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Moo Ho Won

Publications and source records attributed to Moo Ho Won.

123 records · Page 7Linked to original sources

Oxidative DNA damage and alteration of glutamate transporter expressions in the hippocampal Ca1 area immediately after ischemic insult.

Although oxidative stress and excitotoxicity may be interdependent mechanisms that are involved in delayed neuronal death, the temporal participation of these events in the early stage after ischemia-reperfusion insult is unclear. Therefore, in the present study, using the gerbil global ischemic model we investigated whether oxidative stress could be correlated with the expression of the glutamate transporters in the hippocampus, and whether these events are related and cooperate in the events that link ischemia to neuronal death in vivo. Thirty minutes after ischemia, the intensities of glutamate transporter-1 (GLT-1), glutamate/aspar-tate transporter (GLAST), and 8-hydroxy2'-deoxy-guanosine (8-OHdG) immunoreactivities were markedly increased in the hippocampal CA1 area. In contrast, excitatory amino acid carrier-1 (EAAC-1) immunoreactivity was 30% lower in the CA1 area than in the sham level. At 3 h post-reperfusion, the EAAC-1 expression began to increase in the CA1 area. Twelve hours after reperfusion, the reduction of both GLT-1 and GLAST immunoreactivity was salient, while the EAAC-1 immunoreactivity level intensified significantly. The 8-OHdG immunoreactivity peaked at this time point. These findings suggest that oxidative stress and alterations in the glutamate transporter expression in the CA1 area may simultaneously trigger neuronal damages very early after ischemia.

Amino Acid Transport System X-AG↗

Carnosine and related dipeptides protect human ceruloplasmin against peroxyl radical-mediated modification.

Ceruloplasmin (CP) is the major plasma antioxidant and copper transport protein. In a previous study, we showed that the aggregation of human ceruloplasmin was induced by peroxyl radicals. We investigated the effects of antioxidant dipeptides carnosine, homocarnosine and anserine on peroxyl radical-mediated ceruloplasmin modification. Carnosine, homocarnosine and anserine significantly inhibited the aggregation of CP induced by peroxyl radicals. When CP was incubated with peroxyl radicals in the presence of three compounds, ferroxidase activity, as measured by the activity staining method, was protected. All three compounds also inhibited the formation of dityrosine in peroxyl radicals-treated CP. The results suggest that carnosine and related compounds act as peroxyl radical scavenger to protect the protein modification. It is proposed that carnosine and related peptides might be explored as potential therapeutic agents for pathologies that involve CP modification mediated by peroxyl radicals generated in the lipid peroxidation.

Anserine↗

Chronological changes in pyridoxine-5'-phosphate oxidase immunoreactivity in the seizure-sensitive gerbil hippocampus.

To identify the roles of pyridoxine-5'-phosphate (PNP) oxidase in epileptogenesis and the recovery mechanisms in spontaneous seizure, a chronological and comparative analysis of PNP oxidase expression was conducted. PNP oxidase immunoreactivity in a preseizure group of seizure-sensitive (SS) gerbils was detected more strongly than that in a seizure-resistant (SR) group. The density of PNP oxidase immunoreactivity in a 30 min postictal group was significantly lower than that in a preseizure group. In a 12 hr postictal group, PNP oxidase immunodensity had recovered to a preseizure level. The overexpression of PNP oxidase in the hippocampus of preseizure SS gerbils suggests that PNP or pyridoxal 5'-phosphate plays an important role in the modulation of glutamic acid decarboxylase activity and gamma-aminobutyric acid reuptake as mediated by membrane transporter via neurons. In addition, this change in the PNP oxidase immunoreactivity following seizure may be a compensatory response designed to reduce epileptic activity in this animal.

Animals↗

9-polylysine protein transduction domain: enhanced penetration efficiency of superoxide dismutase into mammalian cells and skin.

Antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), have been considered to have a beneficial effect against various diseases that are mediated by the reactive oxygen species (ROS). Although a variety of modified recombinant antioxidant enzymes have been generated to protect against oxidative stresses, the lack of their transduction ability into cells resulted in a limited ability to detoxify intracellular ROS. To render the SOD enzyme capable of detoxifying intracellular ROS when added extracellularly, cell-permeable recombinant SOD proteins were generated. A human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene was fused with a gene fragment that encodes the 9 amino acids Tat protein transduction domain (RKKRRQRRR) of HIV-1 and lysine rich peptide (KKKKKKKKK) in a bacterial expression vector in order to produce a genetic in-frame Tat-SOD and 9Lys-SOD fusion protein, respectively. The expressed and purified Tat-SOD and 9Lys-SOD fusion proteins can transduce into human fibroblast cells, and they were enzymatically active and stable for 24 h. The cell viability of the fibroblast cells that were treated with paraquat, an intracellular superoxide anion generator, was increased by the transduced Tat-SOD or 9Lys-SOD. The transduction efficacy of 9Lys-SOD was more efficient than that of Tat-SOD. We evaluated the ability of the SOD fusion pmteins to transduce into animal skin. This analysis showed that Tat-SOD and 9Lys-SOD fusion proteins efficiently penetrated into the epidermis as well as the dermis of the subcutaneous layer, when sprayed on mice skin (judged by the immunohistochemistry and specific enzyme activities). The enzymatic activity of the transduced 9Lys-SOD was higher than that of Tat-SOD, indicating that the penetration of 9Lys-SOD was more efficient when put into the skin. These results suggest Tat-SOD and 9Lys-SOD fusion proteins can be used as anti-aging cosmetics, or in protein therapy, for various disorders that are related to this antioxidant enzyme and ROS.

Animals↗

Protective effects of carnosine, homocarnosine and anserine against peroxyl radical-mediated Cu,Zn-superoxide dismutase modification.

Carnosine (beta-alanyl-L-histidine), homocarnosine (gamma-amino-butyryl-L-histidine) and anserine (beta-alanyl-1-methyl-L-histidine) have been proposed to act as anti-oxidants in vivo. The protective effects of carnosine and related compounds against the oxidative damage of human Cu,Zn-superoxide dismutase (SOD) by peroxyl radicals generated from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) were studied. The oxidative damage to Cu,Zn-SOD by AAPH-derived radicals led to protein fragmentation, which is associated with the inactivation of enzyme. Carnosine, homocarnosine and anserine significantly inhibited the fragmentation and inactivation of Cu,Zn-SOD by AAPH. All three compounds also inhibited the release of copper ions from the enzyme and the formation of carbonyl compounds in AAPH-treated Cu,Zn-SOD. These compounds inhibited the fragmentation of other protein without copper ion. The results suggest that carnosine and related compounds act as the copper chelator and peroxyl radical scavenger to protect the protein fragmentation. Oxidation of amino acid residues in Cu,Zn-SOD induced by AAPH were significantly inhibited by carnosine and related compounds. It is proposed that carnosine and related dipeptides might be explored as potential therapeutic agents for pathologies that involve Cu,Zn-SOD modification mediated by peroxyl radicals.

Amidines↗

Aggregation of alpha-synuclein induced by the Cu,Zn-superoxide dismutase and hydrogen peroxide system.

Alpha-synuclein is a major component of the abnormal protein aggregation in Lewy bodies of Parkinson's disease (PD) and senile plaques of Alzheimer's disease (AD). Previous studies have shown that the aggregation of alpha-synuclein was induced by copper (II) and H(2)O(2) system. Since copper ions could be released from oxidatively damaged Cu,Zn-superoxide dismutase (SOD), we investigated the role of Cu,Zn-SOD in the aggregation of alpha-synuclein. When alpha-synuclein was incubated with both Cu,Zn-SOD and H(2)O(2), alpha-synuclein was induced to be aggregated. This process was inhibited by radical scavengers and spin trapping agents such as 5,5'-dimethyl 1-pyrolline N-oxide and tert-butyl-alpha-phenylnitrone. Copper chelators, diethyldithiocarbamate and penicillamine, also inhibited the Cu,Zn-SOD/H(2)O(2) system-induced alpha-synuclein aggregation. These results suggest that the aggregation of alpha-synuclein is mediated by the Cu,Zn-SOD/H(2)O(2) system via the generation of hydroxyl radical by the free radical-generating function of the enzyme. The Cu,Zn-SOD/H(2)O(2)-induced alpha-synuclein aggregates displayed strong thioflavin-S reactivity, reminiscent of amyloid. These results suggest that the Cu,Zn-SOD/H(2)O(2) system might be related to abnormal aggregation of alpha-synuclein, which may be involved in the pathogenesis of PD and related disorders.

