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A Study of Endothelium-dependent Pulmonary Arterial Relaxation and the Role of Nitric oxide on Acute Hypoxic Pulmonary Vasoconstriction in Rats (흰쥐 폐동맥의 내피세포의존성 혈관이완과 급성 저산소성 폐동맥수축에서 산화질소의 역할)

  • In, Kwang-Ho;Lee, Jin-Goo;Cho, Jae-Youn;Shim, Jae-Jung;Kang, Kyung-Ho;Yoo, Se-Hwa
    • Tuberculosis and Respiratory Diseases
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    • v.41 no.3
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    • pp.231-238
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    • 1994
  • Backgroud: Since the demonstration of the fact that vascular relaxation by acetylcholine(Ach) results from the release of relaxing factor from the endothelium, the identity and physiology of this endothelium-derived relaxing factor(EDRF) has been the target for many researches. EDRF has been identified as nitric oxide(NO). With the recent evidences that EDRF is an important mediator of vascular tone, there have been increasing interests in defining the role of the EDRF as a potential mediator of hypoxic pulmonary vasoconstriction. But the role of EDRF in modulating the pulmonary circulation is not compeletely clarified. To investigate the endothelium-dependent pulmonary vasodilation and the role of EDRF during hypoxic pulmonary vasoconstriction, we studied the effects of $N^G$-monomethyl-L-arginine(L-NMMA) and L-arginine on the precontracted pulmonary arterial rings of the rat in normoxia and hypoxia. Mothods: The pulmonary arteries of male Sprague Dawley(300~350g) were dissected free of surrounding tissue, and cut into rings. Rings were mounted over fine rigid wires, in organ chambers filled with 20ml of Krebs solution bubbled with 95 percent oxygen and 5 percent carbon dioxide and maintained at $37^{\circ}C$. Changes in isometric tension were recorded with a force transducer(FT.03 Grass, Quincy, USA) Results: 1) Precontraction of rat pulmonry artery with intact endothelium by phenylephrine(PE, $10^{-6}M$) was relaxed completely by acetylcholine(Ach, $10^{-9}-10^{-5}M$) and sodium nitroprusside(SN, $10^{-9}-10^{-5}M$), but relaxing response by Ach in rat pulmonary artery with denuded endothelium was significantly decreased. 2) L-NMMA($10^{-4}M$) pretreatment inhibited Ach($10^{-9}-10^{-5}M$)-induced relaxation, but L-NMMA ($10^{-4}M$) had no effect on relaxation induced by SN($10^{-9}-10^{-5}M$). 3) Pretreatment of the L-arginine($10^{-4}M$) significantly reversed the inhibition of the Ach ($10^{-9}-10^{-5}M$)-induced relaxation caused by L-NMMA($10^{-4}M$) 4) Pulmonary arterial contraction by PE($10^{-6}M$) was stronger in hypoxia than normoxia but relaxing response by Ach($10^{-9}-10^{-5}M$) was decreased, 5) With pretreatment of L-arginine($10^{-4}M$), pulmonary arterial relaxation by Ach($10^{-9}-10^{-5}M$) in hypoxia was reversed to the level of relaxation in normoxia. Conclusion: It is concluded that rat pulmonary arterial relaxation by Ach is dependent on the intact endothelium and is largely mediated by NO. Acute hypoxic pulmonary vasoconstriction is related to the suppression on NO formation in the vascular endothelium.

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TNF-$\alpha$ TGF-$\beta$ and Fibrinolytic Parameters in Tuberculous and Malignant Pleural Effusions (결핵성 및 악성 흉막염에서 TNF-$\alpha$, TGF-$\beta$ 및 섬유소용해계의 역할)

