• Applied Biological Chemistry
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• v.43 no.2
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• pp.86-94
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• 2000

In order to develop the enzymatic hydrolysis system concerned with taste and flavor, strains having the high hydrolyzing activity on the soy protein were selected from some traditional Mejus. Two molds and one bacterium producing enzymes which were different in character of hydrolysis were isolated and identified. Leucine and azodye enzyme activities of both M4 and M5 were relatively high among in the isolated molds. And, leucine enzyme activity of B16 was the lowest in the isolated bacteria. These strains were isolated as microorganisms having a dissimilar hydrolysis pattern on the soy protein by enzymatic reactions. Mold M4 on the culture solid media was mycelium colors of white and its sclerotia colors were changed from white to black. According to the result of slide culture, radial conidial head, subclavate vesicle, conidia of subglobose, stipes of uncolored with smooth walls and metula and phialides were existed. Because M4 was taxonomically similar to the characteristics of Aspergillus oryzae (ahlburg) species, M4 was identified and named as Aspergillus oryzae M4.Mold M5 showed white and black mycelium on the MEA medium. Mold M5 colony exhibited grayish-green color and have long(7 mm) sporangiophores at slide culture. Sporangia became brownish-gray and the wall of larger sporangia was broken to form small collars, and smaller sporangia were fomed continually from large basal membrane. Columella is globose and hyaline, and sporangiospores are ellipsoidal of small diameter$(80\;{\mu}m)$. Because M5 was taxonomically similar to the Mucor circinelloides of zygomycetes, M5 was was identified and named as Mucor circinelloides M5. Bacteria B16 colony was opaque white, circular and lobate, and had rod shaped endospore. B16 was found positive in stain, catalase, ${\beta}-glucosidse$ and V-P tests. B16 was found to utilize D-fructose, ${\alpha}-D-glucose$, maltose, D-mannose, D-raffinose, stachyose and sucrose. By the morphological and physiological results, the characteristics of B16 was thought to correspond to that of Bacillus megaterium. However, fatty acid composition was similar to Paenibacillus marcerans, requiring further study for the definite identification. Accordingly, Bacteria B16 was provisionally classified and named as Bacillus megaterium B16.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.151-158
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• 2007

The possibility of inadvertent introduction of therapeutic gene expressing viral vectors has raised safety concerns about germ-line infection. Particularly, for indications such as prostate cancer and ovarian cancer, the proximity of the point of viral administration to organs of the reproductive system raises concerns regarding inadvertent germ-line transmission of genes carried by the virus vector. To evaluate the safety of in vivo adenovirus mediated gene transfer, we explored the biodistribution, persistance and potential germ-line transmission of p53-expressing adenovirus (Ad-CMV-p53). Both male and female Balb/c mice were injected with $1{\times}10^9$ PFU of Ad-CMV-p53. The PCR analysis showed that there were detectable vector sequences in liver, kidney, spleen, seminal vesicle, epididymis, prostate, ovary, and uterus. The RT-PCR analysis for detecting inserted gene, p53 showed that Ad-CMV-p53 viral RNA were present in spleen, prostate and ovary. Direct injected male and female mice of adenovirus vector into testis and ovary were mated and their of offspring were evaluated for germ-line transmission of the adenoviral vector. The PCR and RT-PCR analysis showed no evidence of germline transmission, although vector sequences were detected in DNA extracted from gonadal tissues. Real-time PCR result confirmed a significant decrease of adenovirus in gonad tissues 1 week after injection. We have also analysed the cell specific localization of viral DNA in gonad tissues by using in-situ PCR. Positive signals were detected in interstitial tissue but not in seminiferous tubule in sperm. In the case of ovary, adenovirus signal were localized to the stromal tissue, but no follicular signals were observed. Together, these data provide strong evidence that the risk of the Inadvertent germ-line transmission of vector sequences following intraperitoneal or direct injection into genito-urinary system of adenovirus is extremely low.

• Development and Reproduction
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• v.11 no.3
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• pp.263-272
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• 2007

