Pectate lyase gene regulatory mutants of Erwinia chrysanthemi.

The pelB gene, which encodes one of the five pectate lyase isoenzymes of Erwinia chrysanthemi 3937, was mutagenized with a mini-Mu transposable element that can form gene fusions to the neomycin phosphotransferase-encoding region. Secondary mutants resistant to kanamycin in the absence of polygalacturonate, an inducer of wild-type pectate lyase activities, were selected. Such mutants produced other pectate lyase isoenzymes in the absence of the inducer.     

Modular control analysis of effects of chronic hypoxia on mouse heart

Modular control analysis (MoCA; Diolez P, Deschodt-Arsac V, Raffard G, Simon C, Santos PD, Thiaudiere E, Arsac L, Franconi JM. Am J Physiol Regul Integr Comp Physiol 293: R13-R19, 2007) was applied here on perfused hearts to describe the modifications of the regulation of heart energetics induced in mice exposed to 3-wk chronic hypoxia. MoCA combines 31P-NMR spectroscopy and modular (top down) control analysis to describe the integrative regulation of energy metabolism in the intact beating heart, on the basis of two modules [ATP/phosphocreatine (PCr) production and ATP/PCr consumption] connected by the energetic intermediates. In contrast with previous results in rat heart, in which all control of contraction was on ATP demand, mouse heart energetics presented a shared control of contraction between ATP/PCr-producing and -consuming modules. In chronic hypoxic mice, the decrease in heart contractile activity and PCr-to-ATP ratio was surprisingly associated with an important and significant higher response of ATP/PCr production (elasticity) to PCr changes compared with control hearts (-10.4 vs. -2.46). By contrast, no changes were observed in ATP/PCr consumption since comparable elasticities were observed. Since elasticities determine the regulation of energetics of heart contraction, the present results show that this new parameter may be used to uncover the origin of the observed dysfunctions under chronic hypoxia conditions. Considering the decrease in mitochondrial content reported after exposure to chronic hypoxia, it appears that the improvement of ATP/PCr production response to ATP demand may be viewed as a positive adaptative mechanism. It now appears crucial to understand the very processes responsible for ATP/PCr producer elasticity toward the energetic intermediates, as well as their regulation.     

Inhibition of cardiac contractility by 5-hydroxydecanoate and tetraphenylphosphonium ion: a possible role of mitoKATP in response to inotropic stress

This study investigates the role of the mitochondrial ATP-sensitive K+ channel (mitoKATP) in response to positive inotropic stress. In Langendorff-perfused rat hearts, inotropy was induced by increasing perfusate calcium to 4 mM, by adding 80 microM ouabain or 0.25 microM dobutamine. Each of these treatments resulted in a sustained increase in rate-pressure product (RPP) of approximately 60%. Inhibition of mitoKATP by perfusion of 5-hydroxydecanoate (5-HD) or tetraphenylphosphonium before induction of inotropic stress resulted in a marked attenuation of RPP. Inhibition of mitoKATP after induction of stress caused the inability of the heart to maintain a high-work state. In human atrial fibers, the increase in contractility induced by dobutamine was inhibited 60% by 5-HD. In permeabilized fibers from the Langendorff-perfused rat hearts, inhibition of mitoKATP resulted, in all cases, in an alteration of adenine nucleotide compartmentation, as reflected by a 60% decrease in the half-saturation constant for ADP [K1/2 (ADP)]. We conclude that opening of cardiac mitoKATP is essential for an appropriate response to positive inotropic stress and propose that its involvement proceeds through the prevention of stress-induced decrease in mitochondrial matrix volume. These results indicate a physiological role for mitoKATP in inotropy and, by extension, in heart failure.     

Ablation of Succinate Production from Glucose Metabolism in the Procyclic Trypanosomes Induces Metabolic Switches to the Glycerol 3-Phosphate/Dihydroxyacetone Phosphate Shuttle and to Proline Metabolism

Trypanosoma brucei is a parasitic protist that undergoes a complex life cycle during transmission from its mammalian host (bloodstream forms) to the midgut of its insect vector (procyclic form). In both parasitic forms, most glycolytic steps take place within specialized peroxisomes, called glycosomes. Here, we studied metabolic adaptations in procyclic trypanosome mutants affected in their maintenance of the glycosomal redox balance. T. brucei can theoretically use three strategies to maintain the glycosomal NAD⁺/NADH balance as follows: (i) the glycosomal succinic fermentation branch; (ii) the glycerol 3-phosphate (Gly-3-P)/dihydroxyacetone phosphate (DHAP) shuttle that transfers reducing equivalents to the mitochondrion; and (iii) the glycosomal glycerol production pathway. We showed a hierarchy in the use of these glycosomal NADH-consuming pathways by determining metabolic perturbations and adaptations in single and double mutant cell lines using a combination of NMR, ion chromatography-MS/MS, and HPLC approaches. Although functional, the Gly-3-P/DHAP shuttle is primarily used when the preferred succinate fermentation pathway is abolished in the Δpepck knock-out mutant cell line. In the absence of these two pathways (Δpepck/^RNAiFAD-GPDH.i mutant), glycerol production is used but with a 16-fold reduced glycolytic flux. In addition, the Δpepck mutant cell line shows a 3.3-fold reduced glycolytic flux compensated by an increase of proline metabolism. The inability of the Δpepck mutant to maintain a high glycolytic flux demonstrates that the Gly-3-P/DHAP shuttle is not adapted to the procyclic trypanosome context. In contrast, this shuttle was shown earlier to be the only way used by the bloodstream forms of T. brucei to sustain their high glycolytic flux.     