  • Shim, Tae-Sun;Yang, Sung-Eun;Chi, Hyun-Sook;Kim, Mi-Jung;Chung, Hun;JeGal, Yang-Jin;Lim, Chae-Man;Lee, Sang-Do;Koh, Youn-Suck;Kim, Woo-Sung;Kim, Dong-Soon;Kim, Won-Dong
    • Tuberculosis and Respiratory Diseases
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    • v.49 no.2
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    • pp.149-161
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    • 2000
  • Background : Residual pleural thickening (RPT) develops in about 50% of tuberculous pleurisy ($PL_{TB}$). Some reports have suggested that elevated TNF-$\alpha$ and impaired fibrinolysis could be the cause of RPT, but until now, the mechanism and predictors of RPT have not been well known. TGF-$\beta$ has been known to promote fibrogenesis and is increased in tuberculous pleural fluid (PF). $PL_{TB}$ and malignant pleurisy ($PL_{MAL}$) manifest lymphocyte-dominant exudative pleural effusion, and it has clinical implications in the differentiation of the two diseases based on the findings of pleural effusion. We performed this study to compare pleural fluid TNF-$\alpha$ TGF-$\beta$, and fibrinolytic parameters between $PL_{TB}$ and $PL_{MAL}$, and to find the predictors of RPT in $PL_{TB}$. Methods : Thirty-five $PL_{TB}$ and 14 $PL_{MAL}$ patients who were admitted to the Asan Medical Center from February 1997 to August 1999 were enrolled. All $PL_{TB}$ patients were prescribed a primary, short-course, anti-tuberculosis regimen. INF-$\alpha$ tissue plasminogen activator (tPA), plasminogen activator inhibitor 1 (PAI-1), plasminogen, $\alpha$2-antiplasmin, and D-dimer were measured in both PF and PB. TGF-$\beta$was measured only in PF. Clinical characteristics, TNF-$\alpha$ TGF-$\beta$ and fibrinolytic parameters were compared between patients with RPT less than 2 mm and patients with more than 2 mm of the thirty patients who completed the anti-tuberculosis treatment. Results : The levels of TNF-$\alpha$ tPA, PAI-1, plasminogen, $\alpha$2-antiplasmin, and D-dimer in PF were higher than those in peripheral blood (PB) in $PL_{TB}$, whereas only plasminogen, $\alpha$2-antiplasmin, and D-dimer were higher in PF than in PB in $PL_{MAL}$. Pleural fluid TNF-$\alpha$ TGF-$\beta$, PAI-1, plasminogen, $\alpha$2-antiplasmin were increased in $PL_{TB}$ compared with $PL_{MAL}$, but these factors did not show any further advantages over ADA in differentiation between $PL_{TB}$ and $PL_{MAL}$. TNF-$\alpha$ TGF-$\beta$ and fibrinolytic parameters did not show any differences between patients with RPT less than 2 mm and patients with RPT more than 2 mm. Conclusion : Our data suggest that TNF-$\alpha$, TGF-$\beta$ and fibrinolytic parameters may play some role for the development of RPT in $PL_{TB}$, but they failed to predict the occurrence of RPT in $PL_{TB}$. Also these parameters did not seem to have any advantages over ADA in differentiating between two diseases.

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Establishment and Application of Molecular Genetic Techniques for Preimplantation Genetic Diagnosis of Osteogenesis Imperfecta (골형성부전증의 착상전 유전진단을 위한 분자유전학적 방법의 조건 확립과 적용)

  • Kim, Min-Jee;Lee, Hyoung-Song;Choi, Hye-Won;Lim, Chun-Kyu;Cho, Jae-Won;Kim, Jin-Young;Song, In-Ok;Kang, Inn-Soo
    • Clinical and Experimental Reproductive Medicine
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    • v.35 no.2
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    • pp.99-110
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    • 2008
  • Objectives: Preimplantation genetic diagnosis (PGD) has become an assisted reproductive technique for couples carrying genetic conditions that may affect their offspring. Osteogenesis imperfecta (OI) is an autosomal dominant disorder of connective tissue characterized by bone fragility and low bone mass. At least 95% of cases are caused by dominant mutations in the COL1A1 or COL1A2. In this study, we report on our experience clinical outcomes with 5 PGD cycles for OI in two couples. Methods: Before clinical PGD, we assessed the amplification rate and allele drop-out (ADO) rate of alkaline lysis and nested PCR protocol using heterozygous patient's single lymphocytes in the pre-clinical diagnostic tests for OI. We performed 5 cycles of PGD for OI by nested PCR for the causative mutation loci, COL1A1 c.2452G>A and c.3226G>A, in case 1 and case 2, respectively. The PCR products were analyzed by agarose gel electrophoresis, restriction fragment length polymorphism (RFLP) analysis with HaeIII restriction enzyme in the case 1 and direct DNA sequencing. Results: We confirmed the causative mutation loci, COL1A1 c.2452G>A in case 1 and c.3226G>A in case 2. In the pre-clinical tests, the amplification rate was 94.2% and ADO rate was 22.5% in case 1, while 98.1% and 1.9% in case 2, respectively. In case 1, a total of 34 embryos were analyzed and 31 embryos (91.2%) were successfully diagnosed in 3 PGD cycles. Eight out of 19 embryos diagnosed as unaffected embryos were transferred in all 3 cycles, and in the third cycle, pregnancy was achieved and a healthy baby was delivered without any complications in July, 2005. In case 2, all 19 embryos (100.0%) were successfully diagnosed and 4 out of 11 unaffected embryos were transferred in 2 cycles. Pregnancy was achieved in the second cycle and the healthy baby was delivered in March, 2008. The causative locus was confirmed as a normal by amniocentesis and postnatal diagnosis. Conclusions: To our knowledge, these two cases are the first successful PGD for OI in Korea. Our experience provides a further demonstration that PGD is a reliable and effective clinical techniques and a useful option for many couples with a high risk of transmitting a genetic disease.

