Application of the yeast Yarrowia lipolytica as a model to analyse human pathogenic mutations in mitochondrial complex I (NADH:ubiquinone oxidoreductase)

While diagnosis and genetic analysis of mitochondrial disorders has made remarkable progress, we still do not understand how given molecular defects are correlated to specific patterns of symptoms and their severity. Towards resolving this dilemma for the largest and therefore most affected respiratory chain enzyme, we have established the yeast Yarrowia lipolytica as a eucaryotic model system to analyse respiratory chain complex I. For in vivo analysis, eYFP protein was attached to the 30-kDa subunit to visualize complex I and mitochondria. Deletions strains for nuclear coded subunits allow the reconstruction of patient alleles by site-directed mutagenesis and plasmid complementation. In most of the pathogenic mutations analysed so far, decreased catalytic activities, elevated K(M) values, and/or elevated I(50) values for quinone-analogous inhibitors were observed, providing plausible clues on the pathogenic process at the molecular level. Leigh mutations in the 49-kDa and PSST homologous subunits are found in regions that are at the boundaries of the ubiquinone-reducing catalytic core. This supports the proposed structural model and at the same time identifies novel domains critical for catalysis. Thus, Y. lipolytica is a useful lower eucaryotic model that will help to understand how pathogenic mutations in complex I interfere with enzyme function.     

Functional significance of conserved histidines and arginines in the 49-kDa subunit of mitochondrial complex I

We have studied the ubiquinone-reducing catalytic core of NADH:ubiquinone oxidoreductase (complex I) from Yarrowia lipolytica by a series of point mutations replacing conserved histidines and arginines in the 49-kDa subunit. Our results show that histidine 226 and arginine 141 probably do not ligate iron-sulfur cluster N2 but that exchanging these residues specifically influences the properties of this redox center. Histidines 91 and 95 were found to be essential for ubiquinone reductase activity of complex I. Mutations at the C-terminal arginine 466 affected ubiquinone affinity and inhibitor sensitivity but also destabilized complex I. These results provide further support for a high degree of structural conservation between the 49-kDa subunit of complex I and its ancestor, the large subunit of water-soluble [NiFe] hydrogenases. In several mutations of histidine 226, arginine 141, and arginine 466 the characteristic EPR signatures of iron-sulfur cluster N2 became undetectable, but specific, inhibitor-sensitive ubiquinone reductase activity was only moderately reduced. As we could not find spectroscopic indications for a modified cluster N2, we concluded that these complex I mutants were lacking most of this redox center but were still capable of catalyzing inhibitor-resistant ubiquinone reduction at near normal rates. We discuss that this at first surprising scenario may be explained by electron transfer theory; after removal of a single redox center in a chain, electron transfer rates are predicted to be still much faster than steady-state turnover of complex I. Our results question some of the central mechanistic functions that have been put forward for iron-sulfur cluster N2.     

The effects of time of day-specific resistance training on adaptations in skeletal muscle hypertrophy and muscle strength: A systematic review and meta-analysis

The present paper endeavored to elucidate the topic on the effects of morning versus evening resistance training on muscle strength and hypertrophy by conducting a systematic review and a meta-analysis of studies that examined time of day-specific resistance training. This systematic review was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines with searches conducted through PubMed/MEDLINE, Scopus, and SPORTDiscus databases. The Downs and Black checklist was used for the assessment of the methodological quality of the included studies. Studies that examined the effects of time of day-specific resistance training (while equating all other training variables, such as training frequency and volume, between the groups) on muscle strength and/or muscle size were included in the present review. The random effects model was used for the meta-analysis. Meta-analyses explored (1) the differences in strength expression between morning and evening hours at baseline; (2) the differences in strength within the groups training in the morning and evening by using their post-intervention strength data from the morning and evening strength assessments; (3) the overall differences between the effects of morning and evening resistance training (with subgroup analyses conducted for studies that assessed strength in the morning hours and for the studies that assessed strength in the evening hours). Finally, a meta-analysis was also conducted for studies that assessed muscle hypertrophy. Eleven studies of moderate and good methodological quality were included in the present review. The primary findings of the review are as follows: (1) at baseline, a significant difference in strength between morning and evening is evident, with greater strength observed in the evening hours; (2) resistance training in the morning hours may increase strength assessed in the morning to similar levels as strength assessed in the evening; (3) training in the evening hours, however, maintains the general difference in strength across the day, with greater strength observed in the evening hours; (4) when comparing the effects between the groups training in the morning versus in the evening hours, increases in strength are similar in both groups, regardless of the time of day at which strength assessment is conducted; and (5) increases in muscle size are similar irrespective of the time of day at which the training is performed.     

