• 한국동물생명공학회지
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• 제39권3호
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• pp.179-193
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• 2024

Background: Because oxidative stress can induce decreased quality of caprine semen during the storage, there has been limitation for the use of stored semen in the assisted reproductive technologies. The present study, therefore, assesses the potential of Annona muricata (A. muricata) to reduce semen storage associated-damages. Methods: Semen was collected by electro-ejaculation from ten bucks, and extended with Tris-egg yolk (TEY) supplemented with A. muricata leaf aqueous extract (SAE) at 20 (SAE20), 40 (SAE40), and 80 (SAE80) ㎍/mL. Sperm variables including motility, motion characteristics, viability, membrane functionality, and DNA integrity were assessed at different storage periods (6, 24, 48, and 72 hr). In addition, oxidative stress indicators in the extender supplemted with SAE were also assessed for each group. Results: By adding SAE at 80 ㎍/mL in TEY, the storage of goat buck semen was improved, resulting in reduced loss of sperm motility, viability, DNA fragmentation, and membrane integrity during chilled storage at 4℃ for up to 72 hr. In addition, enrichment of TEY extender with SAE significantly (p < 0.05) reduced malondialdehyde, an indicator of oxidative stress, compared to the negative control. Conclusions: Supplementation of SAE in TEY extender can reduce buck spermatozoa liquid storage associated damages due to oxidative stress.

• 생명과학회지
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• 제27권5호
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• pp.517-523
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• 2017

본 실험은 항산화제인 melatonin, silymarin, curcumin 및 vitamin E가 aresenite에 의해 손상된 돼지의 정자성상능력에 미치는 영향을 조사한 연구이다. 돼지정자는 채취 후 희석하여 실험에 이용하였으며, $100{\mu}M$ arsenite는 인위적으로 정자를 손상시키는데 사용하였다. 또한 100 nM melatonin, $2{\mu}M$ silymarin, $10{\mu}M$ curcumin 및 $500{\mu}M$ Vitamin E를 희석된 돼지정자에 첨가하여 3, 6 및 9시간 동안 배양 후 정자의 운동성, 원형질막 온전성, 미토콘드리아 기능성 및 원형질막 지질과산화를 검토하였다. 그 결과, 정자의 운동성 및 원형질막 온전성은 $100{\mu}M$ arsenite 처리구에서 유의적으로 감소하였으며, 항산화제 단독처리구는 유의적으로 증가하였다. 또한, arsenite에 의해 손상된 처리구는 항산화제에 의해 유의적으로 증가하였다(p<0.05). 정자의 미토콘드리아 기능성은 $100{\mu}M$ arsenite 처리구에서 유의적으로 감소하였고, 정자의 원형질막 지질과산화는 $100{\mu}M$ arsenite 처리구에서 유의적으로 증가하는 것으로 나타났다(p<0.05). 결론적으로, arsenite의해 감소된 돼지정자의 운동성과 원형질막은 melatonin, silymarin, curcumin 및 vitamin E와 같은 항산화제에 의하여 예방될 수 있다고 판단되며, arsenite에 의해 감소된 정자의 수정능력은 항산화제에 의해 회복될 수 있을 것이라 생각된다.

• 한국임상수의학회지
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• 제30권6호
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• pp.442-448
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• 2013

개 정액 동결을 위한 glucose가 첨가된 glycerol-free TRIS 희석액을 개발하기 위해 glycerol-free TRIS내 알맞은 glucose의 양과 0.3 M glucose가 첨가된 glycerol-free TRIS내 정자의 적정 노출시간을 조사하였다. 여섯 마리의 수캐의 사출액을 0.04 M glucose가 첨가된 glycerol-free TRIS내에서 $4^{\circ}C$까지 100분 동안 냉각한 후 서로 다른 glucose농도 (0 M, 0,04 M, 0.1 M, 0.2 M, 0.3 M)의 glycerol-free TRIS에서 30분 동안 냉각하여 동결하였다. $37^{\circ}C$에서 25 초 동안 융해한 후 정자의 막 고유성과 첨단체 고유성을 검사하였다. 부가적으로 0.3 M glucose가 첨가된 glycerol-free TRIS내 정자의 적정 노출시간에 따른 정자의 동결 후 운동성, 생존성, DNA 고유성을 확인하였다. 막 고유성과 첨단체 고유성은 각각 6-carboxyfluoresceindiacetate(6-CFDA)/propidium iodide(PI) fluorescent staining와 Pisum sativum agglutinin conjugated-fluorescein isothiocyanate 방법에 의해 검사하였다. DNA 고유성은 terminal deoxynucleotidyl transferase dUTP nick end labeling로 염색하여 flow cytometry로 검사하였다. 0.2 M 또는 0.3 M glucose가 첨가된 glycerol-free TRIS에서 동결된 정자가 낮은 농도의 glucose가 첨가된 희석액에서 동결된 정자보다 막 고유성이 높게 나타났으며(p<0.05), 첨단체 고유성은 0.3 M 군에서 높게 나타났다(p<0.05). 운동성은 50 분 군에서 높게 나타났으나(p<0.05), DNA fragmentation index는 노출시간에 따라 차이가 없었다. 본 연구 결과 개정자가 0.3 M glucose가 첨가된 glycerol-free TRIS에서 $4^{\circ}C$, 50 분간 냉각 후 동결과 융해 후 더 높은 생존성을 나타냈다.