Protoplast Fusion of Nicotiana glauca and Solanum tuberosum Using Selectable Marker Genes (표식유전자를 이용한 담배와 감자의 원형질체 융합)

  • Park, Tae-Eun;Chung, Hae-Joun
    • The Journal of Natural Sciences
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    • v.4
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    • pp.103-142
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    • 1991
  • These studies were carried out to select somatic hybrid using selectable marker genes of Nicotiana glauca transformed by NPTII gene and Solanum tuberosum transformed by T- DNA, and to study characteristics of transformant. The results are summarized as follows. 1. Crown gall tumors and hairy roots were formed on potato tuber disc infected by A. tumefaciens Ach5 and A. rhizogenes ATCC15834. These tumors and roots could be grown on the phytohormone free media. 2. Callus formation from hairy root was prompted on the medium containing 2, 4 D 2mg/I with casein hydrolysate lg/l. 3. The survival ratio of crown gall tumor callus derived from potato increased on the medium containing the activated charcoal 0. 5-2. 0mg/I because of the preventions on the other hand, hairy roots were necrosis on the same medium. 4. Callus derived from hairy root were excellently grown for a short time by suspension culture on liquid medium containing 2, 4-D 2mg/I and casein hydrolysate lg/l. 5. The binary vector pGA643 was mobilized from E. coli MC1000 into wild type Agrobacteriurn tumefaciens Ach5, A. tumefaciens $A_4T$ and disarmed A. tuniefaciens LBA4404 using a triparental mating method with E. ccli HB1O1/pRK2013. Transconjugants were obtained on the minimal media containing tetracycline and kanamycin. pGA643 vectors were confirmed by electrophoresis on 0.7% agarose gel. 6. Kanamycin resistant calli were selected on the media supplemented with 2, 4-D 0.5mg/1 and kanamycin $100\mug$/ml after co- cultivating with tobacco stem explants and A. tumefaciens LBA4404/pGA643, and selected calli propagated on the same medium. 7. The multiple shoots were regenerated from kanamycin resistant calli on the MS medium containing BA 2mg/l. 8. Leaf segments of transformed shoot were able to grow vigorusly on the medium supplemented with high concentration of kanamycin $1000\mug$/ml. 9. Kanamycin resistant shoots were rooting and elongated on medium containing kanamycin $100\mug$/ml, but normal shoot were not. 10. For the production of protoplast from potato calli transformed by T-DNA and mesophyll tissue transformed by NPTII gene, the former was isolated in the enzyme mixture of 2.0% celluase Onozuka R-10, 1.0% dricelase, 1.0% macerozyme. and 0.5M mannitol, the latter was isolated in the enzyme mixture 1.0% Celluase Onozuka R-10, 0.3% macerozyme, and 0.7M mannitol. 11. The optimal concentrationn of mannitol in the enzyme mixture for high protoplast yield was 0.8M at both transformed tobacco mesophyll and potato callus. The viabilities of protoplast were shown above 90%, respectively. 12. Both tobacco mesophyll and potato callus protoplasts were fused by using PEG solution. Cell walls were regenerated on hormone free media supplemented with kanamycin after 5 days, and colonies were observed after 4 weeks culture.