Ureteral stent-associated infection and sepsis: pathogenesis and prevention: a review

Ureteral stents are commonly used devices in hospital settings. However, their usage is often complicated by associated urinary tract infections as a result of bacterial adhesion onto the indwelling implant surfaces, followed by the formation of layers of biofilm. Once formed, the biofilm is exceedingly difficult to remove, potentially leading to further morbidity and even urosepsis. Urosepsis, where pathogens from the urinary tract enter the bloodstream, has a mortality rate of up to 50% of severely infected patients. Hence, it is important to understand its pathogenesis. In this review, ureteral stent-associated urinary tract infection and urosepsis will be addressed. In particular, the bacterial mechanisms involved, as well as the prevention and treatment of these infections will be discussed.     

Stimulation of ligninolytic enzyme production in Phanerochaete chrysosporium by polyoxyalkanes

AIMS: Poly(ethylene glycol) (PEG) and some substances similar to PEG in chemical structure were tested as stimulators of ligninolytic enzyme production in shaken culture of Phanerochaete chrysosporium. METHODS AND RESULTS: The substances that caused high enzymatic activity were linear polymers [poly(ethylene glycol), poly(propylene glycol), poly(butylene glycol) and poly(vinyl alcohol)] and cyclic polymers (crown ether). They can have terminal groups other than -OH [PEG (di)methyl ether, PEG sulphate, PEG derivative with the amino group and xanthate]. The maximum lignin peroxidase activities were compared with the surface pressure caused by the stimulator. Addition of polymers composed of charged monomer units did not increase the enzymatic activity and the fungi did not grow at all on addition of polymers having a fixed positive charge. CONCLUSIONS: Lignin peroxidase activity was increased after the addition of polymers with uncharged monomer units. It was higher and its maximum was reached in a shorter time on addition of polymers with higher molecular weights. SIGNIFICANCE AND IMPACT OF STUDY: Beside Tweens there are several polymers that stimulate ligninolytic enzyme production in shaken culture of P. chrysosporium. Their characteristics are: similarity to PEG in chemical structure, having uncharged monomer units and high molecular weight.     

Cluster N1 of complex I from Yarrowia lipolytica studied by pulsed EPR spectroscopy

After reduction with nicotinamide adenine dinucleotide (NADH), NADH:ubiquinone oxidoreductase (complex I) of the strictly aerobic yeast Yarrowia lipolytica shows clear signals from five different paramagnetic iron-sulfur (FeS) clusters (N1-N5) which can be detected using electron paramagnetic resonance (EPR) spectroscopy. The ligand environment and the assignment of several FeS clusters to specific binding motifs found in several subunits of the complex are still under debate. In order to characterize the hyperfine interaction of the surrounding nuclei with FeS cluster N1, one- and two-dimensional electron spin echo envelope modulation experiments were performed at a temperature of 30 K. At this temperature only cluster N1 contributes to the overall signal in a pulsed EPR experiment. The hyperfine and quadrupole tensors of a nitrogen nucleus and the isotropic and dipolar hyperfine couplings of two sets of protons could be determined by numerical simulation of the one- and two-dimensional spectra. The values obtained are in perfect agreement with a ferredoxin-like binding structure by four cysteine amino acid residues and allow the assignment of the nitrogen couplings to a backbone nitrogen nucleus and the proton couplings to the beta-protons of the bound cysteine residues.     

The Redox-Bohr group associated with iron-sulfur cluster N2 of complex I

Proton pumping respiratory complex I (NADH:ubiquinone oxidoreductase) is a major component of the oxidative phosphorylation system in mitochondria and many bacteria. In mammalian cells it provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme cause numerous human disorders. Yet the mechanism of complex I is still elusive. A group exhibiting redox-linked protonation that is associated with iron-sulfur cluster N2 of complex I has been proposed to act as a central component of the proton pumping machinery. Here we show that a histidine in the 49-kDa subunit that resides near iron-sulfur cluster N2 confers this redox-Bohr effect. Mutating this residue to methionine in complex I from Yarrowia lipolytica resulted in a marked shift of the redox midpoint potential of iron-sulfur cluster N2 to the negative and abolished the redox-Bohr effect. However, the mutation did not significantly affect the catalytic activity of complex I and protons were pumped with an unchanged stoichiometry of 4 H(+)/2e(-). This finding has significant implications on the discussion about possible proton pumping mechanism for complex I.