Inhibition of cytochrome c oxidase activity in rat cerebral cortex and human skeletal muscle by D-2-hydroxyglutaric acid in vitro.

L-2-Hydroxyglutaric (LGA) and D-2-hydroxyglutaric (DGA) acids are the characteristic metabolites accumulating in the neurometabolic disorders known as L-2-hydroxyglutaric aciduria and D-2-hydroxyglutaric aciduria, respectively. Although these disorders are predominantly characterized by severe neurological symptoms, the neurotoxic mechanisms of brain damage are virtually unknown. In this study we have evaluated the role of LGA and DGA at concentrations ranging from 0.01 to 5.0 mM on various parameters of energy metabolism in cerebral cortex slices and homogenates of 30-day-old Wistar rats, namely glucose uptake, CO(2) production and the respiratory chain enzyme activities of complexes I to IV. DGA significantly decreased glucose utilization (2.5 and 5.0 mM) by brain homogenates and CO(2) production (5 mM) by brain homogenates and slices, whereas LGA had no effect on either measurement. Furthermore, DGA significantly inhibited cytochrome c oxidase activity (complex IV) (EC 1.9.3.1) in a dose-dependent manner (35-95%) at doses as low as 0.5 mM, without compromising the other respiratory chain enzyme activities. In contrast, LGA did not interfere with these activities. Our results suggest that the strong inhibition of cytochrome c oxidase activity by increased levels of DGA could be related to the neurodegeneration of patients affected by D-2-hydroxyglutaric aciduria.     

Neuromuscular control of a single twitch muscle in wild type and mutant <Emphasis Type="Italic">Drosophila</Emphasis>, measured with an ergometer

How do deficits in neuronal growth, aging or synaptic function affect the final, mechanical output of a single muscle twitch? We address this in vivo (indeed in situ) with a novel ergometer that records the output of a large specialised muscle, the Drosophila jump muscle. Here, we describe in detail the ergometer, its construction and use. We evaluated the ergometer by showing that adult fly jump muscle output varies little between 3 h and 7 days; but newly eclosed flies produce only 65%. In a mutant with little octopamine (Tβh), jump muscle performance is reduced by 28%. The initial responses of synaptic growth mutants (highwire and spinster) do not differ from wild type, as expected on the homeostatic hypothesis. However, responses in highwire mutations gradually decline following repeated stimuli, suggesting physiological as well as anatomical abnormalities. We conclude that the assay is robust, sensitive and reliable with a good throughput.     

Folding intermediates of SNARE complex assembly

SNARE (soluble NSF attachment protein receptor) proteins assemble into a stable complex essential for vesicle-membrane fusion. To further understand SNARE function we have used solution nuclear magnetic resonance (NMR) spectroscopy to characterize three assembly states of a yeast SNARE complex: first, the 'closed' conformation of Sso1; second, the binary complex of Sso1 and Sec9; and third, the ternary complex of Sso1, Sec9 and Snc1. Sec9 and Snc1 are unstructured in isolation. Sso1 likely consists of a four helix bundle formed by part of the C-terminal Hcore domain and the N-terminal H(A)H(B)H(C) domain, and this bundle is flanked on both sides by large flexible regions. Sso1 switches to an 'open' state when its Hcore domain binds Sec9. Conformational switching of the Hcore domain, via H(A)H(B)H(C), may provide a key regulatory mechanism in SNARE assembly. Formation of binary and ternary complexes induces additional alpha-helical structure in previously unstructured regions. Our data suggest a directed assembly process beginning distal to the membrane surfaces and proceeding toward them, bringing membranes into close proximity and possibly leading to membrane fusion.     

