purification-membrane-compartment-for-endoplasmic-reticulum

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

Takasaki University of Health and Welfare

The effects of a standardized extract of Ginkgo biloba L. leaves (EGb) and its terpene constituents, bilobalide and ginkgolides, on the activities of detoxification enzymes, i.e., glutathione S-transferases (GSTs) and DT-diaphorase, and glutathione contents, were investigated in the mouse liver. Oral treatment with EGb (100-1,000 mg/kg) and bilobalide (10-30 mg/kg) once a day for 4 days caused a dose-dependent elevation in GST activity. Ginkgolide A (30 mg/kg, for 4 days) also significantly elevated GST activity, whereas ginkgolide B and ginkgolide C at the same dose had no effects. EGb significantly increased the protein level of GST pi, and bilobalide significantly increased those of GST alpha and GST mu Moreover, EGb-treatment and bilobalide-treatment caused significant elevations in DT-diaphorase activity and in hepatic glutathione contents.

Effects of extract of Ginkgo biloba leaves and its constituents on carcinogen-metabolizing enzyme activities and glutathione levels in mouse liver.

Ion channels play important roles in vital cellular signaling processes in both excitable and nonexcitable cells. Since 1987, a large number of channel genes have been cloned, and their biophysical properties, subunit stoichiometries, channel assemblies, and modulation by second messengers and ligands have been gradually elucidated. At present, more than ten ion channel genes have been identified as causing human hereditary diseases. Molecular techniques such as the positional cloning method are indispensable for finding new genes for channel-related diseases. Ion channels participate in the excitation-restoration of neurons and myocytes. Mutations of ion channels in these cells cause abnormal excitation and diseases such as long QT syndrome and ataxia. The second physiological function of ion channels, in addition to their regulation of cell excitability, is ion transport. Bartter's syndrome and Liddle's syndrome are due to abnormalities of ion transport. Most of these ion channel diseases are caused by loss of function, although some mutations are known to result in gain of function. The number of identified channel-related diseases is growing rapidly. Elucidation of the molecular basis of an ion channel disease not only provides new opportunities for early diagnosis and therapy for the disease but also provides clues to determine a previously unknown function of the ion channel.

Ion channels and diseases.

We have previously demonstrated that 4-day-treatment of mice with bilobalide, a sesquiterpene of Ginkgo biloba L., increases GABA levels in mouse brain, but, effects of chronic treatment with it are not clear. To study effects of chronic treatment of mice with bilobalide on amino acid levels in the brain, we determined the levels of aspartate, glutamate, serine, glutamine, glycine, taurine and GABA in the hippocampus, striatum and cortex. Bilobalide (3 mg/kg/day) was administered orally to 4-week-old mice for 40 days. Bilobalide treatment resulted in a significant increase in the levels of glutamate, aspartate, gamma-aminobutyric acid (GABA), and glycine in the hippocampus of mice compared with the control. An increased level of glycine after bilobalide treatment was also detected in the striatum. In the cortex, bilobalide increased the GABA level, whereas it decreased the level of aspartate. These changes in the levels of various amino acids may be involved in the broad spectrum of pharmacological activities of the extract of Ginkgo biloba on the central nervous system.

Effects of chronic administration of bilobalide on amino acid levels in mouse brain.

Dendritic cells (DCs) are capable of processing and presenting exogenous antigens using MHC class I molecules. This pathway is called antigen cross-presentation and plays an important role in the stimulation of naïve CD8(+) T cells for infectious and tumor immunity. Our previous studies in DC2.4 cells and bone marrow-derived DCs revealed that exogenously added ovalbumin (OVA) is processed through endoplasmic reticulum (ER)-associated degradation (ERAD) for cross-presentation. In this study, we aimed to further confirm these results by purification of the subcellular compartment in which exogenous antigens undergo ERAD from homogenates of DC2.4 cells pretreated with biotinylated OVA (bOVA). bOVA-containing vesicles were purified using streptavidin (SA)-magnetic beads from cell homogenates and were found to contain ER chaperones and ERAD components together with proteins for antigen presentation. In purified microsomes, bOVA was retained in membranous fractions and degraded by the ubiquitin proteasome system in presence reticulocyte lysates and ATP. These results strongly suggested that DCs processed and degraded exogenous antigens through ERAD for cross-presentation in this purified subcellular compartment.