• 대한의용생체공학회:의공학회지
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• 제26권2호
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• pp.101-110
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• 2005

Periosteum and periosteum-derived progenitor cells have demonstrated the potential for stimulative applications in repairs of various musculoskeletal tissues. It has been found that the periosteum contains mesenchymal progenitor cells capable of differentiating into either osteoblasts or chondrocytes depending on the culture conditions. Anatomically, the periosteum is a heterogeneous multi-layered membrane, consisting of an inner cambium and an outer fibrous layer. The present study was designed to elucidate the cellular phenotypic characteristics of cambium and fibrous layer cells in vitro, and to assess whether structural integrity of the tendon in the bone tunnel can be improved by periosteal augmentation of the tendon­bone interface. It was found the cells from each layer showed distinct phenotypic characteristics in a primary monolayer culture system. Specifically, the cambium cells demonstrated higher osteogenic characteristics (higher alkaline phosphatase and osteocalcin levels), as compared to the fibrous cells. Also in vivo animal model showed that a periosteal augmentation of a tendon graft could enhance the structural integrity of the tendon-bone interface, when the periosteum is placed between the tendon and bone interface with the cambium layer facing toward the bone. These findings suggest that extra care needs to be taken in order to identify and maintain the intrinsic phenotypes of the heterogeneous cell types within the periosteum. This will improve our understanding of periosteum in applications for musculoskeletal tissue repairs and tissue engineering.

• Biomolecules & Therapeutics
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• 제3권3호
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• pp.242-244
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• 1995

In vitro cell culture system has been proposed as a promising alternative model to in vivo skin irritation test. These studies were performed to screen the cytotoxicity effects of surfactants using normal human skin fibroblasts. Cell membrane integrity assessed by the leakage of lactate dehydrogenase (LDH) and mitochondrial integrity by MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromides reduction test were affected in a dose dependent manner. The irritation potential of surfactants to human skin patch test, and the changes of capillary permeability by rabbit intradermal safety test were assessed as in vivo methods. Our results suggest that LDH leakage assay and MTT reduction test using cultured human fibroblasts could be predictive for the irritancy of various surfactants in human, and LDH assay is superior correlated with in vivo test (r=0.886) to MTT test with in vivotest (r=0.757).

• 한국동물생명공학회지
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• 제37권1호
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• pp.55-61
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• 2022

The acrosome cap allows sperm to penetrate the egg membrane and produce male pronuclei within female chicken eggs, facilitating successful fertilization. Given this, it is important to establish practical methods for evaluating the integrity of the acrosome cap and thus the quality of the rooster's sperm. There are several established methods for evaluating the acrosomes of mammalian sperm, but none of these methods are suitable for evaluating the acrosome status of rooster spermatozoa. Therefore, a simplified method for evaluating the rooster acrosome is needed. Here we evaluated the usefulness of CBB (coomassie brilliant blue) staining of the acrosome at concentrations of 0.04%, 0.08%, and 0.3% CBB solutions. Our data revealed a clear staining pattern for intact acrosome caps at 0.04% and 0.08% CBB but not at 0.3% CBB. This protocol revealed differences in acrosome integrity between fresh and frozen rooster sperm smears suggesting that CBB staining may facilitate easier semen evaluation in roosters. This protocol allows for the accurate differential staining of acrosome cap in rooster spermatozoa.