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A Study on Corrosion according to Distance between Amalgam and Dissimilar Metals (아말감과 이종(異種)금속의 거리에 따른 부식에 대한 고찰)

  • Kim, Ju-won;Jeong, Eun-gyeong
    • Journal of dental hygiene science
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    • v.4 no.3
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    • pp.103-109
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    • 2004
  • The present study prepared 72 test samples - 24 made of amalgam alloy, 24 of Verabond (Ni-Cr alloy) for crown and 24 of Talladium $^{TM}alloy$ for denture - according to the manufacturers' manuals and general method in consideration of the width of the mesial-distal dental crown of the lower $1^{st}$ molar and MOD cavity in clinics, put them in a 200 ml beaker containing 80 ml of artificial saliva, and measured their galvanic corrosion at distances of 0 mm, 7 mm and 40 mm after 7 days. Isolated metals in the electrolyte such as Cu, Ag, Ni, Cr, Sn, Zn and Hg were quantitatively analyzed with Inductively Coupled Plasma - Atomic Emission Spectrometer (ICP-AES, JY-50P, VG Elemental Co. France), and from the results were drawn conclusions as follows. First, Cu, Sn, Ag, Hg and Zn were highly advantageous when amalgam contacted gold alloy compared to Ni-Cr alloy for crown and Talladium alloy for denture. In addition, although gold alloy was finest in terms of oral tissue and biocompatibility, it was most disadvantageous when it was with amalgam. Second, when amalgam contacted gold alloy, heavy metals such as Ni and Cr were not isolated at all because gold alloy did not contain such elements but Sn was isolated as much as $227.1{\pm}18.0035{\mu}g/cm^2$ although it was not included in the composition either. Hg was also isolated. These elements are assumed to have been isolated from amalgam itself. Third, when amalgam alloy was apart from gold alloy 0 mm, 7 mm and 40 mm, Cu and Ag showed significance but Hg did not. This suggests that gold alloy must not be used together with amalgam, and must not be used between dissimilar prostheses regardless of distance. Fourth, when amalgam alloy contacted Ni-Cr alloy for crown, Ag was not isolated from the amalgam, but Zn, Ni, Sn, Hg and Cu were isolated in order of quantity. Significance was observed according to distance - 0 mm, 7 mm and 40 mm. Hg was not isolated but heavy metals Ni and Cr were isolated. If amalgam alloy was in the opposite arch or it was apart from Ni-Cr alloy for crown, the isolation Hg was less than that when amalgam alloy contacted Ni-Cr alloy for crown. Fifth, when amalgam alloy contacted Talladium alloy for denture, significance was observed at distances of 0mm, 7 mm and 40 mm. Hg was not isolated but heavy metals Ni and Cr were isolated. If amalgam alloy was in the opposite arch or it was apart from Talladium alloy for denture, the isolation Hg was less than that when amalgam alloy contacted Talladium alloy for denture. Sixth, according to the result of ICPES test on Cu, Sn, Ag, Hg, Zn, Ni and Cr of amalgam alloy, gold ally, Verabond and Talladium alloy when these alloys contacted artificial saliva, significance was observed in Cu and Hg. Seventh, when amalgam alloy contracted two non-precious metals Ni-Cr alloy for crown and Talladium alloy for denture in artificial saliva, significance was observed in the isolated by-products of Hg, Ni and Cr according to distance.

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Clinical Study on Thoracic Actinomycosis (흉부 방선균종의 임상적 고찰)

  • Hong, Sang-Bum;Kim, Woo-Sung;Lee, Jae-Hwan;Bang, Sung-Jo;Shim, Tae-Son;Lim, Chae-Man;Lee, Sang-Do;Koh, Youn-Suck;Lee, In-Chul;Kim, Dong-Soon;Kim, Won-Dong
    • Tuberculosis and Respiratory Diseases
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    • v.45 no.5
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    • pp.1058-1066
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    • 1998
  • Background: Actinomycotic infection is uncommon and primary actinomycosis of the lung and chest wall has been less frequently reported. This disease may present as chronic debilitating illness with radiologic manifestation simulating lung tumor, pulmonary infiltrating lesion, or chronic suppuration. Diagnosis of choice was not definded yet and role of bronchoscopy on diagnosis was not described yet. Methods: From 1989 to 1998, we experienced 17 cases of thoracic actinomycosis. We have reviewed the case notes of 17 patients with thoracic actinomycosis. The mean age at presentation was $53{\pm}13$ years, 11 were male. Results: Cough, hemoptysis, sputum production, chest pain and weight loss were the commonest symptoms. The mean delay between presentation and diagnosis was $6.6{\pm}7.8$ months. There were six patients who presented with a clinical picture of a suppurative lesion and eleven patients were suspected of having primary lung tumor initially. In no cases was made an accurate diagnosis at the time of hospital admission. Associated diseases were emphysema (1 case), bronchiectasis (2 cases) and tuberculosis (2 cases). Bronchoscopic findings were mucosal swelling and stenosis(n=4), mucosal swelling, stenosis and necrotic covering (n=2), mass (n=3), mass and necrotic covering (n=1) and normal(n=6). Radiologic findings were mass lesion(n=8), pneumonitis(n=3), atelectasis(n=3), pleural effusion(n=2), and normal(n=3). Final diagnosis was based on percutaneous needle aspiration and biopsy (n=3), bronchoscopic biopsy specimens (n=9), mediastinoscopic biopsy (n=1) and histologic examination of resected tissue in the remaining patients(n=4) who received surgical excision. Among 17 patients, 13 were treated medically and the other 4 received surgical intervention followed by antibiotic treatment. Regarding the surgically treated patients, suspected malignancy is the most common indication for operation. However. both medically and surgically treated patients achieved good clinical results. Conclusion: Thoracic actinomycosis is rare. but should still be considered in the differential diagnosis of a chrinic, localized pulmonary lesion. Thoracic actinomycosis may co-exist with pulmonary tuberculosis or lung cancer. If the lesion is located in the central of the lung. the bronchoscopy is recommanded for the diagnosis.

