NMR Structural and Biophysical Analysis of the Disease-Linked Inner Mitochondrial Membrane Protein MPV17

MPV17 is an integral inner mitochondrial membrane protein, whose loss-of-function is linked to the hepatocerebral form of the mitochondrial-DNA-depletion syndrome, leading to a tissue-specific reduction of mitochondrial DNA and organ failure in infants. Several disease-causing mutations in MPV17 have been identified and earlier studies with reconstituted protein suggest that MPV17 forms a high conductivity channel in the membrane. However, the molecular and structural basis of the MPV17 functionality remain only poorly understood. In order to make MPV17 accessible to high-resolution structural studies, we here present an efficient protocol for its high-level production in E. coli and refolding into detergent micelles. Using biophysical and NMR methods, we show that refolded MPV17 in detergent micelles adopts a compact structure consisting of six membrane-embedded α-helices. Furthermore, we demonstrate that MPV17 forms oligomers in a lipid bilayer that are further stabilized by disulfide-bridges. In line with these findings, MPV17 could only be inserted into lipid nanodiscs of 8–12 nm in diameter if intrinsic cysteines were either removed by mutagenesis or blocked by chemical modification. Using this nanodisc reconstitution approach, we could show that disease-linked mutations in MPV17 abolish its oligomerization properties in the membrane. These data suggest that, induced by oxidative stress, MPV17 can alter its oligomeric state from a properly folded monomer to a disulfide-stabilized oligomeric pore which might be required for the transport of metabolic DNA precursors into the mitochondrial matrix to compensate for the damage caused by reactive oxygen species.     

Adaptation of the tetrazolium method for testing the seed viability, and scanning electron microscopy study of some Western European orchids

A modified tetrazolium method was formulated for use with seeds of Western European orchids. The sequence of treatments which gave the highest percentage of coloured (i.e. viable) embryos was: (1) pretreatment in a solution of 5% (w/v) Ca(OCl)₂+ 1% (v/v) Tween-80, (2) soaking for 1 day in sterile water, (3) the classical tetrazolium test. The optimal duration of the pretreatment in Ca(OCl)₂+ Tween-80 depends upon the species, and to investigate the effect a scanning electron microscopy study was performed on the testa of 3 species. For a given species, the optimal pretreatment period was not affected by the year of harvest or the source of the seed lots.     

Animal models for respiratory chain disease

Elucidation of the pathogenesis in respiratory chain diseases is of great importance for developing specific treatments. The limitations inherent to the use of patient material make studies of human tissues often difficult and the mouse has therefore emerged as a suitable model organism for studies of respiratory chain diseases. In this review, we present an overview of the field and discuss in depth a few examples of animal models reproducing pathology of human disease with primary and secondary respiratory chain involvement.     

Genetic markers for identification of Patagonian toothfish and Antarctic toothfish

Fillet samples of the toothfish Dissostichus eleginoides and D. mawsoni can be distinguished readily by muscle proteins revealed by isoelectric focusing and mitochondrial DNA markers. The proteins also distinguish toothfish from other species marketed under similar trade names.     

Phosphorylation and ubiquitination of dynamin-related proteins (AtDRP3A/3B) synergically regulate mitochondrial proliferation during mitosis

The balance between mitochondrial fission and fusion is disrupted during mitosis, but the mechanism governing this phenomenon in plant cells remains enigmatic. Here, we used mitochondrial matrix‐localized Kaede protein (mt‐Kaede) to analyze the dynamics of mitochondrial fission in BY‐2 suspension cells. Analysis of the photoactivatable fluorescence of mt‐Kaede suggested that the fission process is dominant during mitosis. This finding was confirmed by an electron microscopic analysis of the size distribution of mitochondria in BY‐2 suspension cells at various stages. Cellular proteins interacting with Myc‐tagged dynamin‐related protein 3A/3B (AtDRP3A and AtDRP3B) were immunoprecipitated with anti‐Myc antibody‐conjugated beads and subsequently identified by microcapillary liquid chromatography–quadrupole time‐of‐flight mass spectrometry (CapLC Q‐TOF) MS/MS. The identified proteins were broadly associated with cytoskeletal (microtubular), phosphorylation, or ubiquitination functions. Mitotic phosphorylation of AtDRP3A/AtDRP3B and mitochondrial fission at metaphase were inhibited by treatment of the cells with a CdkB/cyclin B inhibitor or a serine/threonine protein kinase inhibitor. The fate of AtDRP3A/3B during the cell cycle was followed by time‐lapse imaging of the fluorescence of Dendra2‐tagged AtDRP3A/3B after green‐to‐red photoconversion; this experiment showed that AtDRP3A/3B is partially degraded during interphase. Additionally, we found that microtubules are involved in mitochondrial fission during mitosis, and that mitochondria movement to daughter cell was limited as early as metaphase. Taken together, these findings suggest that mitotic phosphorylation of AtDRP3A/3B promotes mitochondrial fission during plant cell mitosis, and that AtDRP3A/3B is partially degraded at interphase, providing mechanistic insight into the mitochondrial morphological changes associated with cell‐cycle transitions in BY‐2 suspension cells.     

