Non-local thermodynamic effects and efficiency of oxidative phosphorylation

A non-equilibrium thermodynamic model of oxidative phosphorylation is formulated, which allows us to take into account some non-local effects. In this way, we compute the influence of the tangential resistivity of the inner mitochondrial membrane to proton current, as well as that of the distance between active sites, on the stoichiometry and efficiency of energy conversion.     

Bacteriophage MS2 RNA: A correlation between the stability of the codon: Anticodon interaction and the choice of code words

Summary The non-random distribution of degenerate code words in Bacteriophage MS2 RNA can be explained partially by considerations of the stability of the codon-anticodon complex in prokaryotic systems. Supporting this hypothesis we note that wobble codons are positively selected in codons having G and/or C in the first two positions. In contrast, wobble codons are statitically less likely in codons composed of A and U in the first two positions. Analyses of nucleotides adjacent to 5 and 3 ends of codons indicate a nonrandom distribution as well. It is thus likely that some elements of RNA evolution are independent of the structural needs of the RNA itself and of the translated protein product.This work was supported by grants from the Belgian Government Actions concertées - Gekon-certéerde Acties, N.F.W.O. and F.K.F.O. as well as from le Ministère de l'éducation du Québec. A preliminary report of this work was given at the EMBO ribosome workshop, Brussels 1976     

Hapten-specific B cell repertoire probed by hybridoma technology: selection and characterization of representative clonotypes from the antibody-forming cell pool

In the course of this study, more than 3000 phthalate-specific antibody-forming cell hybrids were identified using the hybridoma technology. With the aid of a rapid screening assay and an extensive library of phthalate analogs, it was possible to assign selected hapten-specific clones to one of 11 distinct fine-specificity sets. This compartmentalization of the phthalate-specific hybridomas has made it possible to focus attention upon a single manageable portion of the phthalate-specific repertoire. Fourteen clones from a single fine-specificity set were selected for further immunochemical characterization. Five of these clones were found to secrete an antibody that was indistinguishable in isoelectric focusing. Affinity-purified, high-resolution anti-idiotype antibodies were prepared with specificity for the antibodies produced by one of these clones (i.e., 4C7). A major portion of the serologically defined private idiotype (4C7 IdI) was shown to be associated with the ligand-combining site. Our results indicate that the five clones that share a common spectrotype also express the 4C7 IdI. Two other independently derived clones from two distinct fusions also share this idiotype. The 4C7 IdI was also identified in affinity-purified anti-phthalate antibodies derived from a pool of phthalate-immune serum (conventional antibody) and from affinity-purified antibodies derived from a pool of serum from unimmunized BALB/c mice (natural antibody). The 4C7 IdI is thus considered to represent a repeating clonotype in the phthalate-specific repertoire of BALB/c mice, and will serve as one of several useful clonal markers that are being developed for studies of the mechanism regulating idiotype expression.     

mtDNA T8993G Mutation-Induced F1F0-ATP Synthase Defect Augments Mitochondrial Dysfunction Associated with hypoxia/reoxygenation: The Protective Role of Melatonin

Background

F1F0-ATP synthase (F1F0-ATPase) plays important roles in regulating mitochondrial function during hypoxia, but the effect of F1F0-ATPase defect on hypoxia/reoxygenation (H/RO) is unknown. The aim of this study was to investigate how mtDNA T8993G mutation (NARP)-induced inhibition of F1F0-ATPase modulates the H/RO–induced mitochondrial dysfunction. In addition, the potential for melatonin, a potent antioxidant with multiple mitochondrial protective properties, to protect NARP cells exposed to H/RO was assessed.

Methods And Findings

NARP cybrids harboring 98% of mtDNA T8993G genes were established as an in vitro model for cells with F1F0-ATPase defect; their parental osteosarcoma 143B cells were studied for comparison. Treating the cells with H/RO using a hypoxic chamber resembles ischemia/reperfusion in vivo. NARP significantly enhanced apoptotic death upon H/RO detected by MTT assay and the trypan blue exclusion test of cell viability. Based on fluorescence probe-coupled laser scanning imaging microscopy, NARP significantly enhanced mitochondrial reactive oxygen species (mROS) formation and mitochondrial Ca²⁺ (mCa²⁺) accumulation in response to H/RO, which augmented the depletion of cardiolipin, resulting in the retardation of mitochondrial movement. With stronger H/RO stress (either with longer reoxygenation duration, longer hypoxia duration, or administrating secondary oxidative stress following H/RO), NARP augmented H/RO-induced mROS formation to significantly depolarize mitochondrial membrane potential (ΔΨm), and enhance mCa²⁺ accumulation and nitric oxide formation. Also, NARP augmented H/RO-induced mROS oxidized and depleted cardiolipin, thereby promoting permanent mitochondrial permeability transition, retarded mitochondrial movement, and enhanced apoptosis. Melatonin markedly reduced NARP-augmented H/RO-induced mROS formation and therefore significantly reduced mROS-mediated depolarization of ΔΨm and accumulation of mCa²⁺, stabilized cardiolipin, and then improved mitochondrial movement and cell survival.