Purification of the subcellular compartment in which exogenous antigens undergo endoplasmic reticulum-associated degradation from dendritic cells.

The hydroxyl radical (OH) possesses the strongest oxidation potential among reactive oxygen species (ROS). Hydroxyl radicals react nonpreferentially with proteins, lipids, and nucleic acids. Additionally, mitochondrial localization of OH causes dysfunction in the mitochondria. The cytoplasmic targets of OH-induced oxidation are unknown. No cytoplasm-specific OH scavenger is available; thus, elucidating the cytoplasmic targets of OH-induced oxidation has proven difficult. Accordingly, we developed a cytoplasm-specific OH-targeted scavenger, TA293, and a mitochondrion-specific scavenger, mitoTA293. Both TA293 and mitoTA293 scavenged OH but not O2(-) or H2O2. We then examined the intracellular localization of both scavengers in vitro and in vivo. TA293 scavenged pyocyanin-induced cytoplasmic OH but not antimycin A-induced mitochondrial oxidation. mitoTA293 scavenged antimycin A-induced mitochondrial OH but not cytoplasmic OH. TA293 but not mitoTA293 suppressed pyocyanin-induced oxidative damage in the lungs and kidneys of mice. Additionally, TA293 suppressed the expression of inflammatory signaling pathway components and mediators and suppressed OH-induced cellular senescence and apoptosis. These data suggested that TA293 could be used as a novel tool for studying the effects of hydroxyl radical damage within the cytoplasm.

The novel antioxidant TA293 reveals the role of cytoplasmic hydroxyl radicals in oxidative stress-induced senescence and inflammation.

In the present study, to investigate the possibility that chronic ethanol treatment might alter Ca(2+) -inhibited type 5 adenylyl cyclase (AC) activity, we examined the effect of chronic ethanol treatment on striatal dopaminergic signal transduction, especially the AC system, in mice. We fed male C57BL/6 mice for 7 days with a 5% ethanol-containing or control liquid diet. Basal and forskolin-stimulated AC activities were reduced in striatal membranes of ethanol-treated mice. 5'-guanylylimidodiphosphate-stimulated AC activity was also decreased in ethanol-treated mice. But no significant differences were observed in the levels of the guanine nucleotide binding protein subunits Gsα and Gi1&2α determined by immunoblotting, between ethanol-treated and control mice. These results indicated that the function of the catalytic subunit of AC was decreased in the straitum of chronically ethanol-treated mice. We further examined the inhibitory regulation of AC activity in the context of a change of type 5 AC. Inhibition of forskolin-stimulated AC activity by 10 μM free Ca(2+) was smaller in ethanol-treated mice than in control mice. However, the protein level of type 5 AC in the striatum, determined by immunoblotting, was not significantly different between ethanol-treated and control mice. These findings suggest that Ca(2+) -inhibited, presumably type 5, AC activity is reduced in mouse striatum by chronic ethanol treatment, and that this reduction is not due to a decrease in type 5 AC expression.

Shinichi Hatta

Laboratory of Physiological Chemistry,
Faculty of Pharmacy,
Laboratory of Physiological Chemistry, Faculty of Pharmacy

Takasaki University of Health and Welfare

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Shinichi Hatta

Laboratory of Physiological Chemistry,Faculty of Pharmacy,Laboratory of Physiological Chemistry, Faculty of Pharmacy

Takasaki University of Health and Welfare

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Laboratory of Physiological Chemistry,
Faculty of Pharmacy,
Laboratory of Physiological Chemistry, Faculty of Pharmacy