• BMB Reports
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• v.40 no.5
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• pp.723-730
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• 2007

Kinesin is an ATP-driven microtubule motor protein that plays important roles in control of microtubule dynamics, intracellular transport, cell division and signal transduction. The kinesin superfamily is composed of numerous members that are classified into 14 subfamilies. Animal kinesins have been well characterized. In contrast, plant kinesins have not yet to be characterized adequately. Here, a novel plant-specific kinesin gene, GhKCH2, has been cloned from cotton (Gossypium hirsutum) fibers and biochemically identified by prokaryotic expression, affinity purification, ATPase activity assay and microtubule-binding analysis. The putative motor domain of GhKCH2, $M_{396-734}$ corresponding to amino acids Q396-N734 was fused with 6$\times$His-tag, soluble-expressed in E. coli and affinity-purified in a large amount. The biochemical analysis demonstrated that the basal ATPase activity of $M_{396-734}$ is not activated by $Ca^{2+}$, but stimulated 30-fold max by microtubules. The enzymatic activation is microtubule-concentration-dependent, and the concentration of microtubules that corresponds to half-maximum activation was about 11 ${\mu}M$, much higher than that of other kinesins reported. The cosedimentation assay indicated that $M_{396-734}$ could bind to microtubules in vitro whenever the nucleotide AMP-PNP is present or absent. As a plant-specific microtubule-dependent kinesin with a lower microtubule-affinity and a nucleotide-independent microtubule-binding ability, cotton GhKCH2 might be involved in the function of microtubules during the deposition of cellulose microfibrils in fibers or the formation of cell wall.

• Preventive Nutrition and Food Science
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• v.22 no.3
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• pp.184-190
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• 2017

Matrix metalloproteinases (MMPs) are endopeptidases that take significant roles in extracellular matrix degradation and therefore linked to several complications such as metastasis of cancer progression, oxidative stress, and hepatic fibrosis. Hizikia fusiformis, a brown algae, was reported to possess bioactivities, including but not limited to, antiviral, antimicrobial, and anti-inflammatory partly due to bioactive polysaccharide contents. In this study, the potential of H. fusiformis against cancer cell invasion was evaluated through the MMP inhibitory effect in HT1080 fibrosarcoma cells in vitro. H. fusiformis crude extract was fractionated with organic solvents, $H_2O$, n-BuOH, 85% aqueous MeOH, and n-hexane (n-Hex). The non-toxicity of the fractions was confirmed by MTT assay. All fractions inhibited the enzymatic activities of MMP-2 and MMP-9 according to the gelatin zymography assay. Cell migration was also significantly inhibited by the n-Hex fraction. In addition, both gene and protein expressions of MMP-2 and -9, and tissue inhibitor of MMPs (TIMPs) were evaluated by reverse transcription-polymerase chain reaction and Western blotting, respectively. The fractions suppressed the mRNA and protein levels of MMP-2, MMP-9 while elevating the TIMP-1 and TIMP-2, with the $H_2O$ fraction being the least effective while n-Hex fraction the most. Collectively, the n-Hex fraction from brown algae H. fusiformis could be a potential inhibitor of MMPs, suggesting the presence of various derivatives of polysaccharides in high amounts.

• Journal of Microbiology and Biotechnology
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• v.28 no.2
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• pp.275-283
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• 2018

Ischemic stroke can result from blockage of blood vessels, forming fibrin clots in the body and causing irreparable brain damage. Remedial thrombolytic agents or anticoagulants have been studied; however, because the FDA-approved tissue plasminogen activator has low efficacy and side effects, it is necessary to develop safer and more effective treatment candidates. This study aimed at assessing the fibrinolytic and anticoagulation features of a novel serine protease extracted and purified from Diopatra sugokai, a polychaeta that inhabits tidal flats. The purified serine protease was obtained through ammonium sulfate precipitation, affinity chromatography, and ion-exchange chromatography. Its molecular size was identified via SDS-PAGE. To characterize its enzymatic activities, the protease activity at various pH and temperatures, and in the presence of various inhibitors, was measured via azocasein assay. Its fibrinolytic activity and anticoagulant effect were assessed by fibrin zymography, fibrin plate assay, and fibrinogenolytic activity assays. The novel 38 kDa serine protease had strong indirect thrombolytic activity rather than direct activity over broad pH (4-10) and temperature ($37^{\circ}C-70^{\circ}C$) ranges. In addition, the novel serine protease exhibited anticoagulant activity by degrading the ${\alpha}$-, ${\beta}$-, and ${\gamma}$-chains of fibrinogen. In addition, it did not produce cytotoxicity in endothelial cells. Therefore, this newly isolated serine protease is worthy of further investigation as a novel alkaline serine protease for thrombolytic therapy against brain ischemia.

