• Preventive Nutrition and Food Science
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• v.13 no.3
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• pp.182-189
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• 2008

In this study, the effect of pH on the antioxidative activities of melanoidins formed as a result of the reaction between sugars, glucose (Glc) or fructose (Fru), and amino acids, L-asparagine (L-Asn) and D-asparagine (D-Asn) are examined. For this purpose, antioxidative activities were evaluated on the basis of reducing power, including ferric reducing/antioxidant power (FRAP) and free radical scavenging activity includes 1,1-diphenyl-2-picryl- hydrazil (DPPH) and 2,2'-azinobis(3-ethylbenothiazoline-6-sulfonic acid) diammonium salt (ABTS) and ferrous ion chelating activity. Ethylene diamine tetraacetate (EDTA) and trolox, a water-soluble analog of tocopherol, were used as reference antioxidant compounds. The antioxidative activities of the melanoidins at a pH of 7.0 were greater than those with a pHs of 4.0 and pH 10.0. Especially, it was found that the melanoidins formed from D-isomers are more effective antioxidants in different in vitro assays. The reducing power and chelating activity of the melanoidins formed from the Fru systems were higher than those of the melanoidins formed from the Glc systems. However, the ABTS radical scavenging activity of the melanoidins formed from the Glc systems were higher than those of the melanoidins formed from the Fru systems. In particular, the DPPH radical scavenging activity and the FRAP of the melanoidins showed different antioxidative activities according to pH level.

• Korean Journal of Microbiology
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• v.36 no.1
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• pp.14-19
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• 2000

Algal lytic enzyme, an extracellular enzyme, was purified from the culture filtrate of Penicillium oxalicum(HCLF-34) by ultrafiltration, gel filtration chromatography, and anion exchange chromatography. The enzyme has a molecular mass of approximately 22 kDa, an it is a monomer by renaturation SDS-PAGE. The amino acid sequences of the enzyme was revealed to be NH2-Glu-Ser-Tyr-Ser-Ser-Asn-Ala-Ala-Gly-Ala-Val-Leu-Ile---, had about 84% identity with the mature light chain of aspergillopepsin II precursor and 81% identity with the mature protein of the acid proteinase EapC precursor.

• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1291-1299
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• 2007

Two ${\beta}$-1,4-glucanases (DI and DIII fractions) were purified to homogeneity from the culture filtrate of a cellulolytic bacteria, Cellulomonas sp. CS 1-1, which was classified as a novel species belonging to Cellulomonas uda based on chemotaxanomic and phylogenetic analyses. The molecular mass was estimated as 50,000 Da and 52,000 Da for DI and DIII, respectively. Moreover, DIII was identified as a glycoprotein with a pI of 3.8, and DI was identified as a non-glycoprotein with a pI of 5.3. When comparing the ratio of the CMC-saccharifying activity and CMC-liquefying activity, DI exhibited a steep slope, characteristic of an endoglucanase, whereas DIII exhibited a low slope, characteristic of an exoglucanase. The substrate specificity of the purified enzymes revealed that DI efficiently hydrolyzed CMC as well as xylan, whereas DIII exhibited a high activity on microcrystalline celluloses, such as Sigmacells. A comparison of the hydrolysis patterns for pNP-glucosides (DP 2-5) using an HPLC analysis demonstrated that the halosidic bond 3 from the nonreducing end was the preferential cleavage site for DI, whereas bond 2, from which the cellobiose unit is split off, was the preferential cleavage site for DIII. The partial N-terminal amino acid sequences for the purified enzymes were $^1Ala-Gly-Ser-Thr-Leu-Gln-Ala-Ala-Ala-Ser-Glu-Ser-Gly-Arg-Tyr^{15}$-for DI and $^1Ala-Asp-Ser-Asp-Phe-Asn-Leu-Tyr-Val-Ala-Glu-Asn-Ala-Met-Lys^{15}$-for DIII. The apparent sequences exhibited high sequence similarities with other bacterial ${\beta}$-1,4-glucanases as well as ${\beta}$-1,4-xylanases.