Conclusion

NARP-induced inhibition of F1F0-ATPase enhances mROS formation upon H/RO, which augments the depletion of cardiolipin and retardation of mitochondrial movement. Melatonin may have the potential to rescue patients with ischemia/reperfusion insults, even those associated with NARP symptoms.     

Coenzyme Q10 deficiency in mitochondrial DNA depletion syndromes

We evaluated coenzyme Q₁₀ (CoQ) levels in patients studied under suspicion of mitochondrial DNA depletion syndromes (MDS) (n = 39). CoQ levels were quantified by HPLC, and the percentage of mtDNA depletion by quantitative real-time PCR. A high percentage of MDS patients presented with CoQ deficiency as compared to other mitochondrial patients (Mann–Whitney-U test: p = 0.001). Our findings suggest that MDS are frequently associated with CoQ deficiency, as a possible secondary consequence of disease pathophysiology. Assessment of muscle CoQ status seems advisable in MDS patients since the possibility of CoQ supplementation may then be considered as a candidate therapy.     

Modulation of endocytic traffic in polarized Madin-Darby canine kidney cells by the small GTPase RhoA

Efficient postendocytic membrane traffic in polarized epithelial cells is thought to be regulated in part by the actin cytoskeleton. RhoA modulates assemblies of actin in the cell, and it has been shown to regulate pinocytosis and phagocytosis; however, its effects on postendocytic traffic are largely unexplored. To this end, we expressed wild-type RhoA (RhoAWT), dominant active RhoA (RhoAV14), and dominant inactive RhoA (RhoAN19) in Madin-Darby canine kidney (MDCK) cells expressing the polymeric immunoglobulin receptor. RhoAV14 expression stimulated the rate of apical and basolateral endocytosis, whereas RhoAN19 expression decreased the rate from both membrane domains. Polarized basolateral recycling of transferrin was disrupted in RhoAV14-expressing cells as a result of increased ligand release at the apical pole of the cell. Degradation of basolaterally internalized epidermal growth factor was slowed in RhoAV14-expressing cells. Although apical recycling of immunoglobulin A (IgA) was largely unaffected in cells expressing RhoAV14, transcytosis of basolaterally internalized IgA was severely impaired. Morphological and biochemical analyses demonstrated that a large proportion of IgA internalized from the basolateral pole of RhoAV14-expressing cells remained within basolateral early endosomes and was slow to exit these compartments. RhoAN19 and RhoAWT expression had little effect on these postendocytic pathways. These results indicate that in polarized MDCK cells activated RhoA may modulate endocytosis from both membrane domains and postendocytic traffic at the basolateral pole of the cell.     

Functional compensation by particular nucleotide substitutions of a critical G*U wobble base-pair during aminoacylation of transfer RNA

Expression of the genetic code depends on precise tRNA aminoacylation by cognate aminoacyl-tRNA synthetase enzymes. The G.U wobble base-pair in the acceptor helix of Escherichia coli alanine tRNA is the primary aminoacylation determinant of this molecule. Previous work on the process of synthetase recognition of the G.U pair showed that replacing G.U by a G.C Watson-Crick base-pair inactivates alanine acceptance by the tRNA, but that C.A and G.A wobble pair replacements preserve acceptance. Work by another group reported that the effects of a G.C replacement were reversed by a distal wobble base-pair in the anticodon helix. This result is potentially interesting because it suggests that distant regions in alanine tRNA are functionally coupled during synthetase recognition and more generally because recognition determinants of many other tRNAs lie in both the acceptor helix and anticodon helix region. Here, we have conducted an extensive in vivo analysis of the distal wobble pair in alanine tRNA and report that it does not behave like a compensating mutation. Restoration of alanine acceptance was not detected even when the synthetase enzyme was overproduced. We discuss the previous experimental evidence and suggest how the distal wobble pair was incorrectly analyzed. The available data indicate that all principal recognition determinants of alanine tRNA lie in the molecule's acceptor helix.