Humans↗

Protection by carnosine-related dipeptides against hydrogen peroxide-mediated ceruloplasmin modification.

Carnosine, homocarnosine, and anserine are present in high concentrations in the muscle and brain of many animals and humans. Previous studies showed that these compounds have an antioxidant function. We investigated the protective effects of carnosine and related compounds on the modification of human ceruloplasmin that is induced by H2O2. Carnosine, homocarnosine, and anserine significantly inhibited the fragmentation and inactivation of ceruloplasmin that is induced by H2O2. All three compounds also inhibited the release of copper ion from protein, and the formation of hydroxyl radicals in the ceruloplasmin/H2O2 system. These compounds inhibited the fragmentation of human serum albumin that is induced by the copper-catalyzed oxidation system, as well as by the iron-catalyzed oxidation system. These results suggest that carnosine, homocarnosine, and anserine might protect ceruloplasmin against H2O2-mediated oxidative damage through a combination of copper chelation and free radical scavenging.

Animals↗

Production of monoclonal antibodies and immunohistochemical studies of brain myo-inositol monophosphate phosphatase.

Five monoclonal antibodies that recognize porcine brain myo-inositol monophosphate phosphatase (IMPase) have been selected and designated as mAb IMPP 9, IMPP 10, IMPP 11, IMPP 15, and IMPP 17. These antibodies recognize different epitopes of the enzyme and one of these inhibited the enzyme activity. When the total proteins of the porcine brain homogenate separated by SDS-PAGE were probed with monoclonal antibodies, a single reactive protein band of 29 kDa, co-migrating with the purified porcine brain IMPase, was detected. Using the anti-IMPase antibodies as probes, the cross reactivities of the brain IMPase from human and other mammalian tissues, as well as from avian sources, were investigated. Among the human and animal tissues tested, the immunoreactive bands on Western blots appeared to have the same molecular mass of 29 kDa. In addition, there was IMPase immunoreactivity in the various neuronal populations in the rat brain. These results indicate that mammalian brains contain only one major type of immunologically similar IMPase, although some properties of the enzymes that were previously reported differ from each another. The first demonstration of the IMPase localization in the brain may also provide useful data for future investigations on the function of this enzyme in relation to various neurological diseases.

5'-Nucleotidase↗

Immunohistochemical studies of brain pyridoxine-5'-phosphate oxidase.

A total of six hybridoma cell lines, which produce monoclonal antibodies (mAbs) against the sheep brain pyridoxine-5'-phosphate oxidase (PNP oxidase), were established. Isotype analysis revealed that all antibodies corresponded to the IgG 2B kappa subclass. Immunoblotting with various tissue homogenates indicated that all the mAbs specifically recognize a single protein band of 30 kDa. They also appear to be extensively cross-reactive among different mammalian and avian sources. These results demonstrated that only one type of immunologically similar PNP oxidase is present in all of the mammalian tissues tested. When the purified PNP oxidase was incubated with the mAbs, the enzyme activity was inhibited up to a maximum of 81%. Furthermore, these antibodies were successfully applied in immunohistochemistry in order to detect PNP oxidase in various regions of rat brain tissues. The immunoreactive neurons in PNP oxidase were found in cerebellar cortex, hippocampus, amygdala, paraventricular nucleus, cerebral cortex and ependyma. This result suggests that PNP oxidase may play an important role in the neuronal metabolism.

