New insights into the membrane-binding and activation mechanism of pyruvate oxidase from Escherichia coli

Pyruvate oxidase from Escherichia coli (EcPOX) is a thiamin diphosphate- (ThDP) and FAD-dependent peripheral membrane protein that carries out the irreversible oxidative decarboxylation of pyruvate to acetate and carbon dioxide. Concomitant two-electron reduction of the flavin cofactor was suggested to induce a structural rearrangement of the C-terminus triggering recruitment of the protein from the cytosol to the cell membrane, where the electrons are eventually transferred to final electron acceptor ubiquinone 8. Binding to the membrane, or alternatively, mild proteolytic digestion leads to a multifold enhancement of both the catalytic activity and substrate affinity. Recent X-ray crystallographic studies on EcPOX in the resting state and on a C-terminal truncation variant mimicking the membrane-bound activated form have fueled our understanding of the membrane-binding mechanism and concomitant catalytic activation. In the resting state, the auto-inhibitory C-terminal membrane anchor adopts a half-barrel/helix fold that occludes the active site. Upon activation, the C-terminus is expelled and becomes structurally flexible thereby freeing the active site. Circular dichroism spectroscopic analysis revealed the isolated C-terminus to be disordered, however, formation of a helical structure was observed in the presence of micelles. Limited proteolysis experiments indicate that activation of EcPOX involves at least two sequential structural transitions: the first occurring after binding of pyruvate to ThDP and the second after two-electron reduction of the flavin.     

Coagulation factors and their inhibitors

A comprehensive three-dimensional picture of the coagulation process is beginning to emerge. Crystallographic structure determinations of prothrombin, factor Xa, factor IXa, tissue factor and factor XIII represent important advances in our understanding of the coagulation cascade. Similarly, structures of antithrombin, tissue factor pathway inhibitor and thrombomodulin provide details of endogenous anticoagulatory mechanisms. NMR spectroscopy of multiple domains of coagulation proteins represents an important contribution to the analysis of flexibility and rigidity of modular proteins. Thrombin, as the prime candidate for antithrombotic drug design, continues to be an object of intense efforts in applied crystallography.     

Frequency and the brain

The brain is an information handling device. The information is coded as the frequency of nerve action potentials. Mechanisms of the encoding and manipulation of information are discussed. A general discussion of the cortex as a frequency generating and frequency sensitive device is given.     

Forced Exercise Preconditioning Attenuates Experimental Autoimmune Neuritis by Altering Th1 Lymphocyte Composition and Egress

A short-term exposure to moderately intense physical exercise affords a novel measure of protection against autoimmune-mediated peripheral nerve injury. Here, we investigated the mechanism by which forced exercise attenuates the development and progression of experimental autoimmune neuritis (EAN), an established animal model of Guillain–Barré syndrome. Adult male Lewis rats remained sedentary (control) or were preconditioned with forced exercise (1.2 km/day × 3 weeks) prior to P2-antigen induction of EAN. Sedentary rats developed a monophasic course of EAN beginning on postimmunization day 12.3 ± 0.2 and reaching peak severity on day 17.0 ± 0.3 (N = 12). By comparison, forced-exercise preconditioned rats exhibited a similar monophasic course but with significant (p < .05) reduction of disease severity. Analysis of popliteal lymph nodes revealed a protective effect of exercise preconditioning on leukocyte composition and egress. Compared with sedentary controls, forced exercise preconditioning promoted a sustained twofold retention of P2-antigen responsive leukocytes. The percentage distribution of pro-inflammatory (T_h1) lymphocytes retained in the nodes from sedentary EAN rats (5.1 ± 0.9%) was significantly greater than that present in nodes from forced-exercise preconditioned EAN rats (2.9 ± 0.6%) or from adjuvant controls (2.0 ± 0.3%). In contrast, the percentage of anti-inflammatory (T_h2) lymphocytes (7–10%) and that of cytotoxic T lymphocytes (∼20%) remained unaltered by forced exercise preconditioning. These data do not support an exercise-inducible shift in T_h1:T_h2 cell bias. Rather, preconditioning with forced exercise elicits a sustained attenuation of EAN severity, in part, by altering the composition and egress of autoreactive proinflammatory (T_h1) lymphocytes from draining lymph nodes.     

Artemia hemoglobins: Increase in net synthesis of the β-polypeptide (relative to the α-polypeptide) in hypoxia

Previous studies have shown that in the brine shrimp there are three dimeric hemoglobins with polypeptide composition α₂, αβ, β₂. Concentrations of the α- and β-polypeptides increase in hypoxia. We now report a two-dimensional electrophoretic method for assay of radiolabelled polypeptidesin each hemoglobin. Net synthesis (synthesis minus degradation) of the β-chain, relative to that of the α-chain, increases more than 3-fold (in male and female adults) within 3 days following a downshift in oxygen concentration from 0.2 to 0.1 mM in the culture medium. 3 days after downshift (2 days after in vivo incorporation of radiolabelled leucine), the β-homodimer contained 10–20% of the radiolabel in the three hemoglobins although β₂ was usually not detectable in the protein stain of an overloaded gel. The amount of radioactive leucine incorporated per unit amount of protein was more than 300-times greater in the β₂ homodimer than in the β-subunit of the heterodimer, suggesting that β₂ does not dissociate rapidly during electrophoresis on the first dimension non-denaturing gel. This evidence for stable association of the two β-monomers and the 5–8 heme-binding domains within each monomer (in vivo and during electrophoresis on non-denaturing gels) allows us to exclude one of two alternative interpretations of genetic data published previously. We present an independent line of evidence for the dimer model of the native hemoglobins (which states that each polypeptide has many heme-binding domains).     

The ecology of a Mediterranean tortoise (Testudo hermanni): Reproduction

A three-year study on reproduction in wild populations of Testudo hermanni has shown that:
( a ) sexual activity occurs between March and October, except the nesting season (May and June), and is most frequent in August and September,
( b ) tortoises are promiscuous in their mating habits, there is no evidence of mate selection in relation to size,
( c ) nest site selection is correlated to ground temperature and nesting itself mainly occurs in the early evening before dark, when the ground temperature characteristics are most indicative of a specific site's relative temperature,
( d ) incubation takes about three months and is probably temperature dependent,
( e ) most nests are preyed upon by mammalian predators within a few days after laying (> 90% being destroyed), the main cue being the smell of the eggs,
( f ) average clutch size in Greece is higher than in France, while mean egg weight in France is higher than in Greece, and there is no strong relationship between body size or weight and egg or clutch weight in either country,
( g ) in contrast to other studies, there is no apparent correlation between rainfall and reproduction parameters,
( h ) some females nest in successive years, and more than once in a year, with a laying interval of 10–20 days,
( i ) mean size of sexually active individuals is not significantly different from the mean size of adults, although nesting females are significantly bigger; the minimum size for sexually active males is about 120 mm and for females 130 mm.
The discussion examines these observations in an evolutionary context.