Kainate is a glutamate analog that has been widely used in pharmacological studies of neuronal injury related to ischemic conditions and epilepsy. While altered lipid metabolism has been implicated in kainate action, no study has yet investigated the associated changes in lipid metabolites on a systems scale. Here we describe a mass spectrometry-based approach for profiling of lipid mixtures in a nontargeted fashion. Combined with tandem mass spectrometry, this method aims to identify lipids that are altered between two conditions, the kainate-treated and the control hippocampal tissues. In addition to reductions in major phospholipids with mainly polyunsaturated fatty acyl chains, we find elevated levels of ions that correspond to acylated forms of phosphatidylethanolamines and ceramides. Acylated phosphatidylethanolamines are neuroprotective lipids and precursors for anandamide, which signals via cannabinoid receptors. Quantitative analysis of ceramides shows that many molecular species with different acyl compositions are increased during kainate treatment. This increase is mainly restricted to neurons rather than other brain cells in the hippocampus as revealed by immunohistochemistry of brain slices.

Mycobacteria store triacylglycerols (TGs) in the form of intracellular lipid droplets (LDs) during hypoxia-induced nonreplicating persistence. These bacteria are phenotypically drug-resistant and therefore are believed to be the cause for prolonged tuberculosis treatment. LDs are also associated with bacilli in tuberculosis patient sputum and hypervirulent strains. Although proteins bound to LDs are well characterized in eukaryotes, the identities and functions of such proteins have not been described in mycobacteria. Here, we have identified five proteins: Tgs1 (BCG3153c), Tgs2 (BCG3794c), BCG1169c, BCG1489c, and BCG1721, which are exclusively associated with LDs purified from hypoxic nonreplicating Mycobacterium bovis bacillus Calmette-Guérin (BCG). Disruption of genes tgs1, tgs2, BCG1169c, and BCG1489c in M. bovis BCG revealed that they are indeed involved in TG metabolism. We also characterized BCG1721, an essential bi-functional enzyme capable of promoting buildup and hydrolysis of TGs, depending on the metabolic state. Nonreplicating mycobacteria overexpressing a BCG1721 construct with an inactive lipase domain displayed a phenotype of attenuated TG breakdown and regrowth upon resuscitation. In addition, by heterologous expression in baker's yeast, these mycobacterial proteins also co-localized with LDs and complemented a lipase-deficient yeast strain, indicating that neutral lipid deposition and homeostasis in eukaryotic and prokaryotic microorganisms are functionally related. The demonstrated functional role of BCG1721 to support growth upon resuscitation makes this novel LD-associated factor a potential new target for therapeutic intervention.Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB) 5 infects more than one-third of the world population, resulting in two million deaths per year. The success of this pathogen hinges on its ability to enter into a nonreplicating persisting (NRP) state upon infecting the human host. In this state, the mycobacteria survive quiescently for decades (1) and reactivate opportunistically, such as during co-infection with human immunodeficiency virus, to cause disease (2). However, metabolic processes and enzymes necessary for the tubercle bacilli to transit into and out of the dormant state are not well characterized yet.It is widely recognized that M. tuberculosis utilizes fatty acids (FAs) as a carbon source during persistent infection (3). Mycobacteria store FAs in the form of triacylglycerols (TGs) during dormancy (4, 5) and in TB patient sputum (6, 7). Moreover, a direct correlation between the buildup of TGs and phenotypic drug resistance in M. tuberculosis was established (5), highlighting the importance of these lipids during latency and pathogenicity. TG utilization occurs when M. tuberculosis is subjected to a nutrient-deprived environment (8) and when Mycobacterium bovis bacillus Calmette-Guérin (BCG) is resuscitated from a hypoxic, nongrowing state (9), which underscores the important functional role of intracellular lipid storage during NRP...

The ability of aurones to modulate the efflux activities of ABCG2 and ABCB1 was investigated by quantifying their effects on the accumulation of pheophorbide A (PhA) in ABCG2-overexpressing MDA-MB-231/R cells and calcein AM in ABCB1-overexpressing MDCKII/MDR1 cells. Key structural features for interactions at both ABCG2 and ABCB1 are a methoxylated ring A, an intact exocyclic double bond, and the location of the carbonyl bond on ring C. Modifications on rings B and C were less critical and served primarily to moderate activity and selectivity for one or both transporters. These SAR trends were quantified by Free-Wilson analyses and are reflected in a pharmacophore model for PhA accumulation. Several compounds were found to be equipotent with fumitremorgin C (FTC) in promoting PhA accumulation, and they also demonstrated strong affinities for ABCB1. These compounds were disubstituted on ring B with methoxy or a combination of methoxy and hydroxy groups. Taken together, our findings highlight the versatility of the aurone template as a lead scaffold for the design of dual-targeting ABCG2 and ABCB1 modulators.

No abstract