• Nutrition Research and Practice
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• 제7권1호
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• pp.3-8
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• 2013

Buckwheat is known as a health food but is one of the major food allergens triggering potentially fatal anaphylaxis in Asia, especially in Japan and Korea. This study was conducted to investigate the characteristic of enzymatic resistance of buckwheat protein and allergenic potential. Enzymatic resistance of buckwheat protein was performed with in vitro digestibility test in simulated gastric fluid (SGF), pH 1.2, using pepsin and simulated intestinal fluid (SIF) using chymotrypsin. Reactivity of buckwheat proteins to human IgE was performed using six allergic patients sensitized to buckwheat. Buckwheat's IgE levels were measured using the Phadia UniCAP-system. Buckwheat protein, 16 kDa, still remained after 30 min treatment of pepsin on SDS-PAGE. Even though 16 kDa almost disappeared after 60 min treatment, two out of the six buckwheat patients' sera showed reactivity to hydrolysate after 60 min treatment, indicating that allergenicity still remained. In simulated intestinal fluid (SIF) using chymotrypsin, buckwheat protein, 24 kDa, showed resistance to hydrolysis with chymotrypsin on SDS-PAGE, and still had allergenicity based on the result of ELISA. Our results suggest that buckwheat proteins have strong resistance to enzyme degradation. This may be attributed in part to the allergenic potential of buckwheat. Further study should be continued regarding buckwheat allergy.

• Clinical and Experimental Reproductive Medicine
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• 제25권2호
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• pp.207-216
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• 1998

Certain types of somatic cells, as well as follicular cumulus cells associating with mammalian oocytes, are known to produce beneficial effects on in vitro fertilization and pre implantation development of mammalian eggs when they are present in oocyte culture medium. To investigate the nature of the effects of somatic cells, the resistance of mouse oocytes against chymotrypsin treatment was examined after culture within various cell conditioned media. When mouse oocytes matured for 17-18 hr in the presence of cumulus cells were treated with 1 % chymotrypsin, half of them remained still alive even after 240 min $(t_{50}>240.0)$. In contrast half of mouse oocytes cultured without cumulus cells underwent degeneration within 65.0 min $(t_{50}=65.0{\pm}13.2min)$ of the same treatment. To see if the effects were duc to the secretory products of cumulus cells, mouse cumulus cells were cultured for 20 hr in medium containing 0.4% BSA and the supernatant of culture medium (conditioned medium) was taken. After maturation in the cumulus cell conditioned medium, mouse oocytes exhibited $t_{50}=190.0{\pm}10.8$ min upon chymotrypsin treatment whereas half of oocytes cultured without conditioned medium degenerated within 25.5 min. Human granulosa cell conditioned medium gave similar effects such that oocytes matured in conditioned medium exhibited $t_{50}=183.3{\pm}19.1$ min while $t_50$ of control group oocytes was $60.0{\pm}6.8$ min, Oocytes matured in vero cell conditioned medium exhibited $t_{50}=196.7{\pm}8.8$ min. On the other hand, amniotic cell conditioned medium resulted in the chymotrypsin resistance of $t_{50}=80.0{\pm}8.4$ min which was not statistically different from the control value of $t_{50}=48.0{\pm}13.2$ min. Based upon these results, it is suggested that certain somatic cell types including cumulus cells might change the biochemical properties of mouse oocyte membrane during meiotic maturation as revealed by the enhanced resistance against chymotrypsin treatment. Such effects of somatic cells appear to be mediated via the secretory products rather than direct communication between somatic cells and oocytes.

• 농업과학연구
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• 제25권1호
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• pp.52-67
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• 1998

