Influence of mitochondrial DNA level on cellular energy metabolism: implications for mitochondrial diseases

The total amount of cellular mitochondrial DNA (mtDNA) varies widely and seems to be related to the nature and metabolic state of tissues and cells in culture. It is not known, however, whether this variation has any significance in vivo, and to which extent it regulates energy production. To better understand the importance of the cellular mtDNA level, we studied the influence of a gradual reduction of mtDNA copy number on oxidative phosphorylation in two models: (a) a control human cell line treated with different concentrations of 2′, 3′-dideoxycytidine, a nucleoside analogue that inhibits mtDNA replication by interfering with mitochondrial DNA polymerase γ, and (b) a cell line derived from a patient presenting mtDNA depletion. The two models were used to construct biochemical and phenotypic threshold curves. Our results show that oxidative phosphorylation activities are under a tight control by the amount of mtDNA in the cell, and that the full complement of mtDNA molecules are necessary to maintain a normal energy production level.     

Presence and isolation of 2 lipoproteins immunologically related to apolipoprotein A I in human serum

Very high density lipoproteins d : 1.23--1.25 g/ml (VHDL2) have been isolated from human serum by preparative ultracentrifugation. They contain 80 per cent proteins and 20 per cent lipids. Lipids are mainly phospholipids (80 per cent). The proportion of lysolecithin (50 per cent) is higher than that of lecithin (40 per cent). The quantity of cholesterol is low, the free cholesterol: total cholesterol ratio is 0.35. VHDL2 consisted principally in lipoprotein D and two lipoproteins immunologically apparented to apolipoprotein A I, called LP A I1 and LP A I2. The LP A I1 has a molecular weight slightly higher and a hydrated density lower than that of LP AI2. Our experiments suggest that LP A I1 exists in the serum before ultracentrifugation while LP A I 2 comes from HDL degradation during ultracentrifugation. The immunological heterogeneity of apo A I forming different protein-lipid complexes is discussed.     

Phospholipase A2 action on planar lipid bilayers generates a small, transitory current that is voltage independent.

Addition of either bee venom or Trimeresurus flavoviridis phospholipase A2 (PLA2) to the solution bathing the front side of a voltage-clamped, planar lipid bilayer consistently produced a transitory current lasting approximately 100 s. This current is consistent with anions moving through the membrane to the rear side. The peak current is independent of holding potential. PLA2 activity on phospholipid membranes not only produced a current but also led to membrane rupture within 300 s. The current depends on Ca2+ and lipid type. Addition of PLA2 in the absence of Ca2+ or to membranes made of nonsubstrate lipids (e.g., glycerol monooleate or lysophosphatidylcholine) produced no current and did not break the bilayer. Peak current height, signal decay time, and time to membrane rupture all depended on PLA2 dose, whereas total charge produced was constant. This current does not flow through ion channels because there are no channels present and the current is not voltage dependent. The evidence is consistent with the hypothesis that the current is generated by the movement of ionized fatty acid produced by PLA2 action. These results demonstrate a simple method to measure enzyme activity in the presence of different substrates and varied environmental conditions.     

Structural modifications of oxidized insulin B-chain induced by glycerol addition

Summary ¹H-NMR studies of the bovine insulin S-sulfonated B-chain are reported in H₂O/D₂O (9/1) and in glycerol-d ₅ (5 M) using two-dimensional NMR spectroscopy. The first results show that the oxidized insulin B-chain secondary structure differs from that of native insulin by a loss of the α-helix between the two disulfide bridges and that the glycerol favours the structuring of the peptide.     

State-Dependent Mate-Assessment and Mate-Selection Behavior in Female Threespine Sticklebacks (Gasterosteus aculeatus, Gasterosteiformes: Gasterosteidae)

Most investigations of mate-choice have focused on the outcomes of mate-choice (i.e. which mates are chosen), and thus we generally know very little about how mates are chosen (i.e. how mates are found, assessed, and selected). Mate-choice by females has been shown to be dependent on the state of the female, with females being less selective when limited by time or energy. This result could be caused by changes in female mate-assessment or mate-selection behavior. We examined whether manipulations of time and energy affected the mate-choice behavior of female threespine stickleback ( Gasterosteus aculeatus ). We found that female mate-choice behavior, when not divided into stages, was affected by both of the manipulations. This matches previous findings. When we divided female courtship behavior into six stages, we found that the two manipulations affected different portions of the mate-choice process. The holding-time manipulation changed female behavior at the beginning and end of the mate-choice process and the swimming manipulation changed female behavior only at the end of the mate-choice process. Neither of the manipulations significantly affected female behavior in the middle portion of the mate-choice process. Thus, the mate-choice process appears to be composed of multiple state-dependent decisions. We discuss how a better mechanistic understanding of how female state affects female mate-choice behavior can produce testable predictions and provide a basis for investigating the evolution of mating systems.     