• 농업과학연구
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• 제49권2호
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• pp.259-267
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• 2022

Although pesticides are recognized as necessary substances to improve agricultural production, exposure to pesticides is known to have a direct or indirect adverse effect on the reproductive function of mammals. The present study examines the effects of mancozeb, a well-known fungicide, on the fertility capacity of spermatozoa. Boar spermatozoa exposed to varying concentrations of mancozeb (0.01 - 0.5 µM) were evaluated for motility, motion kinetic parameters, viability, acrosome integrity and the generation of intracellular reactive oxygen species (ROS) after 30 min or 2 hrs of incubation. A significant reduction in the motility of spermatozoa was observed upon exposure to mancozeb. Similarly, there was a significant reduction of the motion kinematics of sperm treated with mancozeb as compared to untreated controls (p < 0.05). The sperm viability percentage and acrosome integrity also showed dose-dependent decreases upon exposure to mancozeb. High concentrations of mancozeb (0.2 - 0.5 µM) induced higher levels of intracellular ROS production, which resulted in the loss of the sperm membrane and decreased sperm motility due to oxidative stress. Taken together, the results here indicate that direct exposure to mancozeb affects the sperm fertility capacity. Hence, careful research that examines the interaction between reproduction and environmental toxins is crucial to prevent fertility disorders in animals.

• Journal of Microbiology and Biotechnology
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• 제22권4호
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• pp.470-478
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• 2012

Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane ${\beta}$-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies.

• The Plant Pathology Journal
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• 제32권2호
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• pp.85-94
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• 2016

Studies were conducted to determine the role of 3-methylthioproprionic acid (MTPA) in the pathogenicity of potato stem canker, Rhizoctonia solani, and the concentrations required to inhibit growth of R. solani under laboratory and plant house-based conditions. The experiments were laid out in a completely randomized design with five treatments and five replications. The treatments were 0, 1, 2, 4, and 8 mM concentrations of MTPA. The purified toxin exhibited maximal activity at pH 2.5 and $30^{\circ}C$. MTPA at 1, 2, 4, and 8 mM levels reduced plant height, chlorophyll content, haulm fresh weight, number of stolons, canopy development, and tuber weight of potato plants, as compared to the control. MTPA significantly affected mycelial growth with 8 mM causing the highest infection. The potato seedlings treated with MTPA concentrations of 1.0-8.0 mM induced necrosis of up to 80% of root system area. Cankers were resulted from the injection of potato seedling stems with 8.0 mM MTPA. The results showed the disappearance of cell membrane, rough mitochondrial and cell walls, change of the shape of chloroplasts, and swollen endoplasmic reticulum. Seventy-six (76) hours after toxin treatment, cell contents were completely broken, cytoplasm dissolved, and more chromatin were seen in the nucleus. The results suggested that high levels of the toxin concentration caused cell membrane and cytoplasm fracture. The integrity of cellular structure was destroyed by the phytotoxin. The concentrations of the phytotoxin were significantly correlated with pathogenicity and caused damage to the cell membrane of potato stem base tissue.

• Applied Biological Chemistry
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• 제32권1호
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• pp.23-29
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• 1989

soybean hypocotyl에서 분리한 미토콘드리아로부터 submitochondrial particles와 matrix 단백질 추출액을 준비하고 전기분해법으로 제조한 superoxide를 처리하여 malate dehydrogenase 및 cytochrome C oxidase의 불활성화와 mitochondrial membrane의 과산화를 각각 조사하였다. 막에 결합되어 있는 cytochrome C oxidase나 수용액상태의 malate dehydrogenas는 모두 $O^{-}_{2}$에 대해 매우 민감하게 불활성화되었다. 즉 dismutation 반응이 빠르게 진행되어 실질적으로 효소의 불활성화에 기여하게 될 농도는 대단히 낮을 것으로 추정되는 $O^{-}_{2}$의 명목상 처리농도 1.4mM 전후에서 두 효소는 그 활성을 완전히 상실하였다. 한편 malondialdehyde의 생성을 지표로 하여 측정된 membrane 과산화는 인지질로서만 이루어진 liposome의 경우보다는 다소 낮은 수준이었으나 무시할 수는 없는 정도였다. mitochondrial membrane의 과산화가 상대적으로 억제된 것은 막에 결합되어 있는 항산화제 및 단백질들에 의한 $O^{-}_{2}$소거효과에 기인하였으리라 해석된다. 식물 미토콘드리아의 대표적인 대사과정인 TCA cycle과 호흡전자전달반응의 성분효소들인 malate dehydrogenase와 cytochrome C oxidase가 불활성화되었고 membrane이 과산화 되었다는 사실은, 식물의 냉해와 광피해 발현기작에서 공히공통적인 화학적 인자로 인정되는 $O^{-}_{2}$의 과잉생성 및 축적이 그것의 주 생성처인 미토콘드리아의 생화학적 기능과 구조에 미칠수 있는 파괴적 효과를 적절히 지시하는 것이다.