Pharmaceutical Analysis in the Plant Endomembrane System.

Vesicle trafficking is an essential cellular process conserved in eukaryotes to precisely transport proteins to their destinations. The plant endomembrane system plays a pivotal role in orchestrating this vesicle-mediated protein transport process, making its study essential for a comprehensive understanding of plant growth and development. Pharmaceutical analysis proves highly useful in investigating the plant endomembrane system. To facilitate further studies in this area, we present a summary of several commonly used chemical inhibitors in this chapter, providing a practical resource for researchers interested in the plant endomembrane system.

Mfn2 regulates mitochondria and mitochondria-associated endoplasmic reticulum membrane function in neurodegeneration induced by repeated sevoflurane exposure.

Repeated sevoflurane exposure in neonatal mice can leads to neuronal apoptosis and mitochondrial dysfunction. The mitochondria are responsible for energy production to maintain homeostasis in the central nervous system. The mitochondria-associated endoplasmic reticulum membrane (MAM) is located between the mitochondria and endoplasmic reticulum (ER), and it is critical for mitochondrial function and cell survival. MAM malfunction contributes to neurodegeneration, however, whether it is involved in sevoflurane-induced neurotoxicity remains unknown. Our study demonstrated that repeated sevoflurane exposure induced mitochondrial dysfunction and dampened the MAM structure. The upregulated ER-mitochondria tethering enhanced Ca2+ transition from the cytosol to the mitochondria. Overload of mitochondrial Ca2+ contributed to opening of the mitochondrial permeability transition pore (mPTP), which caused neuronal apoptosis. Mitofusin 2(Mfn2), a key regulator of ER-mitochondria contacts, was found to be suppressed after repeated sevoflurane exposure, while restoration of Mfn2 expression alleviated cognitive dysfunction due to repeated sevoflurane exposure in the adult mice. These evidences suggest that sevoflurane-induced MAM malfunction is vulnerable to Mfn2 suppression, and the enhanced ER-mitochondria contacts promotes mitochondrial Ca2+ overload, contributing to mPTP opening and neuronal apoptosis. This paper sheds light on a novel mechanism of sevoflurane-induced neurotoxicity. Furthermore, targeting Mfn2-mediated regulation of the MAM structure and mitochondrial function may provide a therapeutic advantage in sevoflurane-induced neurodegeneration.

Organelle-Targeted Laurdans Measure Heterogeneity in Subcellular Membranes and Their Responses to Saturated Lipid Stress.

Organelles feature characteristic lipid compositions that lead to differences in membrane properties. In cells, membrane ordering and fluidity are commonly measured using the solvatochromic dye Laurdan, whose fluorescence is sensitive to lipid packing. As a general lipophilic dye, Laurdan stains all hydrophobic environments in cells; therefore, it is challenging to characterize membrane properties in specific organelles or assess their responses to pharmacological treatments in intact cells. Here, we describe the synthesis and application of Laurdan-derived probes that read out the membrane packing of individual cellular organelles. The set of organelle-targeted Laurdans (OTL) localizes to the ER, mitochondria, lysosomes, and Golgi compartments with high specificity while retaining the spectral resolution needed to detect biological changes in membrane ordering. We show that ratiometric imaging with OTLs can resolve membrane heterogeneity within organelles as well as changes in lipid packing resulting from inhibition of trafficking or bioenergetic processes. We apply these probes to characterize organelle-specific responses to saturated lipid stress. While the ER and lysosomal membrane fluidity is sensitive to exogenous saturated fatty acids, that of mitochondrial membranes is protected. We then use differences in ER membrane fluidity to sort populations of cells based on their fatty acid diet, highlighting the ability of organelle-localized solvatochromic probes to distinguish between cells based on their metabolic state. These results expand the repertoire of targeted membrane probes and demonstrate their application in interrogating lipid dysregulation.

Inhibition of PLA2G4A attenuated valproic acid- induced lysosomal membrane permeabilization and restored impaired autophagic flux: Implications for hepatotoxicity.

Valproic acid (VPA) has broad efficacy against several seizures but causes liver injury limiting its prolonged clinical use. Some studies have demonstrated that VPA-induced hepatotoxicity is characterized by microvesicular hepatic steatosis. However, novel detailed mechanisms to explain VPA-induced hepatic steatosis and experimentally rigorously validated protective agents are still lacking. In this study, 8-week-old C57BL/6J mice were gavaged with VPA (500 mg/kg/d) for 4 weeks to establish an in vivo model of VPA-induced chronic liver injury. Quantitative proteomic and non-targeted lipidomic analyses were performed to explore the underlying mechanisms of VPA-induced hepatotoxicity. As a result, VPA-induced hepatotoxicity is associated with impaired autophagic flux, which is attributed to lysosomal dysfunction. Further studies revealed that VPA-induced lysosomal membrane permeabilization (LMP), allows soluble lysosomal enzymes to leak into the cytosol, which subsequently led to impaired lysosomal acidification. A lower abundance of glycerophospholipids and an increased abundance of lysophospholipids in liver tissues of mice in the VPA group strongly indicated that VPA-induced LMP may be mediated by the activation of phospholipase PLA2G4A. Metformin (Met) acted as a potential protective agent attenuating VPA-induced liver dysfunction and excessive lipid accumulation. Molecular docking and cellular thermal shift assays demonstrated that Met inhibited the activity of PLA2G4A by directly binding to it, thereby ameliorating VPA-induced LMP and autophagic flux impairment. In conclusion, this study highlights the therapeutic potential of targeting PLA2G4A-mediated lysosomal dysfunction in VPA-induced hepatotoxicity.