Electron cryomicroscopy structure of N-ethyl maleimide sensitive factor at 11 A resolution

N-ethyl maleimide sensitive factor (NSF) belongs to the AAA family of ATPases and is involved in a number of cellular functions, including vesicle fusion and trafficking of membrane proteins. We present the three-dimensional structure of the hydrolysis mutant E329Q of NSF complexed with an ATP-ADP mixture at 11 A resolution by electron cryomicroscopy and single-particle averaging of NSF.alpha-SNAP.SNARE complexes. The NSF domains D1 and D2 form hexameric rings that are arranged in a double-layered barrel. Our structure is more consistent with an antiparallel orientation of the two rings rather than a parallel one. The crystal structure of the D2 domain of NSF was docked into the EM density map and shows good agreement, including details at the secondary structural level. Six protrusions corresponding to the N domain of NSF (NSF-N) emerge from the sides of the D1 domain ring. The density corresponding to alpha-SNAP and SNAREs is located on the 6-fold axis of the structure, near the NSF-N domains. The density of the N domain is weak, suggesting conformational variability in this part of NSF.     

Sympatric Plasmodium falciparum isolates from Venezuela have structured var gene repertoires

Background

Plasmodium falciparum antigenic diversity and polymorphism confuses the issue of antimalarial vaccine development. Merozoite surface protein (MSP)-1 and -2 are two highly polymorphic vaccine candidates. Characterisation of their precise polymorphism in endemic regions may facilitate the design of an effective vaccine.

Methods

Isolates obtained in 52 Gabonese children presenting with uncomplicated malaria were genotyped by nested-PCR of msp -1 block 2, and msp -2 block 3, to analyze both parasite population polymorphism and clone fluctuations.

Results

Twenty-five and 19 different alleles were respectively obtained for msp-1 and msp-2 loci, the RO33 family of msp-1 being poorly polymorphic. Four cases of non-random distribution of alleles were reported of the FC27, and/or 3D7 families of msp-2. All but two isolates were composed of more than one genotype, and the multiplicity of infection (MOI) was 4.0. Neither parasite density nor age was related to MOI. Clone fluctuations were studied for ten subjects who were sampled again at reappearance of parasites in blood. Disappearance and reappearance of alleles were observed following treatment, suggesting difficulties in assessing polymorphism and in distinguishing reinfection from recrudescence.

Conclusion

P. falciparum polymorphism is extensive in Southeast Gabon, and most of infections are composed of multiple clones. The fluctuation of clones contributes to parasite diversity.     

Crystal structures of a Rab protein in its inactive and active conformations

We have determined crystal structures of Sec4, a member of the Rab family in the G protein superfamily, in two states: bound to GDP, and to a non-hydrolyzable GTP analog, guanosine-5'-(beta, gamma)-imidotriphosphate (GppNHp). This represents the first structure of a Rab protein bound to GDP. Sec4 in both states grossly resembles other G proteins bound to GDP and GppNHp. In Sec4-GppNHp, structural features common to active Rab proteins are observed. In Sec4-GDP, the switch I region is highly disordered and displaced relative to the switch I region of Ras-GDP. In two of the four molecules of Sec4-GDP in the asymmetric unit of the Sec4-GDP crystals, the switch II region adopts a conformation similar to that seen in the structure of the small G protein Ran bound to GDP. This allows residues threonine 76, glutamate 80, and arginine 81 of Sec4 to make contacts with other conserved residues and water molecules important for nucleotide binding. In the other two molecules in the asymmetric unit, these interactions do not take place. This structural variability in both the switch I and switch II regions of GDP-bound Sec4 provides a possible explanation for the high off-rate of GDP bound to Sec4, and suggests a mechanism for regulation of the GTPase cycle of Rab proteins by GDI proteins.     

Version 1.2 of the Crystallography and NMR system

Version 1.2 of the software system, termed Crystallography and NMR system (CNS), for crystallographic and NMR structure determination has been released. Since its first release, the goals of CNS have been (i) to create a flexible computational framework for exploration of new approaches to structure determination, (ii) to provide tools for structure solution of difficult or large structures, (iii) to develop models for analyzing structural and dynamical properties of macromolecules and (iv) to integrate all sources of information into all stages of the structure determination process. Version 1.2 includes an improved model for the treatment of disordered solvent for crystallographic refinement that employs a combined grid search and least-squares optimization of the bulk solvent model parameters. The method is more robust than previous implementations, especially at lower resolution, generally resulting in lower R values. Other advances include the ability to apply thermal factor sharpening to electron density maps. Consistent with the modular design of CNS, these additions and changes were implemented in the high-level computing language of CNS.     

