Direct Demonstration of Half-of-the-sites Reactivity in the Dimeric Cytochrome bc1 Complex: ENZYME WITH ONE INACTIVE MONOMER IS FULLY ACTIVE BUT UNABLE TO ACTIVATE THE SECOND UBIQUINOL OXIDATION SITE IN RESPONSE TO LIGAND BINDING AT THE UBIQUINONE REDUCTION SITE*

We previously proposed that the dimeric cytochrome bc₁ complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc₁ monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289–22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc₁ complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10–16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c₁ by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wild-type dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.     

Design, synthesis, and biological evaluation of pirenzepine analogs bearing a 1,2-cyclohexanediamine and perhydroquinoxaline units in exchange for the piperazine ring as antimuscarinics

Pirenzepine (2) is one of the most selective muscarinic M(1) versus M(2) receptor antagonists known. A series of 2 analogs, in which the piperazyl moiety was replaced by a cis- and trans-cyclohexane-1,2-diamine (3-6) or a trans- and cis-perhydroquinoxaline rings (7 and 8) were prepared, with the aim to investigate the role of the piperazine ring of 2 in the interaction with the muscarinic receptors. The structural change leading to compounds 3-6 abolished in binding assays the muscarinic M(1)/M(2) selectivity of 2, due to an increased M(2) affinity. Rather, compounds 3-6 displayed a reversed selectivity showing more affinity at the muscarinic M(2) receptor than at all the other subtypes tested.     

Adenoviral-mediated overexpression of catalase inhibits endothelial cell proliferation

Although hydrogen peroxide (H(2)O(2)) induces proliferation of vascular smooth muscle cells, its role in endothelial cell proliferation is unclear. Our aim was to study the role of hydrogen peroxide in endothelial cell proliferation by overexpressing catalase. Human aortic endothelial cells were transduced with adenoviral vectors encoding beta-galactosidase (Adbetagal) or catalase (AdCat) or were exposed to diluent alone (control). Transgene expression was demonstrated by beta-galactosidase staining, Western analysis, and significantly increased enzyme activity in AdCat-transduced cells. Overexpression of catalase decreased DNA synthesis in AdCat compared with control and Adbetagal-transduced cells (536.8 +/- 31 vs. 1,875.1 +/- 132.9 vs. 1,347.5 +/- 93.7 dpm/well, respectively; P < 0.05 vs. control and Adbetagal). Six days after transduction with AdCat (multiplicity of infection = 50), cell numbers were significantly reduced (AdCat: 38 +/- 1.8% of cell counts in control, P < 0.05; and 45 +/- 2% of cell count in Adbetagal, P < 0.05). Incubation with aminotriazole 10 mmol/l, an inhibitor of catalase, prevented this effect. The number of apoptotic cells was increased one- and threefold 2 and 4 days, respectively, after transduction with AdCat. Exogenous administration of low concentrations of H(2)O(2) (50 microM) significantly increased cell proliferation, whereas it was inhibited by higher concentrations. These results suggest that H(2)O(2) is an important modulator of endothelial cell proliferation.     

Endogenous ghrelin is an orexigenic peptide acting in the arcuate nucleus in response to fasting

Ghrelin, a circulating growth-hormone releasing peptide derived from stomach, stimulates food intake through neuropeptide Y (NPY) neurons of the arcuate nucleus in the hypothalamus (ARC). We examined the effect of ghrelin microinjected into the ARC and the influence of intracerebroventricular (i.c.v.) pretreatment with a GHRH or NPY receptor antagonist on ghrelin-induced food intake in free-feeding male rats. Ghrelin (0.1-1 microg) stimulated food intake in a dose-dependent manner, and this effect was reduced by 55-60% by the Y(5) NPY receptor antagonist (10 microg i.c.v.), but not by the GHRH receptor antagonist MZ-4-71 (10 microg i.c.v.). We also evaluated the effects of passive ghrelin immunoneutralization by the microinjection of anti-ghrelin immunoglobulins (IgGs) intracerebroventricularly or directly into the ARC on food intake in free-feeding and fasted male rats. i.c.v. administration of anti-ghrelin IgGs decreased cumulative food intake over 24 h, whereas microinfusion of anti-ghrelin IgGs into the ARC induced only a short-lived (2 and 6 h) effect. Collectively, these data would indicate that centrally derived ghrelin has a major role in the control of food intake in rats and, in this context, blood-born ghrelin would be effective only in relation to its ability to reach the ARC, which is devoid of blood-brain barrier.     

Leber's hereditary optic neuropathy (LHON) pathogenic mutations induce mitochondrial-dependent apoptotic death in transmitochondrial cells incubated with galactose medium

Leber's hereditary optic neuropathy (LHON), a maternally inherited form of central vision loss, is associated with mitochondrial DNA pathogenic point mutations affecting different subunits of complex I. We here report that osteosarcoma-derived cytoplasmic hybrids (cybrid) cell lines harboring one of the three most frequent LHON pathogenic mutations, at positions 11778/ND4, 3460/ND1, and 14484/ND6, undergo cell death when galactose replaces glucose in the medium, contrary to control cybrids that maintain some growth capabilities. This is a well known way to produce a metabolic stress, forcing the cells to rely on the mitochondrial respiratory chain to produce ATP. We demonstrate that LHON cybrid cell death is apoptotic, showing chromatin condensation and nuclear DNA laddering. Moreover, we also document the mitochondrial involvement in the activation of the apoptotic cascade, as shown by the increased release of cytochrome c into the cytosol in LHON cybrid cells as compared with controls. Cybrids bearing the 3460/ND1 and 14484/ND6 mutations seemed more readily prone to undergo apoptosis as compared with the 11778/ND4 mutation. In conclusion, LHON cybrid cells forced by the reduced rate of glycolytic flux to utilize oxidative metabolism are sensitized to an apoptotic death through a mechanism involving mitochondria.     

