenotypes and to signal the end of proliferation; in tumor cells thereby, low H(2O(2 due to defective mitochondrial metabolism can contribute to sustain proliferation. Mitogen-activated protein kinases (MAPKs orchestrate signal transduction and recent data indicate that are present in mitochondria and regulated by the redox state. On these bases, we investigated the mechanistic connection of tumor mitochondrial dysfunction, H(2O(2 yield, and activation of MAPKs in LP07 murine tumor cells with confocal microscopy, in vivo imaging and directed mutagenesis. Two redox conditions were examined: low 1 microM H(2O(2 increased cell proliferation in ERK1/2-dependent manner whereas high 50 microM H(2O(2 arrested cell cycle by p38 and JNK1/2 activation. Regarding the experimental conditions as a three-compartment model (mitochondria, cytosol, and nuclei, the different responses depended on MAPKs preferential traffic to mitochondria, where a selective activation of either ERK1/2 or p38-JNK1/2 by co-localized upstream kinases (MAPKKs facilitated their further passage to nuclei. As assessed by mass spectra, MAPKs activation and efficient binding to cognate MAPKKs resulted from oxidation of conserved ERK1/2 or p38-JNK1/2 cysteine domains to sulfinic and sulfonic acids at a definite H(2O(2 level. Like this, high H(2O(2 or directed mutation of redox-sensitive ERK2 Cys(214 impeded binding to MEK1/2, caused ERK2 retention in mitochondria and restricted shuttle to nuclei. It is surmised that selective cysteine oxidations adjust the electrostatic forces that participate in a particular MAPK-MAPKK interaction. Considering that tumor mitochondria are dysfunctional, their inability to

History of genome editing in yeast.

Science.gov (United States)

Fraczek, Marcin G; Naseeb, Samina; Delneri, Daniela

2018-05-01

For thousands of years humans have used the budding yeast Saccharomyces cerevisiae for the production of bread and alcohol; however, in the last 30-40 years our understanding of the yeast biology has dramatically increased, enabling us to modify its genome. Although S. cerevisiae has been the main focus of many research groups, other non-conventional yeasts have also been studied and exploited for biotechnological purposes. Our experiments and knowledge have evolved from recombination to high-throughput PCR-based transformations to highly accurate CRISPR methods in order to alter yeast traits for either research or industrial purposes. Since the release of the genome sequence of S. cerevisiae in 1996, the precise and targeted genome editing has increased significantly. In this 'Budding topic' we discuss the significant developments of genome editing in yeast, mainly focusing on Cre-loxP mediated recombination, delitto perfetto and CRISPR/Cas. © 2018 The Authors. Yeast published by John Wiley & Sons, Ltd.

21 CFR 172.898 - Bakers yeast glycan.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Bakers yeast glycan. 172.898 Section 172.898 Food... Multipurpose Additives § 172.898 Bakers yeast glycan. Bakers yeast glycan may be safely used in food in accordance with the following conditions: (a) Bakers yeast glycan is the comminuted, washed, pasteurized, and...

The peptidyl prolyl cis/trans isomerase Pin1/Ess1 inhibits phosphorylation and toxicity of tau in a yeast model for Alzheimer’s disease

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Ann De Vos

2015-04-01

Full Text Available Since hyperphosphorylation of protein tau is a crucial event in Alzheimer’s disease, additional mechanisms besides the interplay of kinase and phosphatase activities are investigated, such as the effect of the peptidyl prolyl cis/trans isomerase Pin1. This isomerase was shown to bind and isomerize phosphorylated protein tau, thereby restoring the microtubule associated protein function of tau as well as promoting the dephosphorylation of the protein by the trans-dependent phosphatase PP2A. In this study we used models based on Saccharomyces cerevisiae to further elucidate the influence of Pin1 and its yeast ortholog Ess1 on tau phosphorylation and self-assembly. We could demonstrate that in yeast, a lack of Pin1 isomerase activity leads to an increase in phosphorylation of tau at Thr231, comparable to AD brain and consistent with earlier findings in other model organisms. However, we could also distinguish an effect by Pin1 on other residues of tau, i.e. Ser235 and Ser198/199/202. Furthermore, depletion of Pin1 isomerase activity results in reduced growth of the yeast cells, which is enhanced upon expression of tau. This suggests that the accumulation of hyperphosphorylated and aggregation-prone tau causes cytotoxicity in yeast. This study introduces yeast as a valuable model organism to characterize in detail the effect of Pin1 on the biochemical characteristics of protein tau, more specifically its phosphorylation and aggregation.

The wine and beer yeast Dekkera bruxellensis.

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Schifferdecker, Anna Judith; Dashko, Sofia; Ishchuk, Olena P; Piškur, Jure

2014-09-01

Recently, the non-conventional yeast Dekkera bruxellensis has been gaining more and more attention in the food industry and academic research. This yeast species is a distant relative of Saccharomyces cerevisiae and is especially known for two important characteristics: on the one hand, it is considered to be one of the main spoilage organisms in the wine and bioethanol industry; on the other hand, it is 'indispensable' as a contributor to the flavour profile of Belgium lambic and gueuze beers. Additionally, it adds to the characteristic aromatic properties of some red wines. Recently this yeast has also become a model for the study of yeast evolution. In this review we focus on the recently developed molecular and genetic tools, such as complete genome sequencing and transformation, to study and manipulate this yeast. We also focus on the areas that are particularly well explored in this yeast, such as the synthesis of off-flavours, yeast detection methods, carbon metabolism and evolutionary history. © 2014 The Authors. Yeast published by John Wiley & Sons, Ltd.

Biotechnology of non-Saccharomyces yeasts-the basidiomycetes.

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Johnson, Eric A

2013-09-01

Yeasts are the major producer of biotechnology products worldwide, exceeding production in capacity and economic revenues of other groups of industrial microorganisms. Yeasts have wide-ranging fundamental and industrial importance in scientific, food, medical, and agricultural disciplines (Fig. 1). Saccharomyces is the most important genus of yeast from fundamental and applied perspectives and has been expansively studied. Non-Saccharomyces yeasts (non-conventional yeasts) including members of the Ascomycetes and Basidiomycetes also have substantial current utility and potential applicability in biotechnology. In an earlier mini-review, "Biotechnology of non-Saccharomyces yeasts-the ascomycetes" (Johnson Appl Microb Biotechnol 97: 503-517, 2013), the extensive biotechnological utility and potential of ascomycetous yeasts are described. Ascomycetous yeasts are particularly important in food and ethanol formation, production of single-cell protein, feeds and fodder, heterologous production of proteins and enzymes, and as model and fundamental organisms for the delineation of genes and their function in mammalian and human metabolism and disease processes. In contrast, the roles of basidiomycetous yeasts in biotechnology have mainly been evaluated only in the past few decades and compared to the ascomycetous yeasts and currently have limited industrial utility. From a biotechnology perspective, the basidiomycetous yeasts are known mainly for the production of enzymes used in pharmaceutical and chemical synthesis, for production of certain classes of primary and secondary metabolites such as terpenoids and carotenoids, for aerobic catabolism of complex carbon sources, and for bioremediation of environmental pollutants and xenotoxicants. Notwithstanding, the basidiomycetous yeasts appear to have considerable potential in biotechnology owing to their catabolic utilities, formation of enzymes acting on recalcitrant substrates, and through the production of unique primary

