• Archives of Pharmacal Research
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• v.29 no.12
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• pp.1164-1170
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• 2006

A triptolide-lysozyme (TP-LZM) conjugate was synthesized to achieve renal specific delivery and to reduce the side effects of triptolide. Triptolide was coupled to lysozyme through succinic via an ester bond with an average coupling degree of 1 mol triptolide per 1 mol lysozyme. The lysozyme can specifically accumulate in the proximal tubular cells of the kidney, making it a potential carrier for targeting drugs to the kidney. The structure of triptolide succinate (TPS) was confirmed by IR, $^{1}H-NMR$, MS and UV. The concentrations of triptolide in various samples were determined by reversed-phase high-performance liquid chromatography (HPLC). In this study, the physicochemical and stability profiles of TP-LZM under various conditions were investgated the stability and releasing profiles of triptolide-lysozyme (TP-LZM) under various conditions. In vitro release trails showed triptolide-lysozyme was relatively stable in plasma (less than 30% of free triptolide released) and could release triptolide quickly in lysosome (more than 80% of free triptolide released) at $37^{\circ}C$ for 24 h. In addition, the biological activities of the conjugate on normal rat kidney proximal tubular cells (NRK52E) were also tested. The conjugate can effectively reduce NO production in the medium of NRK52E induced by lipopolysaccharide (LPS) but with much lower toxicity. These studies suggest the possibility to promote curative effect and reduce its extra-renal toxicity of triptolide by TP-LZM conjugate.

• Journal of Life Science
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• v.28 no.3
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• pp.300-306
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• 2018

Lysozymes are innate immune factors that play a critical role in the defense against pathogens in various invertebrate animals including spoon worms. In this study, an invertebrate-type lysozyme was isolated from the body wall of spoon worm, Urechis unicinctus. The acidified body wall extract was partially separated using a Sep-Pak C18 cartridge. Among the fractions, the materials that were eluted with 60% methanol/0.1% trifluoroacetic acid showed the most potent antimicrobial activity against Bacillus subtilis KCTC 1021. A series of high performance liquid chromatography (HPLC) steps were then utilized to isolate a single antimicrobial absorbance peak. The molecular weight of the antimicrobial peak was approximated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which was approximately 13 to 14 kDa. The partial primary structure of this antimicrobial protein that was analyzed, using LC-MS/MS, was CTGGRPPTCEDYAK (1611.69 Da). Homology search of these fourteen residues, using the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST), revealed that the isolated protein was similar to the invertebrate-type lysozymes described in other animals. Then, the antimicrobial and lysozyme enzymatic (muramidase) activities of this protein were assessed. The isolated protein possessed antimicrobial activity and potent muramidase activity, which were comparable to those of hen egg white lysozyme. Therefore, the isolated protein was designated as Urechis unicinctus invertebrate-type lysozyme from the body wall, Uu-iLysb.

• Korean Journal of Food Science and Technology
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• v.28 no.3
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• pp.399-404
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• 1996

Encapsulation of lysozyme using lecithin vesicles and its stimilated release properties were studied. Lecithin vesicles were prepared by the dehydration-rehydration (DR)method. The highest encapsulation efficiency (EE) value of 80.1% was obtained by sonicating the multilamellar vesicles (MLVs) at 100 KHz for 120 min in bath sonicator. The value of entrapment progressively increased with the concentration of lysozyme, while the EE value decreased with the increase of enzyme concentration up to 50mg per 100mg per 100mg of lecithin, and then became nearly constant. At the pH of 5.9, only a small amount of lysozyme was released from DR vesicles during incubation at $37^{\circ}C$ As the pH decreased to 3.0, lysozyme was released more rapidly. Lysozyme release was accelerated for 24h and reached a plateau after 72h incubation with 1% Tween 80, $Ca^{2+}$ gave a pulse-like-release in the first hour, which was followed by a slow release.

• KSBB Journal
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• v.14 no.3
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• pp.279-283
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• 1999

We have compared the breakthrough behavior of lysozyme contained in fresh han egg white on various cation exchagers, and the adsorbent, known by the trade name Cellufine C-200 (Amicon), has shown the best performance. The effects of ion strength, pH, and linear flow rate on the breakthrough behavior were examined using the Cellufine C-200 adsorbent. The optimal conductivity, pH and linear flow rate were determined from the breakthrough behavior and found to be 2.75 mS/cm, 7.0, and 0.635 cm/min, respectively.

