• YAKHAK HOEJI
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• v.58 no.1
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• pp.21-27
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• 2014

We have developed 8 peptide derivatives as potential MC1R antagonists and their inhibitory effects on ${\alpha}$-MSH induced cell growth in cultured normal human melanocytes (NHM) were investigated. From these experiments, the two most potent peptide derivatives, 5-phenylvaleric acid-(D)His-Arg-Trp-$(Lys)_6NH_2$ (P 6) and 5-phenylvaleric acid-(D)His-Arg-Trp-$(Lys)_9NH_2$ (P 7) were selected for further studies. In ${\alpha}$-MSH depleted NHM cells, we have found that the treatment with 1 ${\mu}M$ of these two peptide derivatives, P 6 and P 7, inhibited the cell proliferation induced by the addition of 1 nM ${\alpha}$- MSH by 70% and 72%, respectively. In NHM cells without previous ${\alpha}$-MSH depletion, 1 ${\mu}M$ treatment in the presence of 10 nM ${\alpha}$-MSH resulted in 70% (P 6) and 80% (P 7) decrease in cell growth and 64% (P 6) and 71% (P 7) reduction in melanin synthesis, respectively. The peptide derivatives P 6 and P 7 were proved to have no apparent cytotoxicity and inhibited the elevation of intracellular cAMP concentration triggered by ${\alpha}$-MSH. In conclusion, our data suggest that the peptide derivatives reported in this study, 5-phenylvaleric acid-(D)His-Arg-Trp-$(Lys)_6NH_2$ (P 6) and 5-phenylvaleric acid-(D)His- Arg-Trp-$(Lys)_9NH_2$ (P 7) strongly antagonize ${\alpha}$-MSH, inhibit cell proliferation and melanin synthesis, and lower the intracellular cAMP concentration, hence have a promising potential as a novel skin lightening agent.

• Proceedings of the Korea Crystallographic Association Conference
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• 2003.05a
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• pp.18-18
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• 2003

Thioredoxin (Trx) is a small redox protein that is ubiquitously distributed from achaes to human. In diverse organisms, the protein is involved in various physiological roles by acting as electron donor and regulators of transcription and apoptosis as well as antioxidants. Sequences of Trx within various species are 27～69% identical to that of E. coli and all Trx proteins have the same overall fold, which consists of central five β strands surrounded by four α helices. The N-terminal cysteine in WCGPC motif of Trx is redox sensitive and the motif is highly conserved. Compared with general cysteine, the N-terminal cysteine has low pKa value. The result leads to increased reduction activity of protein. Recently, novel thio.edoxin-related protein (TRP14) was found from rat brain. TRP14 acts as disulfide reductase like Trx1, and its redox potential and pKa are similar to those of Trx1. However, TRP14 takes up electrons from cytosolic thioredoxin reductase (TrxR1), not from the mitochondrial thioredoxin reductase (TrxR2). Biological roles of TES14 were reported to be involved in regulating TNF-α induced signaling pathways in different manner with Trx1. In depletion experiments, depletion of TRP14 increased TNF-α induced phosphorylation and degradation of IκBα more than the depletion Trx1 did. It also facilitated activation of JNK and p38 MAP kinase induced by TNF-α. Unlike Trx1, TRP14 shows neither interaction nor interference with ASK1. Here, we determined three-dimensional crystal structure of TRP14 by MAD method at 1.8Å. The structure reveals that the conserved cis-Pro (Pro90) and active site-W-C-X-X-C motif, which may be involved in substrate recognition similar to Trx1 , are located at the beginning position of strand β4 and helix α2, respectively. The TRP14 structure also shows that surface of TRP14 in the vicinity of the active site, which is surrounded by an extended flexible loop and an additional short a helix, is different from that of Trx1. In addition, the structure exhibits that TRP14 interact with a distinct target proteins compared with Trx1 and the binding may depend mainly on hydrophobic and charge interactions. Consequently, the structure supports biological data that the TRP14 is involved in regulating TNF-α induced signaling pathways in different manner with Trx1.

