• Journal of the East Asian Society of Dietary Life
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• v.7 no.3
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• pp.289-300
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• 1997

Newborn dog chymosin was extracted from the stomachs of dogs of 2 weeks of age, and was purified by ion exchange chromatography. Half of the sequence was determined by amino acid sequencing and the complete sequence was deduced from a cloned chymosin cDNA Results showed that the zymogen showed 79% sequence identity with calf prochymosin and 54% identity with porcine pepsinogen A The size of the propart and location of the residue which becomes the amino-terminus in the active enzyme was the same in the prochymosins. The maximum general proteolytic activity at pH 3.2 of newborn dog chymosin was 3-4% of that of porcine pepsin A at pH 2, whereas the milk clotting activity relative to the general proteolytic activity of newborn dog chymosin was much higher than that of calf chymosin. Agar gel electrophoresis at pH 5.2 of stomach extracts of individual dogs showed the existence of two predominant genetic variants of zymogen and enzyme. The two variants could not be distinguished by amino acid composition or amino-terminal sequencing, and no differences in the enzymatic properties of the genetic variants were observed. It was concluded that of the residues that participate in the substrate binding, calf and newborn dog chymosin differ in the following positions (porcine pepsin numbering, subsites in parentheses) : Ser 12 Thr(S$_4$), Leu 30 Val(S$_1$/S$_3$), His 74 Gln(S'$_2$), Val 111 Ile(S$_1$/S$_3$), Lys 220 Met(S$_4$). With regard to the low general proteolytic activity of newborn dog chymosin, the substitution Asp303 Val relative to calf chymosin may contribute to an explanation of this.

• Journal of Genetic Medicine
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• v.14 no.2
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• pp.71-74
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• 2017

Mutations in the DNA methyltransferase 1 gene (DNMT1) were reported to cause two phenotypes: OMIM 604121 and OMIM 614116. The first phenotype includes autosomal dominant cerebellar ataxia, deafness, and narcolepsy, which were reported to be caused by mutations in exon 21. The second phenotype includes hereditary sensory and autonomic neuropathy type 1E, which was suggested to be caused by mutations in exon 20 and 21. In this article, we report a novel heterozygous missense variant c.898A>C, p.(Lys300Gln) in exon 12 of DNMT1 in a young woman who presented with pure cerebellar ataxia. This report indicates that a mutation in exon 12 may lead to pure cerebellar ataxia. Another possibility is that the patient is currently in an early stage of the disease, and as the disease progresses, she will have more manifestations. To confirm or exclude this possibility, a subsequent follow-up study reporting the disease progression in this patient may be needed. Further reports of cases with the same mutation are needed to confirm the phenotype of this mutation.

• Korean Journal of Environmental Agriculture
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• v.24 no.3
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• pp.245-252
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• 2005

The objective of the present work was to determine the corresponding uptake and assimilation of ${NO_3}^-$ in roots and shoots of leafy radish by applying of mixed amino acids (MAA). The amino acids used in this experiment were alanine (Ala), ${\beta}-alanine\;({\beta}-Ala)$, aspartic acid (Asp), asparagines (Asn), glutamic acid (Glu), glutamine (Gln), and glycine (Gly). Leafy radish was grown by conventional fertilization with macro- and micronutrients under controlled conditions. The 15-day-old seedlings were treated 0, 0.3 and 3.0 mM of MAA containing 5 mM ${NO_3}^-$ in growth medium. Nitrate uptake was determined by following ${NO_3}^-$ depletion from the uptake solution. The activity of the enzymes related to the process of ${NO_3}^-$ reduction (NR: nitrate reductase; NiR: nitrite reductase; GS: glutamine synthetase) and the content of ${NO_2}^-\;and\;{ND_3}^-$ were analyzed in shoots and roots. The results of this study showed that ${NO_3}^-$ uptake was inhibited 38% with treatment of 0.3 mM of MAA. However, there was more than three times increase of N03- uptake in 3.0 mM MAA. In addition, the enzymatic activities were positively affected by the high MAA rate. Finally, the ${NO_3}^-$ content was increased slightly both in shoots and roots of leafy radish by MAA treatments.

