• Journal of Ginseng Research
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• v.47 no.3
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• pp.408-419
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• 2023

Background: Ginsenoside compound K (CK), the main active metabolite in Panax ginseng, has shown good safety and bioavailability in clinical trials and exerts neuroprotective effects in cerebral ischemic stroke. However, its potential role in the prevention of cerebral ischemia/reperfusion (I/R) injury remains unclear. Our study aimed to investigate the molecular mechanism of ginsenoside CK against cerebral I/R injury. Methods: We used a combination of in vitro and in vivo models, including oxygen and glucose deprivation/reperfusion induced PC12 cell model and middle cerebral artery occlusion/reperfusion induced rat model, to mimic I/R injury. Intracellular oxygen consumption and extracellular acidification rate were analyzed by Seahorse multifunctional energy metabolism system; ATP production was detected by luciferase method. The number and size of mitochondria were analyzed by transmission electron microscopy and MitoTracker probe combined with confocal laser microscopy. The potential mechanisms of ginsenoside CK on mitochondrial dynamics and bioenergy were evaluated by RNA interference, pharmacological antagonism combined with co-immunoprecipitation analysis and phenotypic analysis. Results: Ginsenoside CK pretreatment could attenuate mitochondrial translocation of DRP1, mitophagy, mitochondrial apoptosis, and neuronal bioenergy imbalance against cerebral I/R injury in both in vitro and in vivo models. Our data also confirmed that ginsenoside CK administration could reduce the binding affinity of Mul1 and Mfn2 to inhibit the ubiquitination and degradation of Mfn2, thereby elevating the protein level of Mfn2 in cerebral I/R injury. Conclusion: These data provide evidence that ginsenoside CK may be a promising therapeutic agent against cerebral I/R injury via Mul1/Mfn2 mediated mitochondrial dynamics and bioenergy.

• Journal of Ecology and Environment
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• v.37 no.4
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• pp.395-410
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• 2014

The genus Callophyllis is recorded as six separate species with imprecise species delimitation in Korea. To elucidate the species boundaries of Korean Callophyllis, we performed morphological observations and molecular analyses, and included three Japanese Callophyllis species from the type locality. From the results of molecular analyses using plastid rbcL and mitochondrial COI-5P genes, we confirmed ten Callophyllis species, including five cryptic ones: C. adhaerens, C. adnata, C. crispata, and C. japonica from Korea and Japan; C. hayamensis as an unrecorded species from Korea; C. cartilaginea, C. mollitia, C. repens, C. serratifolia, and C. undulata as new species from Korea. There were no Korean specimens that matched C. adnata or C. crispata from Japan, except Korean C. japonica, which formed a genetic group with the Japanese species. We obtained the interspecific divergences among the five cryptic species as 0.6-4.5% in rbcL and 2.8-8.4% in COI-5P. We recognized that the species diversity of Callophyllis has been underestimated from the northwestern Pacific region. The species boundary of Callophyllis from Korea and Japan will be a cornerstone to revealing the phylogenetic affinity of the genus distributed in both hemispheres of the western Pacific.

• BMB Reports
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• v.31 no.4
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• pp.307-327
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• 1998

Cytochrome c peroxidase (CcP) is a yeast mitochondrial enzyme which catalyzes the reduction of hydrogen peroxide to water using two equivalents of ferrocytochrome c. The CcP/cytochrome c system has many features which make it a very useful model for detailed investigation of heme protein structure/function relationships including activation of hydrogen peroxide, protein-protein interactions, and long-range electron transfer. Both CcP and cytochrome c are single heme, single subunit proteins of modest size. High-resolution crystallographic structures of both proteins, of one-to-one complexes of the two proteins, and a number of active-site mutants are available. Site-directed mutagenesis studies indicate that the distal histidine in CcP is primarily responsible for rapid utilization of hydrogen peroxide implying significantly different properties of the distal histidine in the peroxidases compared to the globins. CcP and cytochrome c bind to form a dynamic one-to-one complex. The binding is largely electrostatic in nature with a small, unfavorable enthalpy of binding and a large positive entropy change upon complex formation. The cytochrome c-binding site on CcP has been mapped in solution by measuring the binding affinities between cytochrome c and a number of CcP surface mutations. The binding site for cytochrome c in solution is consistent with the crystallographic structure of the one-to-one complex. Evidence for the involvement of a second, low-affinity cytochrome c-binding site on CcP in long-range electron transfer between the two proteins is reviewed.

