• 한국식물생명공학회:학술대회논문집
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• 한국식물생명공학회 2005년도 추계학술대회 및 한일 식물생명공학 심포지엄
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• pp.426-426
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• 2005

• Asian-Australasian Journal of Animal Sciences
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• 제29권10호
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• pp.1515-1521
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• 2016

Anethole and garlic have an immune modulatory effects on avian coccidiosis, and these effects are correlated with gene expression changes in intestinal epithelial lymphocytes (IELs). In this study, we integrated gene expression datasets from two independent experiments and investigated gene expression profile changes by anethole and garlic respectively, and identified gene expression signatures, which are common targets of these herbs as they might be used for the evaluation of the effect of plant herbs on immunity toward avian coccidiosis. We identified 4,382 and 371 genes, which were differentially expressed in IELs of chickens supplemented with garlic and anethole respectively. The gene ontology (GO) term of differentially expressed genes (DEGs) from garlic treatment resulted in the biological processes (BPs) related to proteolysis, e.g., "modification-dependent protein catabolic process", "proteolysis involved in cellular protein catabolic process", "cellular protein catabolic process", "protein catabolic process", and "ubiquitin-dependent protein catabolic process". In GO analysis, one BP term, "Proteolysis", was obtained. Among DEGs, 300 genes were differentially regulated in response to both garlic and anethole, and 234 and 59 genes were either up- or down-regulated in supplementation with both herbs. Pathway analysis resulted in enrichment of the pathways related to digestion such as "Starch and sucrose metabolism" and "Insulin signaling pathway". Taken together, the results obtained in the present study could contribute to the effective development of evaluation system of plant herbs based on molecular signatures related with their immunological functions in chicken IELs.

• Mass Spectrometry Letters
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• 제7권1호
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• pp.1-11
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• 2016

Various efforts have been developed to improve sample preparation steps, which strongly depend on hands-on processes for accurate and sensitive quantitative proteome analysis. In this study, we carried out heating the sample prior to trypsin digestion using an instrument to improve the tryptic digestion process. The heat shock generated by the system efficiently denatured proteins in the sample and increased the reproducibility in quantitative proteomics based on peptide abundance measurements. To demonstrate the effectiveness of the protocol, three cell lines (A human lung cancer cell line (A549), a human embryonic kidney cell line (HEK293T), and a human colorectal cancer cell line (HCT-116)) were selected and the effect of heat shock was compared to that of normal tryptic digestion processes. The tryptic digests were desalted and analysed by LC-MS/MS, the results showed 57 and 36% increase in the number of identified unique peptides and proteins, respectively, than conventional digestion. Heat shock treated samples showed higher numbers of shorter peptides and peptides with low inter-sample variation among triplicate runs. Quantitative LC-MS/MS analysis of heat shock treated sample yielded peptides with smaller relative error percentage for the triplicate run when the peak areas were compared. Exposure of heat-shock to proteomic samples prior to proteolysis in conventional digestion process can increase the digestion efficiency of trypsin resulting in production of increased number of peptides eventually leading to higher proteome coverage.

• Journal of Microbiology and Biotechnology
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• 제24권6호
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• pp.795-802
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• 2014

The aim of this study was to determine the effects of temperature and supplementation with skim milk powder (SMP) on the microbial and proteolytic properties during the storage of cottage cheese. Cottage cheese was manufactured using skim milk with 2% SMP and without SMP as the control, and then stored at $5^{\circ}C$ or $12^{\circ}C$ during 28 days. The chemical composition of the cottage cheese and the survival of the cheese microbiota containing starter lactic acid bacteria (SLAB) and non-starter culture lactic acid bacteria (NSLAB) were evaluated. In addition, changes in the concentration of lactose and lactic acid were analyzed, and proteolysis was evaluated through the measurement of acid soluble nitrogen (ASN) and non-protein nitrogen (NPN), as well as electrophoresis profile analysis. The counts of SLAB and NSLAB increased through the addition of SMP and with a higher storage temperature ($12^{\circ}C$), which coincided with the results of the lactose decrease and lactic acid production. Collaborating with these microbial changes, of the end of storage for 28 days, the level of ASN in samples at $12^{\circ}C$ was higher than those at $5^{\circ}C$. The NPN content was also progressively increased in all samples stored at $12^{\circ}C$. Taken together, the rate of SLAB and NSLAB proliferation during storage at $12^{\circ}C$ was higher than at $5^{\circ}C$, and consequently it led to increased proteolysis in the cottage cheese during storage. However, it was relatively less affected by SMP fortification. These findings indicated that the storage temperature is the important factor for the quality of commercial cottage cheese.