본 연구는 포유동물의 젖에 함유되어 있는 lactoferrin(LF)의 생리적 특성을 밝혀 기능성 식품 특히 항균작용을 이용한 식품제조업에의 이용을 위한 기초자료를 얻고자 실시하였다. 따라서 젖소의 초유로부터 lactoferrin을 분리 정제하여, 소화부위에 따른 lactoferrin의 항균활성을 분석하고자 pepsin, trypsin 및 chymotrypsin으로 분해하여 lactoferrin의 항균력을 측정하고, gel-filtration으로 분리 정제된 각각의 fraction별로 단백질을 정량하여 Escherichia coli와 Staphylococcus aureus에 접종하여 항균효과를 지니는 각 효소별 fraction의 분자량과 peptide fragment를 검토하였다. 1. 젖소의 초유로부터 분리 정제된 lactoferrin(LF)을 단백질 분해 효소인 pepsin, trypsin 및 chymotrypsin으로 분해한 결과 bovine lactoferrin은 SDS-PAGE를 수행하여 band를 확인할 수 있었다. pepsin으로 분해된 lactoferrin은 분자량 14KDa까지에서도 band를 확인할 수가 없었으며, trypsin과 chymotrypsin으로 분해한 lactoferrin은 분해되지 않은 lactoferrin이 존재함을 나타내고, 33KDa에서도 band를 확인할 수 있었다. 2. Sephadex G-50 column을 사용하여 bovine lactoferrin를 효과적으로 정제하였다. Sephadex G-50 column chromatography를 수행 한 결과 bovine lactoferrin은 Tris-HCl사이에서 용출되었으며, pepsin, trypsin 및 chymotrypsin으로 분해한 lactoferrin은 각각 2, 3, 2 개의 peak를 보였고, HPLC 분석결과 첫번째 peak는 주로 분해되지 않은 lactoferrin 수용체가 존재하는 것으로 확인되었으며, trypsin과 chymotrypsin처리에서 항균효과도 유사점을 관찰할 수 있었다. 3. 효소처리한 lactoferrin의 항균효과를 알아보기 위하여 Escherichia coli와 Staphylococcus aureus를 접종하여 항균활성을 비교하였다. 그 결과 각 효소에 대하여 pepsin으로 처리한 lactoferrin을 접종한 시험구가 대조구에 비하여 낮은 생장율을 보여 현저한 항균활성을 지님을 알 수 있었다. 그리고 trypsin과 chymotrysin에 의한 분해물도 미생물 배양 8시간까지는 효소 처리전 bovine lactoferrin보다는 항균활성을 나타냄을 알 수 있었다. 4. Sephadex G-50 column을 사용하여 분리 정제된 bovine lactoferrin fraction별로 SDS-PAGE를 실시한 결과 lactoferrin fraction의 경우는 chromatography 수행 결과와 비교하여, pepsin과 chymotrypsin 분해물은 저분자량임을 알 수 있었고, trypsin에 의한 lactoferrin만이 단일 band를 보임으로서 단백질 분해의 특징을 볼 수 있었다.

• Asian-Australasian Journal of Animal Sciences
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• 제16권12호
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• pp.1822-1829
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• 2003

Porcine colostrum and milk were separated into the acid-soluble and casein fractions by acidification followed by centrifuge. The acid-soluble fraction of porcine colostrum was further separated by liquid chromatography and anisotropic membrane filtration. Trypsin and chymotrypsin inhibitory capacity in porcine colostrum, milk and their components was determined by incubating bovine trypsin or chymotrypsin in a medium containing their corresponding substrates with or without addition of various amounts of porcine colostrum, porcine milk or their components. The inhibition of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) degradation in pig small intestinal contents by porcine colostrum was measured by incubating iodinated IGF-I or EGF with the intestinal contents with or without addition of porcine colostrum. Degradation of labeled IGF-I or EGF was determined by monitoring the generation of radioactivity soluble in 30% trichloroacetic acid (TCA). The results showed that porcine colostrum had high levels of trypsin and chymotrypsin inhibitory activity and increased the stability of IGF-I and EGF in pig intestinal contents. The inhibitory activity declined rapidly during lactation. It was also found that trypsin and chymotrypsin inhibitory activity and the inhibition on IGF-I and EGF degradation in the acid-soluble fraction were higher than that in the casein fraction. Heat-resistance study indicated that trypsin inhibitors in porcine colostrum survived heat treatments of $100^{\circ}C$ water bath for up to 10 min, but exposure to boiling water bath for 30 min significantly decreased the inhibitory activity. Compared with the trypsin inhibitors, the chymotrypsin inhibitors were more heatsensitive. Separation of the acid-soluble fraction of porcine colostrum by liquid chromatography and anisotropic membrane filtration revealed that the trypsin and chymotrypsin inhibitory capacity was mainly due to a group of small proteins with molecular weight of 10,000-50,000. In conclusion, the present study confirmed the existence of high levels of protease inhibitors in porcine colostrum, and the inhibition of porcine colostrum on degradation of milk-borne growth factors in the pig small intestinal tract was demonstrated for the first time.

• 약학회지
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• 제51권3호
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• pp.199-205
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• 2007

Thermostable asparaginase was purified to homogeneity from mesophilic Strepfomyces lincolnensis M-20 by 30${\sim}$70% ammonium sulfate precipitation and asparagine-Sepharose CL 6B affinity column chromatography, The apparent molecular mass of L-asparaginase by SDS-PAGE was found to be 47 kDa, whereas by its mobility on Sephacryl S-300 column was around 180 kDa, indicating that the enzyme at the native stage acts as tetramer, The purified enzyme showed a single band on acrylamide gel electrophoresis. The optimum pH and temperature were pH 9.5 and 55${\circ}$C, respectively. Chemical modification experiments of purified asparagines implied the existence cystein residue located at or near active site. Purified asparaginase retained the 85% of the initial activity after incubation at 90${\circ}$C for 30 min. A correlation between themostability and resistance to proteolysis of commercial asparaginase and purified asparaginase from Strepfomyces lincolnensis M-20 was investigated. Purified thermostable asparaginase was resistant to trypsin and chymotrypsin treatment, while the commercial asparaginase was not themostable and was susceptible to proteolytic treatment with trypsin and chymotrypsin.

