• The Journal of Internal Korean Medicine
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• v.27 no.4
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• pp.888-894
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• 2006

Objectives : For the purpose of developing new anti-HIV agents from natural sources, the extracts of Ricinus communis were tested for their inhibitory effects on essential enzymes reverse transcriptase (RT), protease and alpha-glucosidase. Inhibition activity of major compounds of Ricinus communis were predicted from quantitative structure activity relationships (QSAR) in silico. Methods and Results : In the anti-HIV-1 RT using enzyme-linked oligonucleotide sorbent assay (ELOSA) method, water and methanol extracts (100ug/ml) of Ricinus communis showed strong activity of 94.2% and 82.7%, respectively. In the HIV-1 protease and alpha-glucosidase inhibition assay, neither water nor methanol extracts of Ricinus communis inhibited the activity of the enzyme to cleave any substrates as oligopeptides and oligosaccharides. Conclusions : We found that for these samples it is possible that the inhibition of the RT in vitro is due to the secondary metabolites of Ricinus communis such as ricinine and quercetin. It would beof great interest to identify the compounds which are responsible for this inhibition, since all therapeutically useful agents up to date are RT inhibitors.

• Electrical & Electronic Materials
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• v.14 no.12
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• pp.3-6
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• 2001

In common globular proteins, the native form is n its most stable state. However, the native form of inhibitory serpins (serine protease inhibitors) and some viral membrane fusion proteins is in a metastable state. Metastability in these proteins is critical to their biological functions. Our previous studies revealed that unusual interactions, such as side-chain overpacking, buried polar groups, surface hydrophobic pockets, ad internal cavities are the structural basis of the native metastability. To understand the mechanism by which these structural defects regulate protein functions, cavity-filling mutations of $\alpha$1-antitrypsin, a prototype serpin, were characterized. Increasing conformational stability is correlated with decreasing inhibitory activity. Moreover, the activity loss appears to correlate with the decrease in the rate of the conformational switch during complex formation with a target protease. We also increased the stability of $\alpha$1-antitrypsin greatly via combining various stabilizing single amino acid substitutions that were distributed throughout the molecule. The results showed that a substantial increase of stability, over 13 kcal/mol, affected the inhibitory activity with a correlation of 11% activity loss per kcal/mol. The results strongly suggest that the native metastability of proteins is indeed a structural design that regulates protein functions and that the native strain of $\alpha$1-antitrypsin distributed throughout the molecule regulates the inhibitory function in a concerted manner.

• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.3
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• pp.187-191
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• 2003

Proteolytic enzymes existing in plant cell cultured media are the major reason for the loss of secreted human granulocyte-macrophage colony-stimulating factor (hGM-CSF). The addition of pepstatin, aprotinin and PMSF relatively decreased the proteolytic degradation of hGM-CSF in a conditioned medium, but sufficient prevention against the proteolytic activity could not be obtained with chemical protease inhibitors. Gelatin, as a competitive substrate for protease, showed a stabilizing effect in a conditioned medium. Compared to the initial hGM-CSF concentration in a conditioned medium. with 10 g/L of gelatin, 68% of the hGM-CSF remained after 5 days. In a cell culture experiment, 5 g/L of gelatin significantly stimulated the hGM-CSF production and accumulation in culture media, with no growth inhibition. compared to the controls (4.72 $\mu\textrm{g}$/L), the extracellular hGM-CSF level could be increased to 39.78 $\mu\textrm{g}$/L with the addition of 5 g/L of gelatin.

• Proceedings of the Korean Institute of Electrical and Electronic Material Engineers Conference
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• 2001.11a
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• pp.3-6
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• 2001