• 한국미생물·생명공학회지
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• 제12권4호
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• pp.285-291
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• 1984

액상발효유 제조시에 유산균 starter의 단백질 분해능이 산생성과 제품의 단백질 안정성에 미치는 영향을 조사하였다. L. bulgarious CH 2, L. helviticus IAM 1042, L. jugurti 3048은 우유에 배양할 때에 비교적 높은 단백질 분해능을 나타내었으나 L. casei YIT 9018은 단백질 분해능이 아주 약했다. 또 단백질 분해능이 높은 균들은 낮은 균들보다 우유에서의 증식과 산생성 속도가 빨랐다. 유단백질의 분해가 가장 심하게 이루어지는 시기는 균의 대수증식기이었고, 배양개시로부터 24∼48시간 이후에서는 유단백질의 분해가 거의 더 진행되지 않았다. L. bulgaricus CH 2, L. helviticus IAM 1042, L. jugurti 3048, L. acidophilus L 54, L. casei 3012로 제조된 발효유들은 모두 유단백질 분해가 비교적 심하게 일어났고, pH 3.5에서의 유단백질 용해도가 낮았고, 제품의 저장시에 유단백질 침전이 심하게 생성되었다. 그러나 artificial acidification이나 L. casei YIT 9018에 의하여 제조된 제품들은 유단백질의 분해가 거의 없었고, pH 3.5 에서의 단백질 용해도도 높았고, 저장시에 침전도 전혀 발생되지 않았다.

• Animal cells and systems
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• 제16권4호
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• pp.280-288
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• 2012

Targeted degradation of proteins through ubiquitin-mediated proteolysis is an important control mechanism in various cellular processes. The process of ubiquitin conjugation is achieved by three enzyme complexes, among which the ubiquitin ligase complex (E3) is in charge of substrate specificity. The SCF (SKP1-CUL1-F-box) family portrays the largest and the most characterized member of the E3 ligases. For each SCF complex, the ubiquitination target is recognized by the F-box protein subunit, which interacts with the substrate through a unique C-terminal domain. We have characterized a novel F-box protein CFL-1 that represents a single LRR-type F-box (FBXL) in the Caenorhabditis elegans genome. CFL-1 is highly homologous to FBXL20 and FBXL2 of mammals, which are known to regulate synaptic vesicle release and cell cycle, respectively. A green fluorescence protein (GFP)-reporter gene fused to the cfl-1 promoter showed restricted expression around the amphid and the anus. Modulation of CFL-1 activity by RNAi affected the time interval between defecations. RNAi-treated worms also exhibited reduced tendency to form dauer when exposed to daumone. The potential involvement of CFL-1 in the control of defecation and pheromone response adds to the ever expanding list of cellular processes controlled by ubiquitin-mediated proteolysis in C. elegans. We suggest that CFL-1, as a single LRR-type F-box protein in C. elegans, may portray a prototype gene exerting diverse functions that are allocated among multiple FBXLs in higher organisms.

• 한국작물학회지
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• 제47권2호
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• pp.85-94
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• 2002

Antibodies raised against the purified p-subunit of $\beta$-conglycinin were used in immunohistochemical studies to monitor the pattern of $\beta$-conglycinin mobilization in the cotyledons during soybean [Glycine max (L.) Merr.] seed germination. Western blot analysis revealed that the break down of the $\beta$-subunit of $\beta$-conglycinin commenced as early as 2 days after seed imbibition (DAI). Concurrent with the degradation of the $\beta$-subunit of $\beta$-conglycinin, accumulation of 48, 28, and 26 kD proteolytic intermediates was observed from 2 to 6 DAI. Western blot analysis also revealed that the acidic subunit of glycinin was mobilized earlier than the basic subunit. The basic glycinin subunit was subjected to proteolysis within 2 DAI resulting in the appearance of an intermediate product approximately 2 kD smaller than the native basic glycinin subunit. In contrast to the major seed storage proteins, lipoxygenase was subjected to limited proteolysis and was detected even after 8 DAI. The first sign of $\beta$-conglycinin breakdown was observed near the vascular strands and proceeded from the vascular strands towards the epidermis. Protein A-gold localization studies using thin sections of soybean cotyledons and antibodies raised against the $\beta$-subunit of $\beta$-conglycinin revealed intense labeling over protein bodies. A pronounced decrease in the protein A-gold labeling intensity over protein bodies was observed at later stages of seed germination. The protein bodies, which were converted into a large central vacuole by 8 DAI, contained very little 7S protein as evidenced by sparse protein A-gold labeling in the vacuoles.