Animals↗

Phytol, SSADH inhibitory diterpenoid of Lactuca sativa.

The succinic semialdehyde dehydrogenase (SSADH) inhibitory component was isolated from the EtOAc fraction of Lactuca sativa through repeated column chromatography; then, it was identified as phytol, a diterpenoid, based on the interpretation of several spectral data. Incubation of SSADH with the phytol results in a time-dependent loss of enzymatic activity, suggesting that enzyme modification is irreversible. The inactivation followed pseudo-first-order kinetics with the second-rate order constant of 6.15 x 10(-2) mM(-1) min(-1). Complete protection from inactivation was afforded by the coenzyme NAD+, whereas substrate succinic semialdehyde failed to prevent the inactivation of the enzyme; therefore, it seems likely that phytol covalently binds at or near the active site of the enzyme. It is postulated that the phytol is able to elevate the neurotransmitter GABA levels in central nervous system through its inhibitory action on one of the GABA degradative enzymes, SSADH.

Aldehyde Oxidoreductases↗

Changes in Na(+)-K(+)-Cl(-) cotransporter immunoreactivity in the gerbil hippocampus following transient ischemia.

We examined alterations in Na(+)-K(+)-Cl(-) cotransporter 1 (NKCC1) immunoreactivity following ischemia. Twelve hours after ischemia, NKCC1 immunoreactivity in the CA1 region and in the hilar region was significantly diminished. Twenty-four hours after ischemia, NKCC1 immunoreactivity was intensified in these hippocampal regions as well as CA2-3. Two days after ischemia, NKCC1 immunoreactivity in the CA1 and the hilar neurons had disappeared, although in the CA2-3 and the granule cell layer NKCC1 immunoreactivities had recovered to the sham level. This finding suggests that NKCC1 may play an important role in the ischemic neuronal injury induced by excitotoxicity as well as neuronal edema.

Animals↗

Changes in Na(+)-K(+)-Cl(-) cotransporter immunoreactivity in the gerbil hippocampus following spontaneous seizure.

The immunoreactivity of Na(+)-K(+)-Cl(-) cotransporter (NKCC) in the gerbil hippocampus associated with various sequelae of spontaneous seizures were investigated in order to identify the roles of NKCC in the epileptogenesis and the recovery mechanisms in these animals. The NKCC immunoreactivities in the CA2-3 regions, the subiculum and the entorhinal cortex, were significantly more intensified in the pre-seizure group of seizure sensitive (SS) gerbils than in the seizure resistant (SR) gerbils. Following the on-set of seizure, the immunoreactivity of NKCC was significantly changed. In the hippocampal complex except the CA1 region, NKCC immunoreactivity in GABAergic neurons was significantly decreased 30 min after seizure on-set, versus the pre-seizure group. On the other hand, NKCC immunoreactivity was dramatically elevated in the CA1 regions, and 3 h postictal NKCC immunoreactivity increased significantly in the dentate gyrus and the dendrites of the pyramidal cells in the CA2-3 regions. These findings suggest that altered NKCC expression may be associated with seizure activity, and have an important role in the postictal recovery by regulating GABA-mediated inhibitory circuit in the hippocampal complex of the gerbil.

Animals↗

The decreases in calcium binding proteins and neurofilament immunoreactivities in the Purkinje cell of the seizure sensitive gerbils.

