Expression of the mammalian mitochondrial genome. Role for membrane potential in the production of mature translation products

Protein synthesis was investigated in isolated mitochondria under conditions which either inhibited electron transport or uncoupled oxidative phosphorylation. In a medium containing an exogenous source of ATP and oligomycin, an inhibitor of the ATP synthase complex, incorporation of [35S]methionine into proteins is stimulated in the presence of inhibitors of the electron transport chain; substituting uncouplers of oxidative phosphorylation for the latter leads, in contrast, to a decrease in the rate of incorporation of the labeled amino acid into mitochondrial translation products. Studies on the metabolic stability of mitochondrial translation products revealed that "mature" polypeptides made in isolated mitochondria are stable as indicated by the absence of degradation during a 50 min "chase" period. Under conditions which reduce or dissipate the membrane potential, 50-60% of the newly made polypeptides (pulse) are degraded within 50 min. The kinetics of the degradation process for individual mitochondrial gene products reveal that the largest proportion of polypeptides degraded to an acid-soluble form during the chase period are abnormal proteins, likely the result of premature chain termination. Emerging as a common denominator in these studies is a role for a transmembrane potential across the inner membrane in the production of mature "stable" mitochondrial gene products.     

Normal limb development in conditional mutants of Fgf4

Fibroblast growth factors (FGFs) mediate multiple developmental signals in vertebrates. Several of these factors are expressed in limb bud structures that direct patterning of the limb. FGF4 is produced in the apical ectodermal ridge (AER) where it is hypothesized to provide mitogenic and morphogenic signals to the underlying mesenchyme that regulate normal limb development. Mutation of this gene in the germline of mice results in early embryonic lethality, preventing subsequent evaluation of Fgf4 function in the AER. A conditional mutant of Fgf4, based on site-specific Cre/loxP-mediated excision of the gene, allowed us to bypass embryonic lethality and directly test the role of FGF4 during limb development in living murine embryos. This conditional mutation was designed so that concomitant with inactivation of the Fgf4 gene by excision of all Fgf4-coding sequences, a reporter gene was activated in Fgf4-expressing cells, allowing assessment of the site-specific recombination reaction. Although a large body of evidence led us to predict that ablation of Fgf4 gene function in the AER of developing mice would result in abnormal limb outgrowth and patterning, we found that Fgf4 conditional mutants had normal limbs. Furthermore, expression patterns of Shh, Bmp2, Fgf8 and Fgf10 were normal in the limb buds of the conditional mutants. These findings indicate that the previously proposed FGF4-SHH feedback loop is not essential for coordination of murine limb outgrowth and patterning. We suggest that some of the roles currently attributed to FGF4 during early vertebrate limb development may be performed by other AER factors in vivo.     

Airway Response to Methacholine following Eucapnic Voluntary Hyperpnea in Athletes

Aim

To evaluate the changes in airway responsiveness to methacholine inhalation test (MIT) when performed after an eucapnic voluntary hyperpnea challenge (EVH) in athletes.

Methods

Two MIT preceded (visit 1) or not (visit 2) by an EVH, were performed in 28 athletes and 24 non-athletes. Twelve athletes and 13 non-athletes had airway hyperresponsiveness (AHR) to methacholine, and 11 athletes and 11 non-athletes had AHR to EVH (EVH+).

Results

The MIT PC₂₀ post-EVH was significantly lower compared to baseline MIT PC₂₀ by 1.3±0.7 doubling-concentrations in EVH+ athletes only (p<0.0001). No significant change was observed in EVH- athletes and EVH+/EVH- non-athletes. A significant correlation between the change in MIT PC₂₀ post-EVH and EVH+/EVH- status and athlete/nonathlete status was found (Adjusted R²=0.26 and p<0.001). Three (11%) athletes and one (4%) non-athlete had a change in the diagnosis of AHR when MIT was performed consecutively to EVH.

Conclusion

The responsiveness to methacholine was increased by a previous indirect challenge in EVH+ athletes only. The mechanisms for such increase remain to be determined. MIT and EVH should ideally be performed on separate occasions as there is a small but possible risk to obtain a false-positive response to methacholine when performed immediately after the EVH.

Trial Registration

ClinicalTrials.gov NCT00686491     

Reproducibility of In Vivo Corneal Confocal Microscopy Using an Automated Analysis Program for Detection of Diabetic Sensorimotor Polyneuropathy

Objective

In vivo Corneal Confocal Microscopy (IVCCM) is a validated, non-invasive test for diabetic sensorimotor polyneuropathy (DSP) detection, but its utility is limited by the image analysis time and expertise required. We aimed to determine the inter- and intra-observer reproducibility of a novel automated analysis program compared to manual analysis.