• BMB Reports
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• v.29 no.4
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• pp.300-307
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• 1996

ATP and ADP are potential regulators of mitochondtial respiration and at physiological concentrations they affect the rate of electron transfer between cytochrome c and cytochrome c oxidase. The electron transfer, however, depends on the electrostatic interaction between the two proteins. In order to exclude any nonspecific ionic effects by these polyvalent nucleotides, we used 2'-O-(2,4,6)trinitro(TNP)-derivatives of ATP and ADP which have three orders of magnitude higher affinity for cytochrome c oxidase. A simple titration of the fluorescence intensity of TNP by cytochrome c oxidase showed a binding stoichiometry of 2:1 cytochrome c:cytochrome c oxidase. Higher ionic strength was required for TNP-ATP than for TNP-ADP to be dissociated from cytochrome c oxidase, indicating that the negative charges on the phosphate group are at least partially responsible for the binding. In both spectrophotometric and polarographic assays, addition of ATP (and ADP to a less extent) showed an enhanced cytochrome c oxidase activity. Both electron paramagnetic resonance and fluorescence spectra indicate that there is no Significant change in the cytochrome c-cytochrome c oxidase interaction. Instead, reduction levels of the cytochromes at steadystate suggest that the increased activity of nucleotide-bound cytochrome c oxidase is due to faster electron transfer from cytochrome ${\alpha}$ to cytochrome ${\alpha}_3$, which is known to be the fate limiting step in the oxygen reduction by cytochrome c oxidase.

• BMB Reports
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• v.52 no.3
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• pp.163-164
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• 2019

The ribosomal synthesis of proteins in the eukaryotic cytosol has always been thought to start from the unformylated N-terminal (Nt) methionine (Met). In contrast, in virtually all nascent proteins in bacteria and eukaryotic organelles, such as mitochondria and chloroplasts, Nt-formyl-methionine (fMet) is the first building block of ribosomal synthesis. Through extensive approaches, including mass spectrometric analyses of the N-termini of proteins and molecular genetic techniques with an affinity-purified antibody for Nt-formylation, we investigated whether Nt-formylated proteins could also be produced and have their own metabolic fate in the cytosol of a eukaryote, such as yeast Saccharomyces cerevisiae. We discovered that Nt-formylated proteins could be generated in the cytosol by yeast mitochondrial formyltransferase (Fmt1). These Nt-formylated proteins were massively upregulated in the stationary phase or upon starvation for specific amino acids and were crucial for the adaptation to specific stresses. The stress-activated kinase Gcn2 was strictly required for the upregulation of Nt-formylated proteins by regulating the activity of Fmt1 and its retention in the cytosol. We also found that the Nt-fMet residues of Nt-formylated proteins could be distinct N-terminal degradation signals, termed fMet/N-degrons, and that Psh1 E3 ubiquitin ligase mediated the selective destruction of Nt-formylated proteins as the recognition component of a novel eukaryotic fMet/N-end rule pathway, termed fMet/N-recognin.

• Animal cells and systems
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• v.3 no.4
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• pp.393-397
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• 1999

Length variations in human mitochondrial DNA (mtDNA) offer useful markers in the study of female aspects of human population history. One such length variation is a 9-bp deletion in the small noncoding segment located between the COII and Iysine tRNA genes (COII/tRNA/$^{Lys}$ intergenic region) which usually contain two tandemly arranged copies of a 9-bp sequence (ccccctcta) in human mtDNA. The mtDNA 9-bp deletion and polymorphic variants of expanded 9-bp repeat motif in the intergenic COII/tRNA$^{Lys}$ region have been found at varying frequencies among different human ethnic groups. We have examined the length variation of the mtDNA COII/tRNA$^{Lys}$ intergenic region from a total of 813 individuals in east Asian populations. The occurrence of the 9-bp deletion was found to be relatively homogeneous in northeast Asian populations (Chinese, 14.2%; Japanese, 14.3%: Koreans, 15.5%), with the exception of Mongolians (5.1%). In contrast, Indonesians (25.0%) and Vietnamese (23.2%) of the southeast Asian populations appeared to have relatively high frequencies of the 9-bp deletion. We identified the existence of a new expanded 9-bp repeat motif which likely resulted from a slipped mispairing insertion of six more cytosines in the intergenic COII$^{Lys}$ region. It was present at low frequencies in the Korean (2/349) and Japanese populations (2/147). Based on the results of this study, the Korean population may reflect a close genetic affinity with the Japanese and Chinese populations than the others surveyed east Asian populations.