Contrast to mouse where its in vitro maturation rates are high without specific supplements or presence of the cumulus cells, there are some species, such as porcine, where its in vitro oocyte maturation rates are still very low. This comparative study was conducted to investigate the role of malate dehydrogenase(Mor2) during oocyte maturation by RNAi in the mouse and porcine. The Mor2 double-stranded RNA(dsRNA) was prepared speciesspecifically and microinjected into the cytoplasm of denuded germinal vesicle(GV) oocytes. Oocytes were cultured for 48 h(porcine) and 16 h(mouse) in M199 with 10% porcine follicular fluid, pyruvate, p-FSH, EGF, cystein, and estradiol-$17{\beta}$. We measured changes in oocyte morphology, maturation rates and mRNA levels after Mor2 RNAi. We confirmed gene sequence-specific knock down of Mor2 mRNA in both species after Mor2 RNAi. In contrast to our previous finding that mMor2 RNAi resulted in GV arrest in the mouse, we found that pMor2 RNAi resulted in MI arrest in denuded porcine oocytes(58%), but developed to MII(84.4%) in COCs. To determine whether this difference between mouse and porcine RNAi is due to differences in culture media, we cultured mouse oocytes in the M199 media for 16 h after mMor2 RNAi. Mouse oocytes were developed to MII stage(62%) and there was no statistical difference compared to that of non-injected(76.8%) and buffer-injected(73.3%) control groups. Therefore, we concluded that the mouse and porcine oocytes are having different metabolic systems in relation to malate dehydrogenase for oocyte maturation. This could be a basis for differences in maturation rates in vitro in two species. Further scrutinized studies on the metabolic pathways would led us in finding better culture system to improve oocyte maturation rates in vitro, especially in more challenging species like the porcine.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.20 no.6
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• pp.509-519
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• 1987

Annual reproductive cycle of the Damselfish, Chromis notatus collected monthly at the four coastal areas around Chejudo, Korea are studied on the bases of histological observations of gonadal tissue and various quantitative variables including gonadosomatic index (GSI), fatness, egg diameter composition and the first maturity. The ovary consisted of a pair of saccular structure with many ovarian sacs. Oogonia proliferated along the germinal epithelium of the ovarian sac. Young oocytes with basophilic cytoplasm showed several nucleoli along the nuclear membrane. When the oocytes reached about $450{\mu}m$ in diameter, nucleus migrate toward the animal pole, nuclear membrane disappeared and most of cytoplasm were filled with yolk materials and oil drops. After ovulation, residual follicle and growing oocytes remaining in the ovarian sacs degenerated. But early young oocytes without follicle layer were not degenerated, and growing continuously till the next year. The testis consisted of a pair of lobular structures in the right and left were united in the posterior seminal vesicle. Cortex of testis was composed of many sperm ducts connected with lobuli. GSI began to increase from March, starting season of longer day length and higher water temperature, and reached the maximum value between June and August. It began to decrease from September with the lowest value appearing between October and February without any evident variation. The annual reproductive cycle could be devided into five successive stage : growing(April to Many), mature(May to August), ripe and spent (June to August) and recovery and resting stage(September to March). The spawning peak occurred from June to August. According to the frequency distribution of egg diameter, Chromis notatus was a polycyclic species to spawn twice or more in a spawning season. Fatness, correlated with gonadal phases, was remarkably decreased by spawning. Percentage of the first maturity . in femate and male fish ranging from 7.0 to 7.9 cm were $50\%$ and from 9.0 to 9.9 cm in total length $100\%$.

• Journal of Life Science
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• v.24 no.3
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• pp.242-251
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• 2014

The objective of this study was to isolate a photosensitizer from Pueraria thunbergiana leaves that induces apoptosis in SK-HEP-1 cells. Column chromatography and thin layer chromatography were used to isolate active compounds from extracts of P. thunbergiana leaves. The structures of the isolated compounds were determined by 1D-NMR, 2D-NMR, and FAB-mass spectroscopy. A substance, named M4-3, was purified from the leaves of P. thunbergiana using various chromatography methods, and the absorbance of the substance was measured. The absorbance was highest at 410 nm, suggesting that the M4-3 substance was a different compound from chlorophyll a and b, which absorb at 410, 502, 533, and 607 nm. Further analyses revealed that the M4-3 compound was a $13^2$-hydoxy pheophorbide, a methyl ester with a molecular weight of 662. M4-3 was identified as a derivative compound of pheophorbide, with a structure that magnesium comes away from the porphyrin ring. The results of the analysis of the cytotoxicity of the M4-3 substance against the SK-HEP-1 cells revealed that it inhibited rates of cell growth by 40% and 80% at a concentration of 0.04 ${\mu}M$ and 0.08 ${\mu}M$, respectively. The M4-3 compound was found to be a photosensitizer for cytotoxicity because it was appeared only in light condition as examining activity in different irradiation conditions (light condition and nonlight condition) under the same concentration. Analysis of morphological changes in the cells following cell death induced by exposure to the M4-3 substance reveled representative phenomena of apoptosis (nuclear condensation, vesicle formation, and fragmentation of DNA). The induction of apoptosis was attributed to the compound's photodynamic activity.