Proteome Analysis of Subsarcolemmal Cardiomyocyte Mitochondria: A Comparison of Different Analytical Platforms

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Francesco Giorgianni

2014-05-01

Full Text Available Mitochondria are complex organelles that play critical roles in diverse aspects of cellular function. Heart disease and a number of other pathologies are associated with perturbations in the molecular machinery of the mitochondria. Therefore, comprehensive, unbiased examination of the mitochondrial proteome represents a powerful approach toward system-level insights into disease mechanisms. A crucial aspect in proteomics studies is design of bioanalytical strategies that maximize coverage of the complex repertoire of mitochondrial proteins. In this study, we evaluated the performance of gel-based and gel-free multidimensional platforms for profiling of the proteome in subsarcolemmal mitochondria harvested from rat heart. We compared three different multidimensional proteome fractionation platforms: polymeric reversed-phase liquid chromatography at high pH (PLRP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, and isoelectric focusing (IEF separations combined with liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS, and bioinformatics for protein identification. Across all three platforms, a total of 1043 proteins were identified. Among the three bioanalytical strategies, SDS-PAGE followed by LC-MS/MS provided the best coverage of the mitochondrial proteome. With this platform, 890 proteins with diverse physicochemical characteristics were identified; the mitochondrial protein panel encompassed proteins with various functional roles including bioenergetics, protein import, and mitochondrial fusion. Taken together, results of this study provide a large-scale view of the proteome in subsarcolemmal mitochondria from the rat heart, and aid in the selection of optimal bioanalytical platforms for differential protein expression profiling of mitochondria in health and disease.

Kinetics of depletion interactions

NARCIS (Netherlands)

Vliegenthart, G.A.; Schoot, van der P.P.A.M.

2003-01-01

Depletion interactions between colloidal particles dispersed in a fluid medium are effective interactions induced by the presence of other types of colloid. They are not instantaneous but built up in time. We show by means of Brownian dynamics simulations that the static (mean-field) depletion force

Between science and industry-applied yeast research.

Science.gov (United States)

Korhola, Matti

2018-03-01

I was fortunate to enter yeast research at the Alko Research Laboratories with a strong tradition in yeast biochemistry and physiology studies. At the same time in the 1980s there was a fundamental or paradigm change in molecular biology research with discoveries in DNA sequencing and other analytical and physical techniques for studying macromolecules and cells. Since that time biotechnological research has expanded the traditional fermentation industries to efficient production of industrial and other enzymes and specialty chemicals. Our efforts were directed towards improving the industrial production organisms: minerals enriched yeasts (Se, Cr, Zn) and high glutathione content yeast, baker´s, distiller´s, sour dough and wine yeasts, and the fungal Trichoderma reesei platform for enzyme production. I am grateful for the trust of my colleagues in several leadership positions at the Alko Research Laboratories, Yeast Industry Platform and at the international yeast community.

Aging changes of macromolecular synthesis in the mitochondria of mouse hepatocytes as revealed by microscopic radioautography

Energy Technology Data Exchange (ETDEWEB)

Nagata, Tetsuji [Shinshu University, Matsumoto (Japan). Dept. of Anatomy and Cell Biology

2007-07-01

This mini-review reports aging changes of macromolecular synthesis in the mitochondria of mouse hepatocytes. We have observed the macromolecular synthesis, such as DNA, RNA and proteins, in the mitochondria of various mammalian cells by means of electron microscopic radioautography technique developed in our laboratory. The number of mitochondria per cell, number of labeled mitochondria per cell with 3H-thymidine, 3H-uridine and 3H-leucine, precursors for DNA, RNA and proteins, respectively, were counted and the labeling indices at various ages, from fetal to postnatal early days and several months to 1 and 2 years in senescence, were calculated, which showed variations due to aging. (author)

Yeast cell metabolism investigated by CO{_2} production and soft X-ray irradiation

Science.gov (United States)

Masini, A.; Batani, D.; Previdi, F.; Milani, M.; Pozzi, A.; Turcu, E.; Huntington, S.; Takeyasu, H.

1999-01-01

Results obtained using a new technique for studying cell metabolism are presented. The technique, consisting in CO2 production monitoring, has been applied to Saccharomyces cerevisiae yeast cells. Also the cells were irradiated using the soft X-ray laser-plasma source at Rutherford Appleton Laboratory with the aim of producing a damage of metabolic processes at the wall level, responsible for fermentation, without great interference with respiration, taking place in mitochondria, and DNA activity. The source was calibrated with PIN diodes and X-ray spectrometers and used Teflon stripes as target, emitting X-rays at about 0.9 keV, with a very low penetration in biological material. X-ray doses delivered to the different cell compartments were calculated following a Lambert-Bouguet-Beer law. Immediately after irradiation, the damage to metabolic activity was measured again by monitoring CO2 production. Results showed a general reduction in gas production by irradiated samples, together with non-linear and non-monotone response to dose. There was also evidence of oscillations in cell metabolic activity and of X-ray induced changes in oscillation frequency.

Oxaliplatin-induced Oxidative Stress Provokes Toxicity in Isolated Rat Liver Mitochondria.

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Tabassum, Heena; Waseem, Mohammad; Parvez, Suhel; Qureshi, M Irfan

2015-11-01

Oxaliplatin is a widely employed platinum-derived chemotherapeutic agent commonly used for the treatment of colorectal cancer. Unfortunately, the benefit of this important drug is compromised by severe side effects such as neuropathy, ototoxicity, gastrointestinal toxicity, and hematological toxicity. Recently, few studies have also suggested the occurrence of hepatotoxicity in oxaliplatin-treated patients. Mitochondria have emerged as targets for anticancer drugs in various kinds of toxicity including hepatotoxicity that can lead to neoplastic disease. Oxidative stress is a well-established biomarker of mitochondrial toxicity. The purpose of this study was to investigate the dose-dependent damage caused by oxaliplatin on isolated liver mitochondria under in vitro conditions. The study was conducted in mitochondria isolated from liver of Wistar rats. Oxaliplatin was incubated with mitochondria in a dose-dependent manner under in vitro conditions. Oxidative stress indexes, non-enzymatic and enzymatic antioxidants were evaluated, looking at the overall armamentarium against the toxicity induced by oxaliplatin. Oxaliplatin caused a significant rise in the mitochondrial oxidative stress indexes lipid peroxidation and protein carbonyl. Alterations in the levels of non-enzymatic antioxidants and activities of enzymatic antioxidants were also observed. Oxidative stress plays an important role in the mitochondrial toxicity of oxaliplatin. The integrity of the hepatic tissue is compromised by the reactive oxygen species-mediated lipid peroxidation and protein carbonyl formation. Copyright © 2015 IMSS. Published by Elsevier Inc. All rights reserved.

UV spectrophotometry for monitoring the performance of a yeast-based deoxygenation process to treat ships' ballast water.

