• Mass Spectrometry Letters
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• 제15권1호
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• pp.1-25
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• 2024

Protein glycosylation, a highly significant and ubiquitous post-translational modification (PTM) in eukaryotic cells, has attracted considerable research interest due to its pivotal role in a wide array of essential biological processes. Conducting a comprehensive analysis of glycoproteins is imperative for understanding glycoprotein bio-functions and identifying glycosylated biomarkers. However, the complexity and heterogeneity of glycan structures, coupled with the low abundance and poor ionization efficiencies of glycopeptides have all contributed to making the analysis and subsequent identification of glycans and glycopeptides much more challenging than any other biopolymers. Nevertheless, the significant advancements in enrichment techniques, chromatographic separation, and mass spectrometric methodologies represent promising avenues for mitigating these challenges. Numerous substrates and multifunctional materials are being designed for glycopeptide enrichment, proving valuable in glycomics and glycoproteomics. Mass spectrometry (MS) is pivotal for probing protein glycosylation, offering sensitivity and structural insight into glycopeptides and glycans. Additionally, enhanced MS-based glycopeptide characterization employs various separation techniques like liquid chromatography, capillary electrophoresis, and ion mobility. In this review, we highlight recent advances in enrichment methods and MS-based separation techniques for analyzing different types of protein glycosylation. This review also discusses various approaches employed for glycan release that facilitate the investigation of the glycosylation sites of the identified glycoproteins. Furthermore, numerous bioinformatics tools aiding in accurately characterizing glycan and glycopeptides are covered.

• Current Research on Agriculture and Life Sciences
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• 제11권
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• pp.121-132
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• 1993

대두(大豆)-근류균(根瘤菌)의 공생(共生)에서 숙주식물(宿主植物)의 Root Exudate에 대한 근류균(根瘤菌)의 화학주성(化學走性)이 근류형성(根瘤形性)(숙주식물(宿主植物))에 미치는 영향(影響)을 밝히기 위해 연구(硏究)한 결과(結果)는 다음과 같다. 대두(大豆) Seed Lectin은 품종간(品種間) 차이(差異)가 없었으며 분자량(分子量) 120,000정도(程度)의 4개(個)의 동일(同一)한 subunit를 가지는 물질(物質)이며, Pea Seed Lectin은 분자량(分子量) 44,000정도(程度)이며 15,000과 7,000정도(程度)의 두 종류(種類)의 subunit를 가지는 물질(物質)이며 대두(大豆) lectin은 표준 lectin 항체와 침강선을 형성하였다. 그러나 완두의 lectin은 표준 lectin과 항원-항체반응이 없었으므로 두 lectin은 동일 항원이 되지 않아 서로 다른 물질이었다. 팔달의 Crude Root Exudate에 대(對)한 근류균(根瘤菌)의 화학주성(化學走性) 비(比)는 KCTC 2422는 각각(各各) 3.5이고 LPN-100은 1.4이며 LCR-101은 각각(各各) 1.4이었다. 뿌리분비물의 각 fraction에 대한 근류균의 화학주성은 중성분획이 가장 높고 양성분획이 그다음이며 음성분획이 가장 낮은 화학주성을 나타내어 각물질에 대한 근류균의 친화도에 차이가 있었다. 근류형성(根瘤形性)의 정도(程度)는 KCTC 2422의 경우(境遇)는 접종(接種)후 7日경부터 Nitrogenase Activity가 나타났으며 LCR-101는 15일 이후(以後)에 Nitrogenase Activity를 나타내었다. LPN-100은 Nitrogenase Activity가 전혀 나타나지 않아 근류균(根瘤菌)의 화학주성(化學走性)이 숙주인식과정(宿主認識過程)에 영향(影響)을 미쳤음을 확인(確認)할 수 있었다.