• Journal of Life Science
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• v.23 no.5
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• pp.689-697
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• 2013

In the United States, about 40,000 new cases of oral cancer are diagnosed each year and nearly 7,800 patients died from it in 2012. Omega-3 polyunsaturated fatty acids have been found to have anticancer effects in a variety of cancer cell lines and animal models, but their effect in oral cancer remains unclear. This study was designed to examine the effect of docosahexaenoic acid (DHA, a kind of omega-3 fatty acid) on oral cancer cells and the molecular mechanism of its action. We found that exposure of squamous cell carcinoma-4 (SCC-4) and squamous cell carcinoma-9 (SCC-9) human oral cancer cells to DHA induced growth inhibition in a dose- and time-dependent manner. Meanwhile, in addition to the elevated levels of apoptotic markers, such as cleaved PARP, subG1 portion and TUNEL-positive nuclei, DHA led to autophagic vesicle formation and an increase in autophagic flux, indicating the involvement of both apoptosis and autophagy in the inhibitory effects of DHA on oral cancer cells. Further experiments revealed that the apoptosis and autophagy induced by DHA were linked to inhibition of mammalian target of rapamycin (mTOR) signaling by AKT inhibition and AMP-activated protein kinase (AMPK) activation in SCC-9 cells. Together, our results suggest that DHA induces apoptosis- and autophagy-associated cell death through the AMPK/AKT/mTOR signaling pathway in oral cancer cells. Thus, utilization of omega-3 fatty acids may represent a promising therapeutic approach for chemoprevention and treatment of human oral cancer.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.17 no.5
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• pp.423-435
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• 1984

The reproductive cycle of the small filefish, Rudarius ercodes was investigated based on the annual variations of gonadosomatic index(GSI) and hepatosomatic index(HSI) by electronic and photic microscophy. The specimens used were collected at the coastal area of Benden island, Sizuokagen, Japan, from September 1982 to August 1983. GSI began to increase from March, starting season of longer daylength and higher water temperature, and reached the maximum value between June and August. It began to decrease from September with the lowest value appearing between November and February without any evident variation. The annual variations of HSI were not distinct in male filefish and were negatively related to GSI in female : HSI decreased in the summer season when the ovary was getting mature and reached the maximum in the winter season when the ovary was getting retrogressive. The ovary consisted of a pair of saccular structure with numerous ovarian sacs branched toward the median cavity. Oogonia divided and proliferated along the germinal epithelium of the ovarian sac. Young oocytes with basophile cytoplasm showed several scattering nucleoli along the nuclear membrane. when the oocytes growing to about 300 ${\mu}m$, nuclear membrane to disappear with nucleus migrating toward the animal pole. The regions of protoplasm were extremely confined within the animal hemisphere in which most of cytoplasms were filled with yolk materials and oil drops. After ovulation, residual follicles and growing oocytes remaining in the ovarian sacs degenerated. But perinucleatic young oocytes without follicles formed were not degenerated, and growing continuously still in the next year. Mitochondria and endoplasmic reticula in the cytoplasm remarkably increased with oocytes maturing and yolk accumulating. Those were considered to be functionally related to the yolk accumulation. Five or six layers of possible vitellogenin, oval-shaped disc structures with high electron density, appeared in the apex of follicular processes stretching to the microvilli pits of mature oocytes. Testis consisting of a pair of lobular structures in the right and left were united in the posterior seminal vesicle, Cortex of testis was composed of several seminiferous tubules, and medulla consisting of many sperm ducts connected with tubules. Steroid hormone-secreting cells with numerous endoplasmic reticula and large mitochondria of well developed cristae were recognized in the interstitial cells of the growing testis. Axial filament of spermatozoon invaginated deeply in the central cavity of the nucleus and the head formed U-shape with acrosome severely lacking, mitochondria formed large globular paranuclei at the posterior head, and microtubular axoneme of the tail represented 9+9+2 type. The annual reproductive cycles could be divided into five successive stages : growth(March to July), maturation(May to September), Spawning(mid May to early October) and resting stages(October to February). The spawning peak occurred from June to August.

• Journal of the korean academy of Pediatric Dentistry
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• v.28 no.2
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• pp.219-227
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• 2001