• Culinary science and hospitality research
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• v.22 no.6
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• pp.71-77
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• 2016

To determine the nutritional value of domestic tomatoes, the levels of amino acids, amino acid metabolites, and the bioactive compound ${\gamma}-aminobutyric-acid$ (GABA) were analyzed in three domestic tomato varieties (Rafito, Momotaro, and Medison). Eighteen free amino acids were found, and total free amino acid content was 3,810.21~4,594.56 mg/100 g (dry weight). L-glutamic acid (L-Glu) was the most abundant amino acid, ranging from 1,866.60 mg/100 g for Momotaro to 2,417.45 mg/100 g for Medison. The next most abundant amino acids were L-glutamine (L-Gln) and L-aspartic acid (L-Asp). The three tomato varieties had a good balance of all the essential amino acids except tryptophan. Total essential amino acid content was 274.26~472.71 mg/100 g (dry weight). The following amino acid metabolites were found: L-carnitine (L-Car), hydroxylysine (Hyl), o-phosphoethanolamine (o-Pea), phosphoserine (p-Ser), ${\beta}-alanine$ (${\beta}-Ala$), N-methyl-histidine (Me-His), ethanolamine (EtNH2),and L-citrulline(L-Cit). Large quantities of GABA were found in all three varieties: 666.95-868.48 mg/100g (dry weight). These results support the use of these tomato varieties as nutritious food materials.

• Biomedical Science Letters
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• v.10 no.3
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• pp.187-194
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• 2004

A short peptide corresponding to the nuclear localization signal (NLS) of human immunodeficiency virus (HIV)-l Tat protein, Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, was employed to improve the efficiency of cellular uptake of nucleic acids. The peptide was first mixed with a reporter plasmid and then with cationic liposomes to form a tripartite complex of DNA/peptide/liposomes (DPL). Transfection efficiency of the DPL complex was compared with that of the conventional DNA/liposomes (DL) complex. When the DPL complex was formed with various cationic liposomes, DOTAP/DOPE (DP) liposome exhibited superior transfection efficiency to other liposomes tested in vitro. With the inclusion of the peptide, the DPL complex showed much enhanced transfection in various cancer cell lines. Particularly, transfection of the DPL complex in serum increased cellular uptake of a transgene up to 2 fold when compared with that in a serum free condition. Further, when the DPL complex was infused through the ureteric route of a rat, transfection efficiency was shown to be better in reporter gene expression than that obtained with the DL complex. This study shows that the DPL complex that is easy to formulate can be employed for much enhanced cellular uptake of a trans gene.

• Korean Journal Plant Pathology
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• v.14 no.3
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• pp.229-239
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• 1998

Antibodies were raised against fusion proteins of the N-terminus and a region containing the GDNQ (Gly-Asp-Asn-Gln) polymerase motif of the L (polymerase) protein of sonchus yellow net virus (SYNV). Immunoblot analyses using these antibodies revealed the presence of the L protein in purified SYNV preparations and in nuclear extracts from infected tobacco. The serological analyses and detection in a polyacrylamide gels suggested that the L protein is present in at least a 20 fold lower abundance than the G, N, M1 and M2 proteins, and has size corresponding to a molecular weight of over 200 kDa as predicted from nucleotide sequence data. Electron microscopy with gold-labelled antibodies was used to localize the N, M2, and G proteins of SYNV in thin sections of infected tissue. When sections of SYNV-infected tissue were treated with antisera against total SYNV proteins and N protein, gold label could be detected in both the viroplasms and in virus particles. With the anti-M2 protein antiserum, the gold label was strongly localized in the viroplasms but only limited labelling of the virus particle sonly. Limited labelling of the L protein was observed in the viroplasms and the virus particles, presumably because of the low abundance of L protein in the tissues.

• Food Science of Animal Resources
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• v.38 no.6
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• pp.1168-1178
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• 2018

The present study aimed to characterise anti-oxidant peptides from water-soluble protein extracts of Hanwoo beef and evaluate their anti-proliferative effect on human colorectal carcinoma cells (HCT116). Antioxidant peptides were purified from the low-molecular-weight fraction (<3 kDa) of Hanwoo beef extract. Antioxidant activity of peptide fractions was determined using the oxygen radical absorbance capacity (ORAC) assay. Purified peptide (P3) displayed higher ORAC activity than the low-molecular-weight fraction ($202.66{\mu}M\;TE/g$ vs $167.38{\mu}M\;TE/g$ of dry matter, respectively) (p<0.05). The peptide sequence of P3 was Cys-Cys-Cys-Cys-Ser-Val-Gln-Lys (888.30 Da). The novel peptide P3, at $250{\mu}g/mL$, also significantly inhibited HCT116 cell proliferation up to 25.24% through phosphorylation of ERK, JNK, and p38 kinase (p<0.05). Hence, antioxidant peptide P3 from Hanwoo beef extract can be used as an antioxidative and anticancer agent in the functional food industry.

