Disruption of fusion results in mitochondrial heterogeneity and dysfunction

Mitochondria undergo continual cycles of fusion and fission, and the balance of these opposing processes regulates mitochondrial morphology. Paradoxically, cells invest many resources to maintain tubular mitochondrial morphology, when reducing both fusion and fission simultaneously achieves the same end. This observation suggests a requirement for mitochondrial fusion, beyond maintenance of organelle morphology. Here, we show that cells with targeted null mutations in Mfn1 or Mfn2 retained low levels of mitochondrial fusion and escaped major cellular dysfunction. Analysis of these mutant cells showed that both homotypic and heterotypic interactions of Mfns are capable of fusion. In contrast, cells lacking both Mfn1 and Mfn2 completely lacked mitochondrial fusion and showed severe cellular defects, including poor cell growth, widespread heterogeneity of mitochondrial membrane potential, and decreased cellular respiration. Disruption of OPA1 by RNAi also blocked all mitochondrial fusion and resulted in similar cellular defects. These defects in Mfn-null or OPA1-RNAi mammalian cells were corrected upon restoration of mitochondrial fusion, unlike the irreversible defects found in fzo yeast. In contrast, fragmentation of mitochondria, without severe loss of fusion, did not result in such cellular defects. Our results showed that key cellular functions decline as mitochondrial fusion is progressively abrogated

Alignment of the amino terminal amino acid sequence of human cytochrome c oxidase subunits I and II with the sequence of their putative mRNAs

Thirteen of the first fifteen amino acids from the NH2-terminus of the primary sequence of human cytochrome c oxidase subunit I and eleven of the first twelve amino acids of subunit II have been identified by microsequencing procedures. These sequences have been compared with the recently determined 5'-end proximal sequences of the HeLa cell mitochondrial mRNAS and unambiguously aligned with two of them. This alignment has allowed the identification of the putative mRNA for subunit I, and has shown that the initiator codon for this subunit is only three nucleotides away from the 5'-end of its mRNA; furthermore, the results have substantiated the idea that the translation of human cytochrome c oxidase subunit II starts directly at the 5'-end of its putative mRNA, as had been previously inferred on the basis of the sequence homology of human mitochondrial DNA with the primary sequence of the bovine subunit

Synthese, Charakterisierung und dispergierende Eigenschaften von anionischen und zwitterionischen Polycarboxylat-Fließmitteln hergestellt mittels unterschiedlicher Synthesemethoden

The impact of different synthesis methods of anionic and zwitterionic polycarboxylate superplasticizers (PCEs) on their composition and their dispersing effect in cement was investigated. The used synthesis method influenced the different PCE properties (sulfate and clay tolerance). Furthermore, the impact of the pH-value (pH = 1.5 or 7.0) of a PCE-solution on the dispersing effectiveness was examined. Here acidic, strongly negative PCEs required a smaller dosage for same dispersing effect than their neutralized counterpart.Die Auswirkung von verschiedenen Synthesemethoden von anionischen und zwitterionischen Polycarboxylat-Fließmitteln (PCEs) auf deren Zusammensetzung und deren Dispergierwirkung in Zement wurden untersucht. Je nach verwendeter Synthesemethode wiesen die PCEs unterschiedliche Eigenschaften (Sulfat-, Tonbeständigkeit) auf. Desweiteren wurde der Einfluss des pH-Werts (pH = 1.5 oder 7.0) einer PCE-Lösung auf deren Fließwirkung erforscht. Saure, stark anionische PCEs benötigten eine geringere Dosierung für die gleiche Dispergierwirkung als die neutralisierten Lösungen

The Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like Episode Syndrome-associated Human Mitochondrial tRNALeu(UUR) Mutation Causes Aminoacylation Deficiency and Concomitant Reduced Association of mRNA with Ribosomes

The pathogenetic mechanism of the mitochondrial tRNALeu(UUR) A3243G transition associated with the mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome has been investigated in transmitochondrial cell lines constructed by transfer of mutant mitochondrial DNA (mtDNA)-carrying mitochondria from three genetically unrelated MELAS patients or of isogenic wild-type mtDNA-carrying organelles into human mtDNA-less cells. An in vivo footprinting analysis of the mtDNA segment within the tRNALeu(UUR) gene that binds the transcription termination factor failed to reveal any difference in occupancy of sites or qualitative interaction with the protein between mutant and wild-type mtDNAs. Cell lines nearly homoplasmic for the mutation exhibited a strong (70-75%) reduction in the level of aminoacylated tRNALeu(UUR) and a decrease in mitochondrial protein synthesis rate. The latter, however, did not show any significant correlation between synthesis defect of the individual polypeptides and number or proportion of UUR codons in their mRNAs, suggesting that another step, other than elongation, may be affected. Sedimentation analysis in sucrose gradient showed a reduction in size of the mitochondrial polysomes, while the distribution of the two rRNA components and of the mRNAs revealed decreased association of mRNA with ribosomes and, in the most affected cell line, pronounced degradation of the mRNA associated with slowly sedimenting structures. Therefore, several lines of evidence indicate that the protein synthesis defect in A3243G MELAS mutation-carrying cells is mainly due to a reduced association of mRNA with ribosomes, possibly as a consequence of the tRNALeu(UUR) aminoacylation defect

Mitochondrial Outer Membrane Permeability Change and Hypersensitivity to Digitonin Early in Staurosporine-induced Apoptosis