Dietary galacto-oligosaccharides prevent airway eosinophilia and hyperresponsiveness in a murine house dust mite-induced asthma model

Background

Allergic asthma is strongly associated with the exposure to house dust mite (HDM) and is characterized by eosinophilic pulmonary inflammation and airway hyperresponsiveness (AHR). Recently, there is an increased interest in using dietary oligosaccharides, also known as prebiotics, as a novel strategy to prevent the development of, or reduce, symptoms of allergy.

Aim

We investigated the preventive capacity of dietary galacto-oligosaccharides (GOS) compared to an intra-airway therapeutic treatment with budesonide on the development of HDM-induced allergic asthma in mice.

Methods

BALB/c mice were intranasally sensitized with 1 μg HDM on day 0 followed by daily intranasal challenge with PBS or 10 μg HDM on days 7 to 11. Two weeks prior to the first sensitization and throughout the experiment mice were fed a control diet or a diet containing 1% GOS. Reference mice were oropharyngeally instilled with budesonide (500 μg/kg) on days 7, 9, 11, and 13, while being fed the control diet. On day 14, AHR was measured by nebulizing increasing doses of methacholine into the airways. At the end of the experiment, bronchoalveolar lavage fluid (BALF) and lungs were collected.

Results

Sensitization and challenge with HDM resulted in AHR. In contrast to budesonide, dietary intervention with 1% GOS prevented the development of AHR. HDM sensitization and challenge resulted in a significant increase in BALF leukocytes numbers, which was suppressed by budesonide treatment and dietary intervention with 1% GOS. Moreover, HDM sensitization and challenge resulted in significantly enhanced concentrations of IL-6, CCL17, IL-33, CCL5 and IL-13 in lung tissue. Both dietary intervention with 1% GOS or budesonide treatment significantly decreased the HDM-induced increased concentrations of CCL5 and IL-13 in lung tissue, while budesonide also reduced the HDM-enhanced concentrations of IL-6 and CCL17 in lung tissue.

Conclusion

Not only did dietary intervention with 1% GOS during sensitization and challenge prevent the induction of airway eosinophilia and Th2-related cytokine and chemokine concentrations in the lung equally effective as budesonide treatment, it also prevented AHR development in HDM-allergic mice. GOS might be useful for the prevention and/or treatment of symptoms in asthmatic disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0171-0) contains supplementary material, which is available to authorized users.     

Generation of potent mouse monoclonal antibodies to self-proteins using T-cell epitope “tags”

Immunization of mice or rats with a "non-self" protein is a commonly used method to obtain monoclonal antibodies, and relies on the immune system''s ability to recognize the immunogen as foreign. Immunization of an antigen with 100% identity to the endogenous protein, however, will not elicit a robust immune response. To develop antibodies to mouse proteins, we focused on the potential for breaking such immune tolerance by genetically fusing two independent T-cell epitope-containing sequences (from tetanus toxin (TT) and diphtheria toxin fragment A (DTA)) to a mouse protein, mouse ST2 (mST2). Wild-type CD1 mice were immunized with three mST2 tagged proteins (Fc, TT and DTA) and the specific serum response was determined. Only in mice immunized with the T-cell epitope-containing antigens were specific mST2 serum responses detected; hybridomas generated from these mice secreted highly sequence-diverse IgGs that were capable of binding mST2 and inhibiting the interaction of mST2 with its ligand, mouse interleukin (IL)-33 (mIL-33). Of the hundreds of antibodies profiled, we identified five potent antibodies that were able to inhibit IL-33 induced IL-6 release in a mast cell assay; notably one such antibody was sufficiently potent to suppress IL-5 release and eosinophilia infiltration in an Alternaria alternata challenge mouse model of asthma. This study demonstrated, for the first time, that T-cell epitope-containing tags have the ability to break tolerance in wild-type mice to 100% conserved proteins, and it provides a compelling argument for the broader use of this approach to generate antibodies against any mouse protein or conserved ortholog.