The biomolecule ubiquinone exerts a variety of biological functions

The chemistry of ubiquinone allows reversible addition of single electrons and protons. This unique property is used in nature for aerobic energy gain, for unilateral proton accumulation, for the generation of reactive oxygen species involved in physiological signaling and a variety of pathophysiological events. Since several years ubiquinone is also considered to play a major role in the control of lipid peroxidation, since this lipophilic biomolecule was recognized to recycle alpha-tocopherol radicals back to the chain-breaking form, vitamin E. Ubiquinone is therefore a biomolecule which has increasingly focused the interest of many research groups due to its alternative pro- and antioxidant activity. We have intensively investigated the role of ubiquinone as prooxidant in mitochondria and will present experimental evidences on conditions required for this function, we will also show that lysosomal ubiquinone has a double function as proton translocator and radical source under certain metabolic conditions. Furthermore, we have addressed the antioxidant role of ubiquinone and found that the efficiency of this activity is widely dependent on the type of biomembrane where ubiquinone exerts its chain-breaking activity.     

Selection of control genes for quantitative RT-PCR based on microarray data

Use of internal reference gene(s) is necessary for adequate quantification of target gene expression by RT-PCR. Herein, we elaborated a strategy of control gene selection based on microarray data and illustrated it by analyzing endomyocardial biopsies with acute cardiac rejection and infection. Using order statistics and binomial distribution we evaluated the probability of finding low-varying genes by chance. For analysis, the microarray data were divided into two sample subsets. Among the first 10% of genes with the lowest standard deviations, we found 14 genes common to both subsets. After normalization using two selected genes, high correlation was observed between expression of target genes evaluated by microarray and RT-PCR, and in independent dataset by RT-PCR (r = 0.9, p < 0.001). In conclusion, we showed a simple and reliable strategy of selection and validation of control genes for RT-PCR from microarray data that can be easily applied for different experimental designs and tissues.     

EtpB Is a Pore-Forming Outer Membrane Protein Showing TpsB Protein Features Involved in the Two-Partner Secretion System

Attachment to host tissues is a critical step in the pathogenesis of most bacterial infections. Enterotoxigenic Escherichia coli (ETEC) remains one of the principal causes of infectious diarrhea in humans. The recent identification of additional ETEC surface molecules suggests that new targets may be exploited in vaccine development. The EtpA protein identified in ETEC H10407 is a large glycosylated adhesin secreted via the two-partner secretion system. EtpA requires its putative partner EtpB for translocation across the outer membrane (OM). We investigated the biochemical and electrophysiological properties of purified EtpB. We showed that EtpB is 65-kDa heat-modifiable protein localized to the OM. Electrophysiological experiments indicated that EtpB is able to form pores in planar lipid bilayer membranes with an asymmetric current, suggesting its functional asymmetry. The pore of EtpB frequently assumes an opened conformation and fluctuates between three well-defined conductance states. In silico analysis of the EtpB amino acid sequence and molecular modeling suggest that EtpB is similar to the well-known TpsB protein FhaC from Bordetella pertussis and has a C-terminal transmembrane β-barrel domain that is occluded by an N-terminal α-helix, an extracellular loop, and two periplasmic polypeptide-transport-associated (POTRA) domains. Together, these data confirm that EtpB is a pore-forming protein mainly folded into a β-barrel conformation and indicate that EtpB presents typical features of the OM TpsB proteins.     

Solid-state 2H NMR spectroscopy of retinal proteins in aligned membranes

Solid-state 2H NMR spectroscopy gives a powerful avenue to investigating the structures of ligands and cofactors bound to integral membrane proteins. For bacteriorhodopsin (bR) and rhodopsin, retinal was site-specifically labeled by deuteration of the methyl groups followed by regeneration of the apoprotein. 2H NMR studies of aligned membrane samples were conducted under conditions where rotational and translational diffusion of the protein were absent on the NMR time scale. The theoretical lineshape treatment involved a static axial distribution of rotating C-C2H3 groups about the local membrane frame, together with the static axial distribution of the local normal relative to the average normal. Simulation of solid-state 2H NMR lineshapes gave both the methyl group orientations and the alignment disorder (mosaic spread) of the membrane stack. The methyl bond orientations provided the angular restraints for structural analysis. In the case of bR the retinal chromophore is nearly planar in the dark- and all-trans light-adapted states, as well upon isomerization to 13-cis in the M state. The C13-methyl group at the "business end" of the chromophore changes its orientation to the membrane upon photon absorption, moving towards W182 and thus driving the proton pump in energy conservation. Moreover, rhodopsin was studied as a prototype for G protein-coupled receptors (GPCRs) implicated in many biological responses in humans. In contrast to bR, the retinal chromophore of rhodopsin has an 11-cis conformation and is highly twisted in the dark state. Three sites of interaction affect the torsional deformation of retinal, viz. the protonated Schiff base with its carboxylate counterion; the C9-methyl group of the polyene; and the beta-ionone ring within its hydrophobic pocket. For rhodopsin, the strain energy and dynamics of retinal as established by 2H NMR are implicated in substituent control of activation. Retinal is locked in a conformation that is twisted in the direction of the photoisomerization, which explains the dark stability of rhodopsin and allows for ultra-fast isomerization upon absorption of a photon. Torsional strain is relaxed in the meta I state that precedes subsequent receptor activation. Comparison of the two retinal proteins using solid-state 2H NMR is thus illuminating in terms of their different biological functions.     