• Biomolecules & Therapeutics
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• v.14 no.1
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• pp.30-35
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• 2006

Tissue-type plasminogen activator (t-PA) is a multidomain serine protease containing two kringle domains, TK1-2. Previously, Pichia-derived recombinant human TK1-2 has been reported as an angiogenesis inhibitor although t-PA plays an important role in endothelial and tumor cell invasion. In this work, in order to improve in vivo efficacy of TK1-2 through elimination of immune reactivity, we mutated wild type TK1-2 into non-glycosylated form (NE-TK1-2) and examined whether it retains anti-angiogenic activity. The plasmid expressing NE-TK1-2 was constructed by replacing $Asn^{l17}\;and\;Asn^{184}$ with glutamic acid residues. After expression in Pichia pastoris, the secreted protein was purified from the culture broth using S-sepharose and UNO S1-FPLC column. The mass spectrum of NE-TK1-2 showed closely neighboring two peaks, 19631.87 and 19,835.44 Da, and it migrated as one band in SDS-PAGE. The patterns of CD-spectra of these two proteins were almost identical. Functionally, purified NE-TK1-2 was shown to inhibit endothelial cell migration in response to bFGF stimulation at the almost same level as wild type TK1-2. Therefore, the results suggest that non-glycosylated NETK1-2 can be developed as an effective anti-angiogenic and anti-tumor agent devoid of immune reactivity.

• Progress in Medical Physics
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• v.29 no.1
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• pp.29-41
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• 2018

The objective of this study was to evaluate the chemical exchange saturation transfer (CEST) effect of amino acids and neurotransmitters, which exist in the human brain, depending on the concentration, pH, and amplitude of the saturation radiofrequency field. Phantoms were developed with asparagine (Asn), ${\gamma}-aminobutyric$ acid (GABA), glutamate (Glu), glycine (Gly), and myoinositol (MI). Each chemical had three different concentrations of 10, 30, and 50 mM and three different pH values of 5.6, 6.2, and 7.4. Full Z-spectrum CEST images for each phantom were acquired with a continuous-wave radiofrequency (RF) saturation pulse with two different $B_1$ amplitudes of $2{\mu}T$ and $4{\mu}T$ using an animal 9.4T MRI system. A voxel-based CEST asymmetry was mapped to evaluate exchangeable protons based on amide (-NH), amine ($-NH_2$), and hydroxyl (-OH) groups for the five target molecules. For all target molecules, the CEST effect was increased with increasing concentration and B1 amplitude; however, the CEST effect with varying pH displayed a different trend depending on the characteristics of the molecule. On CEST asymmetric maps, Glu and MI were well visualized around 3.0 and 0.9 ppm, respectively, and were well separated macroscopically at a pH of 7.4. The exchange rates of Asn, Glu, BABA, and Gly usually decreased with increasing pH. The CEST effect was dependent on the concentration, acidity of the target molecules, and B1 amplitude of the saturation RF pulse. The CEST effect for Asn can be observed in a 9.4T MRI system. The results of this study are based on applying the CEST technique in patients with neurodegenerative diseases when proteins in the brain are increased with disease progression.

• Journal of Microbiology and Biotechnology
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• v.25 no.8
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• pp.1371-1379
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• 2015

The Ca_v1.2 Ca²⁺ channel is essential for cardiac and smooth muscle contractility and many physiological functions. We mutated single, double, and quadruple sites of the four potential Asn (N)-glycosylation sites in the rabbit Ca_v1.2 into Gln (Q) to explore the effects of Nglycosylation. When a single mutant (N124Q, N299Q, N1359Q, or N1410Q) or Ca_v1.2/WT was expressed in Xenopus oocytes, the biophysical properties of single mutants were not significantly different from Ca_v1.2/WT. In comparison, the double mutant N124,299Q showed a positive shift in voltage-dependent gating. Furthermore, the quadruple mutant (QM; N124,299,1359,1410Q) showed a positive shift in voltage-dependent gating as well as a reduction of current. We tagged EGFP to the QM, double mutants, and Ca_v1.2/WT to chase the mechanisms underlying the reduced currents of QM. The surface fluorescence intensity of QM was weaker than that of Ca_v1.2/WT, suggesting that the reduced current of QM arises from its lower surface expression than Ca_v1.2/WT. Tunicamycin treatment of oocytes expressing Ca_v1.2/WT mimicked the effects of the quadruple mutations. These findings suggest that Nglycosylation contributes to the surface expression and voltage-dependent gating of Ca_v1.2.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.2
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• pp.154-159
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• 2017

Objective: This study was designed to characterize the DNA polymorphisms of the melanocortin-1 receptor (MC1R) gene in indigenous Saudi Arabian sheep breeds exhibiting different color coats, along with individuals of the Sawaknee breed, an exotic sheep imported from Sudan. Methods: The complete coding region of MC1R gene including parts of 3' and 5' untranslated regions was amplified and sequenced from three the indigenous Saudi sheep; Najdi (generally black, n = 41), Naeimi (generally white with brown faces, n = 36) and Herri (generally white, n = 18), in addition to 13 Sawaknee sheep. Results: Five single nucleotide polymorphisms (SNPs) were detected in the MC1R gene: two led to nonsynonymous mutations (c.218 T>A, p.73 Met>Lys and c.361 G>A, p.121 Asp>Asn) and three led to synonymous mutations (c.429 C>T, p.143 Tyr>Tyr; c.600 T>G, p.200 Leu>Leu, and c.735 C>T, p.245 Ile>Ile). Based on these five SNPs, eight haplotypes representing MC1R $E^d$ and $E^+$ alleles were identified among the studied sheep breeds. The most common haplotype (H3) of the dominant $E^d$ allele was associated with either black or brown coat color in Najdi and Sawaknee sheep, respectively. Two other haplotypes (H6 and H7) of $E^d$ allele, with only the nonsynonymous mutation A218T, were detected for the first time in Saudi indigenous sheep. Conclusion: In addition to investigating the MC1R allelic variation in Saudi indigenous sheep populations, the present study supports the assumption that the two independent nonsynonymous Met73Lys and Asp121Asn mutations in MC1R gene are associated with black or red coat colors in sheep breeds.