Bayesian characterization of uncertainty in species interaction strengths

Considerable effort has been devoted to the estimation of species interaction strengths. This effort has focused primarily on statistical significance testing and obtaining point estimates of parameters that contribute to interaction strength magnitudes, leaving the characterization of uncertainty associated with those estimates unconsidered. We consider a means of characterizing the uncertainty of a generalist predator’s interaction strengths by formulating an observational method for estimating a predator’s prey-specific per capita attack rates as a Bayesian statistical model. This formulation permits the explicit incorporation of multiple sources of uncertainty. A key insight is the informative nature of several so-called non-informative priors that have been used in modeling the sparse data typical of predator feeding surveys. We introduce to ecology a new neutral prior and provide evidence for its superior performance. We use a case study to consider the attack rates in a New Zealand intertidal whelk predator, and we illustrate not only that Bayesian point estimates can be made to correspond with those obtained by frequentist approaches, but also that estimation uncertainty as described by 95% intervals is more useful and biologically realistic using the Bayesian method. In particular, unlike in bootstrap confidence intervals, the lower bounds of the Bayesian posterior intervals for attack rates do not include zero when a predator–prey interaction is in fact observed. We conclude that the Bayesian framework provides a straightforward, probabilistic characterization of interaction strength uncertainty, enabling future considerations of both the deterministic and stochastic drivers of interaction strength and their impact on food webs.     

Structure and modeling studies of the carboxy-terminus region of human tropoelastin.

Elastin macromolecular assembly is a highly complex mechanism involving many steps including coacervation, cross-linking, and probably other (not known) phenomena. In past studies, it has been proposed that the C-terminal part of tropoelastin is also involved in this process and may play a key role in tropoelastin interactions with other proteins of the final elastic fibres scaffold. Presented here are the results of the biophysical studies (biospectroscopy, bioinformatics) of the C-terminal domain of tropoelastin. We report the detailed structures adopted by the oxidized (native) and reduced forms of the free synthetic peptide with sequence encoded by exon 36 of human tropoelastin (GGACLGKACGRKRK) and propose a dynamical interpretation of which structures may be involved in interactions with other extra-cellular matrix proteins. We also suggest that these structures may be retrieved in other proteins sharing a consensus sequence; however no definitive conclusion can be drawn here on a possible structure-function relationship.     

Ribosome biogenesis is temperature‐dependent and delayed in Escherichia coli lacking the chaperones DnaK or DnaJ†

In Escherichia coli strains carrying null mutations in either the dnaK or dnaJ genes, the late stages of 30S and 50S ribosomal subunit biogenesis are slowed down in a temperature‐dependent manner. At high temperature (44°C), 32S and 45S particles (precursors to 50S subunits) and 21S particles (precursors to 30S subunits) accumulate. The latter are shown by 3′5′ rapid amplification of cDNA ends analysis to contain unprocessed or partially processed 16S ribosomal RNA at the 5′ end, but the 3′ end was never processed. This implies that maturation of 16S ribosomal RNA starts at the 5′‐terminus, and that the 3′‐terminus is only trimmed at a later step. At normal temperatures (30°C?37°C), ribosome assembly in both mutants is not arrested but is significantly delayed, as shown by pulse‐chase analysis. Assembly defects are partially compensated by an overexpression of other heat‐shock proteins, which occurs in the absence of their negative regulator DnaK, or by a plasmid‐driven overexpression of GroES/GroEL, suggesting the involvement of a network of chaperones in ribosome biogenesis.     

Requirements for the valid quantification of immunostains on tissue microarray materials using image analysis

Antibody‐based proteomics applied to tissue microarray (TMA) technology provides a very efficient means of visualizing and locating antigen expression in large collections of normal and pathological tissue samples. To characterize antigen expression on TMAs, the use of image analysis methods avoids the effects of human subjectivity evidenced in manual microscopical analysis. Thus, these methods have the potential to significantly enhance both precision and reproducibility. Although some commercial systems include tools for the quantitative evaluation of immunohistochemistry‐stained images, there exists no clear agreement on best practices to allow for correct and reproducible quantification results. Our study focuses on practical aspects regarding (i) image acquisition (ii) segmentation of staining and counterstaining areas and (iii) extraction of quantitative features. We illustrate our findings using a commercial system to quantify different immunohistochemistry markers targeting proteins with different expression patterns (cytoplasmic, nuclear or membranous) in colon cancer or brain tumor TMAs. Our investigations led us to identify several steps that we consider essential for standardizing computer‐assisted immunostaining quantification experiments. In addition, we propose a data normalization process based on reference materials to be able to compare measurements between studies involving different TMAs. In conclusion, we recommend certain critical prerequisites that commercial or in‐house systems should satisfy in order to permit valid immunostaining quantification.