t-Butylhydroperoxide and gliotoxin stimulate Ca2+ release from rat skeletal muscle mitochondria

Summary

Rat liver mitochondria have a specific Ca²⁺ release pathway which operates when NAD⁺ is hydrolysed to nicotinamide and ADPribose. NAD⁺ hydrolysis is Ca²⁺-dependent and inhibited by cyclosporine A (CSA). Mitochondrial Ca²⁺ release can be activated by the prooxidant t-butylhydroperoxide (tbh) or by gliotoxin (GT), a fungal metabolite of the epipolythiodioxopiperazine group. Tbh oxidizes NADH to NAD⁺ through an enzyme cascade consisting of glutathione peroxidase, glutathione reductase, and the energy linked transhydrogenase, whereas GT oxidizes some vicinal thiols to the disulfide form, a prerequisite for NAD⁺ hydrolysis. We report now that rat skeletal muscle mitochondria also contain a specific Ca²⁺ release pathway activated by both tbh and GT. Ca²⁺ release increases with the mitochondrial Ca²⁺ load, is completely inhibited in the presence of CSA, and is paralleled by pyridine nucleotide oxidation. In the presence of tbh and GT, mitochondria do not lose their membrane potential and do not swell, provided continuous release and re-uptake of Ca²⁺ (‘Ca²⁺ cycling’) is prevented. These data support the notion that both tbh- and GT-induced Ca²⁺ release are not the consequence of an unspecific increase of the inner membrane permeability (‘pore’ formation). Tbh induces Ca²⁺ release from rat skeletal muscle less efficiently than from liver mitochondria indicating that the coupling between tbh and NADH oxidation is much weaker in skeletal muscle mitochondria. This conclusion is corroborated by a much lower glutathione peroxidase activity in skeletal muscle than in liver mitochondria. The prooxidant-dependent pathway promotes, under drastic conditions (high mitochondrial Ca²⁺ loads and high tbh concentrations), Ca²⁺ release to about the same extent and rate as the Na⁺/Ca²⁺ exchanger. This renders the prooxidant-dependent pathway relevant in the pathophysiology of mitochondrial myopathies where its activation by an increased generation of reactive oxygen species probably results in excessive Ca²⁺ cycling and damage to mitochondria.     

Type VI collagen represents a major fraction of connective tissue collagens

A new method for the isolation of type VI collagen from peptic tissue digests is presented which gives tenfold higher yields than methods previously reported. From the amounts of purified protein obtained from human placenta, bovine uterus, chicken gizzard and entire mouse bodies we conclude that type VI collagen represents a major fraction of connective tissue collagens.     

Fosfomycin Permeation through the Outer Membrane Porin OmpF

Fosfomycin is a frequently prescribed drug in the treatment of acute urinary tract infections. It enters the bacterial cytoplasm and inhibits the biosynthesis of peptidoglycans by targeting the MurA enzyme. Despite extensive pharmacological studies and clinical use, the permeability of fosfomycin across the bacterial outer membrane is largely unexplored. Here, we investigate the fosfomycin permeability across the outer membrane of Gram-negative bacteria by electrophysiology experiments as well as by all-atom molecular dynamics simulations including free-energy and applied-field techniques. Notably, in an electrophysiological zero-current assay as well as in the molecular simulations, we found that fosfomycin can rapidly permeate the abundant Escherichia coli porin OmpF. Furthermore, two triple mutants in the constriction region of the porin have been investigated. The permeation rates through these mutants are slightly lower than that of the wild type but fosfomycin can still permeate. Altogether, this work unravels molecular details of fosfomycin permeation through the outer membrane porin OmpF of E. coli and moreover provides hints for understanding the translocation of phosphonic acid antibiotics through other outer membrane pores.     

