A-Young Kim

Protection against kainate neurotoxicity by ginsenosides: Attenuation of convulsive behavior, mitochondrial dysfunction, and oxidative stress

We previously demonstrated that kainic acid (KA)‐mediated mitochondrial oxidative stress contributed to hippocampal degeneration and that ginsenosides attenuated KA‐induced neurotoxicity and neuronal degeneration. Here, we examined whether ginsenosides affected KA‐induced mitochondrial dysfunction and oxidative stress in the rat hippocampus. Treatment with ginsenosides attenuated KA‐induced convulsive behavior dose‐dependently. KA treatment increased lipid peroxidation and protein oxidation and decreased the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio to a greater degree in the mitochondrial fraction than in the hippocampal homogenate. KA treatment resulted in decreased Mn‐superoxide dismutase expression anddiminished the mitochondrial membrane potential. Furthermore, KA treatment increased intramitochondrial Ca2+ and promoted ultrastructural degeneration in hippocampal mitochondria. Treatment with ginsenosides dose‐dependently attenuated convulsive behavior and the KA‐induced mitochondrial effects. Protection appeared to be more evident in mitochondria than in tissue homogenates. Collectively, the results suggest that ginsenosides prevent KA‐induced neurotoxicity by attenuating mitochondrial oxidative stress and mitochondrial dysfunction.

Ginsenosides attenuate kainic acid-induced synaptosomal oxidative stress via stimulation of adenosine A2A receptors in rat hippocampus

Treatment with ginsenosides attenuated KA-induced seizures and oxidative stress in the synaptosome, and reduced synaptic vesicles at the presynaptic terminals dose-dependently. The adenosine A(2A) receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl) xanthine reversed the ginsenoside-mediated pharmacological actions. Neither the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine nor the adenosine A(2B) receptor antagonist alloxazine affected the ginsenoside-mediated pharmacological actions. Our results suggest that ginsenosides block KA-induced synaptosomal oxidative stress, associated with hippocampal degeneration, through activation of adenosine A(2A) receptors.

The pathogenic human Torsin A in Drosophila activates the unfolded protein response and increases susceptibility to oxidative stress

BackgroundDystonia1 (DYT1) dystonia is caused by a glutamic acid deletion (ΔE) mutation in the gene encoding Torsin A in humans (HTorA). To investigate the unknown molecular and cellular mechanisms underlying DYT1 dystonia, we performed an unbiased proteomic analysis.ResultsWe found that the amount of proteins and transcripts of an Endoplasmic reticulum (ER) resident chaperone Heat shock protein cognate 3 (HSC3) and a mitochondria chaperone Heat Shock Protein 22 (HSP22) were significantly increased in the HTorAΔE– expressing brains compared to the normal HTorA (HTorAWT) expressing brains. The physiological consequences included an increased susceptibility to oxidative and ER stress compared to normal HTorAWT flies. The alteration of transcripts of Inositol-requiring enzyme-1 (IRE1)-dependent spliced X box binding protein 1(Xbp1), several ER chaperones, a nucleotide exchange factor, Autophagy related protein 8b (ATG8b) and components of the ER associated degradation (ERAD) pathway and increased expression of the Xbp1-enhanced Green Fluorescence Protein (eGFP) in HTorAΔE brains strongly indicated the activation of the unfolded protein response (UPR). In addition, perturbed expression of the UPR sensors and inducers in the HTorAΔEDrosophila brains resulted in a significantly reduced life span of the flies. Furthermore, the types and quantities of proteins present in the anti-HSC3 positive microsomes in the HTorAΔE brains were different from those of the HTorAWT brains.ConclusionTaken together, these data show that HTorAΔE in Drosophila brains may activate the UPR and increase the expression of HSP22 to compensate for the toxic effects caused by HTorAΔE in the brains.

The Biochemical Adaptations of Spotted Wing Drosophila (Diptera: Drosophilidae) to Fresh Fruits Reduced Fructose Concentrations and Glutathione-S Transferase Activities

Abstract Spotted wing drosophila, Drosophila suzukii Matsumura, is an invasive and economically damaging pest in Europe and North America. The females have a serrated ovipositor that enables them to infest almost all ripening small fruits. To understand the physiological and metabolic basis of spotted wing drosophila food preferences for healthy ripening fruits, we investigated the biological and biochemical characteristics of spotted wing drosophila and compared them with those of Drosophila melanogaster Meigen. We found that the susceptibility to oxidative stressors was significantly increased in spotted wing drosophila compared with those of D. melanogaster. In addition, we found that spotted wing drosophila had significantly reduced glutathione-S transferase (GST) activity and gene numbers. Furthermore, fructose concentrations found in spotted wing drosophila were significantly lower than those of D. melanogaster. Our data strongly suggest that the altered food preferences of spotted wing drosophila may stem from evolutionary adaptations to fresh foods accompanied by alterations in carbohydrate metabolism and GST activities.

Characterization of the molecular features and expression patterns of two serine proteases in Hermetia illucens (Diptera: Stratiomyidae) larvae.