In common globular proteins, the native form is in its most stable state. However, the native form of inhibitory serpins (serine protease inhibitors) and some viral membrane fusion proteins is in a metastable state. Metastability in these Proteins is critical to their biological functions. Our previous studies revealed that unusual interactions, such as side-chain overpacking, buried polar groups, surface hydrophobic pockets, and internal cavities are the structural basis of the native metastability. To understand the mechanism by which these structural defects regulate protein functions, cavity-filling mutations of ${\alpha}$1-antitrypsin, a prototype serpin, were characterized. Increasing conformational stability is correlated with decreasing inhibitory activity. Moreover, the activity loss appears to correlate with the decrease in the rate of the conformational switch during complex formation with a target protease. We also increased the stability of ${\alpha}$1-antitrypsin greatly via combining various stabilizing single amino acid substitutions that were distributed throughout the molecule. The results showed that a substantial increase of stability, over 13 kcal/mol, affected the inhibitory activity with a correlation of 11% activity loss per kcal/mol. The results strongly suggest that the native metastability of proteins is indeed a structural design that regulates protein functions and that the native strain of e 1-antitrypsin distributed throughout the molecule regulates the inhibitory function in a concerted manner.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2020.12a
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• pp.14-14
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• 2020

In December 2019, the COVID-19 epidemic was discovered in Wuhan, China, and since has disseminated around the world impacting human health for millions. Herein, in-silico drug discovery approaches were utilized to identify potential candidates as Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) main protease (M^pro) inhibitors. We investigated several databases including natural and natural-like products (>100,000 molecules), DrugBank database (10,036 drugs), major metabolites isolated from daily used spices (32 molecules), and current clinical drug candidates for the treatment of COVID-19 (18 drugs). All tested compounds were prepared and screened using molecular docking techniques. Based on the calculated docking scores, the top ones from each project under investigation were selected and subjected to molecular dynamics (MD) simulations followed by molecular mechanics-generalized Born surface area (MM-GBSA) binding energy calculations. Combined long MD simulations and MM-GBSA calculations revealed the potent compounds with prospective binding affinities against M^pro. Structural and energetic analyses over the simulated time demonstrated the high stabilities of the selected compounds. Our results showed that 4-bis([1,3]dioxolo)pyran-5-carboxamide derivatives (natural and natural-like products database), DB02388 and Cobicistat (DB09065) (DrugBank database), salvianolic acid A (spices secondary metabolites) and TMC-310911 (clinical-trial drugs database) exhibited high binding affinities with SARS-CoV-2 M^pro. In conclusion, these compounds are up-and-coming anti-COVID-19 drug candidates that warrant further detailed in vitro and in vivo experimental estimations.

• Asian-Australasian Journal of Animal Sciences
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• v.6 no.1
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• pp.19-26
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• 1993

The effect heat treatment (autoclave) on nutritional value of winged bean as compared to soybean has been investigated. The winged bean and soybean were obtained from local cultivar grown in Indonesia. The beans were autoclaved at $120^{\circ}C$ for 15, 30, 45, 60 or 90 minutes, respectively before being ground for chemical analysis. Trypsin inhibitors of winged bean and soybean decreased (p < 0.05) along with decreasing of urease activity as heating time increased from 0 to 90 minutes. Heat treatment significantly (p < 0.05) reduced protein solubility in 0.2% potassium hydroxide of winged bean as well as soybean. In vitro protein digestibility was significantly (p < 0.05) improved by heating treatment (15 to 60 min of autoclaving), however, excessive heating (90 min of autoclaving) decreased the digestibility of winged beans. Excessive heating had adverse effect on lysine, cystine and methionine contents of winged beans. The results of this study suggested that autoclaving at $120^{\circ}C$ within 45 minutes should be adequate to remove protease inhibitors and could improve protein digestibility of winged beans.

• Journal of Microbiology and Biotechnology
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• v.27 no.12
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• pp.2190-2198
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• 2017

Thermoactinomyces sp. strain YT06 was isolated from poultry compost and observed to degrade integral chicken feathers completely at $60^{\circ}C$, resulting in the formation of 3.24 mg/ml of free amino acids from 50 ml of culture containing 10 g/l chicken feathers. Strain YT06 could grow and secrete keratinase using feather as the only carbon and nitrogen sources without other supplement, but complementation of 10 g/l sucrose and 4 g/l $NaNO_3$ increased the production of the keratinolytic enzyme. The maximum protease activity obtained was 110 U/ml and for keratinase was 42 U/ml. The keratinase maintained active status over a broad pH (pH 8-11) and temperature ($60-75^{\circ}C$). It was inhibited by serine protease inhibitors and most metal ions; however, it could be stimulated by $Mn^{2+}$ and the surfactant Tween-20. A reductive agent (${\beta}$-mercaptoethanol) was observed to cleave the disulfide bond of keratin and improve the access of the enzyme to the keratinaceous substrate. Zymogram analysis showed that strain YT06 primarily secreted keratinase with a molecular mass of approximately 35 kDa. The active band was assessed by MALDI-TOF mass spectrometry and was observed to be completely identical to an alkaline serine protease from Thermoactinomyces sp. Gus2-1. Thermoactinomyces sp. strain YT06 shows great potential as a novel candidate in enzymatic processing of hard-to-degrade proteins into high-value products, such as keratinous wastes.

