In vivo peroxidative activity of FALS-mutant human CuZnSODs expressed in yeast.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder leading to loss of motor neurons. We previously characterized the enhanced peroxidative activity of the human familial ALS (FALS) mutants of copper-zinc superoxide dismutase (CuZnSOD) A4V and G93A in vitro. Here, a similar activity is demonstrated for human FALS CuZnSOD mutants in an in vivo model system, the yeast Saccharomyces cerevisiae. Spin trap adducts of alpha-(pyridyl-4-N-oxide)-N-tert-butylnitrone (POBN) have been measured by electron paramagnetic resonance (EPR) in yeast expressing mutant (A4V, L38V, G93A, and G93C) and wild type CuZnSOD upon addition of hydrogen peroxide to the culture. The trapped radical is a hydroxyethyl adduct of POBN, identified by spectral parameters. Mutant CuZnSODs produced greater concentrations of the trapped adduct compared to the wild type enzyme. This observation provides evidence for an oxidative radical mechanism, whereby the mutants of CuZnSOD catalyze the formation of reactive oxygen species that may be related to the development or progression of FALS. This study also presents an in vivo model system to study free radical production in FALS-associated CuZnSOD mutations.     

Ligand-specific dose-response of heterologously expressed olfactory receptors.

Primary olfactory neuronal cultures exposed to odorant stimulation have previously exhibited concentration-related effects in terms of intracellular cAMP levels and adenylate cyclase activity [Ronnett, G.V., Parfitt, D.J., Hester, L.D. & Snyder, S.H. (1991) PNAS88, 2366-2369]. Maximal stimulation occurred for intermediate concentrations, whereas AC activity declined for both low and high odorant concentrations. We suspected that this behavior might be ascribed to the intrinsic response of the first molecular species concerned by odorant detection, i.e. the olfactory receptor itself. In order to check this hypothesis, we developed an heterologous expression system in mammalian cells to characterize the functional response of receptors to odorants. Two mammalian olfactory receptors were used to initiate the study, the rat I7 olfactory receptor and the human OR17-40 olfactory receptor. The cellular response of transfected cells to an odorant stimulation was tested by a spectrofluorimetric intracellular calcium assay, and proved in all cases to be dose-dependent for the known ligands of these receptors, with an optimal response for intermediate concentrations. Further experiments were carried out with the rat I7 olfactory receptor, for which the sensitivity to an odorant, indicated by the concentration yielding the optimal calcium response, depended on the carbon chain length of the aldehydic odorant. The response is thus both ligand-specific and dose-dependent. We thus demonstrate that a differential dose-response originates from the olfactory receptor itself, which is thus capable of efficient discrimination between closely related agonists.     

TLR2 and TLR4 Mediate Differential Responses to Limb Ischemia through MyD88-Dependent and Independent Pathways

Introduction

The danger signal HMGB1 is released from ischemic myocytes, and mediates angiogenesis in the setting of hindlimb ischemia. HMGB1 is a ligand for innate immune receptors TLR2 and TLR4. While both TLR2 and TLR4 signal through myeloid differentiation factor 88 (MyD88), TLR4 also uniquely signals through TIR-domain-containing adapter-inducing interferon-β (TRIF). We hypothesize that TLR2 and TLR4 mediate ischemic myocyte regeneration and angiogenesis in a manner that is dependent on MyD88 signaling.

Methods

Mice deficient in TLR2, TLR4, MyD88 and TRIF underwent femoral artery ligation in the right hindlimb. Laser Doppler perfusion imaging was used to assess the initial degree of ischemia and the extent of perfusion recovery. Muscle regeneration, necrosis and fat replacement at 2 weeks post-ligation were assessed histologically and vascular density was quantified by immunostaining. In vitro, endothelial tube formation was evaluated in matrigel in the setting of TLR2 and TLR4 antagonism.

Results

While control and TLR4 KO mice demonstrated prominent muscle regeneration, both TLR2 KO and TRIF KO mice exhibited marked necrosis with significant inflammatory cell infiltrate. However, MyD88 KO mice had a minimal response to the ischemic insult with little evidence of injury. This observation could not be explained by differences in perfusion recovery which was similar at two weeks in all the strains of mice. TLR2 KO mice demonstrated abnormal vessel morphology compared to other strains and impaired tube formation in vitro.

Discussion

TLR2 and TRIF signaling are necessary for muscle regeneration after ischemia while MyD88 may instead mediate muscle injury. The absence of TLR4 did not affect muscle responses to ischemia. TLR4 may mediate inflammatory responses through MyD88 that are exaggerated in the absence of TLR2. Additionally, the actions of TLR4 through TRIF may promote regenerative responses that are required for recovery from muscle ischemia.     

Safety and Immunogenicity of the Malaria Vaccine Candidate MSP3 Long Synthetic Peptide in 12–24 Months-Old Burkinabe Children

Background

A Phase Ia trial in European volunteers of the candidate vaccine merozoite surface protein 3 (MSP3), a Plasmodium falciparum blood stage membrane, showed that it induces biologically active antibodies able to achieve parasite killing in vitro, while a phase Ib trial in semi-immune adult volunteers in Burkina Faso confirmed that the vaccine was safe.The aim of this study was to assess the safety and immunogenicity of this vaccine candidate in children aged 12–24 months living in malaria endemic area of Burkina Faso.