To investigate the molecular scavenging capabilities of the larvae of Hermetia illucens, two serine proteases (SPs) were cloned and characterized. Multiple sequence alignments and phylogenetic tree analysis of the deduced amino acid sequences of Hi-SP1 and Hi-SP2 were suggested that Hi-SP1 may be a chymotrypsin- and Hi-SP2 may be a trypsin-like protease. Hi-SP1 and Hi-SP2 3-D homology models revealed that a catalytic triad, three disulfide bonds, and a substrate-binding pocket were highly conserved, as would be expected of a SP. E. coli expressed Hi-SP1 and Hi-SP2 showed chymotrypsin or trypsin activities, respectively. Hi-SP2 mRNAs were consistently expressed during larval development. In contrast, the expression of Hi-SP1 mRNA fluctuated between feeding and molting stages and disappeared at the pupal stages. These expression pattern differences suggest that Hi-SP1 may be a larval specific chymotrypsin-like protease involved with food digestion, while Hi-SP2 may be a trypsin-like protease with diverse functions at different stages.

A Combination of Biochemical and Proteomic Analyses Reveals Bx-LEC-1 as an Antigenic Target for the Monoclonal Antibody 3-2A7-2H5-D9-F10 Specific to the Pine Wood Nematode

Pine wilt disease (PWD) is one of the most devastating forest diseases in Asia and Europe. The pine wood nematode, Bursaphelenchus xylophilus, has been identified as the pathogen underlying PWD, although the pathology is not completely understood. At present, diagnosis and confirmation of PWD are time consuming tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for B. xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically, but not those from other closely related nematodes. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions, or bands isolated from SDS-PAGE, two-dimensional electrophoresis, anion exchange chromatography, and immunoprecipitation via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano-LC-ESI-Q-IT-MS). Peptides of galactose-binding lectin-1 of B. xylophilus (Bx-LEC-1) were commonly detected in several proteomic analyses, demonstrating that this LEC-1 is the antigenic target of MAb-D9-F10. The localization of MAb-D9-F10 immunoreactivities at the area of the median bulb and esophageal glands suggested that the Bx-LEC-1 may be involved in food perception and digestion. The Bx-LEC-1 has two nonidentical galactose-binding lectin domains important for carbohydrate binding. The affinity of the Bx-LEC-1 to d-(+)-raffinose and N-acetyllactosamine were much higher than that to l-(+)-rhamnose. Based on this combination of evidences, MAb-D9-F10 is the first identified molecular biomarker specific to the Bx-LEC-1.

Development of Monoclonal Antibodies Specific to Galectin of Pine Wood Nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle and Their Utilization for Detection of Pine Wood Nematodes

Currently, there is no available tool that rapidly diagnoses pine wood nematode (PWN)-infected pine trees in the field. In this study, we synthesized and purified PWN Galectin, which might be an antigen specific to PWN, using the Baculovirus expression system. We used PWN Galectin as an antigen for generating 1,464 fusion hybridoma cell lines secreting monoclonal antibodies (Mabs). Among them, we selected 62 fusion hybridoma cell lines showing high reactivity to PWN Galectin. We further selected 12 fusion hybridoma cell lines showing high reactivity to the standard PWN-infected pine tree phosphate buffered saline (PBS) extract. Additionally, two fusion hybridoma cell lines showing no or extremely low reactivity were used as controls. The selected fusion hybridoma cell lines were subjected to limiting dilutions for selecting and establishing Mab-secreting cell lines showing higher reactivity to the standard PWN-infected pine tree extract than to the standard normal pine tree PBS extract. Moreover, the selected fusion hybridoma cell lines were further selected based on their higher reactivity to PWN protein extracts than to three non-pathogenic nematode protein extracts. The Mab-secreting cell lines established in this study could be used to develop rapid diagnostic tools that can be used in the field or in laboratories for detecting PWN-infected pine trees or PWN.

Regulation of synaptic architecture and synaptic vesicle pools by Nervous wreck at Drosophila Type 1b glutamatergic synapses

Nervous wreck (Nwk), a protein that is present at Type 1 glutamatergic synapses that contains an SH3 domain and an FCH motif, is a Drosophila homolog of the human srGAP3/MEGAP protein, which is associated with mental retardation. Confocal microscopy revealed that circles in Nwk reticulum enclosed T-shaped active zones (T-AZs) and partially colocalized with synaptic vesicle (SV) markers and both exocytosis and endocytosis components. Results from an electron microscopic (EM) analysis showed that Nwk proteins localized at synaptic edges and in SV pools. Both the synaptic areas and the number of SVs in the readily releasable (RRPs) and reserve (RPs) SV pools in nwk2 were significantly reduced. Synergistic, morphological phenotypes observed from eag1;nwk2 neuromuscular junctions suggested that Nwk may regulate synaptic plasticity differently from activity-dependent Hebbian plasticity. Although the synaptic areas in eag1;nwk2 boutons were not significantly different from those of nwk2, the number of SVs in the RRPs was similar to those of Canton-S. In addition, three-dimensional, high-voltage EM tomographic analysis demonstrated that significantly fewer enlarged SVs were present in nwk2 RRPs. Furthermore, Nwk formed protein complexes with Drosophila Synapsin and Synaptotagmin 1 (DSypt1). Taken together, these findings suggest that Nwk is able to maintain synaptic architecture and both SV size and distribution at T-AZs by interacting with Synapsin and DSypt1.

Author Correction: Regulation of synaptic architecture and synaptic vesicle pools by Nervous wreck at Drosophila Type 1b glutamatergic synapses

After online publication of this article, the authors noticed an error in the ACKNOWLEDGEMENTS section.