• BMB Reports
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• v.40 no.4
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• pp.604-609
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• 2007

An Asp/His catalytic site of 10-formyltetrahydrofolate dehydrogenase (FDH) was suggested to have a similar catalytic topology with the Asp/His catalytic site of serine proteases. Many studies supported the hypothesis that serine protease inhibitors can bind and modulate the activity of serine proteases by binding to the catalytic site of serine proteases. To explore the possibility that soybean trypsin inhibitor (SBTI) can recognize catalytic sites of FDH and can make a stable complex, we carried out an SBTI-affinity column by using rat liver homogenate. Surprisingly, the Rat FDH molecule with two typical liver proteins, carbamoyl-phosphate synthetase 1 (CPS1) and betaine homocysteine S-methyltransferase (BHMT) were co-purified to homogeneity on SBTI-coupled Sepharose and Sephacryl S-200 followed by Superdex 200 FPLC columns. These three liver-specific proteins make a protein complex with 300 kDa molecular mass on the gel-filtration column chromatography in vitro. Immuno-precipitation experiments by using anti-FDH and anti-SBTI antibodies also supported the fact that FDH binds to SBTI in vitro and in vivo. These results demonstrate that the catalytic site of rat FDH has a similar structure with those of serine proteases. Also, the SBTI-affinity column will be useful for the purification of rat liver proteins such as FDH, CPS1 and BHMT.

• YAKHAK HOEJI
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• v.38 no.4
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• pp.440-450
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• 1994

Due to the limited bioavailability of $[D-Ala^6]$LHRH from nonparenteral transmucosal sites of administration, enhancement of mucosal permeability by coadministration of several protease inhibitors and/or penetration enhancers were studied in rabbit mucosa. As a reliable bioassay method for $[D-Ala^6]$LHRH, ovulation-inducing effect were measured after vaginal administration in the rat. The permeation of $[D-Ala^6]$LHRH through the mucosal membrane of rabbit mounted on George-Grass diffusion cells were examined in the presence of polyoxyethylene 9-lauryl ether (POE), ${\beta}$-cyclodextrin$({\beta}-CyD)$ or ethylene diamine tetra acetate disodium salt(EDTA). The vaginal membrane showed higher permeability of $[D-Ala^6]$LHRH than the rectal and nasal membrane. POE and ${\beta}-CyD$ showed a small promoting effect on the membrane permeation of $[D-Ala^6]$LHRH, but EDTA showed significant enhancement. Ovaluation was enhanced by the coadministration of sodium laurate(0.5%), a protease inhibitor but was not enhanced by EDTA, a penetration enhancer. On the other hands, coadministration of sodium tauro 24,25 dihydrofusidate(1%) and EDTA(2%) enhanced the ovulation inducing-effect 2.8 times. These results suggest that the vaginal administration of $[D-Ala^6]$LHRH with STDHF or sodium laurate as a protease inhibitor, and EDTA as a penetration enhancer, may become an elective method for transmucosal delivery of $[D-Ala^6]$ LHRH.

• Archives of Pharmacal Research
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• v.28 no.7
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• pp.816-822
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• 2005

Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gin Asp Phe Val Glu Glu lie Leu. CPM-2 was inactivated by $Cu^{2+}\;and\;Zn^{2+}$ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and ${\alpha}_1$-antitrypsin. However, EDTA, EGTA, cysteine, $\beta$-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved $A{\alpha}-$ and $B{\beta}$-chains of fibrinogen and fibrin, and y-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.