GENETIC STRUCTURE IN POPULATIONS OF FUCUS VESICULOSUS (PHAEOPHYCEAE) OVER SPATIAL SCALES FROM 10 M TO 800 KM1

Recent studies showing consequences of species’ genetic diversity on ecosystem performance raise the concern of how key ecosystem species are genetically structured. The bladder wrack Fucus vesiculosus L. is a dominant species of macroalga in the northern Atlantic, and it is particularly important as a habitat‐forming species in the Baltic Sea. We examined the genetic structure of populations of F. vesiculosus with a hierarchical approach from a within‐shore scale (10 m) to a between‐seas scale (Baltic Sea–Skagerrak, 800 km). Analysis of five microsatellite loci showed that population differentiation was generally strong (average F_ST = 12%), being significant at all spatial scales investigated (10¹, 10³, 10^4–5, 10⁶ m). Genetic differentiation between seas (Baltic Sea and Skagerrak) was substantial. Nevertheless, the effects of isolation by distance were stronger within seas than between seas. Notably, Baltic summer‐reproducing populations showed a strong within‐sea, between‐area (70 km) genetic structure, while Baltic autumn‐reproducing populations and Skagerrak summer‐reproducing populations revealed most genetic diversity between samples within areas (<1 km). Despite such differences in overall structure, Baltic populations of summer‐ and autumn‐reproducing morphs did not separate in a cluster analysis, indicating minor, if any, barriers to gene flow between them. Our results have important implications for management and conservation of F. vesiculosus, and we raise a number of concerns about how genetic variability should be preserved within this species.     

Reinvestigation of the O-antigens of Yersinia pseudotuberculosis: revision of the O2c and confirmation of the O3 antigen structures

Structures of the O-antigens of Yersinia pseudotuberculosis O2c and O3 were reinvestigated by NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, (1)H,(13)C HSQC, and HMBC experiments. The following revised structure of the O2c tetrasaccharide repeating unit was established, which differs from the structure proposed earlier in the glycosylation pattern of the mannose residue at the branching point: where Abe stands for 3,6-dideoxy-d-xylo-hexose. The structure of the Y. pseudotuberculosis O3 antigen reported earlier was confirmed.     

Methionine metabolism and methyltransferases in the regulation of aging and lifespan extension across species

Methionine restriction (MetR) extends lifespan across different species and exerts beneficial effects on metabolic health and inflammatory responses. In contrast, certain cancer cells exhibit methionine auxotrophy that can be exploited for therapeutic treatment, as decreasing dietary methionine selectively suppresses tumor growth. Thus, MetR represents an intervention that can extend lifespan with a complementary effect of delaying tumor growth. Beyond its function in protein synthesis, methionine feeds into complex metabolic pathways including the methionine cycle, the transsulfuration pathway, and polyamine biosynthesis. Manipulation of each of these branches extends lifespan; however, the interplay between MetR and these branches during regulation of lifespan is not well understood. In addition, a potential mechanism linking the activity of methionine metabolism and lifespan is regulation of production of the methyl donor S‐adenosylmethionine, which, after transferring its methyl group, is converted to S‐adenosylhomocysteine. Methylation regulates a wide range of processes, including those thought to be responsible for lifespan extension by MetR. Although the exact mechanisms of lifespan extension by MetR or methionine metabolism reprogramming are unknown, it may act via reducing the rate of translation, modifying gene expression, inducing a hormetic response, modulating autophagy, or inducing mitochondrial function, antioxidant defense, or other metabolic processes. Here, we review the mechanisms of lifespan extension by MetR and different branches of methionine metabolism in different species and the potential for exploiting the regulation of methyltransferases to delay aging.