• Journal of the Korea Convergence Society
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• v.11 no.5
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• pp.61-72
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• 2020

An anti-aging peptide was prepared from fish collagen hydrolysate (FCH) by ultrafiltration (MWCO; 1 kDa) and reversed-phased high performance liquid chromatography (RP-HPLC). Its anti-aging properties were evaluated based on the procollagen-synthesizing and MMP-1-inhibiting activities in Hs68 cells. A potent anti-aging peptide (fraction I-I) increased collagen synthesis by 46% and also inhibited MMP-1 secretion by 77%, compared with unpurified FCH. The amino acid sequence of fraction I-I was identified to be Gly-Arg-Arg-Gly-Asn-Lys (GRRGNK; the repeating Gly-X-Y sequence in collagen), and it had a molecular mass of 686.175 Da. It revealed that the anti-aging activity of GRRGNK was mainly due to skin protective effects. These results demonstrated that fish collagen hydrolysate may be a potential source of anti-aging peptides, which could be utilized in various field, including foods, cosmetics, and pharmaceuticals.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.5
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• pp.625-629
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• 2013

The melanocortin 1 receptor (MC1R) gene is related to the plumage color variations in chicken. Initially, the MC1R gene from 30 individuals was sequenced and nine polymorphisms were obtained. Of these, three and six single nucleotide polymorphisms (SNPs) were confirmed as synonymous and nonsynonymous mutations, respectively. Among these, three selected SNPs were genotyped using the restriction fragment length polymorphism (RFLP) method in 150 individuals from five chicken breeds, which identified the plumage color responding alleles. The neighbor-joining phylogenetic tree using MC1R gene sequences indicated three well-differentiated different plumage pigmentations (eumelanin, pheomelanin and albino). Also, the genotype analyses indicated that the TT, AA and GG genotypes corresponded to the eumelanin, pheomelanin and albino plumage pigmentations at nucleotide positions 69, 376 and 427, respectively. In contrast, high allele frequencies with T, A and G alleles corresponded to black, red/yellow and white plumage color in 69, 376 and 427 nucleotide positions, respectively. Also, amino acids changes at position Asn23Asn, Val126Ile and Thr143Ala were observed in melanin synthesis with identified possible alleles, respectively. In addition, high haplotype frequencies in TGA, CGG and CAA haplotypes were well discriminated based on the plumage pigmentation in chicken breeds. The results obtained in this study can be used for designing proper breeding and conservation strategies for the Korean native chicken breeds, as well as for the developing breed identification markers in chicken.

• Microbiology and Biotechnology Letters
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• v.19 no.1
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• pp.14-20
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• 1991

- The purpose of this work is to investigate the effects of various substrates on biosynthesis of ethylene by the Kudzu strain of Pseudomonas syn'ngae pv. Phaseolicola causing halo blight. In the intact cell of P. sym'ngue, optimal condition for ethylene production was achieved at p1-I 7.5 and $30^{\circ}C$ for 9 to 10 hours of culture. Ethylene was most effectively produced from amino acids such as Asn, Gln, Asp ans Glu, compared to those of various kinds of sugars. While ethylene production from $\alpha$-ketoglutarate ($\alpha$-KG) was gradually increased throughout 51 hours incubation period tested. Ethylene production derived from citrate, $\alpha$-KG and oxalacetate as well as a few amino acids was further enhanced by the addition of histidine or arginine. In cell-free ethylene-forming system, ethylene was most effectively produced from $\alpha$-KG, compared to those from citrate, oxalacetate, Glu, Arg, or Asp, at 0.5 mM among the range from 0.25 mM to 5 mM. Anlinooxyacetate, an inhibitor of a pyridoxal phosphate-linked enzyme, completely inhibited ethylene evolution derived from Glu but not affect that derived from $\alpha$-KG. The results obtained in this work suggest that $\alpha$-KG might be a direct precursor of ethylene production in this organism than any other substrates tested.