Muscle fine structure reflects ecotype in two nototheniids

The fine structure of swimming (pectoral) and myotomal (axial) skeletal muscle and myocardium of two species of Antarctic nototheniid fishes were studied by electron microscopy, comparing the cryopelagic Pagothenia borchgrevinki and the benthic Trematomus bernacchii . Mean fibre size varied by a factor of four among muscles within each species and may have reflected the locomotory power available, being larger in pectoral oxidative (red) and axial glycolytic (white) muscle of P. borchgrevinki . Both species use labriform locomotion, and the more active P. borchgrevinki had a greater capillary supply, expressed as a capillary to fibre ratio, than T. bernacchii to both red (3·48 ± 0·36 v . 1·63 ± 0·14, mean ±  s . e .; P  < 0·01) and white (2·70 ± 0·20 v . 1·53 ± 0·18, mean ±  s . e .; P  < 0·01) regions of the pectoral musculature. The greater aerobic scope of P. borchgrevinki was strikingly demonstrated in the higher mitochondrial content of all skeletal muscle types sampled, and the ventricular myocardium (0·269 ± 0·011 v . 0·255 ± 0·012 mean ±  s . e .; P  < 0·05). Minor differences were found in other elements of fibre composition, with the exception of a five‐fold greater lipid content in pectoral red fibres of P. borchgrevinki (0·074 ± 0·014 mean ±  s . e .) v . T. bernacchii (0·010 ± 0·003; P  < 0·05). Differences in muscle fine structure among species clearly reflected differences in their ecotype.     

Ethological study of early language

Sign language studies of cross-fostered chimpanzees measure the effect of special rearing conditions on the development of very young chimpanzees. Cross-fostered chimpanzees, like human children, develop gradually in a process that takes many years. Here we discuss details of the procedure, the overlap between human and chimpanzee infants in the contents of the first 50-item vocabularies, and the ways in which the signs of the chimpanzees exhibit the fuzziness of natural language categories. We also compare the cross-fostering approach with more traditional modular approaches to the study of language-like behavior in nonhuman animals. Project Washoe was originally supported by grants MH-12154 from the National Institute of Mental Health and GB-7432 from the National Science Foundation. We gratefully acknowledge this support and the support that later sign language studies of chimpanzees have received since then from NIH, NSF, the National Geographic Society, the Grant Foundation, the Spencer Foundation, the University of Nevada, and the UNR Foundation.     

Quercetin-induced yeast apoptosis through mitochondrial dysfunction under the accumulation of magnesium in Candida albicans

Latterly, the upsurge in use of antifungal drugs has brought about the emergence of several drug-resistance strains, making it skeptical to continue relying on current therapeutic regime. In the necessity of resistance-free antifungal agent, flavonoids presented possibilities of replacing existing drugs, displaying antifungal activity against pathogenic fungi. Among them, quercetin, one of the most representative flavonoids, exhibited antifungal activity against Candida albicans. To inspect the further understanding regarding quercetin, the antifungal mode of action of quercetin was investigated. In the initial step, the apoptosis was monitored after quercetin treatment. Moreover, intracellular levels of Mg²⁺ was assessed and was determined that Mg²⁺ increase occurred under the influence of quercetin. In addition, several features of mitochondrial dysfunction were monitored. Mitochondrial dysfunction triggers decrease in mitochondrial redox levels and leads to disruption in mitochondrial antioxidant system. Increased intracellular ROS and decreased intracellular redox levels were also displayed, indicating the occurrence of overall disruption in antioxidant systems. Sequentially, DNA fragmentation was observed and this DNA damage in turn induces apoptosis. In analyses, hexaamminecobalt(III) chloride (Cohex) was applied to inhibit Mg²⁺ transport between cytosol and mitochondria. Cohex attenuated the effects induced by quercetin, which demonstrates that the presence of Mg²⁺ is essential in quercetin-induced apoptosis.