• Radiation Oncology Journal
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• v.35 no.3
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• pp.281-288
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• 2017

Purpose: The serum carcinoembryonic antigen (CEA) level has been recognized as a prognostic factor in colorectal cancer, and associated with response of rectal cancer to radiotherapy. This study aimed to identify CEA-interacting proteins in colon cancer cells and observe post-irradiation changes in their expression. Materials and Methods: CEA expression in colon cancer cells was examined by Western blot analysis. Using an anti-CEA antibody or IgG as a negative control, immunoprecipitation was performed in colon cancer cell lysates. CEA and IgG immunoprecipitates were used for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Proteins identified in the CEA immunoprecipitates but not in the IgG immunoprecipitates were selected as CEA-interacting proteins. After radiation treatment, changes in expression of CEA-interacting proteins were monitored by Western blot analysis. Results: CEA expression was higher in SNU-81 cells compared with LoVo cells. The membrane localization of CEA limited the immunoprecipitation results and thus the number of CEA-interacting proteins identified. Only the Ras-related protein Rab-6B and lysozyme C were identified as CEA-interacting proteins in LoVo and SNU-81 cells, respectively. Lysozyme C was detected only in SNU-81, and CEA expression was differently regulated in two cell lines; it was down-regulated in LoVo but up-regulated in SNU-81 in radiation dosage-dependent manner. Conclusion: CEA-mediated radiation response appears to vary, depending on the characteristics of individual cancer cells. The lysozyme C and Rab subfamily proteins may play a role in the link between CEA and tumor response to radiation, although further studies are needed to clarify functional roles of the identified proteins.

• Journal of the Korean Dietetic Association
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• v.24 no.4
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• pp.330-343
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• 2018

Recently, there has been a growing demand for natural preservatives because of increased consumer interest in health. In this study, we produced Lactobacillus rhamnosus cell-free supernatant (LCFS) and evaluated and compared its antimicrobial activity with existing natural preservatives against pathogenic microorganisms and in chicken breast meat contaminated with Escherichia coli and Staphylococcus aureus. Lactobacillus rhamnosus cell-free supernatant possessed 30 units of lysozyme activity and contained 18,835 mg/L of lactic acid, 2,051 mg/L of citric acid and 5,060 mg/L of acetic acid. Additionally, LCFS inhibited the growth of fourteen pathogenic bacteria, S. aureus, Bacillus cereus, Listeria monocytogenes, Vibrio parahaemolyticus, Listeria innocua, S. epidermidis, L. ivanovii, E. coli, Pseudomonas aeruginosa, Shigella sonnei, Shi. flexneri, Proteus vulgaris, Pseudomonas fluorescens, and Klebsiella pneumoniae. The antibacterial activity of LCFS was stronger than that of egg white lysozyme (EWL), Durafresh (DF) and grapefruit seed extract (GSE). Additionally, LCFS maintained its antimicrobial activity after heat treatment at $50^{\circ}C{\sim}95^{\circ}C$ and at pH values of 3~9. Moreover, LCFS inhibited the growth of E. coli and S. aureus in chicken breast meat. In conclusion, it is expected that LCFS, which contains both lysozyme and three organic acids, will be useful as a good natural preservative in the food industry.

• The Journal of the Korean Society for Microbiology
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• v.12 no.1
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• pp.19-32
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• 1977

The specificity of the N- and C-terminal antigenic determinant($P_{17}$: sequence $Lys^1-{cys-}^6-Asn^{27},\;{Trp^{12}}_2-Cys^{127}-Leu^{129}$) of hen egg-white lysozyme(HL) was studied in more detail. In a Scatchard plot of the binding of $^{14}C$-acetyl HL with guinea pig purified anti-$P_{17}$ antibody experimental values bent sharply aear r=1. This suggests of two antibody populations with different affinities for HL or possible steric hindrance in the binding of a second HL molecule to the second binding site of the antibody molecule. The antigenic activities of various peptides were tested by measuring their inhibition of the binding of $^{14}C-acetyl-P_{17}$ with the antibody, Only $P_{17}$ and $P_{17}t$(sequence $Lys^1-cys^6-Homoser^{12},\;Trp^{123}-Cys^{127}-Leu^{128})$) were inhibitory, with $K_1$ values of $2.0{\times}10^4$ and $8.1{\times}10^3$, respectively. These results indicate that the direct binding site of $P_{17}$ to anti-$P_{17}$ antibody may be located in the terminal portion of $P_{17}$ (sequence $Lys^1-Cys^6-Homoser^{12},\;Trp^{123}-Cys^{127}-Leu^{129})$) while the rest of $P_{17}$ may be important in maintaining the conformation of this determinant. The single disulphide bond involved in this determinant is essential for manifestation of immunological activity.