Science.gov (United States)

Veilleux, Éloïse; de Lafontaine, Yves; Thomas, Olivier

2016-04-01

This study assessed the usefulness of UV spectrophotometry for the monitoring of a yeast-based deoxygenation process proposed for ships' ballast water treatment to prevent the transfer of aquatic invasive species. Ten-day laboratory experiments using three treatment concentrations and different water types were conducted and resulted in complete oxygen depletion of treated waters. The treatment performance and quality of treated waters were determined by measuring the UV-visible absorbance spectra of water samples taken over time. Samples were also used for laboratory analysis of water quality properties. The UV absorbance spectra values were strongly correlated (r = 0.96) to yeast cell density in treated waters. The second-order derivative (D (2)) of the spectra varied greatly over time, and the spectrum profiles could be divided into two groups corresponding to the oxygenated and anoxic phases of the treatment. The D (2) value at 215 nm was strongly correlated (r = 0.94) to ammonia levels, which increased over time. The D (2) value at 225 nm was strongly correlated (r > 0.97) to DO concentration. Our results showed that UV spectrophotometry may provide a rapid assessment of the behavior and performance of the yeast bioreactor over time by quantifying (1) the density of yeast cells, (2) the time at which anoxic conditions were reached, and (3) a water quality index of the treated water related to the production of ammonia. We conclude that the rapidity of the technique confers a solid advantage over standard methods used for water quality analysis in laboratory and would permit the direct monitoring of the treatment performance on-board ships.

The mitochondria-mediate apoptosis of Lepidopteran cells induced by azadirachtin.

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Jingfei Huang

Full Text Available 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.

The mitochondria-mediate apoptosis of Lepidopteran cells induced by azadirachtin.

Science.gov (United States)

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.

First description of a novel mitochondrial mutation in the MT-TI gene associated with multiple mitochondrial DNA deletion and depletion in family with severe dilated mitochondrial cardiomyopathy.

Science.gov (United States)

Alila-Fersi, Olfa; Tabebi, Mouna; Maalej, Marwa; Belguith, Neila; Keskes, Leila; Mkaouar-Rebai, Emna; Fakhfakh, Faiza

2018-03-18

Mitochondria are essential for early cardiac development and impaired mitochondrial function was described associated with heart diseases such as hypertrophic or dilated mitochondrial cardiomyopathy. In this study, we report a family including two individuals with severe dilated mitochondrial cardiomyopathy. The whole mitochondrial genome screening showed the presence of several variations and a novel homoplasmic mutation m.4318-4322delC in the MT-TI gene shared by the two patients and their mother and leading to a disruption of the tRNA Ile secondary structure. In addition, a mitochondrial depletion was present in blood leucocyte of the two affected brother whereas a de novo heteroplasmic multiple deletion in the major arc of mtDNA was present in blood leucocyte and mucosa of only one of them. These deletions in the major arc of the mtDNA resulted to the loss of several protein-encoding genes and also some tRNA genes. The mtDNA deletion and depletion could result to an impairment of the oxidative phosphorylation and energy metabolism in the respiratory chain in the studied patients. Our report is the first description of a family with severe lethal dilated mitochondrial cardiomyopathy and presenting several mtDNA abnormalities including punctual mutation, deletion and depletion. Copyright © 2018 Elsevier Inc. All rights reserved.

[Acetylcholine activation of alpha-ketoglutarate oxidation in liver mitochondria].

Science.gov (United States)

Shostakovskaia, I V; Doliba, N M; Gordiĭ, S K; Babskiĭ, A M; Kondrashova, M N

1986-01-01

Activation of alpha-ketoglutarate oxidation in the rat liver mitochondria takes place 15 and 30 min after intraperitoneal injection of acetyl choline. This mediator in doses of 25, 50 and 100 micrograms per 100 g of body weight causes a pronounced stimulation of phosphorylation respiration rate and calcium capacity of mitochondria with alpha-ketoglutarate oxidation. Acetyl choline is found to have a moderate inhibitory action on oxidation of lower (physiological) concentrations of succinate. Its stimulating action on alpha-ketoglutarate oxidation is associated with activation of M-cholinoreceptors; atropine, a choline-blocker, removes completely this effect. It is supposed that alpha-ketoglutarate and succinate are included into the composition of two reciprocal hormonal-substrate nucleotide systems.

Fully Depleted Charge-Coupled Devices

International Nuclear Information System (INIS)

Holland, Stephen E.

2006-01-01

We have developed fully depleted, back-illuminated CCDs that build upon earlier research and development efforts directed towards technology development of silicon-strip detectors used in high-energy-physics experiments. The CCDs are fabricated on the same type of high-resistivity, float-zone-refined silicon that is used for strip detectors. The use of high-resistivity substrates allows for thick depletion regions, on the order of 200-300 um, with corresponding high detection efficiency for near-infrared and soft x-ray photons. We compare the fully depleted CCD to the p-i-n diode upon which it is based, and describe the use of fully depleted CCDs in astronomical and x-ray imaging applications

Deuterium - depleted water. Achievements and perspectives

International Nuclear Information System (INIS)

Titescu, Gh.; Stefanescu, I.; Saros-Rogobete, I.

2001-01-01

Deuterium - depleted water represents water that has an isotopic content lower than 145 ppm D/(D+H) which is the natural isotopic content of water. The research conducted at ICSI Ramnicu Valcea, regarding deuterium - depleted water were completed by the following patents: - technique and installation for deuterium - depleted water production; - distilled water with low deuterium content; - technique and installation for the production of distilled water with low deuterium content; - mineralized water with low deuterium content and technique to produce it. The gold and silver medals won at international salons for inventions confirmed the novelty of these inventions. Knowing that deuterium content of water has a big influence on living organisms, beginning with 1996, the ICSI Ramnicu Valcea, deuterium - depleted water producer, co-operated with Romanian specialized institutes for biological effects' evaluation of deuterium - depleted water. The role of natural deuterium in living organisms was examined by using deuterium - depleted water instead of natural water. These investigations led to the following conclusions: 1. deuterium - depleted water caused a tendency towards the increase of the basal tone, accompanied by the intensification of the vasoconstrictor effects of phenylefrine, noradrenaline and angiotensin; the increase of the basal tone and vascular reactivity produced by the deuterium - depleted water persists after the removal of the vascular endothelium; -2. animals treated with deuterium - depleted water showed an increase of the resistance both to sublethal and to lethal gamma radiation doses, suggesting a radioprotective action by the stimulation of non-specific immune defence mechanism; 3, deuterium - depleted water stimulates immune defence reactions, represented by the opsonic, bactericidal and phagocyte capacity of the immune system, together with increase in the numbers of polymorphonuclear neutrophils; 4. investigations regarding artificial

The effect of 2,4-D and ABA on respiration of isolated mitochondria from maize coleoptiles

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Ewa Raczek

2014-01-01

Full Text Available The susceptibility of isolated maize mitochondria to the growth regulators: 2,4-dichlorophenoxyacetic acid (2,4-D and abscisic acid (ABA was studied. It was found that 2,4-D (a herbicide inhibits respiration in mitochondria, as do other herbicides or phenoxy-acids. In the entire range of concentrations used (10-3-10-9 M, 2,4-D introduced into the medium before the respiration reaction was begun, or during it, limited the intensity of succinate oxidation. It did not, however, markedly change phosphorylation properties. Uncoupling of oxidative phosphorylation took place only after preincubation of mitochondria with 2,4-D and was the result of the destruction of mitochondrial membranes. ABA (a growth inhibitor of plants caused a similar response in maize mitochondria. Preincubation of mitochondria with ABA lead to the uncoupling of oxidative phosphorylation. Whereas ABA introduced during respiration (state 4 respiration or before its onset, lowered the oxidative potential of mitochondria, it also changed the pattern of state 4-3-4 transition after addition of ADP (it was especially visible at high concentrations, which indicates that the coupling of oxidative phosphorylation with the respiratory chain has faltered. It seems that this negative effect of 2,4-D and ABA on respiration of isolated maize mitochondria is connected with the inhibitory effect of these growth regulators on the growth of maize coleoptiles. Interference in the organization mitochondrial membranes results in a lowered supply of ATP - a source of energy needed in elongation processes.