Apoptogenic effect of the lipophilic o-naphthoquinone CG 10-248 on rat hepatocytes: light and electron microscopy studies

CG 10-248 (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione; CG-NQ), a beta-lapachone analogue, modified the ultrastructure of rat hepatocytes, as demonstrated by light and electron microscopy. After 4 h incubation with 100 microM CG-NQ, the following effects were observed: (a) nuclear chromatin condensation; (b) chromatin fragmentation; (c) displacement of mitochondria, concentrated around the nucleus; (d) disruption or expansion of mitochondrial outer or inner membranes, respectively; (e) displacement and alteration of endoplasmic reticulum (rough and smooth); (f) decrease of microvilli; (g) blebbing of plasma membrane and production of apoptotic bodies formed by folding of plasma membrane fragments around mitochondria or peroxysomes; and (h) production of hydrogen peroxide. Expression of such effects varied according to hepatocyte samples and taken together strongly support an apoptotic action of CG-NQ dependent on reactive oxygen species.

Apoptogenic effect of the lipophilic o-naphthoquinone CG 10-248 on rat hepatocytes: light and electron microscopy studies

CG 10-248 (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione; CG-NQ), a beta-lapachone analogue, modified the ultrastructure of rat hepatocytes, as demonstrated by light and electron microscopy. After 4 h incubation with 100 microM CG-NQ, the following effects were observed: (a) nuclear chromatin condensation; (b) chromatin fragmentation; (c) displacement of mitochondria, concentrated around the nucleus; (d) disruption or expansion of mitochondrial outer or inner membranes, respectively; (e) displacement and alteration of endoplasmic reticulum (rough and smooth); (f) decrease of microvilli; (g) blebbing of plasma membrane and production of apoptotic bodies formed by folding of plasma membrane fragments around mitochondria or peroxysomes; and (h) production of hydrogen peroxide. Expression of such effects varied according to hepatocyte samples and taken together strongly support an apoptotic action of CG-NQ dependent on reactive oxygen species. (AU)

Mitochondrial oxidative damage promoted by a synergism betweem Ca2+ and prooxidants: inner membrane permeabilization and mtDNA breakage

Oxidative damage of mitochondria induced by a synergism between Ca2+ and prooxidants is mediated by the attack of mitochondria-generated reactive oxygen species to membrane proteins, lipids and DNA. This results in mitochondrial DNA fragmentation, lipid peroxidation and oxidation of vicinal protein thiols producing high molecular weight membrane protein aggregates. The membrane protein alterations lead to a condition called mitochondrial membrane permeability transition, characterized by formation of nonspecific membrane protein pores sensitive to cyclosporin A, EGTA, dithiothreitol, Mg2+ and ADP. We propose that these alterations are related to the mechanisms by which cells are killed by a series of toxins, xenobiotics or pathological conditions such as prolonged hypoxia or ischemia/reperfusion.

Calcium homeostasis in Trypanosoma cruzi

By using the fluorescent Ca2+ indicator fura 2, submicromolar levels of intracellular Ca2+ have been detected in Trypanosoma cruzi different stages. The intracellular transport mechanisms involved in maintaining Ca2+ homeostasis in T. cruzi have been characterized by measuring Ca2+ transport in digitonin-permeabilized cells. Two intracellular calcium transport systems have been detected. Ca2+ uptake by the mitochondria occurs by an electrophoretic mechanism, is inhibited by antimycin A, FCCP, and ruthenium red, and stimulated by respiratory substrates, phosphate and acetate. This pool has a high capacity and low affinity for Ca2+ and is able to buffer external Ca2+ at concentrations in the range of 0.6-0.7 microM. Ca2+ uptake by the endoplasmic reticulum is inhibited by high concentrations of vanadate and anticalmodulin agents, and stimulated by ATP. This pool has a low capacity and a high affinity for Ca2+ and is able to buffer external Ca2+ at concentrations in the range of 0.05-1.0 microM. In addition, calmodulin has been purified from T. cruzi epimastigotes and shown to stimulate the homologous plasma membrane Ca(2+)-ATPase and cyclic-AMP phosphodiesterase. The gene encoding this protein has been cloned and sequenced and shown to have a great homology to mammalian calmodulin. The role of the plasma membrane of T. cruzi in the regulation of [Ca2+]i has been studied using fura 2-loaded epimastigotes or plasma membrane vesicles prepared from epimastigotes. Plasma membrane vesicles transport Ca2+ in the presence of Mg2+ and have a high affinity, vanadate-sensitive (Ca(2+)-Mg2+)-ATPase with an apparent Km for free Ca2+ of 0.3 microM.(ABSTRACT TRUNCATED AT 250 WORDS)