Recognition of RNA cap in the Wesselsbron virus NS5 methyltransferase domain: implications for RNA-capping mechanisms in Flavivirus

The mRNA-capping process starts with the conversion of a 5′-triphosphate end into a 5′-diphosphate by an RNA triphosphatase, followed by the addition of a guanosine monophosphate unit in a 5′-5′ phosphodiester bond by a guanylyltransferase. Methyltransferases are involved in the third step of the process, transferring a methyl group from S-adenosyl-l-methionine to N7-guanine (cap 0) and to the ribose 2′OH group (cap 1) of the first RNA nucleotide; capping is essential for mRNA stability and proper replication. In the genus Flavivirus, N7-methyltransferase and 2′O-methyltransferase activities have been recently associated with the N-terminal domain of the viral NS5 protein. In order to further characterize the series of enzymatic reactions that support capping, we analyzed the crystal structures of Wesselsbron virus methyltransferase in complex with the S-adenosyl-l-methionine cofactor, S-adenosyl-l-homocysteine (the product of the methylation reaction), Sinefungin (a molecular analogue of the enzyme cofactor), and three different cap analogues (GpppG, ^N7MeGpppG, and ^N7MeGpppA). The structural results, together with those on other flaviviral methyltransferases, show that the capped RNA analogues all bind to an RNA high-affinity binding site. However, lack of specific interactions between the enzyme and the first nucleotide of the RNA chain suggests the requirement of a minimal number of nucleotides following the cap to strengthen protein/RNA interaction. Our data also show that, following incubation with guanosine triphosphate, Wesselsbron virus methyltransferase displays a guanosine monophosphate molecule covalently bound to residue Lys28, hinting at possible implications for the transfer of a guanine group to ppRNA. The structures of the Wesselsbron virus methyltransferase complexes obtained are discussed in the context of a model for N7-methyltransferase and 2′O-methyltransferase activities.     

Rhabdias rhampholeonis n. sp. and Rhabdias mariauxi n. sp. (Nematoda, Rhabdiasoidea), first lung worms from leaf chameleons: Description, molecular evidence and notes on biology

Rhabdias rhampholeonis n. sp. from Rhampholeon (Rh.) spectrum, Cameroon, and Rhabdias mariauxi n. sp. from Rieppeleon brevicaudatus, Tanzania, are the first lung worms from leaf chameleons. The new species are similar to the majority of species parasitic in chamaeleonids by having a long (≥10 mm) and thick body (≥500 µm), long oesophagus (≥800 µm), wide buccal capsule (≥40 µm) and low buccal ratio (<0.5). They most closely resemble Rhabdias chamaeleonis and Rhabdias cristati parasitic in Trioceros spp. from East Africa and Cameroon, respectively. Main distinctive characters are a buccal capsule composed of two segments and the head shape. The dorso-ventrally flattened buccal capsule of R. mariauxi n. sp. is unique in Rhabdias parasitising Chamaeleonidae. Sequences of the 12S rDNA and mitochondrial cytochrome c oxidase subunit 1 (coxI) genes were obtained and compared to those of Rhabdias okuensis, the only sequences published for chamaeleonid lung worms. The smallest nucleotide interspecific distances were found between R. mariauxi n. sp. and the former species of Trioceros from Cameroon. Hermaphroditism in females in the lungs, and R. mariauxi n. sp. free-living stages are like in other species from Chamaeleonidae, but the number of infective larvae produced per free-living female (one or two) was not fixed.     

Know Thyself: Behavioral Evidence for a Structural Representation of the Human Body

Background

Representing one''s own body is often viewed as a basic form of self-awareness. However, little is known about structural representations of the body in the brain.

Methods and Findings

We developed an inter-manual version of the classical “in-between” finger gnosis task: participants judged whether the number of untouched fingers between two touched fingers was the same on both hands, or different. We thereby dissociated structural knowledge about fingers, specifying their order and relative position within a hand, from tactile sensory codes. Judgments following stimulation on homologous fingers were consistently more accurate than trials with no or partial homology. Further experiments showed that structural representations are more enduring than purely sensory codes, are used even when number of fingers is irrelevant to the task, and moreover involve an allocentric representation of finger order, independent of hand posture.

Conclusions

Our results suggest the existence of an allocentric representation of body structure at higher stages of the somatosensory processing pathway, in addition to primary sensory representation.     

Calcium Dependence of Synaptic Vesicle Recycling Before and After Synaptogenesis

Abstract: Using an immunocytochemical assay to monitor synaptic vesicle exocytosis/endocytosis independently of neurotransmitter release, we have investigated some aspects of vesicle recycling in hippocampal neurons at different developmental stages. A calcium- and depolarization-dependent exocytotic/endocytotic recycling of synaptic vesicles was found to take place in neurons already before the formation of synaptic contacts. The analysis of synaptic vesicle recycling at different calcium concentrations revealed the presence of two release components: the first one activated by low calcium concentrations and sustaining vesicle recycling before synaptogenesis, and a second one activated by high calcium concentrations, which is specifically turned on after the establishment of synaptic contacts. These data suggest that formation of synapses correlates with the activation of a putative low-affinity calcium sensor, which allows synaptic vesicle exocytosis to be triggered and turned off over extremely short time scales, in response to large increases in the level of intracellular calcium.