• Journal of Embryo Transfer
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• v.33 no.2
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• pp.75-84
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• 2018

The cryopreservation has been extensively applied in many cells including spermatozoa (semen) during past several decades. Especially, the canine spermatozoa cryopreservation has contributed on generation of progeny of rare/genetically valuable dog breeds, genome resource banking and transportation of male germplasm at a distant place. However, severe and irreversible damages to the spermatozoa during cryopreservation procedures such as the thermal shock (cold shock), formation of intracellular ice crystals, osmotic shock, stress of cryoprotectants and generator of reactive oxygen species (ROS) have been addressed. According as a number of researches have been conducted to overcome these problems and to advance cryopreservation technique, several analytical methods have been employed to evaluate the quality of the fresh or cryopreserved canine spermatozoa in regards to the motility, morphology, integrity of membrane and DNA, mitochondrial activity, ROS generation, binding affinity to oocytes, in vitro fertilization potential and fertility potential by artificial insemination. Because the study designs with certain application of analytical methods are selective and varied depending on each experimental objective and laboratory condition, it is necessary to establish the normal reference data of the fresh or cryopreserved canine spermatozoa for each analytical method to monitor experimental procedure, to translate raw data and to discuss results. Here, we reviewed the recent articles to introduce various analytical methods for the canine spermatozoa as well as to establish the normal reference data for each analytical method in the fresh or cryopreserved canine spermatozoa, based on the results of the previous articles. We hope that this review contributes to the advancement of cryobiology in canine spermatozoa.

• Dementia and Neurocognitive Disorders
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• v.21 no.2
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• pp.71-78
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• 2022

Background and Purpose: The expression of the 18-kDA mitochondrial translocator protein (TSPO) in the brain is an attractive target to study neuroinflammation. However, the binding properties of TSPO ligands are reportedly dependent on genetic polymorphism of the TSPO gene (rs6971). The objective of this study is to investigate the rs6971 gene polymorphism in the Korean population. Methods: We performed genetic testing on 109 subjects including patients with mild cognitive impairment, Alzheimer's disease (AD) dementia, non-AD dementia, and cognitively unimpaired participants. Magnetic resonance imaging scans and detailed neuropsychological tests were also performed, and 29 participants underwent ¹⁸F-DPA714 PET scans. Exon 4 of the TSPO gene containing the polymorphism rs6971 (Ala or Thr at position 147) was polymerase chain reaction amplified and sequenced using the Sanger method. The identified rs6971 genotype codes (C/C, C/T, or T/T) of the TSPO protein generated high-, mixed-, or low-affinity binding phenotypes (HABs, MABs, and LABs), respectively. Results: We found that 96.3% of the study subjects were HAB (105 out of 109 subjects), and 3.7% of the subjects were MAB (4 out of 109 subjects). ¹⁸F-DPA-714 PET scans showed nonspecific binding to the thalamus and brainstem, and increased tracer uptake throughout the cortex in cognitively impaired patients. The participant with the MAB polymorphism had a higher DPA714 signal throughout the cortex. Conclusions: The majority of Koreans are HAB (aprox. 96%). Therefore, the polymorphism of the rs6971 gene would have a smaller impact on the availability of second-generation TSPO PET tracers.

• Journal of Life Science
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• v.22 no.2
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• pp.209-219
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• 2012

The properties of lactate dehydrogenase (LDH, EC 1.1.1.27) eye-specific $C_4$ isozyme were studied by polyacrylamide gel electrophoresis, Western blotting, immunoprecipitation, and enzyme kinetics. Furthermore, we proposed the optimal conditions for measuring the activity of LDH eye-specific $C_4$ isozyme. The isozymes were detected in the cytosol of eye tissues from Lepomis macrochirus and Micropterus salmoides and were more similar to the $A_4$ than the $B_4$ isozyme. LDH/CS in the eye tissue of L. macrochirus was increased in September, so the ratio of anaerobic metabolism was high. The electrophoretic patterns of mitochondrial LDH were similar to those of cytosolic LDH in the eye tissues of L. macrochirus and Micropterus salmoides. LDH eye-specific $C_4$ isozyme from eye tissue was purified by preparative native-PAGE. The activities of LDH eye-specific $C_4$ isozymes in L. macrochirus and M. salmoides were reduced at concentrations greater than 0.2 mM and 0.1 mM of pyruvate, respectively. These concentrations remained at 5.2% and 15.8% as a result of the inhibition by 10 mM of pyruvate, so the degree of inhibition was very high. The LDH activities of eye tissues were reduced at concentrations greater than 22 mM and 24 mM of lactate, respectively, in L. macrochirus and M. salmoides. The ${K_m}^{PYR}$ of eye-specific $C_4$ was 0.088 mM in L. macrochirus and it was 0.033 mM in M. salmoides. The activities of cytosolic and mitochondrial eye-specific $C_4$ isozymes were high in ${\alpha}$-ketobutyric acid. Furthermore, the activities of eye tissue and eye-specific $C_4$ isozyme had to be measured with 0.5 mM of pyruvate and a buffer solution of pH 7.5. As a conclusion, the eye-specific $C_4$ isozyme in M. salmoides has a high affinity for pyruvate and exhibits maximum activity at a lower concentration of pyruvate and at higher concentration of lactate than that in L. macrochirus. Therefore, it seems that the energy produced by the LDH eye-specific $C_4$ isozyme in M. salmoides was used at the first stage of predatory behavior.