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TEMPOROSPATIAL PATTERNS OF PROGRAMMED CELL DEATH DURING EARLY DEVELOPMENT OF THE MOUSE EMBRYOS (생쥐 배자발생초기의 세포자기사 발현 양상에 관한 연구)

  • Baik, Byeong-Ju;Lee, Seung-Ik;Kim, Jae-Gon;Park, Byung-Yong;Park, Byung-Keon
    • Journal of the korean academy of Pediatric Dentistry
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    • v.28 no.4
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    • pp.709-727
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    • 2001
  • The pattern of programmed cell death(PCD) has been examined during the early developmental period of development in mouse embryos, from embryonic day 4.5(E4.5) to E11.5 Embryos from Balb/c breedings were harvested at various embryonic stages between E4.5 and El1.5. Cell death was analysed by in situ terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL) staining in tissue sections and whole embryos. At the blastocyst stage(E4.5), a very few apoptotic cells were found in the inner cell mass of the blastocyst. In the early egg cylinder stage(35.0-5.5), a few apoptotic cells were detected in the embryonic ectoderm, the embryonic endoerm and the proamniotic cavity. In the advanced egg cylinder stage(E5.5-6.5), TUNEL-posifive cells were observed in the extra-embryonic ectoderm and extra-embryonic endoderm as well as in the embryonic ectoderm, embryonic visceral endoderm and proamniotic cavity. In the streak stage(E6.75-7.75), many TUNEL-positive cells were found in the ectoplacental cone. In contrast, only very few apoptotic cells were found in the chorion and extra-embryonic endoderm in extra-embryonic regions. In intra-embryonic region, a few apoptotic cells were randomly found in the embryonic ectoderm, mesoderm and visceral endoderm. At the early somitogenesis stage(E8.0-8.5), most apoptotic cells were observed in the most cranial portion of neural fold (neural ectoderm and adjacent ectoderm). At the mid somitogenesis stage(39.0-9.5), the otic placode first showed TUNEL-positive at this stage. Small number of TUNEL-positive cells were also first seen around optic placode and branchial arches. Three streams of TUNEL-positive cells were clearly seen in the cranial region at 59.5-9.75. At E10.5, apoptotic cells were localized in the developing eye, the junctional portion of medial nasal, lateral nasal and maxillary processes, the lateral portion of branchial arches, the junction of bilateral mandibular processes, and apical ectodermal ridges of limb buds. At E11.5, apoptotic cells were noticeably decreased in most area, except the developing limbs and several somites in the tail region. In this study, the global temporospatial pattern of PCD throughout early development of mouse embryos was discussed. It may provide the basis for further studies on its role in the morphogenesis of the embryo.

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Changes in Biochemical Components of Several Tissues of the Hard Clam, Meretrix petechialis, in Relation to Gonad Developmental Phases (말백합, Meretrix petechialis의 생식소 발달단계에 따른 일부 조직의 생화학적 성분 변화)