• 한국수산과학회지
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• 제33권3호
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• pp.198-204
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• 2000

수산가공공장에서 원료어 처리시 대량으로 발생하는 비가식부의 하나인 대구의 고니부분을 효율적으로 이용하기 위하여 단백질을 효소로 가수분해시킨 후 한외여과막을 사용하여 분자량별로 분획하였으며, 이들 가수분해물 중 항산화활성이 뛰어난 펩타이드를 이온교환 크로마토그래피, 겔크로마토그래피 및 HPLC로 분리${\cdot}$정제하여 그 아미노산 서열을 결정하였다. 여러 가지 단백질 분해효소로 분해시켜 얻은 가수분해물 중에서 항산화활성이 가장 우수한 것은 Alcalase로 천연 항산화제인 ${\alpha}-tocopherol$보다 $5{\%}$정도 뛰어난 효과를 나타내었으며, 이 가수분해물을 한외여과막으로 분자량 10 kDa, 5 kDa 및 1 kDa의 세 종류로 분리하여 항산화활성을 측정한 결과, 1 kDa의 막을 통과하여 분리된 가수분해물이 가장 높은 활성을 나타내었으며, 이는 천연항산화제인 ${\alpha}-tocopherol$보다 $10{\%}$정도 높았다. 이 획분으로 Spsephadex C-25를 사용하여 이온교환 크로마토그래피를 한 결과, $0.5{\~}1.0 M$ NaCl 용액으로 용출시 킨 분획물에 서 ${\alpha}-tocopherol$보다 약 $17{\%}$ 활성이 높게 나타났으며, 이것을 다시 Sephadex G-15로 겔여과하여 3개의 획분을 얻었으며 이 중 획분 II에서 ${\alpha}-tocopherol$보다 약 $45{\%}$가 높은 항산화활성을 보인 획분을 얻었다. 이것을 역상 HPLC를 이용하여 5차의 획분을 얻었으며, 항산화활성은 획분 A에서 ${\alpha}-tocopherol$보다 약 $53{\%}$정도 높게 나타나 가장 우수하였다. 이 획분을 capillary electrophoresis로 순도를 확인하여 아미노산 서열을 결정한 결과 Ser-Asn-Pro-Glu-Trp-Ser-Trp-Asn였다.

• Molecular & Cellular Toxicology
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• 제1권3호
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• pp.196-202
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• 2005

Nitric oxide (NO) is a small, diffusible, and highly reactive molecule, which plays dichotomous regulatory roles under physiological and pathological conditions. NO promotes apoptosis in some cells, and inhibits apoptosis in other cells. In the present study, we attempted to characterize the NO signaling pathway and cellular response in PC12 cells treated with cytokines. $IFN{\gamma}\;and\;TNF{\alpha}$ treatment resulted in a synergistic increase of nitrite accumulation, with the induction of inducible nitric oxide synthase (iNOS) in the PC12 cells. Moreover, as nitrite concentration increased, cell viability decreased. In order to explore MAP kinase involvement in nitric oxide production resultant from $IFN{\gamma}\;and\;TNF{\alpha}$ stimulation, we measured the activation of MAP kinase using specific MAP kinase inhibitors. PC12 cells pretreated with SB203580, a p38 MAP kinase-specific inhibitor, resulted in the inhibition of iNOS expression and NO production. However, PD98059, an ERK/MAP kinase-specific inhibitor, was not observed to exert such an effect. In addition, Stat1 activated by $IFN{\gamma}\;and\;TNF{\alpha}$ was interacted with p38 MAPK. These data suggest that p38 MAP kinase mediates cytokine-mediated iNOS expression in the PC12 cells, and Jak/Stat pathway interferes with p38 MAPK signaling pathway.

• 한국근적외분광분석학회:학술대회논문집
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• 한국근적외분광분석학회 2001년도 NIR-2001
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• pp.1156-1156
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• 2001

This work aimed to prove the feasibility of NIR spectroscopy to detect vegetable protein isolates (soy, pea and wheat) in milk powder. Two hundred and thirty-nine samples of genuine and adulterated milk powder (NIZO, Ede, NL) were analysed by NIRS using an InfraAlyzer 500 (Bran+Luebbe). NIR spectra were collected at room temperature, and data were processed by using Sesame Software (Bran+Luebbe). Separated calibrations for each non-milk protein added, in the range of 0-5%, were calculated. NIR data were processed by using Sesame Software (Bran+Luebbe). Prediction and validation were made by using a set of samples not included into the calibration set. The best calibrations were obtained by the PLSR. The type of data pre-treatment (normalisation, 1$\^$st/ derivative, etc..) was chosen to optimize the calibration parameters. NIRS technique was able to predict with good accuracy the percentage of each vegetable protein added to milk powder (soy: R$^2$ 0.994, SEE 0.193, SEcv 0.301, RMSEPall 0.148; pea: R$^2$ 0.997, SEE 0.1498, SEcv 0.207, RMSEPall 0.148, wheat: R$^2$ 0.997, SEE 0.1418, SEcv 0.335, RMSEPall 0.149). Prediction results were compared to those obtained using other two techniques: capillary electrophoresis and competitive ELISA. On the basis of the known true values of non-vegetable protein contents, the NIRS was able to determine more accurately than the other two techniques the percentage of adulteration in the analysed samples.