Mitochondria-associated endoplasmic reticulum membranes allow adaptation of mitochondrial metabolism to glucose availability in the liver.

Science.gov (United States)

Theurey, Pierre; Tubbs, Emily; Vial, Guillaume; Jacquemetton, Julien; Bendridi, Nadia; Chauvin, Marie-Agnès; Alam, Muhammad Rizwan; Le Romancer, Muriel; Vidal, Hubert; Rieusset, Jennifer

2016-04-01

Mitochondria-associated endoplasmic reticulum membranes (MAM) play a key role in mitochondrial dynamics and function and in hepatic insulin action. Whereas mitochondria are important regulators of energy metabolism, the nutritional regulation of MAM in the liver and its role in the adaptation of mitochondria physiology to nutrient availability are unknown. In this study, we found that the fasted to postprandial transition reduced the number of endoplasmic reticulum-mitochondria contact points in mouse liver. Screening of potential hormonal/metabolic signals revealed glucose as the main nutritional regulator of hepatic MAM integrity both in vitro and in vivo Glucose reduced organelle interactions through the pentose phosphate-protein phosphatase 2A (PP-PP2A) pathway, induced mitochondria fission, and impaired respiration. Blocking MAM reduction counteracted glucose-induced mitochondrial alterations. Furthermore, disruption of MAM integrity mimicked effects of glucose on mitochondria dynamics and function. This glucose-sensing system is deficient in the liver of insulin-resistant ob/ob and cyclophilin D-KO mice, both characterized by chronic disruption of MAM integrity, mitochondrial fission, and altered mitochondrial respiration. These data indicate that MAM contribute to the hepatic glucose-sensing system, allowing regulation of mitochondria dynamics and function during nutritional transition. Chronic disruption of MAM may participate in hepatic mitochondrial dysfunction associated with insulin resistance. © The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

Simulations and observations of plasma depletion, ion composition, and airglow emissions in two auroral ionospheric depletion experiments

International Nuclear Information System (INIS)

Yau, A.W.; Whalen, B.A.; Harris, F.R.; Gattinger, R.L.; Pongratz, M.B.; Bernhardt, P.A.

1985-01-01

In an ionospheric depletion experiment where chemically reactive vapors such as H 2 O and CO 2 are injected into the O + dominant F region to accelerate the plasma recombination rate and to reduce the plasma density, the ion composition in the depleted region is modified, and photometric emissions are produced. We compare in situ ion composition, density, and photometric measurements from two ionospheric depletion experiments with predictions from chemical modeling. The two injections, Waterhole I and III, were part of an auroral perturbation experiment and occurred in different ambient conditions. In both injections a core region of greater than fivefold plasma depletion was observed over roughly-equal5-km diameter within seconds of the injection, surrounded by an outer region of less drastic and slower depletion. In Waterhole I the plasma density was depleted tenfold over a 30-km diamter region after 2 min. The ambient O + density was drastically reduced, and the molecular O + 2 abundance was enhanced fivehold in the depletion region. OH airglow emission associated with the depletion was observed with a peak emission intensity of roughly-equal1 kR. In Waterhole III the ambient density was a decade lower, and the plasma depletion was less drastic, being twofold over 30 km after 2 min. The airglow emissions were also much less intense and below measurement sensitivity (30 R for the OH 306.4-nm emission; 50 R for the 630.0-nm emission)

Yeast-based biosensors: design and applications.

Science.gov (United States)

Adeniran, Adebola; Sherer, Michael; Tyo, Keith E J

2015-02-01

Yeast-based biosensing (YBB) is an exciting research area, as many studies have demonstrated the use of yeasts to accurately detect specific molecules. Biosensors incorporating various yeasts have been reported to detect an incredibly large range of molecules including but not limited to odorants, metals, intracellular metabolites, carcinogens, lactate, alcohols, and sugars. We review the detection strategies available for different types of analytes, as well as the wide range of output methods that have been incorporated with yeast biosensors. We group biosensors into two categories: those that are dependent upon transcription of a gene to report the detection of a desired molecule and those that are independent of this reporting mechanism. Transcription-dependent biosensors frequently depend on heterologous expression of sensing elements from non-yeast organisms, a strategy that has greatly expanded the range of molecules available for detection by YBBs. Transcription-independent biosensors circumvent the problem of sensing difficult-to-detect analytes by instead relying on yeast metabolism to generate easily detected molecules when the analyte is present. The use of yeast as the sensing element in biosensors has proven to be successful and continues to hold great promise for a variety of applications. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

Increased heme synthesis in yeast induces a metabolic switch from fermentation to respiration even under conditions of glucose repression.

Science.gov (United States)

Zhang, Tiantian; Bu, Pengli; Zeng, Joey; Vancura, Ales

2017-10-13

Regulation of mitochondrial biogenesis and respiration is a complex process that involves several signaling pathways and transcription factors as well as communication between the nuclear and mitochondrial genomes. Under aerobic conditions, the budding yeast Saccharomyces cerevisiae metabolizes glucose predominantly by glycolysis and fermentation. We have recently shown that altered chromatin structure in yeast induces respiration by a mechanism that requires transport and metabolism of pyruvate in mitochondria. However, how pyruvate controls the transcriptional responses underlying the metabolic switch from fermentation to respiration is unknown. Here, we report that this pyruvate effect involves heme. We found that heme induces transcription of HAP4 , the transcriptional activation subunit of the Hap2/3/4/5p complex, required for growth on nonfermentable carbon sources, in a Hap1p- and Hap2/3/4/5p-dependent manner. Increasing cellular heme levels by inactivating ROX1 , which encodes a repressor of many hypoxic genes, or by overexpressing HEM3 or HEM12 induced respiration and elevated ATP levels. Increased heme synthesis, even under conditions of glucose repression, activated Hap1p and the Hap2/3/4/5p complex and induced transcription of HAP4 and genes required for the tricarboxylic acid (TCA) cycle, electron transport chain, and oxidative phosphorylation, leading to a switch from fermentation to respiration. Conversely, inhibiting metabolic flux into the TCA cycle reduced cellular heme levels and HAP4 transcription. Together, our results indicate that the glucose-mediated repression of respiration in budding yeast is at least partly due to the low cellular heme level. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Bir1 Deletion Causes Malfunction of the Spindle Assembly Checkpoint and Apoptosis in Yeast

International Nuclear Information System (INIS)