Methods

The study was a double-blind, randomized, controlled, dose escalation phase Ib trial, designed to assess the safety, reactogenicity and immunogenicity of three doses of either 15 or 30 µg of MSP3-LSP adsorbed on aluminum hydroxide in 45 children 12 to 24 months of age randomized into three equal groups. Each group received 3 vaccine doses (on days 0, 28 and 56) of either 15 µg of MSP3-LSP, 30 µg of MSP3-LSP or of the Engerix B hepatitis B vaccine. Children were visited at home daily for the 6 days following each vaccination to solicit symptoms which might be related to vaccination. Serious adverse events occurring during the study period (1 year) were recorded. Antibody responses to MSP3-LSP were measured on days 0, 28, 56 and 84.

Results

All 45 enrolled children received three MSP3 vaccine doses. No serious adverse events were reported. Most of the adverse events reported were mild to moderate in severity. The only reported local symptoms with grade 3 severity were swelling and induration, with an apparently dose related response. All grade 3 adverse events resolved without any sequelae. Both MSP3 doses regimens were able to elicit high levels of anti-MSP3 specific IgG1 and IgG3 antibodies in the volunteers with very little or no increase in IgG2, IgG4 and IgM classes: i.e. vaccination induced predominantly the isotypes involved in the monocyte-dependent mechanism of P. falciparum parasite-killing.

Conclusion

Our results support the promise of MSP3-LSP as a malaria vaccine candidate, both in terms of tolerability and of immunogenicity. Further assessment of the efficacy of this vaccine is recommended.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00452088","term_id":"NCT00452088"}}NCT00452088     

Comparison of the Arylamine <Emphasis Type="Italic">N-</Emphasis>Acetyltransferase from <Emphasis Type="Italic">Mycobacterium marinum</Emphasis> and <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Arylamine N-acetyltansferase (NAT) from Mycobacterium tuberculosis (TBNAT) is a potential drug target for anti-tubercular therapy. Recombinant TBNAT is much less soluble and is produced in lower yields than the closely related NAT from Mycobacterium marinum (MMNAT). In order to explore MMNAT as a model for TBNAT in drug discovery, we compare the two mycobacterial NAT enzymes. Two site-directed mutants of MMNAT have been prepared and characterised: MMNAT71, Tyr → Phe and MMNAT209, Met → Thr, in which residues within 6 Å of the active-site cysteine have been replaced with the corresponding residue from TBNAT. Two chimeric proteins have also been produced in which the third domain of MMNAT has been replaced by the third domain of TBNAT and vice versa. The activity profile of the chimeric proteins suggests a role for the third domain in the evolutionary divergence of NAT between these closely related mycobacterial species.     

Characterization of the bovine ampkγ1 gene

AMP-activated protein kinase (AMPK) represents the mammalian form of the core component of a kinase cascade that is conserved between fungi, plants, and animals. AMPK plays a major role in protecting mammalian cells from metabolic stress by switching off biosynthetic pathways that require ATP and switching on ATP-regenerating pathways. In this report, we describe the isolation and characterization of the gene for the noncatalytic bovine gamma1 subunit of AMPK. The bovine ampkgamma1 (PRKAG1) gene spans in excess of 14 kb and is located at BTA 5q21-q22. It consists of 12 exons ranging in size from 38 b to 166 b, interspersed with 11 introns that range between 97 b and 6753 b in length. The coding region of the bovine gene shares 93% and 90% nucleotide sequence similarity with its human and rat counterparts, and the bovine AMPKgamma1 protein is 98% and 95% identical to its human and rat homologs, respectively, in amino acid sequence. SNP discovery using a cattle DNA panel revealed a number of polymorphisms that may be useful for the evaluation of ampkgamma1 as a candidate gene for energy metabolism-related production traits.     

Amphibians as a model system for the investigation of respiratory control development

Recent medical advances have made it possible for babies to survive premature birth at increasingly earlier developmental stages. This population requires costly and sophisticated medical care to address the problems associated with immaturity of the respiratory system. In addition to pulmonary complications, respiratory instability and apnea reflecting immaturity of the respiratory control system are major causes of hospitalization and morbidity in this highly vulnerable population. These medical concerns, combined with the curiosity of physiologists, have contributed to the expansion of research in respiratory neurobiology. While most researchers working in this field commonly use rodents as an animal model, recent research using in vitro brainstem preparation from bullfrogs (Rana catesbeiana) have revealed the technical advantages of this animal model, and shown that the basic principles underlying respiratory control and its ontogeny are very similar between these two groups of vertebrates. The present review highlights the recent advances in the area of research with a focus on intermittent (episodic) breathing and the role of serotonergic and GABAergic modulation of respiratory activity during development.     

Molecular tools for a molecular medicine: analyzing genes, transcripts and proteins using padlock and proximity probes

Procedures and reagents are needed to specifically detect all the macromolecules that are being identified in the course of genome projects. We discuss how this challenge may be met using a set of ligation-based reagents termed padlock probes and proximity ligation probes. These probes include elements with affinity for specific nucleic acid and protein molecules, respectively, along with unique identifier DNA sequence elements that encode the identity of the recognized target molecules. The information content of DNA strands that form in the detection reactions are recorded after amplification, allowing the recognized target molecules to be identified. The procedures permit highly specific solution-phase or localized analyses of large sets of target molecules as required in future molecular analyses.