  • Kim, Yong-Min;Park, Kwan-Ha;Chung, Ee-Yung;Kim, Jong-Bae;Lee, Chang-Hoon
    • The Korean Journal of Malacology
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    • v.22 no.2
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    • pp.125-134
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    • 2006
  • We investigated the reproductive cycle of the hard clam, Meretrix petechialis with its gonadal development by histological observations. The seasonal changes in biochemical component of the adductor muscle, visceral mass, foot muscle and mantle of the clam were studied by biochemical analysis, from January to December, 2002. The reproductive cycle of this species can be divided into five successive stages: early stage (January to March), late active stage (February to May), ripe stage (April to August), partially spawned stage (July to August) and spent/inactive stage (September to January). Total protein content in the visceral mass was over two times higher than that in the adductor muscle. Monthly changes of total protein content in the adductor muscle were not statistically significant (ANOVA, p = 0.071), while the changes in the visceral mass were significant (p < 0.001). Total protein content in visceral mass was higher during the early active, late active, and ripe stages (from January to May), while the lowest in July. Glycogen content in the adductor muscle was higher than that in the visceral mass. Monthly changes in glycogen contents were statistically significant in both adductor muscle (F = 237.2, p < 0.001) and the visceral mass (F = 64.04, p < 0.001). Glycogen content in the adductor muscle was the highest in the ripe stage (April). Its content was lower in the partially spawned and the spent/inactive stages (June-September). Glycogen contents in the visceral mass were relatively lower until the early active stage, while the highest in the late active stage. RNA content was higher in visceral mass than that in the adductor muscle. Monthly changes in RNA contents were significant in both adductor muscle (F = 195.2, p < 0.001) and visceral mass (F = 78.85, p < 0.001). RNA content in the adductor muscle was high in the early active stage (January-February), and then it decreased rapidly in the late active stage (March-April), thereafter, slightly increased during the partially spawned stage (June-July). RNA content in the visceral mass reached a maximum during the ripe stage (May), and then it decreased rapidly during the partially-spawned stage (June-July). There was significant positive correlation in total protein contents between adductor muscle and visceral mass (r = 0.715, p = 0.020). However, there was no correlation between adductor muscle and visceral mass in glycogen (p = 0.550), while a negative correlation was found between the adductor muscle and visceral mass in RNA (p = 0.518) contents. Especially, changes in RNA content showed a negative correlation between the adductor muscle tissue and visceral mass. Therefore, these results suggest that the nutrient content of the adductor muscle, visceral muscle and foot muscle changed in response to gonadal energy needs.

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Sesquiterpenoids Bioconversion Analysis by Wood Rot Fungi

  • Lee, Su-Yeon;Ryu, Sun-Hwa;Choi, In-Gyu;Kim, Myungkil
    • 한국균학회소식:학술대회논문집
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    • 2016.05a
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    • pp.19-20
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    • 2016
  • Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

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Effect of Sanitation Treatment of Extending Shelf-life on Fresh Poultry Meats (계육(鷄肉)의 유통기간연장(流通期間延長)을 위(爲)한 위생처리방법(衛生處理方法)에 관(關)한 연구(硏究))

  • Cho, M.J.;Jang, P.H.;Park, K.B.;Lee, B.M.
    • Korean Journal of Food Science and Technology
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    • v.14 no.4
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    • pp.291-300
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    • 1982
  • In order to develop effective and simple sanitation method for the extention of shelf-life of fresh poultry meat, the effect of sanitizers, sanitation methods and packaging materials on the extention of shelf-life of poultry meats was observed at the $4^{\circ}C$ and room temp$(10{\sim}20^{\circ}C)$. The results are summarized as follows: 1. The autochonous skin microflora of poultry, before processing, were believed to be removed or killed during the scalding and plucking, and exposed dermal tissue was contaminated by microorganisms from the subsequent stages of processing. 2. In the final stage of poultry processing, total viable counts of microorganisms and coliforms were averaged to $3.5{\times}10^4/cm^2$ and $400/cm^2$, respectively. 3. The refrigerated shelf-life of fresh whole poultry carcasses at $3\;to\;4^{\circ}C$ was extended to 7 to 16 days compared to control with the various treatments of some sanitizers by dipping freshly chilled carcasses for 5 min or spraying 1 liter of sanitizers per carcasses. In the case of storage at $10\;to\;15^{\circ}C$, the shelf-life of poultry carcasses was extended to one to two days by the sanitation treatments compared to control. 4. Spraying sanitation was more effective than dipping sanitation, and 5 minutes dipping and one liter spraying per carcass were enough for effective sanitation of poultry carcasses in most sanitizers. 5. The packaging with an oxygen impermeable polyvinylidene chloride extended the shelf-life to 10 days and 5 days with polyethylene compared to control. When poultry carcasses were sanitized by continuous spraying with one liter of 30 ppm of chlorine and another one liter of 5% of potassium sorbate, packaged with polyvinylidene chlorlde were extended to about 30 days compared to control.

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