Ren, Qun; Liou, Liang-Chun; Gao, Qiuqiang; Bao, Xiaoming; Zhang, Zhaojie

2012-01-01

Cell division in yeast is a highly regulated and well studied event. Various checkpoints are placed throughout the cell cycle to ensure faithful segregation of sister chromatids. Unexpected events, such as DNA damage or oxidative stress, cause the activation of checkpoint(s) and cell cycle arrest. Malfunction of the checkpoints may induce cell death. We previously showed that under oxidative stress, the budding yeast cohesin Mcd1, a homolog of human Rad21, was cleaved by the caspase-like protease Esp1. The cleaved Mcd1 C-terminal fragment was then translocated to mitochondria, causing apoptotic cell death. In the present study, we demonstrated that Bir1 plays an important role in spindle assembly checkpoint and cell death. Similar to H 2 O 2 treatment, deletion of BIR1 using a BIR1-degron strain caused degradation of the securin Pds1, which binds and inactivates Esp1 until metaphase-anaphase transition in a normal cell cycle. BIR1 deletion caused an increase level of ROS and mis-location of Bub1, a major protein for spindle assembly checkpoint. In wild type, Bub1 was located at the kinetochores, but was primarily in the cytoplasm in bir1 deletion strain. When BIR1 was deleted, addition of nocodazole was unable to retain the Bub1 localization on kinetochores, further suggesting that Bir1 is required to activate and maintain the spindle assembly checkpoint. Our study suggests that the BIR1 function in cell cycle regulation works in concert with its anti-apoptosis function.

Citrate and succinate uptake by potato mitochondria

International Nuclear Information System (INIS)

Jung, D.W.; Laties, G.G.

1979-01-01

Potato mitochondria, in the absence of respiration, have a very low capacity for uptake by exchange with endogenous anions, taking up only 2.4 nanomoles citrate and 2.0 nanomoles succinate per milligram protein. Maximum citrate uptake of over 17 nanomoles per milligram protein occurs in the presence of inorganic phosphate, a dicarboxylic acid, and an external energy source (NADH), conditions where net anion accumulation proceeds, mediated by the interlinking of the inorganic phosphate, dicarboxylate, and tricarboxylate carriers. Maximum succinate uptake in the absence of respiratory inhibitors requires only added inorganic phosphate. Compounds which inhibit respiration (antimycin), the exchange carriers (mersalyl and benzylmalonate), or the establishment of the membrane proton motive force (uncouplers) reduce substrate accumulation. A potent inhibitor of the citrate carrier in animal mitochondria, 1,2,3-benzenetricarboxylic acid, does not inhibit citrate uptake in potato mitochondria. Citrate uptake is reduced by concurrent ADP phosphorylation and this reduction is sensitive to oligomycin. The initiation of state 3 after a 3-minute substrate state results in a reduction of the steady-state of citrate uptake by approximately 50%. Accumulation of succinate initially is inhibited by increasing sucrose concentration in the reaction medium from 50 to 400 millimolar. Limited substrate uptake is one of the factors responsible for the often observed depressed initial state 3 respiration rates in many mitochondrial preparations. Since nonlimiting levels of substrate in the matrix cannot be attained by energy-independent exchange, a dependence on respiration for adequate uptake results. Substrate limitation therefore occurs in the matrix for the period of time needed for energy-dependent accumulation of nonlimiting levels

New Insights in the Amyloid-Beta Interaction with Mitochondria

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Carlos Spuch

2012-01-01

Full Text Available Biochemical and morphological alterations of mitochondria may play an important role in the pathogenesis of Alzheimer’s disease (AD. Particularly, mitochondrial dysfunction is a hallmark of amyloid-beta-induced neuronal toxicity in Alzheimer’s disease. The recent emphasis on the intracellular biology of amyloid-beta and its precursor protein (APP has led researchers to consider the possibility that mitochondria-associated and mitochondrial amyloid-beta may directly cause neurotoxicity. Both proteins are known to localize to mitochondrial membranes, block the transport of nuclear-encoded mitochondrial proteins to mitochondria, interact with mitochondrial proteins, disrupt the electron transport chain, increase reactive oxygen species production, cause mitochondrial damage, and prevent neurons from functioning normally. In this paper, we will outline current knowledge of the intracellular localization of amyloid-beta. Moreover, we summarize evidence from AD postmortem brain as well as animal AD models showing that amyloid-beta triggers mitochondrial dysfunction through a number of pathways such as impairment of oxidative phosphorylation, elevation of reactive oxygen species production, alteration of mitochondrial dynamics, and interaction with mitochondrial proteins. Thus, this paper supports the Alzheimer cascade mitochondrial hypothesis such as the most important early events in this disease, and probably one of the future strategies on the therapy of this neurodegenerative disease.

Molecular Strategies for Targeting Antioxidants to Mitochondria: Therapeutic Implications

Science.gov (United States)

2015-01-01

Abstract Mitochondrial function and specifically its implication in cellular redox/oxidative balance is fundamental in controlling the life and death of cells, and has been implicated in a wide range of human pathologies. In this context, mitochondrial therapeutics, particularly those involving mitochondria-targeted antioxidants, have attracted increasing interest as potentially effective therapies for several human diseases. For the past 10 years, great progress has been made in the development and functional testing of molecules that specifically target mitochondria, and there has been special focus on compounds with antioxidant properties. In this review, we will discuss several such strategies, including molecules conjugated with lipophilic cations (e.g., triphenylphosphonium) or rhodamine, conjugates of plant alkaloids, amino-acid- and peptide-based compounds, and liposomes. This area has several major challenges that need to be confronted. Apart from antioxidants and other redox active molecules, current research aims at developing compounds that are capable of modulating other mitochondria-controlled processes, such as apoptosis and autophagy. Multiple chemically different molecular strategies have been developed as delivery tools that offer broad opportunities for mitochondrial manipulation. Additional studies, and particularly in vivo approaches under physiologically relevant conditions, are necessary to confirm the clinical usefulness of these molecules. Antioxid. Redox Signal. 22, 686–729. PMID:25546574

Research in fermentation

Energy Technology Data Exchange (ETDEWEB)

Mills, A K

1966-01-01

The following aspects of the biochemistry of fermentation were discussed: carbohydrate, amino acid, S, and phosphate metabolisms in the yeast cell; pantothenic acid and biotin as the essential growth factors in yeast metabolisms; effects of different aeration conditions on yeast growth, mitochondria development, and lipid contents. Gas chromatographic studies of fermentation products are also discussed.

Cellular characterization of human dermal fibroblasts, focus on mitochondria and maple syrup urine disease

DEFF Research Database (Denmark)

Fernandez-Guerra, Paula

and functions are expressed in HDFs’ culture environment. Studies of molecular disease mechanisms often point to the involvement of mitochondria. Mitochondria are involved in the regulation of cell cycle and programmed cell death as well as cellular stress responses because they are the main producers...

EST analysis on pig mitochondria reveal novel expression differences between developmental and adult tissues

DEFF Research Database (Denmark)

Scheibye-Alsing, Karsten; Cirera, Susanna; Gilchrist, Michael J.

2007-01-01

BACKGROUND: The mitochondria are involved in many basic functions in cells of vertebrates, and can be considered the power generator of the cell. Though the mitochondria have been extensively studied there appear to be only few expression studies of mitochondrial genes involving a large number...