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.652-655
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• 2005

This paper describes the development of an enzymatic assay system for the identification of inhibitors of isocitrate lyase (ICL), one of the key enzymes of the glyoxylate cycle that is considered as a new target for antifungal drugs. A 1.6 kb DNA fragment encoding the isocitrate lyase from Candida albicans ATCC10231 was amplified by PCR, cloned into a vector providing His-Patch-thioredoxin-tag at the N-terminus, expressed in Escherichia coli, and purified by metal chelate affinity chromatography. The molecular mass of the purified ICL was approximately 62 kDa, as determined by SDS-PAGE, and the enzyme activity was directly proportional to incubation time and enzyme concentration. The effects of itaconate-related compounds on ICL activity were also investigated. Among them, itaconic acid, 3-nitropropionate, and oxalate had strong inhibitory activities with $IC_{50}$ values of 5.8, 5.4 and $8.6\;{mu}g/ml$, respectively. These inhibitors also exhibited antifungal activity on YPD agar media containing acetate as a sole carbon source, albeit at high concentration. The results indicate that the C. albicans ICL may be a regulatory enzyme playing a crucial role in fungal growth and is a prime target for antifungal agents.

• Korean Journal of Food Science and Technology
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• v.32 no.3
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• pp.720-725
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• 2000

To reduce the antigenicity of egg white (EW), EW was treated with several proteolytic enzymes, trifluoromethanesulfonic acid (TFMS), heat, and NaOH. Competitive indirect enzyme-linked immunosorbent assay (ciELISA), using rabbit anti-EW antibody, was performed to examine the antigenicity of the treated EW's. Enzymatic hydrolysis gave no good effect on the reduction of the antigenicity of EW. Neither did the pretreatment with ${\gamma}-irradiation$ before the hydrolysis reduce the antigenicity. TFMS treatment removed the antigenicity of EW. The antigenicity of EW heated at $120^{\circ}C$ for 30 min or treated with NaOH at 0.3% (w/v) and more, decreased to less than 1/10,000 as compared with that of native EW. The combinatory treatment with NaOH, followed by heat at $70^{\circ}C$ for 15 min had a synergic effect on the reduction of the antigenicity of EW.

• Korean Journal of Microbiology
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• v.36 no.2
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• pp.84-90
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• 2000

Biodegradation of vanous medium-chain-length polyhydroxyalkanoates (MCL-PHAs) by an extracellular depolymerase system from Pseudomonas sp. RY-1 was investigated under laboratoly conditions. The degradation rate of the polymers was determined by quantitative clem zone technique, enzyme (turbidity) assay, and respirometry assay. Although the enzyme system secreted by Pscudomor~as sp. RY-1 was capable of degrading all MCL-PHAs tested. its secretion was influenced by the availability of secondary carbon sources. The rate of enzymatic degradation of MCL-PHAs was dependent upou the monomeric composition of the polyesters and reduced as the chain lengths of the monomer m t s in the polyesters increased. MCL-PHAs containing C-even monomer units showed faster degradation rate than MCL-PHAs containing C-odd monomer units. Respiration rates of MCL-PHAs with C-even monomer uuts were also much faster than those of MCL-PHAs with C-odd monomer units. The degmdation rate of MCL-PHAs bearing unsaturated substituents was faster than that of mcl-PHAs without functional substituents, which is suggesting the correlation between the degradation rate and the crystallinity of MCL-PHAs.

• IMMUNE NETWORK
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• v.6 no.2
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• pp.102-110
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• 2006

Background: Dendritic cells (DC) are professional antigen-presenting cells in the immune system and can induce T cell response against virus infections, microbial pathogens, and tumors. Therefore, immunization using DC loaded with tumor-associated antigens (TAAs) is a powerful method of inducing anti-tumor immunity. For induction of effective anti-tumor immunity, antigens should be efficiently introduced into DC and presented on MHC class I molecules at high levels to activate antigen-specific $CD8^+$ T cells. We have been exploring methods for loading exogenous antigens into APC with high efficiency of Ag presentation. In this study, we tested the effect of the cationic liposome (Lipofectin) for transferring and loading exogenous model antigen (OVA protein) into BM-DC. Methods: Bone marrow-derived DC (EM-DC) were incubated with OVA-Lipofectin complexes and then co-cultured with B3Z cells. B3Z activation, which is expressed as the amount of ${\beta}$-galactosidase induced by TCR stimulation, was determined by an enzymatic assay using ${\beta}$-gal assay system. C57BL/6 mice were immunized with OVA-pulsed DC to monitor the in vivo vaccination effect. After vaccination, mice were inoculated with EG7-OVA tumor cells. Results: BM-DC pulsed with OVA-Lipofectin complexes showed more efficient presentation of OVA-peptide on MHC class I molecules than soluble OVA-pulsed DC. OVA-Lipofectin complexes-pulsed DC pretreated with an inhibitor of MHC class I-mediated antigen presentation, brefeldin A, showed reduced ability in presenting OVA peptide on their surface MHC class I molecules. Finally, immunization of OVA-Lipofectin complexes-pulsed DC protected mice against subsequent tumor challenge. Conclusion: Our data provide evidence that antigen-loading into DC using Lipofectin can promote MHC class I- restricted antigen presentation. Therefore, antigen-loading into DC using Lipofectin can be one of several useful tools for achieving efficient induction of antigen-specific immunity in DC-based immunotherapy.