Understanding Voltage Gating of Providencia stuartii Porins at Atomic Level

Bacterial porins are water-filled β-barrel channels that allow translocation of solutes across the outer membrane. They feature a constriction zone, contributed by the plunging of extracellular loop 3 (L3) into the channel lumen. Porins are generally in the open state, but undergo gating in response to external voltages. To date the underlying mechanism is unclear. Here we report results from molecular dynamics simulations on the two porins of Providenica stuartii, Omp-Pst1 and Omp-Pst2, which display distinct voltage sensitivities. Voltage gating was observed in Omp-Pst2, where the binding of cations in-between L3 and the barrel wall results in exposing a conserved aromatic residue in the channel lumen, thereby halting ion permeation. Comparison of Omp-Pst1 and Omp-Pst2 structures and trajectories suggests that their sensitivity to voltage is encoded in the hydrogen-bonding network anchoring L3 onto the barrel wall, as we observed that it is the strength of this network that governs the probability of cations binding behind L3. That Omp-Pst2 gating is observed only when ions flow against the electrostatic potential gradient of the channel furthermore suggests a possible role for this porin in the regulation of charge distribution across the outer membrane and bacterial homeostasis.     

Fluorescence Quenching as a Tool to Investigate Quinolone Antibiotic Interactions with Bacterial Protein OmpF

The outer membrane porin OmpF is an important protein for the uptake of antibiotics through the outer membrane of gram-negative bacteria; however, the possible binding sites involved in this uptake are still not recognized. Determination, at the molecular level, of the possible sites of antibiotic interaction is very important, not only to understand their mechanism of action but also to unravel bacterial resistance. Due to the intrinsic OmpF fluorescence, attributed mainly to its tryptophans (Trp²¹⁴, Trp⁶¹), quenching experiments were used to assess the site(s) of interaction of some quinolone antibiotics. OmpF was reconstituted in different organized structures, and the fluorescence quenching results, in the presence of two quenching agents, acrylamide and iodide, certified that acrylamide quenches Trp⁶¹ and iodide Trp²¹⁴. Similar data, obtained in presence of the quinolones, revealed distinct behaviors for these antibiotics, with nalidixic acid interacting near Trp²¹⁴ and moxifloxacin near Trp⁶¹. These studies, based on straightforward and quick procedures, show the existence of conformational changes in the protein in order to adapt to the different organized structures and to interact with the quinolones. The extent of reorganization of the protein in the presence of the different quinolones allowed an estimate on the sites of protein/quinolone interaction.     

O-glycosylation of the non-canonical T-cadherin from rabbit skeletal muscle by single mannose residues

O-mannosylation is a vital protein modification. In humans, defective O-mannosylation of α-dystroglycan results in severe congenital muscular dystrophies. However, other proteins bearing this modification in vivo are still largely unknown. Here, we describe a highly reliable method combining glycosidase treatment with LC–MS analyses to identify mammalian O-mannosylated proteins from tissue sources. Our workflow identified T-cadherin (H-cadherin, CDH13) as a novel O-mannosylated protein. In contrast to known O-mannosylated proteins, single mannose residues (Man-α-Ser/Thr) are attached to this cell adhesion molecule. Conserved O-glycosylation sites in T-, E- and N-cadherins from different species, point to a general role of O-mannosyl glycans for cadherin function.     

Thermal environment shapes cuticle melanism and melanin-based immunity in the ground cricket Allonemobius socius

The thermal melanin hypothesis posits that ectothermic individuals of larger size or from colder environments exhibit darker cuticles due to melanin’s efficacy in absorbing solar radiation. However, melanin is also a crucial component of arthropod immunity. Thus, thermal selection for increased cuticle darkness may profoundly influence melanin-based immune function. In this study, we address the relationships between the thermal environment (season length), cuticular melanism and two aspects of melanin-based immunity across nine thermally distinct populations of the cricket Allonemobius socius. We found that season length (i.e. degree days) and body size had a positive association with cuticle melanism in both sexes across populations, supporting the thermal melanism hypothesis. Despite their smaller size, males were found to have darker cuticles and superior melanin-based immunity. This pattern may be the result of additional selection on males due to sex-specific temperature-dependent activities, such as male calling song. Perhaps most interestingly, we found that short season length populations (i.e. colder) exhibited a greater phenoloxidase activity (aspect of the melanin-based immune system) in addition to darker cuticles in both sexes. This pattern is consistent with direct thermal selection on cuticular color, coupled with indirect selection on melanin-based immunity due to pleiotropy. Thus, thermal selection on cuticle darkness appears to indirectly shape the evolution of pathogen resistance in this system, and potentially for other terrestrial arthropod systems whose ranges encompass a significant thermal gradient.