• Clinical and Experimental Reproductive Medicine
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• 제26권3호
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• pp.407-417
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• 1999

Objective: Mammalian follicle cells are the most important somatic cells which help oocytes grow, mature and ovulate and thus are believed to provide oocytes with various functional and structural components. In the present study we have examined whether cumulus or granulosa cells might playa role in establishing the plasma membrane structure of mouse oocytes during meiotic maturation. Design: In particular the differential resistances of mouse oocytes against chymotrypsin treatment were examined following culture with or without cumulus or granulosa cells, or in these cell-conditioned media. Results: When mouse denuded oocytes, freed from their surrounding cumulus cells, were cultured in vitro for $17{\sim}18hr$ and then treated with 1% chymotrypsin, half of the oocytes underwent degeneration within 37.5 min ($t_{50}=37.5{\pm}7.5min$) after the treatment. In contrast cumulus-enclosed oocytes showed $t_{50}=207.0$. Similarly, when oocytes were co-cultured with cumulus cells which were not associated with the oocytes but present in the same medium, the $t_{50}$ of co-cultured oocytes was $177.5{\pm}13.1min$. Furthermore, when oocytes were cultured in the cumulus cell-conditioned medium, $t_{50}$ of these oocytes was $190.0{\pm}10.8min$ whereas $t_{50}$ of the oocytes cultured in M16 alone was $25.5{\pm}2.9min$. Granulosa cell-conditioned medium also increased the resistance of oocytes against chymotrypsin treatment such that $t_{50}$ of oocytes cultured in granulosa cell-conditioned medium was $152.5{\pm}19.0min$ while that of oocytes cultured in M16 alone was $70.0{\pm}8.2min$. To see what molecular components of follicle cell-conditioned medium are involved in the above effects, the granulosa cell-conditioned medium was separated into two fractions by using Microcon-10 membrane filter having a 10 kDa cut-off range. When denuded oocytes were cultured in medium containing the retentate, $t_{50}$ of the oocytes was $70.0{\pm}10.5min$. In contrast, $t_{50}$ of the denuded oocytes cultured in medium containing the filtrate was $142.0{\pm}26.5min$. $T_{50}$ of denuded oocytes cultured in medium containing both retentate and filtrate was $188.0{\pm}13.6min$. However, $t_{50}$ of denuded oocytes cultured in M16 alone was $70.0{\pm}11.0min$ and that of oocytes cultured in whole granulosa cell-conditioned medium was $156.0{\pm}27.9min$. When surface membrane proteins of oocytes were electrophoretically analyzed, no difference was found between the protein profiles of oocytes cultured in M16 alone and of those cultured in the filtrate. Conclusions: Based upon these results, it is concluded that mouse follicle cells secrete a factor(s) which enhance the resistance of mouse oocytes against a proteolytic enzyme treatment. The factor appears to be a small molecules having a molecular weight less than 10 kDa.

• 한국식품영양과학회지
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• 제28권2호
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• pp.438-443
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• 1999

A livetin solution(LS) containing yolk immunoglobulins(IgY) was separated by treating the egg yolk with natural gum, carrageenan. Carrageenan has been used as a food ingredient. Relative absorbance of IgY LS after proteolysis was investigated. IgY LS was fairly stable to pepsin digestion at pH 3.0. However, IgY LS appeared to be susceptible to pepsin digestion at pH 2.0, showing 18% of relative absorbance and complete breakdown H chain after 30 min exposure. Relative absorbance of IgY LS was considerably high after exposure to trypsin and chymotrypsin for 8 hr. IgY LS showed especially good stability to chymotrypsin digestion.

• Journal of Microbiology and Biotechnology
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• 제16권2호
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• pp.312-317
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• 2006

The chromosomal sprD gene encoding Streptomyces griseus protease D (SGPD), a chymotrypsin-like protease, was disrupted in Streptomyces griseus by insertion of the neomysin-resistance gene. The production of chymotrypsin activity of sprD disruptant was not completely abolished, but delayed by 24 h, compared with that of wild-type strain. The aerial mycelial formation of sprD disruptant was retarded, and specifically the formation of spores was not observed in the central region of colonies. However, normal morphological development into spores was observed in the marginal region of colonies. In addition, the production of yellow pigment that might be dependent on A-factor was also decreased in the sprD disruptant, compared with that of the wild-type strain. Introduction of the sprD gene, which was placed on a high copy-numbered plasmid into S. griseus ${\Delta}sprD$, partially restored the ability of morphological development, and a significant level of sporulation was observed. When the overexpression vector for sprD, pWHM3-D, was introduced in S. griseus, there was no significant change in the chymotrypsin activity or colonial morphology, in contrast to Streptomyces lividans, indicating the presence of a tight regulation system for the overexpression of the sprD gene in S. griseus.