• Korean Journal of Food Science and Technology
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• v.23 no.3
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• pp.366-369
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• 1991

In order to elucidate texturization mechanism of extrudated protein, egg white lysozyme was heated under high pressure conditions, and its solubility and changes of molecular weight were investigated. Under high pressure conditions of $100,\;300\;and\;600\;kg/cm^2$, solubility decreased gradually with increasing temperature in the samples heated at $70,\;120\;and\;150^{\circ}C$ and decreased notably with increasing pressure at $200^{\circ}C$. Polymerization was found in the samples heated at $150\;and\;200^{\circ}C$ while a band which located below monomer(low-molecular) could be recognized. Molecular weight of the low-molecular was estimated to be about $6,000{\sim}9,000$ and no smaller peptide was recognized. The polymerization may have occured by disulfide crosslinking in the samples heated at $120^{\circ}C$ but other crosslinking may have played a role in those at $150\;and\;200^{\circ}C$.

• Animal cells and systems
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• v.15 no.1
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• pp.29-36
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• 2011

Insect lysozymes are basic, cationic proteins synthesized in fat body and hemocytes in response to bacterial infections and depolymerize the bacterial cell wall. The c-type lysozyme of the insect Spodoptera litura (SLLyz) is a single polypeptide chain of 121 residues with four disulfide bridges and 17 rare codons and is approximately 15 kDa. The full-length SLLyz cDNA is 1039 bp long with a poly(A) tail, and contains an open reading frame of 426 bp long (including the termination codon), flanked by a 54 bp long 5' UTR and a 559 bp long 3' UTR. As a host for the production of high-level recombinant proteins, E. coli is used most commonly because of its low cost and short generation time. However, the soluble expression of heterologous proteins in E. coli is not trivial, especially for disulfide-bonded proteins. In order to prevent inclusion body formation, GST was selected as a fusion partner to enhance the solubility of recombinant protein, and fused to the amplified products encoding mature SLLyz. The expression vector pGEX-4T-1/rSLLyz was then transformed into E. coli BL21(DE3)pLysS for soluble expression of rSLLyz, and the soluble fusion protein was purified successfully. Inhibition zone assay demonstrated that rSLLyz showed antibacterial activity against B. megaterium. These results demonstrate that the GST fusion expression system in E. coli described in this study is efficient and inexpensive in producing a disulfide-bonded rSLLyz in soluble, active form, and suggest that the insect lysozyme is an interesting system for future structural and functional studies.

• Korean Journal of Microbiology
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• v.28 no.1
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• pp.47-54
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• 1990

In order to investigate physiological characteristics of fusants by interspecific protoplast fusion of the genus Cellulomonas, protoplasts of Cellulomonas flavigena NCIB 12901 and Cellulomonas bibula NCIB 8142 were fused and cell wall regenerated. To give gene maker, C. bibula was treated with 500 ug/ml NTG for 1 hr and arginine requiring auxotrophic mutants were isolated. Protoplasts of the genus Cellulomonas were obtained by treatment with $600{\mu}{\textrm{g}}$/ml lysozyme, and 0.5M sorbitol was optimal for osmotic stabilizer on protoplast fromation. Protoplast fusion was enhanced by 40% PEG)M.W.6,000) containing 25 mM $CaCl_{2}$ at $30^{\circ}C$ for 30 min and fusion frequency between C. bibula and C. flavigena was $5\times 10^{-4}$. Processes of protoplast formation, cell wall regeneration and protoplast fusion were obsdrved by scanning electron microscope. By comparing enzyme activities of cellulase, exocellobiohydrolase, .betha.-glucosidase of the parent strains of Cellulomonas with those of thier mutants and fusants, fusants with increased enzyme activity were obtained. By the studies on nutritional requirement, antibiotic resistance, cellulolytic enzyme activities, type of peptidoglycan and motility of two mutants and fusants, fusants were proved to be recombinant of both mutant strains.