• Microbiology and Biotechnology Letters
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• v.18 no.1
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• pp.89-93
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• 1990

To improve the stability of recombinant pBR322-trip$^+$ plasmid (pLTW24) in E. coli culture, a positive selection system was devised. A DNA fragment containing pheW$^+$ gene (a structural gene for tRNA$^{phe}$ was isolated and inserted into the pBR322-trip$^+$ plasmid(pLTP24). A temperature sensitive host strain. LC901-pheS$^{-ts}$, was constructed for this plasmid by transducing pheS$^{-ts}$ allele (phenylalanyl-tRNA synthetase) to E. coli LC901 using P1kc bacteriophage. The LC901-pheS$^{-ts}$ cells were unable to grow at a restrictive temperature when they had lost the pBR322 :: pheW$^+$ (pLTP24) plasmid. The effects of pheW$^+$ gene on the plasmid stability and the expression level of trip$^+$ gene in LC901-pheS$^{-ts}$ strain were investigated. The proportion of Trip$^+$ colonies among LC901-pheS$^{-ts}$ strain carrying plasmid pLTP24 was 99%, whereas that of LC901 strain carrying plasmid pLTW24 was 7% at the end of 20 generations. After 100 generations of growth, the strain LC901-pheS$^{-ts}$ carrying plasmid pLTP24 showed little loss of plasmids. While the majority of plasmid pLTW24 in LC901 strain were lost in the same period. The activities of tryptophan synthetase (T. Sase) and anthranilate synthetase (A. Sase) in LC901 strain carrying pLTW24 were about 1.2 times and 1.8 times respectively of those in LC901-pheS$^{-ts}$ strain carrying pLTP24.

• Journal of Applied Biological Chemistry
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• v.48 no.3
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• pp.133-137
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• 2005

In vitro antimutagenicity of Bacillus natto islolated from Natto, Japanese traditional fermented food, was investigated using umu-test. Mutagenicity of S9-activated metabolites of Trp-P-2 and IQ for Salmonella typhimurium TA 1535/pSK1002 was remarkably inhibited by addition of bacterial cells and their cytoplasmic fraction. Desmutagenicity by cytoplasmic fraction increased with increasing concentration of the fraction. Bioantimutagenic effect of cytoplasm on Salmonella typhimurium SD-100 did not show bioantimutagenic activity against mutated bacterial cells induced by Trp-P-2. Cytoplasmic fraction exhibited 17% bioantimutagenicity due to desmutation caused by IQ.

• Korean journal of food and cookery science
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• v.14 no.5
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• pp.468-474
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• 1998

This study was performed to determine the effects of antimutagenicity from Barley (Hordeum vulgare). In Salmonella typhimurium reversion assay (In vitro test), the extract of Barley (Hordeum vulgare) inhibited mutagenic activity of 4-NQO and Trp-p-1 with 59 mix. in Salmonella typhimurium TA98 and TA100. In Micronucleus test (In vivo test), the methanol extract of Barley (Hordeum vulgare) inhibited micronucleus formation in bone marrow by cyclophosphamide. The $\beta$-glucan of Barley (Hordeum vulgare) showed inhibitory effects of 59-77% in mutagenic activity of 4-NQO by Salmonella typhimurium TA100. The mutagenicity of Trp-p-1 with S9 mix. by Salmonella typhimurium TA98 showed inhibitory effects of 24-56%. The methanl extract (M) was fractionated with ether (MI), ethylacetate (M2), buthanol (M3) and water (M4). The Antimutagenicity of Trp-p-1 with 59 mix. by Salmonella typhimurium TA98 in Barley fraction showed the following: methanol extract (99.58%)>ether fraction (98.05%)>buthanol fraction (56.90%)>water fraction (56.72%)>ethyl acetate fraction (28.72%). Among them, ether fraction in TA 98 showed strong antimutagenicity effects (85.56%, 98.05%) against mutation induced by 4-NQO and Trp-p-1. As concentration of the methanol extract increased (1.25~5 g/kg/10 cc), micronucleus formation in bone marrow by chemical mutagen (CP) showed inhibitory effects of 50% (p< 0.05).