• 한국근적외분광분석학회:학술대회논문집
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• 한국근적외분광분석학회 2001년도 NIR-2001
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• pp.1158-1158
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• 2001

Fast and dynamic biochemical, enzymatic and morphological changes occur during the so-called generative development and during the vegetative processes in seeds. The most characteristic biochemical and compositional changes of this period are the formation and decline of storage components or their precursors, the change of their degree in polymerization and an extensive change in water content. The aim of the present study was to detect the maturation processes in seed nondestructively and to verify the applicability of near infrared spectroscopic methods in the measurement of physiological, chemical and biochemical changes in wheat seed. The amount and variation of different water “species” has been changed intensively during maturation. Characteristic changes of three water absorption bands (1920, 1420 and 1150 nm) during maturation were analysed. It was concluded that the free/bound transition of water molecules could be followed sensitively in different region of NIR spectra. Kinetic changes of carbohydrate reserves were characteristic during maturation. An intensive formation and decline of carbohydrate reserves were observed during early stage of maturation (0 -13 days, high energy demand). An accelerated formation of storage carbohydrates (starch) was detected in the second phase of maturation. Five characteristic absorption bands were analysed which were sensitive indicators the changes of carbohydrates occurred during maturation. Precursors of protein synthesis and the synthesis of reserve proteins and their kinetic changes during maturation were followed from NIR spectra qualitative and qualitatively. Dynamic formation of amino acids and the changes of N forms were detected by spectroscopic, chromatographic and by capillary electrophoresis methods. Calibration equations were developed and validated in order to measure the optimal maturation time protein and moisture content of developing wheat seeds. The spectroscopic methods are offering chance and measurement potential in order to detect fine details of physiological processes. The spectra have many hidden details, which can help to understand the biochemical background of processes.

• BMB Reports
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• 제34권3호
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• pp.194-200
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• 2001

Secreted arginase from Evernia prunastri thallus has been purified 616-fold from the incubation medium. Purified arginase was resolved as only one peak in a capillary electrophoresis with a pI value of 5.35. The protein contained high amounts of acidic amino acids, such as Asx and Glx, and a relatively high quantity of Ser and Gly. The molecular mass of native, purified arginase was estimated as about 26 kDa by SE-HPLC. Substrate saturated kinetic showed a typical Michaelis-Menten relationship with a K_m value of 3.3 mM L-arginine. Atranorin behaved as a mixed activator of the enzyme (apparent $K_m$ = 0.96 mM); whereas evernic and usnic acid were revealed as non competitive inhibitors (apparent $K_m$ values were 3.16 mM and 3.05 mM, respectively). Kinetics of atranorin binding indicated that saturation was reached from 0.18 ${\mu}mol$ of the total atranorin and the occurrence of multiple sites for the ligand. This agrees with a possible aggregation of several enzyme subunits during the interaction process. A value of binding sites of about 12 was obtained. The binding of evernic acid was saturated from 23 nmol of total phenol. The number of binding sites was about 5. The loss of the binding ability of evernic acid could be interpreted as a single negative cooperatively. Usnic acid behaves in a similar way to evernic acid, although the binding saturation occurs at $0.14\;{\mu}moles$ of the ligand. This binding appears to be unspecific, and has 28 usnic acid binding sites to the protein.