• Journal of Veterinary Science
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• v.25 no.2
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• pp.31.1-31.8
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• 2024

Background: Recently, there has been a growing interest in stem cells for human medicine. Limited feline endometrial mesenchymal stem cell (fEM-MSC) research in veterinary medicine necessitates reporting for future feline disease research and therapy. Objectives: This study aimed to isolate fEM-MSCs from feline endometrial tissues and evaluate their morphology, proliferative ability, differentiation ability, and immunophenotype. Methods: Feline endometrial tissues were obtained from the ovariohysterectomies of healthy cats and isolated using an enzymatic method. The morphology and proliferative ability of the isolated cells were assessed using a doubling time (DT) assay from passages 3 to 6 (P3 - P6). We measured pluripotency gene expressions of cells in P2 using quantitative real-time polymerase chain reaction (qRT-PCR). To investigate MSC characteristics, a trilineage differentiation assay was conducted in P4, and cells in P4 were immunophenotyped using flow cytometry. Results: fEM-MSCs showed a typical spindle-shaped morphology under a microscope, and the DT was maintained from P3 to P6. fEM-MSCs could differentiate into adipocytes, osteoblasts, and chondrocytes, and expressed three pluripotency markers (OCT4, SOX2, and NANOG) by qRT-PCR. Immunophenotypic analysis showed that the fEM-MSCs were CD14 ^-, CD34 ^-, CD45 ^-, CD9⁺, and CD44⁺. Conclusions: In this study, the feline endometrium was a novel source of MSCs, and to the best of our knowledge, this is the first report on the isolation method and characteristics of fEM-MSCs.

• Journal of Oral Medicine and Pain
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• v.34 no.3
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• pp.227-235
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• 2009

Many of the protective functions of saliva can be attributed to the biological, physical, structural, and rheological characteristics of salivary glycoproteins. Therefore, the development of ideal saliva substitutes requires understanding of the rheological as well as biological properties of human saliva. In the present study, we investigated the changes of salivary enzymatic activities by saliva substitutes and compared viscosity of saliva substitutes with human saliva. Five kinds of saliva substitutes such as Moi-Stir, Stoppers4, MouthKote, Saliva Orthana, and SNU were used. Lysozyme activity was determined by the turbidimetric method. Peroxidase activity was determined with an NbsSCN assay. $\alpha$-Amylase activity was determined using a chromogenic substrate, 2-chloro-p-nitrophenol linked with maltotriose. The pH values of saliva substitutes were measured and their viscosity values were measured with a cone-and-plate digital viscometer at six different shear rates. Various types of saliva substitutes affected the activities of salivary enzymes in different ways. Stoppers4 enhanced the enzymatic activities of hen egg-white lysozyme, bovine lactoperoxidase (bLP), and $\alpha$-amylase. Saliva Orthana and SNU inhibited bLP activity and enhanced $\alpha$-amylase activity. MouthKote inhibited $\alpha$-amylase activity. Moi-Stir inhibited the enzymatic activities of bLP and $\alpha$-amylase. The pH values were very different according to the types of saliva substitutes. Stoppers4, MouthKote, and Saliva Orthana showed lower values of viscosity at low shear rates and higher values of viscosity at high shear rates compared with unstimulated and stimulated whole saliva. Moi-Stir and SNU displayed much higher values of viscosity than those of natural whole saliva. Collectively, our results indicate that each saliva substitute has its own biological and rheological characteristics. Each saliva substitute affects the enzymatic activity of salivary enzyme and finally oral health in different ways.

• Korean Journal of Plant Resources
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• v.26 no.6
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• pp.678-685
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• 2013

The objective of this study was to examine the effect of 7 traditional Korean formulations (7TKFs) on intimal thickening of rat carotid artery injured by balloon catheter in vivo and on the proliferation of human smooth muscle cells (HASMCs) and secretion of matrix metalloproteinase-2 (MMP-2) in vitro. 7TKFs (400 mg/kg) were administered orally for 4 weeks from the day of balloon injury in the rats. HASMC proliferation was assessed by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) assay while enzymatic action of MMP-2 was carried out by gelatin zymography. Among 7TKFs, Samhwang-sasim-tang (SST), Banha-sasim-tang (BST) and Kegi-honghwa-tang (KHT) significantly reduced the intimal thickening by suppressing HASMC proliferation and MMP-2 expression in both extracellular and intracellular levels. Thus, the results suggest that SST, BST and KHT can be considered as a therapeutic value in the prevention of atherosclerosis because restenosis after PTCA (percutaneous transluminal coronary angioplasty) is supposed to be 'accelerated atherosclerosis'.