Mitochondria: Target organelles for estrogen action

Directory of Open Access Journals (Sweden)

Małgorzata Chmielewska

2017-06-01

Full Text Available Estrogens belong to a group of sex hormones, which have been shown to act in multidirectional way. Estrogenic effects are mediated by two types of intracellular receptors: estrogen receptor 1 (ESR1 and estrogen receptor 2 (ESR2. There are two basic mechanisms of estrogen action: 1 classical-genomic, in which the ligand-receptor complex acts as a transcriptional factor and 2 a nongenomic one, which is still not fully understood, but has been seen to lead to distinct biological effects, depending on tissue and ligand type. It is postulated that nongenomic effects may be associated with membrane signaling and the presence of classical nuclear receptors within the cell membrane. Estrogens act in a multidirectional way also within cell organelles. It is assumed that there is a mechanism which manages the migration of ESR into the mitochondrial membrane, wherein the exogenous estrogen affect the morphology of mitochondria. Estrogen, through its receptor, can directly modulate mitochondrial gene expression. Moreover, by regulating the level of reactive oxygen species, estrogens affect the biology of mitochondria. The considerations presented in this paper indicate the pleiotropic effects of estrogens, which represent a multidirectional pathway of signal transduction.

Too Depleted to Try? Testing the Process Model of Ego Depletion in the Context of Unhealthy Snack Consumption.

Science.gov (United States)

Haynes, Ashleigh; Kemps, Eva; Moffitt, Robyn

2016-11-01

The process model proposes that the ego depletion effect is due to (a) an increase in motivation toward indulgence, and (b) a decrease in motivation to control behaviour following an initial act of self-control. In contrast, the reflective-impulsive model predicts that ego depletion results in behaviour that is more consistent with desires, and less consistent with motivations, rather than influencing the strength of desires and motivations. The current study sought to test these alternative accounts of the relationships between ego depletion, motivation, desire, and self-control. One hundred and fifty-six undergraduate women were randomised to complete a depleting e-crossing task or a non-depleting task, followed by a lab-based measure of snack intake, and self-report measures of motivation and desire strength. In partial support of the process model, ego depletion was related to higher intake, but only indirectly via the influence of lowered motivation. Motivation was more strongly predictive of intake for those in the non-depletion condition, providing partial support for the reflective-impulsive model. Ego depletion did not affect desire, nor did depletion moderate the effect of desire on intake, indicating that desire may be an appropriate target for reducing unhealthy behaviour across situations where self-control resources vary. © 2016 The International Association of Applied Psychology.

Prevalence of candida and non-candida yeasts isolated from patients with yeast fungal infections in Tehran labs

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Hashemi SJ

2011-04-01

Full Text Available "n 800x600 Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman","serif";} Background: Infections caused by opportunistic yeasts such as Candida species, Trichosporon, Rhodotorula and Saccharomyces have increased in immunocompromis-ed patients and their identification is crucial as intrinsic and acquired resistance of some yeast species to antifungal agents are on the rise. The aim of this study was to identify the organisms to the species level in order to suggest accurate and effective antifungal therapies."n"nMethods: In this study that carried out in Tehran, Iran in 2009, 200 patients with yeast infection were medically examined and clinical specimens were prepared for direct examination and culture on Sabouraud dextrose agar. Subsequently, the isolated yeast colonies were identified using various tests including culture on Corn Meal agar with Tween 80, CHROMagar Candida and casein agar. For the definite identification of organisms some biochemical tests were done based on carbohydrate assimilation by RapID Yeast Plus System kit, and, finally, a molecular method, PCR-RFLP, using Hpa II enzyme, was performed for the remaining unknown yeast species."n"nResults: A total of 211 yeast isolates were identified in 200 patients with yeast infections. The most frequent isolated yeasts were Candida albicans, 124 (58.77%, followed by Candida parapsilosis, 36 (17.06%, Candida tropicalis, 17 (8.06%, Candida glabrata, 13 (6.16%, Candida krusei, 8 (3.79%, Candida guilliermondii, 2 (0.96%, Trichosporon, 3 (1.14%, Rhodotorula, 1 (0.47%, Saccaromyces cerevisiae, 1 (0.47% and other

Oral yeast colonization throughout pregnancy.

Science.gov (United States)

Rio, R; Simões-Silva, L; Garro, S; Silva, M-J; Azevedo, Á; Sampaio-Maia, B

2017-03-01

Recent studies suggest that placenta may harbour a unique microbiome that may have origin in maternal oral microbiome. Although the major physiological and hormonal adjustments observed in pregnant women lead to biochemical and microbiological modifications of the oral environment, very few studies evaluated the changes suffered by the oral microbiota throughout pregnancy. So, the aim of our study was to evaluate oral yeast colonization throughout pregnancy and to compare it with non-pregnant women. The oral yeast colonization was assessed in saliva of 30 pregnant and non-pregnant women longitudinally over a 6-months period. Demographic information was collected, a non-invasive intra-oral examination was performed and saliva flow and pH were determined. Pregnant and non-pregnant groups were similar regarding age and level of education. Saliva flow rate did not differ, but saliva pH was lower in pregnant than in non-pregnant women. Oral yeast prevalence was higher in pregnant than in non-pregnant women, either in the first or in the third trimester, but did not attain statistical significance. In individuals colonized with yeast, the total yeast quantification (Log10CFU/mL) increase from the 1st to the 3rd trimester in pregnant women, but not in non-pregnant women. Pregnancy may favour oral yeast growth that may be associated with an acidic oral environment.

Biotechnological Applications of Dimorphic Yeasts

Science.gov (United States)

Doiphode, N.; Joshi, C.; Ghormade, V.; Deshpande, M. V.

The dimorphic yeasts have the equilibrium between spherical growth (budding) and polarized (hyphal or pseudohyphal tip elongation) which can be triggered by change in the environmental conditions. The reversible growth phenomenon has made dimorphic yeasts as an useful model to understand fungal evolution and fungal differentiation, in general. In nature dimorphism is clearly evident in plant and animal fungal pathogens, which survive and most importantly proliferate in the respective hosts. However, number of organisms with no known pathogenic behaviour also show such a transition, which can be exploited for the technological applications due to their different biochemical make up under different morphologies. For instance, chitin and chitosan production using dimorphic Saccharomyces, Mucor, Rhizopus and Benjaminiella, oil degradation and biotransformation with yeast-form of Yarrowia species, bioremediation of organic pollutants, exopolysac-charide production by yeast-phase of Aureobasidium pullulans, to name a few. Myrothecium verrucaria can be used for seed dressing in its yeast form and it produces a mycolytic enzyme complex in its hyphal-form for the biocontrol of fungal pathogens, while Beauveria bassiana and other entomopathogens kill the insect pest by producing yeast- like cells in the insect body. The form-specific expression of protease, chitinase, lipase, ornithine decarboxylase, glutamate dehydrogenases, etc. make Benjaminiella poitrasii, Basidiobolus sp., and Mucor rouxii strains important in bioremediation, nanobiotechnology, fungal evolution and other areas.

The Toxicity of Depleted Uranium

OpenAIRE

Briner, Wayne

2010-01-01

Depleted uranium (DU) is an emerging environmental pollutant that is introduced into the environment primarily by military activity. While depleted uranium is less radioactive than natural uranium, it still retains all the chemical toxicity associated with the original element. In large doses the kidney is the target organ for the acute chemical toxicity of this metal, producing potentially lethal tubular necrosis. In contrast, chronic low dose exposure to depleted uranium may not produce a c...