• 한국식품과학회지
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• 제18권3호
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• pp.234-240
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• 1986

탈지대두를 파파인 및 ${\alpha}$-아밀라아제를 사용하여 분해시켜 얻은 탈지대두분해물의 흡습특성을 조사하였고 이를 이용하여 모형 중간수분식품을 제조하였다. 탈지 대두분해물의 등온흡혼곡선(等溫吸混曲線)은 단백질 및 전분질이 많이 분해될 수록 동일한 수분함량에서 수분활성도가 저하되었으며 BET 일분자층값(BET monolayer value)은 증가 하였다. 단백질분해정도가 약 78%인 탈지대두분해물의 보수력은 슈크로오스보다 높았으며 소르비틀에 조금 뒤 떨어졌다. 탈지대두분해물의 등온흡습곡선에 가장 적합한 식은 Oswin식이었고 단백질 분해가 많이 진행된 경우에는 Halsey식이 가장 적합하였다. 쌀가루와 탈지대두분해물로 제조한 모형(模型) 중간수분식품(中間水分食品)은 Grover식과 Lang 및 Steinberg식으로 수분활성도를 예측할 수 있었다. 탈지대두분해물을 첨가하여 제조한 식빵에서는 저장성이 향상(向上)됨을 관찰하였다.

• Molecules and Cells
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• 제42권11호
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• pp.783-793
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• 2019

When endoplasmic reticulum (ER) functions are perturbed, the ER induces several signaling pathways called unfolded protein response to reestablish ER homeostasis through three ER transmembrane proteins: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Although it is important to measure the activity of ATF6 that can indicate the status of the ER, no specific cell-based reporter assay is currently available. Here, we report a new cell-based method for monitoring ER stress based on the cleavage of $ATF6{\alpha}$ by sequential actions of proteases at the Golgi apparatus during ER stress. A new expressing vector was constructed by using fusion gene of GAL4 DNA binding domain (GAL4DBD) and activation domain derived from herpes simplex virus VP16 protein (VP16AD) followed by a human $ATF6{\alpha}$ N-terminal deletion variant. During ER stress, the GAL4DBD-VP16AD(GV)-$hATF6{\alpha}$ deletion variant was cleaved to liberate active transcription activator encompassing GV-$hATF6{\alpha}$ fragment which could translocate into the nucleus. The translocated GV-$hATF6{\alpha}$ fragment strongly induced the expression of firefly luciferase in HeLa Luciferase Reporter cell line containing a stably integrated 5X GAL4 site-luciferase gene. The established double stable reporter cell line HLR-GV-$hATF6{\alpha}$(333) represents an innovative tool to investigate regulated intramembrane proteolysis of $ATF6{\alpha}$. It can substitute active pATF6(N) binding motif-based reporter cell lines.

• Molecules and Cells
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• 제45권10호
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• pp.695-701
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• 2022

Homeostatic regulation of meristematic stem cells accomplished by maintaining a balance between stem cell self-renewal and differentiation is critical for proper plant growth and development. The quiescent center (QC) regulates root apical meristem homeostasis by maintaining stem cell fate during plant root development. Cell cycle checkpoints, such as anaphase promoting complex/cyclosome/cell cycle switch 52 A2 (APC/C^CCS52A2), strictly control the low proliferation rate of QC cells. Although APC/C^CCS52A2 plays a critical role in maintaining QC cell division, the molecular mechanism that regulates its activity remains largely unknown. Here, we identified SCF^FBS1, a ubiquitin E3 ligase, as a key regulator of QC cell division through the direct proteolysis of CCS52A2. FBS1 activity is positively associated with QC cell division and CCS52A2 proteolysis. FBS1 overexpression or ccs52a2-1 knockout consistently resulted in abnormal root development, characterized by root growth inhibition and low mitotic activity in the meristematic zone. Loss-of-function mutation of FBS1, on the other hand, resulted in low QC cell division, extremely low WOX5 expression, and rapid root growth. The 26S proteasome-mediated degradation of CCS52A2 was facilitated by its direct interaction with FBS1. The FBS1 genetically interacted with APC/C^CCS52A2-ERF115-PSKR1 signaling module for QC division. Thus, our findings establish SCF^FBS1-mediated CCS52A2 proteolysis as the molecular mechanism for controlling QC cell division in plants.