We have shown here that the apoptosis inducer staurosporine causes an early decrease in the endogenous respiration rate in intact 143B.TK- cells. On the other hand, the activity of cytochrome c oxidase is unchanged for the first 8 h after staurosporine treatment, as determined by oxygen consumption measurements in intact cells. The decrease in the endogenous respiration rate precedes the release of cytochrome c from mitochondria. Moreover, we have ruled out caspases, permeability transition, and protein kinase C inhibition as being responsible for the decrease in respiration rate. Furthermore, overexpression of the gene for Bcl-2 does not prevent the decrease in respiration rate. The last finding suggests that Bcl-2 acts downstream of the perturbation in respiration. The evidence of normal enzymatic activities of complex I and complex III in staurosporine-treated 143B.TK- osteosarcoma cells indicates that the cause of the respiration decrease is probably an alteration in the permeability of the outer mitochondrial membrane. Presumably, the voltage-dependent anion channel closes, thereby preventing ADP and oxidizable substrates from being taken up into mitochondria. This interpretation was confirmed by another surprising finding, namely that, in staurosporine-treated 143B.TK- cells permeabilized with digitonin at a concentration not affecting the mitochondrial membranes in naive cells, the outer mitochondrial membrane loses its integrity; this leads to a reversal of its impermeability to exogenous substrates. The loss of outer membrane integrity leads also to a massive premature release of cytochrome c from mitochondria. Most significantly, Bcl-2 overexpression prevents the staurosporine-induced hypersensitivity of the outer membrane to digitonin. Our experiments have thus revealed early changes in the outer mitochondrial membrane, which take place long before cytochrome c is released from mitochondria in intact cells

Contamined lands: a Canadian perspective

The existence of contaminated sites in Canada has become a problem of nation-wide concern. Actions at civil and common law based on the traditional requirements of showing that property interests have been affected or personal injury has resulted are inadequate to address widespread harms arising from pollution. At the national level programs and policies have been developed to address clean-up of contaminated sites. At the provincial level legislation is being developed, directed at making persons responsible for the pollution they cause. Nonetheless, there are shortcomings under the present system in matters concerning victim redress and clean-up and restoration of contaminated sites. Victims are still struggling to obtain redress and compensation, especially in cases of defendant bankruptcy. It may be necessary as in the U.S. to create a Superfund to ensure compensation when there are orphan sites or when the defendant has become insolvent. There may be merit in establishing at the Federal level and in the other provinces a class action scheme along the lines of the Quebec model with an Assistance Fund to help litigants. In addition, there is a need to develop in legislation comprehensive requirements of clean-up and restoration of contaminated sites, that can be applied consistently nation-wide.La question des sols contaminés au Canada est devenue une question d'intérêt national. Les recours du droit civil et du droit commun, qui requièrent un dommage à la propriété ou un préjudice personnel, ne sont plus adéquats pour faire face aux dommages considérables qui résultent de la pollution. Au niveau national, des politiques et des programmes de décontamination des sols ont été élaborés. Au niveau provincial, des efforts législatifs sont en cours et visent à imputer la responsabilité aux pollueurs. Toutefois, le système en place accuse certaines faiblesses particulièrement en ce qui a trait aux recours des victimes ainsi qu'à la décontamination et restauration des lieux contaminés. Les victimes doivent lutter pour obtenir une indemnisation surtout lorsque le défendeur fait faillite. Il peut s'avérer nécessaire, à l'instar de ce qui s'est fait aux États-Unis, de créer un fonds spécial d'indemnisation dans les cas des sites orphelins ou lorsque le défendeur devient insolvable. Il pourrait être utile au niveau fédéral et dans les autres provinces canadiennes d'instaurer un mécanisme de recours collectif comme celui qui existe au Québec de même qu'un fonds d'aide pour soutenir financièrement ceux qui exercent un tel recours. Au surplus, il importe de légiférer pour établir des standards de décontamination et de restauration des lieux contaminés qui puissent s'appliquer à l'échelle nationale

The site of synthesis of the iron-sulfur subunits of the flavoprotein and iron-protein fractions of human NADH dehydrogenase

The site of synthesis of the iron-sulfur subunits of the flavoprotein and iron-protein fractions of the human respiratory chain NADH dehydrogenase has been investigated to test the possibility that any of them is synthesized in mitochondria. For this purpose, antibodies specific for individual subunits of the bovine enzyme, which cross- reacted with the homologous human subunits in immunoblot assays, were tested against HeLa cell mitochondrial proteins labeled in vivo with [35S]methionine in the absence or presence of inhibitors of mitochondrial or cytoplasmic protein synthesis. The results clearly indicated that all the iron-sulfur subunits of the flavoprotein and iron-protein fractions of human complex I are synthesized in the cytosol and are, therefore, encoded in nuclear genes

Working Through It: Reimagining Grief and Bereavement in the Workplace Post COVID-19

Grief and bereavement in the workplace is a complex endeavour for employees and management to navigate at the best of times. We live in a death-denying society which keeps us from knowing what to say or do in the face of loss, which is complicated by the work-home divide that discourages employees from bringing personal issues into the professional realm. However, the events of the COVID-19 pandemic have affected both these elements because it has put death, loss, and grief at the forefront of consciousness, and blurred the lines between personal and professional as employees moved from the office to working from home. This shift presents an opportunity to rethink how organizations approach grief and bereavement in the workplace. There has not been a great deal of research devoted to this topic, even though most people in the workplace will be affected by a significant loss at some point in their career, and the impacts of grief and loss can have negative consequences on work ability and the workplace. The losses and grief brought on by the pandemic are complicated and likely to have long-standing impacts on the mental health of employees. In order to adequately support employees, build resilience, and continue to operate effectively, organizations need to reconsider and invest in grief and bereavement support strategies. This project utilizes user interviews, systems analysis tools, and a design thinking process to explore possible interventions that can be used in the workplace to enable more effective grief and bereavement support