High level of uncoupling protein 1 expression in muscle of transgenic mice selectively affects muscles at rest and decreases their IIb fiber content

The mitochondrial uncoupling protein of brown adipose tissue (UCP1) was expressed in skeletal muscle and heart of transgenic mice at levels comparable with the amount found in brown adipose tissue mitochondria. These transgenic mice have a lower body weight, and when related to body weight, food intake and energy expenditure are increased. A specific reduction of muscle mass was observed but varied according to the contractile activity of muscles. Heart and soleus muscle are unaffected, indicating that muscles undergoing regular contractions, and therefore with a continuous mitochondrial ATP production, are protected. In contrast, the gastrocnemius and plantaris muscles showed a severely reduced mass and a fast to slow shift in fiber types promoting mainly IIa and IIx fibers at the expense of fastest and glycolytic type IIb fibers. These observations are interpreted as a consequence of the strong potential dependence of the UCP1 protonophoric activity, which ensures a negligible proton leak at the membrane potential observed when mitochondrial ATP production is intense. Therefore UCP1 is not deleterious for an intense mitochondrial ATP production and this explains the tolerance of the heart to a high expression level of UCP1. In muscles at rest, where ATP production is low, the rise in membrane potential enhances UCP1 activity. The proton return through UCP1 mimics the effect of a sustained ATP production, permanently lowering mitochondrial membrane potential. This very likely constitutes the origin of the signal leading to the transition in fiber types at rest.     

Systemic virulence of Erwinia chrysanthemi 3937 requires a functional iron assimilation system.

In Erwinia chrysanthemi, conditions of iron starvation initiate production of a catechol-type siderophore and enhance production of three outer membrane polypeptides. Twenty-two mutants affected in the different stages of this iron assimilation system were isolated by mini-Mu insertion mutagenesis. All of them failed to induce systemic soft rot on axenically grown Saintpaulia plants. From the siderophore auxotrophs and the iron uptake mutants, clones having recovered the missing function(s) were isolated by using the in vivo cloning vector pULB113 (RP4::mini-Mu). An R-prime plasmid containing a ca. 35.5-kilobase-pair DNA insert was identified. Restoration of the iron functions restored partially, if not completely, the virulence of the parental strain.     

Alteration of mitochondrial oxidative phosphorylation in aged skeletal muscle involves modification of adenine nucleotide translocator

The process of skeletal muscle aging is characterized by a progressive loss of muscle mass and functionality. The underlying mechanisms are highly complex and remain unclear. This study was designed to further investigate the consequences of aging on mitochondrial oxidative phosphorylation in rat gastrocnemius muscle, by comparing young (6 months) and aged (21 months) rats. Maximal oxidative phosphorylation capacity was clearly reduced in older rats, while mitochondrial efficiency was unaffected. Inner membrane properties were unaffected in aged rats since proton leak kinetics were identical to young rats. Application of top-down control analysis revealed a dysfunction of the phosphorylation module in older rats, responsible for a dysregulation of oxidative phosphorylation under low activities close to in vivo ATP turnover. This dysregulation is responsible for an impaired mitochondrial response toward changes in cellular ATP demand, leading to a decreased membrane potential which may in turn affect ROS production and ion homeostasis. Based on our data, we propose that modification of ANT properties with aging could partly explain these mitochondrial dysfunctions.     

Effect of bovine serum albumin on membrane potential in plant mitochondria

The membrane potential in highly coupled potato ( Solanum tuberosum L.) mitochondria, as measured by changes in safranine absorbance, was significantly increased by addition of bovine serum albumin. Purification of potato mitochondria on Percoll, in removing 50% of free unsaturated fatty acids, decreased the BSA-de-pendent membrane potential. The effect of added linoleic acid and of the natural accumulation of fatty acids during aging was studied. The response of membrane potential to addition of bovine serum albumin appeared to be directly correlated to the amount of free unsaturated fatty acids. Aging in vitro, in releasing free fatty acids, decreased respiratory control and ADP:O ratios and collapsed the membrane potential. During 2–3 h of incubation, addition of BSA completely restored membrane potential and oxidative phosphorylation.
It is concluded that both in fresh and in aged potato mitochondria the effect of bovine serum albumin on oxidative phosphorylation can be ascribed to an effect on membrane permeability to ions. BSA, in binding free unsaturated fatty acids, restored maximal membrane potential. The bovine serum albumin-dependent membrane potential appears to be a sensitive criterion of the functional integrity of the inner mitochondrial membrane.