In previous studies, it has been reported that Purkinje cell degeneration during seizure is evoked by excitotoxicity due to an increase in the intracellular Ca(2+) level, though calbindin D-28k (CB) and parvalbumin (PV), intracellular free calcium buffers, are abundantly colocalized in these cells. In the present study, we investigated the expressions of CB, PV, neurofilament (NF) 68, 150, 200, and polyphosphorylated epitope in NF (RT 97), in the cerebellum of gerbils to identify the mechanism of Purkinje cell damages induced by seizure. In seizure resistant gerbils, nearly all the Purkinje cells showed CB, PA, NF 150, NF 200 and RT 97 immunoreactivity. In SS gerbils, however, a clear decrease in the number of CB(+) and PV(+) Purkinje cells was observed. The NF and RT 97 immunoreactivities, in the Purkinje cells was also lower (except NF 68), but not absent. These results suggest several points. First, the decrease in the concentrations of CB and PV may render the Purkinje cells more susceptible to intermittent Ca(2+) fluctuations and more prone to accumulating intolerable quantities of Ca(2+). Second, during the Ca(2+)-PV interaction PV plays an important role in facilitating donations of Mg(2+), which is a potent enzyme activator in phosphorylation. Thus the decline in PV concentration also implicated the defects of phosphorylation in the NF. Third, increases in both the intracellular Ca(2+) level and dephosphorylation trigger the degradation of the NF, particularly NF 200. Finally, these degradations in the NF induce the functional defects in Purkinje cell, which then cause Purkinje cell degeneration.

Animals↗

The ceruloplasmin and hydrogen peroxide system induces alpha-synuclein aggregation in vitro.

Alpha-synuclein is a key component of Lewy bodies in the brain of patients with Parkinson's disease (PD) and recent studies suggest that oxidative stress reactions might contribute to abnormal aggregation of this molecule. Since hydrogen peroxide-mediated ceruloplasmin (CP) modification can induce the formation of free radicals and release of copper ions, we investigated the role of CP in the aggregation of alpha-synuclein. When alpha-synuclein was incubated with both CP and H(2)O(2), alpha-synuclein concomitantly was induced to be aggregated. Thioflavin-S staining of alpha-synuclein aggregates showed that they displayed characteristic fibrillar structures. Hydroxyl radical scavengers and spin-trapping agent such as 5,5'-dimethyl 1-pyrolline N-oxide and tert-butyl-alpha-phenylnitrone significantly inhibited the aggregation of alpha-synuclein. Copper chelator, penicillamine also inhibited the CP/H(2)O(2) system-induced alpha-synuclein aggregation. This indicates that the aggregation of alpha-synuclein can be mediated by the CP/H(2)O(2) system via the generation of hydroxyl radical. The CP/H(2)O(2) system-induced alpha-synuclein aggregation resulted in the generation of protein carbonyl derivatives. Antioxidant molecules, carnosine, homocarnosine and anserine significantly inhibited the CP/H(2)O(2) system-induced aggregation of alpha-synuclein. These results suggest that the CP/H(2)O(2) system may be related to abnormal aggregation of alpha-synuclein which may be involved in the pathogenesis of PD and related disorders.

Benzothiazoles↗

Transcortical alterations in Na(+)-K+ ATPase and microtubule-associated proteins immunoreactivity in the rat cortical atrophy model induced by hypoxic ischemia.

To identify the chronological transcortical change in the contralateral hemisphere following ischemic insults, we investigated the changes in microtubule associated protein (MAP) and Na(+)-K+ ATPase expressions in the peri-infarct zone and contralateral hemisphere, including the hippocampus. Two days after hypoxic ischemia, Na(+)-K+ ATPase immunoreactivity was significantly enhanced in the contralateral cortex and was maintained up to 7 days after ischemia, whereas Na(+)-K+ ATPase immunoreactivity in the peri- and infarct zones was unaffected by hypoxic ischemia. In contrast, 2 to 7 days after ischemia, MAP1A and MAP2 immunoreactivity in the ipsi- and contralateral cortex significantly decreased, whereas in layer V, MAP1 immunoreactivity obviously accumulated in the neurons and their processes. In the hippocampus, 2 days after insults both MAP1A and MAP2 immunoreactivity was significantly reduced within the ipsi- and contralateral hippocampus. In the contralateral hippocampus, however, the distribution of MAP2 immunoreactivity recovered to the sham level 7 days after ischemia, whereas MAP1A immunoreactive axons remained 2 months after ischemia. The results suggest that the unilateral elevation of Na(+)-K+ ATPase immunoreactivity reflects elevated neuronal activity. In addition, this asymmetric hyperexcitability might play an important role in the recovery or the reorganization of the brain, accompanied by transcortical changes in MAPs expression.

Animals↗