• Clinical and Experimental Reproductive Medicine
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• 제27권1호
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• pp.23-29
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• 2000

Objective: Zona pellucida (ZP) has been thought to be the barrier of egg to sperm penetration before and after fertilization. The phenomenon of ZP hardening has been considered as a post-fertilization event until now, and it is generally accepted that it is caused by the secretory products of cortical granules released during the cortical reaction. Hardening of ZP could occur "spontaneously" in mammalian oocytes in standard culture conditions, and that it is probably not a consequence of cortical reaction. The purpose of our study was to investigate the effect of human amniotic fluid (HAF) on nuclear maturation (NM) and fertilization ability of mouse immature oocytes. Methods: HAF was obtained from patients undergoing amniocentesis at $16{\sim}20$ weeks of gestation. HAF from five to ten patients was centrifuged and the supernatants was pooled. Cumulusenclosed mouse immature oocytes were incubated in the medium containing HAF, and examined to confirm NM and fertilization. Female ICR mice (about 3 weeks old) were stimulated with 7.5 IU PMSG. Immature oocytes were isolated at $48{\sim}52$ hrs post PMSG injection and cultured in TCM-199 supplemented with 20% HAF for 18 hrs. FBS was used as a control for the examination. Matured oocytes (MII) were fertilized with sperms collected from the epididymis of male mice (over 10 weeks old). Fertilization was in conducted T6 medium containing 15 mg/ml BSA, and confirmed at 6 hrs post-insemination. Fertilization rate was assessed in zona-intact or zona-free oocytes (denuded by trypsin). Evaluation of NM and fertilization was carried out by rapid staining method. ZP hardening was evaluated by incubating cumulus cell-free mature oocytes in 0.001% chymotrypsin at $37^{\circ}C$ for 10 min. Results: There was no significant difference between the effects of HAF (86.6%) and FBS (87.7%) supplements on NM of immature oocytes. When maturation medium was supplemented with HAF, total fertilization rates (7%) were significantly lower (p<0.01) than that of FBS (85.1%). In HAF group, fertilization rate was increased (p<0.01) in zona-free oocytes (7% versus 100%). The resistance of mouse oocyte ZP to digestion by chymotrypsin after maturation in vitro was significantly higher (p<0.01) in HAF group (86.7%) than in FBS (6.7%). To culture oocytes in FBS were very effective in preventing ZP hardening. However cultured oocytes in HAF showed high rate of ZP hardening (p<0.01). Conclusions: These results suggest that HAF can be used as a supplement for the NM of mouse immature oocytes in vitro. However, HAF induces spontaneous hardening of ZP of mouse immaure oocytes during maturation in vitro.

• 한국가축번식학회지
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• 제17권2호
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• pp.93-101
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• 1993

These experiments were carried out to investigate whether zona hardening affect the efficiency of in vitro fertilization in mouse oocytes. The soluble properties for zona pellucida of oocytes matured in vivo, aged oocytes, and ovarian oocytes matured in vitro have been analyzed with proteolytic enzyme, 3mg/ml of $\alpha$-chymotrypsin. The mean solubility(t50) for the zona of unfertilized oocytes, oocytes not fertilized at the first inseminati and in vitro produced zygotes were 10.1, 20.3 and 32.3min., respectively. The t50 for zona lysis of fertilized oocytes was significantly difference than those observed for unfertilized oocytes and oocytes not fertilized at the first insemination(P<0.01). In addition, the t50 of zona in ovulated oocytes with and without cumulus cells incubated for 0, 3, 6, 9 and 12 hr in vitro, t50 were 13.9, 11.1, 20.7 and 28.0min., and 22.3, 21.0, 30.0 and 33.5min., respectively. In these experiments, the zona pellucida showed a gradual increase in resistance to dissolution by $\alpha$-chyjotrypsin with in vitro aging for more than 6 hrs. This effect was greater in cumulus-free as compared to cumulus-intact oocytes. Finally, in cumulus-intact and cumulus-free ovarian oocytes matured for 0, 5, 10 and 15 hr in vitro the t50 of zona pellucida were 3.0, 10.6, 18.4 and 24.5 min., and 3.0, 14.0, 26.2 and 32.0 min., respectively. Clear differences in solubility between the zona pellucida of oocytes matured in vivo and in vitro. This data were found suggest that under in vitro conditions there is a gradual change in the soluble properties of the zona pellucida, particularly in the absence of the cumulus cells.