• 대한의생명과학회지
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• 제17권2호
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• pp.151-155
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• 2011

Short tandem repeats (STRs) loci are the genetic markers used for forensic human identity test. With multiplex polymerase chain reaction (PCR) assays, STRs are examined and measured PCR product length relative to sequenced allelic ladders. In the repeat region and the flanking region of the commonly-used STR may have DNA sequence variation. A mismatch due to sequence variation in the DNA template may cause allele drop-out (i.e., a "null" or "silent" allele) when it falls within PCR primer binding sites. The STR markers were co-amplified in a single reaction by using commercial PowerPlex$^{(R)}$ 16 system and AmpFlSTR$^{(R)}$ Identifiler$^{(R)}$ PCR amplification kits. Separation of the PCR products and fluorescence detection were performed by ABI PRISM$^{(R)}$ 3100 Genetic Analyzer with capillary electrophoresis. The GeneMapper$^{TM}$ ID software were used for size calling and analysis of STR profiles. Here, this study described a forensic human identity test in which allelic drop-out occurred in the STR system D18S51. During the course of human identity test, two samples with a homozygous (16, 16 and 21, 21) genotype at D18S51 locus were discovered using the PowerPlex$^{(R)}$ 16 system. The loss of alleles was confirmed when the samples were amplified using AmpFlSTR$^{(R)}$ Identifiler$^{(R)}$ PCR amplification kit and resulted in a heterozygous (16, 20 and 20, 21) genotype at this locus each other. This discrepancy results suggest that appropriate measures should be taken for database comparisons and that allele should be further investigated by sequence analysis and be reported to the forensic community.

• Journal of Animal Science and Technology
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• 제55권2호
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• pp.87-93
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• 2013

We identified four new bovine tri-nucleotide microsatellite loci and analyzed their sequence structures and genetic parameters in 105 randomly selected Korean cattle (Hanwoo). Allele numbers of the loci B17S0808, B15S6253, B8S7996, and B17S4998 were 10, 11, 12, and 29, respectively. These alleles contained a simple or compound repeat sequences with some variations. Allele distributions of all these loci were in Hardy-Weinberg equilibrium (P > 0.05). Observed heterozygosity and expected heterozygosity ranged from 0.54 (B15S6253) to 0.92 (B17S4998) and from 0.599 (B15S6253) to 0.968 (B17S4998), respectively, and two measures of heterozygosity at each locus were highly correlated. Polymorphism information content (PIC) for these 4 loci ranged from 0.551 (B15S6253) to 0.932 (B17S4998), which means that all these loci are highly informative (PIC > 0.5). Other genetic parameters, power of discrimination (PD) and probability of exclusion (PE) ranged from 0.783 (B15S6253) to 0.984 (B17S4998) and from 0.210 (B15S6253) to 0.782 (B17S4998), respectively. Their combined PD and PE values were 0.9999968 and 0.98005176, respectively. Capillary electrophoresis revealed that average peak height ratio for a stutter was 13.89% at B17S0808, 26.67% at B15S6253, 9.09% at B8S7996, and 43.75% at B17S4998. Although the degree of genetic variability of the locus B15S6253 was relatively low among these four microsatellite markers, their favorable parameters and low peak height ratios for stutters indicate that these four new tri-nucleotide microsatellite loci could be useful multiplex PCR markers for the forensic and population genetic studies in cattle including Korean native breed.

• International Journal of Oral Biology
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• 제36권2호
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• pp.59-64
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• 2011

Anthocyanins are naturally occuring phytochemicals and the main components of the coloring of plants, flowers and fruits. They are known to elicit antioxidative, anti-inflammatory and cancer preventive activity. In this study, we investigated anthocyanins in black / yellow soybean seedcoats using different methods of detection - thin layer chromatography (TLC), capillary zone electrophoresis (CZE) and HPLC analysis. The anthocyanins in soybean seedcoats were extracted by five independent methods of extraction and the aglycons (anthocyanidins) of the corresponding anthocyanins were prepared by acid mediated hydrolysis. The anthocyanin / anthocyanidin in black soybean seedcoat showed characteristic TLC mobility, CZE electrophoretic retention and HPLC migration time while little of anthocyanins were detected from yellow soybean seedcoat. The extracted anthocyanins showed pH dependent retention time in CZE and spectral change in UV-Vis spectrum. HPLC analysis of the hydrolyzed extract of black soybean seedcoat identified the presence of four anthocyanidins. The major anthocyanin in black soybean seedcoat was cyanin (cyanidin-3-O-glucoside), with the relative order of anthocyanidin in cyanidin > delphinidin > petunidin > pelargonidin.