Acetohydroxyacid synthase from Mycobacterium avium and its inhibition by sulfonylureas and imidazolinones

Tuberculosis (TB) remains one of the world's leading causes of death from infectious disease. It is caused by infection with Mycobacterium tuberculosis or sometimes, particularly in immune-compromised patients, Mycobacterium avium. The aim of this study was to create a tool that could be used in the search for new anti-TB drugs that inhibit branched-chain amino acid (BCAA) biosynthesis, as these are essential amino acids that are not available to a mycobacterium during growth in an infected organism. To this end, we cloned, overexpressed, purified and characterised for the first time an acetohydroxyacid synthase (AHAS), a key enzyme in the pathway to the biosynthesis of the BCAAs, from the genus Mycobacterium. Nine commercial herbicides of the sulfonylurea and imidazolinone classes were tested for their influence on this enzyme. Four of the sulfonylureas were potent inhibitors of the enzyme. The relative potency of the different inhibitors towards the M. avium enzyme was unlike their potency towards other AHASs whose inhibitor profile has been reported, emphasising the advantage of using a mycobacterial enzyme as a tool in the search for new anti-TB drugs.     

G Protein and β-Arrestin Signaling Bias at the Ghrelin Receptor

The G protein-coupled ghrelin receptor GHSR1a is a potential pharmacological target for treating obesity and addiction because of the critical role ghrelin plays in energy homeostasis and dopamine-dependent reward. GHSR1a enhances growth hormone release, appetite, and dopamine signaling through G_q/11, G_i/o, and G_12/13 as well as β-arrestin-based scaffolds. However, the contribution of individual G protein and β-arrestin pathways to the diverse physiological responses mediated by ghrelin remains unknown. To characterize whether a signaling bias occurs for GHSR1a, we investigated ghrelin signaling in a number of cell-based assays, including Ca²⁺ mobilization, serum response factor response element, stress fiber formation, ERK1/2 phosphorylation, and β-arrestin translocation, utilizing intracellular second loop and C-tail mutants of GHSR1a. We observed that GHSR1a and β-arrestin rapidly form metastable plasma membrane complexes following exposure to an agonist, but replacement of the GHSR1a C-tail by the tail of the vasopressin 2 receptor greatly stabilizes them, producing complexes observable on the plasma membrane and also in endocytic vesicles. Mutations of the contiguous conserved amino acids Pro-148 and Leu-149 in the GHSR1a intracellular second loop generate receptors with a strong bias to G protein and β-arrestin, respectively, supporting a role for conformation-dependent signaling bias in the wild-type receptor. Our results demonstrate more balance in GHSR1a-mediated ERK signaling from G proteins and β-arrestin but uncover an important role for β-arrestin in RhoA activation and stress fiber formation. These findings suggest an avenue for modulating drug abuse-associated changes in synaptic plasticity via GHSR1a and indicate the development of GHSR1a-biased ligands as a promising strategy for selectively targeting downstream signaling events.     

Slow oscillations in neural networks with facilitating synapses

The synchronous oscillatory activity characterizing many neurons in a network is often considered to be a mechanism for representing, binding, conveying, and organizing information. A number of models have been proposed to explain high-frequency oscillations, but the mechanisms that underlie slow oscillations are still unclear. Here, we show by means of analytical solutions and simulations that facilitating excitatory (E _f) synapses onto interneurons in a neural network play a fundamental role, not only in shaping the frequency of slow oscillations, but also in determining the form of the up and down states observed in electrophysiological measurements. Short time constants and strong E _f synapse-connectivity were found to induce rapid alternations between up and down states, whereas long time constants and weak E _f synapse connectivity prolonged the time between up states and increased the up state duration. These results suggest a novel role for facilitating excitatory synapses onto interneurons in controlling the form and frequency of slow oscillations in neuronal circuits.     

ApoB-containing lipoproteins regulate angiogenesis by modulating expression of VEGF receptor 1

Despite the clear major contribution of hyperlipidemia to the prevalence of cardiovascular disease in the developed world, the direct effects of lipoproteins on endothelial cells have remained obscure and are under debate. Here we report a previously uncharacterized mechanism of vessel growth modulation by lipoprotein availability. Using a genetic screen for vascular defects in zebrafish, we initially identified a mutation, stalactite (stl), in the gene encoding microsomal triglyceride transfer protein (mtp), which is involved in the biosynthesis of apolipoprotein B (ApoB)-containing lipoproteins. By manipulating lipoprotein concentrations in zebrafish, we found that ApoB negatively regulates angiogenesis and that it is the ApoB protein particle, rather than lipid moieties within ApoB-containing lipoproteins, that is primarily responsible for this effect. Mechanistically, we identified downregulation of vascular endothelial growth factor receptor 1 (VEGFR1), which acts as a decoy receptor for VEGF, as a key mediator of the endothelial response to lipoproteins, and we observed VEGFR1 downregulation in hyperlipidemic mice. These findings may open new avenues for the treatment of lipoprotein-related vascular disorders.     

Ethanol, the choline requirement, methylation and liver injury

The findings obtained in this laboratory and others over the past decade are discussed in order to formulate a thesis, indicating the adverse action of ethanol on a vital methylation process in the liver. Evidence is shown that the rat may have a means of compensating for this impairment in methylation whereas humans do not have this same ability to protect against this action of ethanol. These considerations may offer a basis of why rats are apparently more resistant to alcoholic liver injury than humans.