Amyloid-β triggers the release of neuronal hexokinase 1 from mitochondria.

Directory of Open Access Journals (Sweden)

Leonardo M Saraiva

2010-12-01

Full Text Available Brain accumulation of the amyloid-β peptide (Aβ and oxidative stress underlie neuronal dysfunction and memory loss in Alzheimer's disease (AD. Hexokinase (HK, a key glycolytic enzyme, plays important pro-survival roles, reducing mitochondrial reactive oxygen species (ROS generation and preventing apoptosis in neurons and other cell types. Brain isozyme HKI is mainly associated with mitochondria and HK release from mitochondria causes a significant decrease in enzyme activity and triggers oxidative damage. We here investigated the relationship between Aβ-induced oxidative stress and HK activity. We found that Aβ triggered HKI detachment from mitochondria decreasing HKI activity in cortical neurons. Aβ oligomers further impair energy metabolism by decreasing neuronal ATP levels. Aβ-induced HKI cellular redistribution was accompanied by excessive ROS generation and neuronal death. 2-deoxyglucose blocked Aβ-induced oxidative stress and neuronal death. Results suggest that Aβ-induced cellular redistribution and inactivation of neuronal HKI play important roles in oxidative stress and neurodegeneration in AD.

Edaravone Decreases Paraquat Toxicity in A549 Cells and Lung Isolated Mitochondria

OpenAIRE

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 int...

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

International Nuclear Information System (INIS)

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

2011-01-01

Highlights: → We screened and identified Tyrphostin A9, a receptor tyrosine kinase inhibitor as a strong mitochondria fission inducer. → Tyrphostin A9 treatment promotes mitochondria dysfunction and contributes to cytotoxicity in cancer cells. → Tyrphostin A9 induces apoptotic cell death through a Drp1-mediated pathway. → 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.

Biotechnological production of high specific activity L-35S-cysteine and L-35S-methionine by using a diploid yeast Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Gajendiran, N.; Jayachandran, N.; Unny, V.K.P.; Thyagarajan, S.; Rao, B.S.

1994-01-01

High specific activity L- 3 5 S-cysteine and L- 35 S-methionine were synthesised by using a wild type diploid strain of baker's yeast-Saccharomyces cerevisiae. Yeast cells were grown in a sulphur depleted synthetic medium in which Na 2 3 5 SO 4 (50 mCi/ml) was supplemented as the sole sulphur source. The level of incorporation was 60% on an average. The protein hydrolysate of the cultured cells was subjected to paper and column chromatographic separations to get the individual L- 3 5 S-aminoacids. The radiochemical yields of cysteine and methionine were 6-7% and 18-20% respectively. The radiochemical purity of the products was >95%. The highest specific activity for the products obtained by employing this method was 1100 Ci/mmole from the starting material, Na 2 35 SO 4 , with a specific activity of 1350 Ci/mmole. (Author)

Interactions between Drosophila and its natural yeast symbionts-Is Saccharomyces cerevisiae a good model for studying the fly-yeast relationship?

Science.gov (United States)

Hoang, Don; Kopp, Artyom; Chandler, James Angus

2015-01-01

Yeasts play an important role in the biology of the fruit fly, Drosophila melanogaster. In addition to being a valuable source of nutrition, yeasts affect D. melanogaster behavior and interact with the host immune system. Most experiments investigating the role of yeasts in D. melanogaster biology use the baker's yeast, Saccharomyces cerevisiae. However, S. cerevisiae is rarely found with natural populations of D. melanogaster or other Drosophila species. Moreover, the strain of S. cerevisiae used most often in D. melanogaster experiments is a commercially and industrially important strain that, to the best of our knowledge, was not isolated from flies. Since disrupting natural host-microbe interactions can have profound effects on host biology, the results from D. melanogaster-S. cerevisiae laboratory experiments may not be fully representative of host-microbe interactions in nature. In this study, we explore the D. melanogaster-yeast relationship using five different strains of yeast that were isolated from wild Drosophila populations. Ingested live yeasts have variable persistence in the D. melanogaster gastrointestinal tract. For example, Hanseniaspora occidentalis persists relative to S. cerevisiae, while Brettanomyces naardenensis is removed. Despite these differences in persistence relative to S. cerevisiae, we find that all yeasts decrease in total abundance over time. Reactive oxygen species (ROS) are an important component of the D. melanogaster anti-microbial response and can inhibit S. cerevisiae growth in the intestine. To determine if sensitivity to ROS explains the differences in yeast persistence, we measured yeast growth in the presence and absence of hydrogen peroxide. We find that B. naardenesis is completely inhibited by hydrogen peroxide, while H. occidentalis is not, which is consistent with yeast sensitivity to ROS affecting persistence within the D. melanogaster gastrointestinal tract. We also compared the feeding preference of D

Polyhydroxybutyrate Targets Mammalian Mitochondria and Increases Permeability of Plasmalemmal and Mitochondrial Membranes

Science.gov (United States)

Elustondo, Pia A.; Angelova, Plamena R.; Kawalec, Michał; Michalak, Michał; Kurcok, Piotr; Abramov, Andrey Y.; Pavlov, Evgeny V.

2013-01-01

Poly(3-hydroxybutyrate) (PHB) is a polyester of 3-hydroxybutyric acid (HB) that is ubiquitously present in all organisms. In higher eukaryotes PHB is found in the length of 10 to 100 HB units and can be present in free form as well as in association with proteins and inorganic polyphosphate. It has been proposed that PHB can mediate ion transport across lipid bilayer membranes. We investigated the ability of PHB to interact with living cells and isolated mitochondria and the effects of these interactions on membrane ion transport. We performed experiments using a fluorescein derivative of PHB (fluo-PHB). We found that fluo-PHB preferentially accumulated inside the mitochondria of HeLa cells. Accumulation of fluo-PHB induced mitochondrial membrane depolarization. This membrane depolarization was significantly delayed by the inhibitor of the mitochondrial permeability transition pore - Cyclosporin A. Further experiments using intact cells as well as isolated mitochondria confirmed that the effects of PHB directly linked to its ability to facilitate ion transport, including calcium, across the membranes. We conclude that PHB demonstrates ionophoretic properties in biological membranes and this effect is most profound in mitochondria due to the selective accumulation of the polymer in this organelle. PMID:24086638

Deuterium-depleted water. Romanian achievements and perspective

International Nuclear Information System (INIS)

Stefanescu, Ion; Saros-Rogobete, Irina; Titescu, Gheorghe

2001-01-01

Deuterium-depleted water has an isotopic content smaller than 145 ppm D/(D+H) which is the natural isotopic content of water. Beginning with 1996 ICSI Rm. Valcea, deuterium-depleted water producer, co-operated with Romanian specialized institutes for biological effect's evaluation of deuterium-depleted water. These investigations lead to the following conclusions: - Deuterium-depleted water caused a tendency towards the increase of the basal tonus, accompanied by the intensification of the vasoconstrictor effects of phenylefrine, noradrenaline and angiotensin; the increase of the basal tonus and vascular reactivity produced by the deuterium-depleted water persist after the removal of the vascular endothelium; - Animals treated with deuterium-depleted water showed an increase of the resistance both to sublethal and to lethal gamma radiation doses, suggesting a radioprotective action; - Deuterium-depleted water stimulates immune defence reactions and increases the numbers of polymorphonuclear neutrophils; - Investigations regarding artificial reproduction of fish with deuterium-depleted water fecundated solutions confirmed favourable influence in embryo growth stage and resistance in subsequent growth stages; - It was studied germination, growth and quantitative character's variability in plants; one can remark the favourable influence of deuterium-depleted water on biological process in plants in various ontogenetic stages; - The deuterium depletion in seawater produces the diminution of the water spectral energy related to an increased metabolism of Tetraselmis Suecica. (authors)

Characterization of DNA-binding proteins from pea mitochondria

DEFF Research Database (Denmark)

Hatzack, F.A.; Dombrowski, S.; Brennicke, A.