• Journal of Microbiology and Biotechnology
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• v.31 no.11
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• pp.1576-1582
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• 2021

Bacterial β-glucuronidase in the intestine is involved in the conversion of 7-ethyl-10-hydroxycamptochecin glucuronide (derived from irinotecan) to 7-ethyl-10-hydroxycamptothecin, which causes intestinal bleeding and diarrhea (side effects of anti-cancer drugs). Twelve compounds (1-12) from Polygala tenuifolia were evaluated in terms of β-glucuronidase inhibition in vitro. 4-O-Benzoyl-3'-O-(O-methylsinapoyl) sucrose (C3) was highly inhibitory at low concentrations. C3 (an uncompetitive inhibitor) exhibited a k_i value of 13.4 μM; inhibitory activity increased as the substrate concentration rose. Molecular simulation revealed that C3 bound principally to the Gln158-Tyr160 enzyme loop. Thus, C3 will serve as a lead compound for development of new β-glucuronidase inhibitors.

• BMB Reports
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• v.31 no.4
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• pp.364-369
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• 1998

Insect plasma protein is abundant in the hemolymph of holometabolous insect larvae and is used as a source of amino acids and energy for construction of adult structures during metamorphosis. In order to understand the mechanism of decomposition of larval plasma proteins by hemocyte protease, we tried to purify a cysteine protease from the hemocyte lysate by using Carbobenzoxy-L-Phenylalanyl-L-Arginine-4-Methyl-Coumaryl-7-Amide (Z-Phe-Arg-MCA) as substrate and to identify plasma proteins that are selectively susceptible to the purified protease. Here, we describe the purification and characterization of a cysteine protease that specifically hydrolyzes the plasma protein of the coleopteran insect, Tenebrio molitor, larvae. The molecular mass of this enzyme was 25 kDa, as determined by SDS-PAGE under reducing conditions. The amino acids sequence of its $NH_{2}-terminus$ was determined to be Leu-Pro-Gly-Gln-Ile-Asp-Trp-Arg-Asp-Lys-Gly. This sequence contained Pro, Asp, and Arg residues, conserved in many papain superfamily enzymes. The specific cysteine protease inhibitors, such as E-64 and leupetin, inhibited its hydrolytic activity. One plasma protein with a molecular mass of 48 kDa was selectively hydrolyzed within 3 h when the purified enzyme and plasma proteins were incubated in vitro. However, the 48 kDa protein was not hydrolyzed by the purified 25 kDa protease in the presence of E-64. Western blotting analysis at various developmental stages showed that the purified enzyme was detected at larvae, pupae, and adult stages, but not the embryo stage.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.639-644
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• 1998

Extracellular serine proteases were isolated from a soil bacterium, alkalophilic coryneform bacterium TU-19, which have been grown in a liquid medium optimized at 3$0^{\circ}C$ and pH 10.0. Three different sizes, 120 kDa (protease I), 80 kDa (protease II), and 45 kDa (protease III), of serine pro teases were purified using Sephadex G-150 and QAE-Sephadex chromatography (Kang et al. 1995. Agric. Chem Biotech. 38: 534-540). SDS-PAGE showed that the 120 kDa protease was degraded into the 80 kDa protease in 20 mM Tris-HCI (pH 8.0) buffer solution. This degradation was enhanced in the presence of 0.5 M NaCl and 5 mM EDTA, but was inhibited in the presence of 5 mM $CaCl_2$. These results indicated that the $Ca^{2＋}$ ion seems to stabilize the 120 kDa protease like other proteases derived from Bacillus species. The $NH_2$-terminal amino acid sequences of the 10 residues of both proteases were completely identical: Met-Asn-Thr-Gln-Asn-Ser-Phe-Leu-Ile-Lys. In contrast to this, the 80 kDa protease has 1.5 times higher specific activity than the 120 kDa protease does (Kang et al. 1995. Agric. Chern. Biotech. 38: 534-540). Therefore the C-terminal of the 120 kDa protease seems to be autolyzed to the 80 kDa protease but this autolysis did not decrease the protease activity. Optimum pH and temperature of both 80 kDa and 120 kDa proteases were pH 10.5 and $45^{\circ}C$, respectively, and pH and thermal stability were almost identical. Several divalent ions except the $Fe^{2＋}$ ion showed similar effects on activities of both proteases, which are similarly resistant to three different detergents.