• Journal of the Korean Society of Food Science and Nutrition
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• v.40 no.1
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• pp.37-46
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• 2011

Polyphenolic content and antimutagenicity of the methanol extracts prepared from 22 cultivars of sweet potato with different flesh colors were investigated using Folin-Ciocalteu's phenol reagent method and Ames test, respectively. There was a remarkable cultivar difference in the polyphenolic content of sweet potato. Su, Hayanmi and Shinhwangmi among 17 cultivars of non-purple sweet potato had higher polyphenolic contents of 21.4, 21.5 and $20.3{\mu}g$ (GAE/g dried sweet potato), respectively, whereas Manami and Yeonhwangmi were very much lower at 4.6 and $4.8{\mu}g$. Mokpo No.62, Borami, Sinjami, Jami and Ayamurasaki had much higher polyphenolic contents of 67.7, 76.9, 44.9, 128.3 and $93.2{\mu}g$, respectively, than non-purple sweet potato. The methanol extract from the sweet potato effectively inhibited the reverse mutation induced by 1-NP, daunomycin, Trp-P-1, Trp-P-2 and 2-AA on S. Typhimurium TA 98, and by 1-NP on S. Typhimurium TA 100. These results suggest that the antimutagencity properties may be influenced by the tested mutagen and strain rather than the polyphenolic content of non-purple and purple sweet potato. However, in the purple sweet potatoes, a high polyphenolic content may influence the antimutagencity properties.

• Bulletin of the Korean Chemical Society
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• v.34 no.2
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• pp.600-608
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• 2013

Amide analogs of tridecapeptide ${\alpha}$-factor (WHWLQLKPGQPMYCONH$_2$) of Saccharomyces cerevisiae, in which Trp at position 1 and 3 were replaced with other residues, were synthesized to ascertain whether cooperative interactions between two Trp residues occurred upon binding with its receptor. Analogs containing Ala or Aib at position 3 of the peptide $[Ala_3]{\alpha}$-factor amide (2) and $[Aib_3]{\alpha}$-factor amide (5) exhibited greater decreases in bioactivity than analogs with same residue at position one $[Ala^1]{\alpha}$-factor amide (1) and $[Aib^1]{\alpha}$-factor amide (4), reflecting that $Trp^3$ may plays more important role than $Trp^1$ for agonist activity. Analogs containing Ala or Aib in both position one and three 3, 6 exhibited complete loss of bioactivity, emphasizing both the essential role and the combined role of two indole rings for triggering cell signaling. In contrast, double substituted analog with D-Trp in both positions 9 exhibited greater activity than single substituted analog with D-Trp 8 or deleted analog 7, reflecting the combined contribution of two tryptophane residues of ${\alpha}$-factor ligand to activation of Ste2p through interaction with residue $Tyr^{266}$ and importance of the proper parallel orientation of two indole rings for efficient triggering of signal G protein coupled activation. Among ten amide analogs, $[Ala^{1,3}]{\alpha}$-factor amide (3), $[Aib^{1,3}]{\alpha}$-factor amide (6), [D-$Trp^3]{\alpha}$-factor amide (8) and [des-$Trp^1,Phe^3]{\alpha}$-factor amide (10) were found to have antagonistic activity. Analogs 3 and 6 showed greater antagonistic activity than analogs 8 and 10.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.48 no.1
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• pp.11-24
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• 2022