Little is known about processing mechanism of pain sensation of the oral cavity at the 1st synapse of trigeminal sensory nuclei. Serial ultrathin sections of tooth pulp afferent terminals, identified by the transganglionic transport of 1% wheatgerm agglutinin conjugated horseradish peroxidase, were investigated with electron microscope. Quantitative ultrastructural analysis was performed on digitizing tablet connected to Macintoshi personal computer (software; NIH Image 1.60, NIH, Bethesda, MD). Labeled boutons could be classified into two types by the shapes of containing vesicles : S bouton, which contained mainly spherical vesicles (Dia. 45-55 nm) and few large dense cored vesicles (Dia, 80-120nm), and LDCV bouton, which contained spherical vesicles as well as large number of large dense cored vesicles. Most of the parameters on the ultrastructural characteristic and synaptic organization of labeled boutons were similar between S and LDCV boutons, except shapes of containing vesicles. Majority of the labeled boutons showed simple synaptic arrangement. The labeled boutons were frequency presynaptic to dendritic spine, and to a lesser extent, dendritic shaft. They rarely synapsed with soma and adjacent proximal dendrite. A small proportion of labeled boutons made synaptic contacts with presynaptic, pleomorphic vesicles containing endings and synaptic triad. Morphometric parameters of labeled boutons including volume and surface area, total apposed area, mitochondrial volume, active zone area, vesicle number and density showed wide variation and these were not significantly different between S and LDCV boutons. The present study revealed characteristic features on ultrastructure and synaptic connection of pulpal afferents which may involved in transmission of oral pain sensation.

• Clinical and Experimental Reproductive Medicine
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• v.34 no.2
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• pp.95-106
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• 2007

Objective: We previously identified differentially expressed genes (DEGs) between germinal vesicle (GV) and metaphase II (MII) mouse oocyte. The present study was accomplished as a preliminary study to elucidate the role of ribose 5-phosphate isomerase A (Rpia), the essential enzyme of the pentose phosphate pathway (PPP), in oocyte maturation. We observed expression of Rpia in the mouse and porcine oocytes. Methods: Expression pattern of the 11 MII-selective DEGs in various tissues was evaluated using RT-PCR and selected 4 genes highly expressed in the ovary. According to the oocyte-selective expression profile, we selected Rpia as a target for this study. We identified the porcine Rpia sequence using EST clustering technique, since it is not yet registered in public databases. Results: The extended porcine Rpia nucleotide sequence was submitted and registered to GenBank (accession number EF213106). We prepared primers for porcine Rpia according to this sequence. In contrast to the oocyte-specific expression in the mouse, Rpia was expressed in porcine cumulus and granulosa cells as well as in oocytes. Conclusion: This is the first report on the characterization of the Rpia gene in the mouse and porcine ovarian cells. Results of the present study suggest that the mouse and porcine COCs employ different mechanism of glucose metabolism. Therefore, the different metabolic pathways during in vitro oocyte maturation (IVM) in different species may lead different maturation rates. It is required to study further regarding the role of Rpia in glucose metabolism of oocytes and follicular cell fore exploring the regulatory mechanism of oocyte maturation as well as for finding the finest culture conditions for in vitro maturation.

• Development and Reproduction
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• v.10 no.3
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• pp.203-209
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• 2006

Ethane 1,2-Dimethane sulfonate(EDS), a toxin which specifically kills Leydig cells(LC), has been widely used to prepare the reversible testosterone(T) depletion rat model. Previous studies including our own clearly demonstrated that the dramatic weight loss of the T-dependent accessory sex organs such as epididymis and seminal vesicle in this 'LC knock-out' rats. These weight loss could be derived from massive and abrupt death of the cells via apoptotic process. The present study was performed to test the effect of EDS administration on the expression of some apoptotic genes in the rat epididymis. Adult male Sprague-Dawley rats($300{\sim}350$ g B.W.) were injected with single dose of EDS(75 mg/kg, i.p.) and sacrificed on Weeks 0, 1, 2, 3, 4, 5, 6 and 7. Tissue weights and the numbers of the epididymal sperm were measured. The transcriptional activities of the bcl-2, bax, Fas and Fas ligand(Fas-L) were evaluated by semi-quantitative RT-PCR. As expected, the weights and the sperm counts of epididymis declined progressively after the EDS treatment during Week 1 and 2. These decrements were discontinued with a gradual return towards normal during Weeks $5{\sim}7$, although the maximal recoveries of the epididymal weights(71%) and sperm count(38%) were subnormal on Week 7. The initial level of bcl-2 transcripts persisted to Week 6 then elevated significantly on Week 7. The level of bax transcripts significantly decreased on Week 6, and no remarkable change was found in the rest of the experimental period. The transcripts for the Fas in epididymis elevated during Weeks $1{\sim}2$, returned to normal on Week 3, and the level persisted to the Week 7. Similarly, the level of Fas-L transcripts elevated during Weeks $1{\sim}3$ and returned to normal after Week 4. Our results demonstrated the transient T depletion by EDS administration could induce the changes in expression of the apoptotic genes in rat epididymis. The activation of Fas and Fas-L in the epididymis of EDS-treated rats might be responsible for the initial apototic process and consequently the tissue damage and the sperm loss. Future studies will attempt to determine the precise molecular mechanism(s) of apoptosis in the rat epididymis.