1998-01-01

We studied transcription initiation in the mitochondria of higher plants, with particular respect to promoter structures. Conserved elements of these promoters have been successfully identified by in vitro transcription systems in different species, whereas the involved protein components are still...

Genomics and the making of yeast biodiversity.

Science.gov (United States)

Hittinger, Chris Todd; Rokas, Antonis; Bai, Feng-Yan; Boekhout, Teun; Gonçalves, Paula; Jeffries, Thomas W; Kominek, Jacek; Lachance, Marc-André; Libkind, Diego; Rosa, Carlos A; Sampaio, José Paulo; Kurtzman, Cletus P

2015-12-01

Yeasts are unicellular fungi that do not form fruiting bodies. Although the yeast lifestyle has evolved multiple times, most known species belong to the subphylum Saccharomycotina (syn. Hemiascomycota, hereafter yeasts). This diverse group includes the premier eukaryotic model system, Saccharomyces cerevisiae; the common human commensal and opportunistic pathogen, Candida albicans; and over 1000 other known species (with more continuing to be discovered). Yeasts are found in every biome and continent and are more genetically diverse than angiosperms or chordates. Ease of culture, simple life cycles, and small genomes (∼10-20Mbp) have made yeasts exceptional models for molecular genetics, biotechnology, and evolutionary genomics. Here we discuss recent developments in understanding the genomic underpinnings of the making of yeast biodiversity, comparing and contrasting natural and human-associated evolutionary processes. Only a tiny fraction of yeast biodiversity and metabolic capabilities has been tapped by industry and science. Expanding the taxonomic breadth of deep genomic investigations will further illuminate how genome function evolves to encode their diverse metabolisms and ecologies. Copyright © 2015 Elsevier Ltd. All rights reserved.

Accelerating Yeast Prion Biology using Droplet Microfluidics

Science.gov (United States)

Ung, Lloyd; Rotem, Assaf; Jarosz, Daniel; Datta, Manoshi; Lindquist, Susan; Weitz, David

2012-02-01

Prions are infectious proteins in a misfolded form, that can induce normal proteins to take the misfolded state. Yeast prions are relevant, as a model of human prion diseases, and interesting from an evolutionary standpoint. Prions may also be a form of epigenetic inheritance, which allow yeast to adapt to stressful conditions at rates exceeding those of random mutations and propagate that adaptation to their offspring. Encapsulation of yeast in droplet microfluidic devices enables high-throughput measurements with single cell resolution, which would not be feasible using bulk methods. Millions of populations of yeast can be screened to obtain reliable measurements of prion induction and loss rates. The population dynamics of clonal yeast, when a fraction of the cells are prion expressing, can be elucidated. Furthermore, the mechanism by which certain strains of bacteria induce yeast to express prions in the wild can be deduced. Integrating the disparate fields of prion biology and droplet microfluidics reveals a more complete picture of how prions may be more than just diseases and play a functional role in yeast.

Capillary electrophoretic analysis reveals subcellular binding between individual mitochondria and cytoskeleton

Science.gov (United States)

Kostal, Vratislav; Arriaga, Edgar A.

2011-01-01

Interactions between the cytoskeleton and mitochondria are essential for normal cellular function. An assessment of such interactions is commonly based on bulk analysis of mitochondrial and cytoskeletal markers present in a given sample, which assumes complete binding between these two organelle types. Such measurements are biased because they rarely account for non-bound ‘free’ subcellular species. Here we report on the use of capillary electrophoresis with dual laser induced fluorescence detection (CE-LIF) to identify, classify, count and quantify properties of individual binding events of mitochondria and cytoskeleton. Mitochondria were fluorescently labeled with DsRed2 while F-actin, a major cytoskeletal component, was fluorescently labeled with Alexa488-phalloidin. In a typical subcellular fraction of L6 myoblasts, 79% of mitochondrial events did not have detectable levels of F-actin, while the rest had on average ~2 zeptomole F-actin, which theoretically represents a ~ 2.5-μm long network of actin filaments per event. Trypsin treatment of L6 subcellular fractions prior to analysis decreased the fraction of mitochondrial events with detectable levels of F-actin, which is expected from digestion of cytoskeletal proteins on the surface of mitochondria. The electrophoretic mobility distributions of the individual events were also used to further distinguish between cytoskeleton-bound from cytoskeleton-free mitochondrial events. The CE-LIF approach described here could be further developed to explore cytoskeleton interactions with other subcellular structures, the effects of cytoskeleton destabilizing drugs, and the progression of viral infections. PMID:21309532

Nucleo-mitochondrial interaction of yeast in response to cadmium sulfide quantum dot exposure

International Nuclear Information System (INIS)

Pasquali, Francesco; Agrimonti, Caterina; Pagano, Luca; Zappettini, Andrea; Villani, Marco; Marmiroli, Marta; White, Jason C.; Marmiroli, Nelson

2017-01-01

Highlights: • CdS QDs induce oxidative stress in yeast. • CdS QDs disrupt mitochondrial membrane potentials and morphology. • CdS QDs do not affect mtDNA content. • CdS QDs modify the expression of genes involved in mitochondrial organization and function. • Deletion of some of these genes induces either tolerant or sensitive phenotypes to CdS QDs. - Abstract: Cell sensitivity to quantum dots (QDs) has been attributed to a cascade triggered by oxidative stress leading to apoptosis. The role and function of mitochondria in animal cells are well understood but little information is available on the complex genetic networks that regulate nucleo-mitochondrial interaction. The effect of CdS QD exposure in yeast Saccharomyces cerevisiae was assessed under conditions of limited lethality (<10%), using cell physiological and morphological endpoints. Whole-genomic array analysis and the screening of a deletion mutant library were also carried out. The results showed that QDs: increased the level of reactive oxygen species (ROS) and decreased the level of reduced vs oxidized glutathione (GSH/GSSG); reduced oxygen consumption and the abundance of respiratory cytochromes; disrupted mitochondrial membrane potentials and affected mitochondrial morphology. Exposure affected the capacity of cells to grow on galactose, which requires nucleo-mitochondrial involvement. However, QDs exposure did not materially induce respiratory deficient (RD) mutants but only RD phenocopies. All of these cellular changes were correlated with several key nuclear genes, including TOM5 and FKS1, involved in the maintenance of mitochondrial organization and function. The consequences of these cellular effects are discussed in terms of dysregulation of cell function in response to these “pathological mitochondria”.

Nucleo-mitochondrial interaction of yeast in response to cadmium sulfide quantum dot exposure

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