Methods

In a cross-sectional diagnostic study, 20 non-diabetes controls (mean age 41.4±17.3y, HbA1c 5.5±0.4%) and 26 participants with type 1 diabetes (42.8±16.9y, 8.0±1.9%) underwent two separate IVCCM examinations by one observer and a third by an independent observer. Along with nerve density and branch density, corneal nerve fibre length (CNFL) was obtained by manual analysis (CNFL_MANUAL), a protocol in which images were manually selected for automated analysis (CNFL_{SEMI-AUTOMATED}), and one in which selection and analysis were performed electronically (CNFL_{FULLY-AUTOMATED}). Reproducibility of each protocol was determined using intraclass correlation coefficients (ICC) and, as a secondary objective, the method of Bland and Altman was used to explore agreement between protocols.

Results

Mean CNFL_Manual was 16.7±4.0, 13.9±4.2 mm/mm² for non-diabetes controls and diabetes participants, while CNFL_{Semi-Automated} was 10.2±3.3, 8.6±3.0 mm/mm² and CNFL_{Fully-Automated} was 12.5±2.8, 10.9 ± 2.9 mm/mm². Inter-observer ICC and 95% confidence intervals (95%CI) were 0.73(0.56, 0.84), 0.75(0.59, 0.85), and 0.78(0.63, 0.87), respectively (p = NS for all comparisons). Intra-observer ICC and 95%CI were 0.72(0.55, 0.83), 0.74(0.57, 0.85), and 0.84(0.73, 0.91), respectively (p<0.05 for CNFL_{Fully-Automated} compared to others). The other IVCCM parameters had substantially lower ICC compared to those for CNFL. CNFL_{Semi-Automated} and CNFL_{Fully-Automated} underestimated CNFL_Manual by mean and 95%CI of 35.1(-4.5, 67.5)% and 21.0(-21.6, 46.1)%, respectively.

Conclusions

Despite an apparent measurement (underestimation) bias in comparison to the manual strategy of image analysis, fully-automated analysis preserves CNFL reproducibility. Future work must determine the diagnostic thresholds specific to the fully-automated measure of CNFL.     

Chitooligosaccharide sensing and downstream signaling: contrasted outcomes in pathogenic and beneficial plant–microbe interactions

In plants, short chitin oligosaccharides and chitosan fragments (collectively referred to as chitooligosaccharides) are well-known elicitors that trigger defense gene expression, synthesis of antimicrobial compounds, and cell wall strengthening. Recent findings have shed new light on chitin-sensing mechanisms and downstream activation of intracellular signaling networks that mediate plant defense responses. Interestingly, chitin receptors possess several lysin motif domains that are also found in several legume Nod factor receptors. Nod factors are chitin-related molecules produced by nitrogen-fixing rhizobia to induce root nodulation. The fact that chitin and Nod factor receptors share structural similarity suggests an evolutionary conserved relationship between mechanisms enabling recognition of both deleterious and beneficial microorganisms. Here, we will present an update on molecular events involved in chitooligosaccharide sensing and downstream signaling pathways in plants and will discuss how structurally related signals may lead to such contrasted outcomes during plant–microbe interactions.     

Epsin: inducing membrane curvature

Epsin was originally discovered by virtue of its binding to another accessory protein, Eps15. Members of the epsin family play an important role as accessory proteins in clathrin-mediated endocytosis. Epsin isoforms have been described that differ in intracellular site of action and/or in tissue distribution, although all epsins essentially contribute to membrane deformation. Besides inducing membrane curvature, epsin also plays a key function as adaptor protein, coupling various components of the clathrin-assisted uptake and fulfils an important role in selecting and recognizing cargo. Furthermore, epsin possesses the ability to block vesicle formation during mitosis. To perform all these functions, epsin, apart from interacting with PtdIns(4,5)P2 via its ENTH domain, also engages in several protein interactions with different components of the clathrin-mediated endocytic system. Recently, RNA interference has successfully been exploited to generate a cell line constitutively silencing epsin expression, which can be used to study internalization of multiple ligands.     

Short rotation coppice culture of willows and poplars as energy crops on metal contaminated agricultural soils

Phytoremediation, more precisely phytoextraction, has been placed forward as an environmental friendly remediation technique, that can gradually reduce increased soil metal concentrations, in particular the bioavailable fractions. The aim of this study was to investigate the possibilities of growing willows and poplars under short rotation coppice (SRC) on an acid, poor, sandy metal contaminated soil, to combine in this way soil remediation by phytoextraction on one hand, and production of biomass for energy purposes on the other. Above ground biomass productivities were low for poplars to moderate for willows, which was not surprising, taking into account the soil conditions that are not very favorable for growth of these trees. Calculated phytoextraction efficiency was much longer for poplars than these for willows. We calculated that for phytoextraction in this particular case it would take at least 36 years to reach the legal threshold values for cadmium, but in combination with production of feedstock for bioenergy processes, this type of land use can offer an alternative income for local farmers. Based on the data of the first growing cycle, for this particular case, SRC of willows should be recommended.