It has been reported that the ethanolic extract of the root of Piper methysticum (P. methysticum) inhibits melanogenesis in melanocyte stimulating hormone (MSH)-activated B16 melanoma cells. Flavokawain B (FKB) and Flavokawain C (FKC) isolated from this extract have been found to inhibit melanin production based on anti-melanogenesis activity. This study was designed to find out the inhibition and its process of FKB and FKC on melanin synthesis in melan-a melanocytes. FKB and FKC inhibited melanogenesis at 10 μM, 5 μM respectively in melan-a melanocytes. However, they did not inhibit extracellular tyrosinase activity from melan-a melanocytes. FKB reduced the protein level of tyrosinase (Tyr), tyrosinase-related protein 1 (TRP-1), tyrosinase-related protein 2 (TRP-2), microphthalmia-associated transcription factor (MITF) and the mRNA level of Tyr and TRP-1. FKC reduced the protein level of TRP-2 and MITF and the mRNA level of TRP-1 and Tyr. The reduced expression of Tyr and TRP-1 might be resulted from the decreased MITF which regulates major melanogenic proteins. However, since the mRNA expression of MITF did not change by FKB and FKC treatment, the effects of FKB and FKC on extracellular signal regulating kinase (ERK)/AKT phosphorylation, known to regulate the degradation of MITF, were confirmed. FKB and FKC significantly increased the phosphorylation of ERK1/2, not in AKT. These results suggest that FKB and FKC may be helpful as a potential depigmenting agent for various hyper-pigmentary disorders.

• Korean Journal of Veterinary Research
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• v.58 no.1
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• pp.1-7
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• 2018

Genus Artemisia occurs as a hardy plant and has a wide range of culinary and medicinal features. In this study, we aimed to describe the melanin inhibitory activity of one Artemisia species, i.e., Artemisia capillaris Thunb. Ethanol extracts of fermented Artemisia capillaris (Art.EtOH.FT) and non-fermented Artemisia capillaris (Art.EtOH.CT) were tested for their ability to inhibit tyrosinase activity and melanin pigmentation. Both extracts showed dose-dependent inhibition against ${\alpha}$-melanocyte stimulating hormone-stimulated melanin formation and tyrosinase activity, without cytotoxicity. At $100{\mu}g/mL$, both extracts showed greater inhibition than kojic acid, the positive control. Protein expressions of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2) at the transcriptional level were determined by using real-time and semi-quantitative polymerase chain reaction. To complete the mechanistic study, presences of upstream elements of MITF, the phosphorylated-extracellular signal-regulated kinase (p-ERK), and phosphorylated-mitogen-activated protein kinase kinase (p-MEK) were confirmed by using western blot analysis. Expressions of p-TYR, p-TRP-1 and p-TRP-2, downstream factors for p-ERK and p-MITF, were translationally inhibited by both extracts. Art.EtOH.FT induced more potent effects than Art.EtOH.CT, especially signal transduction effects. In summary, Artemisia capillaris extracts appear to act as potent hypopigmentation agents.

• Journal of Life Science
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• v.27 no.12
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• pp.1403-1409
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• 2017

In this study, the xylitol dehydrogenase (XYL2) gene was expressed in Saccharomyces cerevisiae as a host cell for ease of use in the degradation of lignocellulosic biomass (xylose). To select suitable expression systems for the S.XYL2 gene from S. cerevisiae and the P.XYL2 gene from Pichia stipitis, $pGMF{\alpha}-S.XYL2$, $pGMF{\alpha}-P.XYL2$, $pAMF{\alpha}-S.XYL2$ and $pAMF{\alpha}-P.XYL2$ plasmids with the GAL10 promoter and ADH1 promoter, respectively, were constructed. The mating factor ${\alpha}$ ($MF{\alpha}$) signal sequence was also connected to each promoter to allow secretion. Each plasmid was transformed into S. cerevisiae $SEY2102{\Delta}trp1$ strain and the xylitol dehydrogenase activity was investigated. The GAL10 promoter proved more suitable than the ADH1 promoter for expression of the XYL2 gene, and the xylitol dehydrogenase activity from P. stipitis was twice that from S. cerevisiae. The xylitol dehydrogenase showed $NAD^+$-dependent activity and about 77% of the recombinant xylitol dehydrogenase was secreted into the periplasmic space of the $SEY2102{\Delta}trp1/pGMF{\alpha}-P.XYL2$ strain. The xylitol dehydrogenase activity was increased by up to 41% when a glucose/xylose mixture was supplied as a carbon source, rather than glucose alone. The expression system and culture conditions optimized in this study resulted in large amounts of xylitol dehydrogenase using S. cerevisiae as the host strain, indicating the potential of this expression system for use in bioethanol production and industrial applications.