• Korean Journal of Microbiology
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• v.30 no.3
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• pp.160-163
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• 1992

Killer toxin from killer yeast fusant FWKS 260 developed by protoplast fusion between the wild killer yeast and alcohol-fermenting yeast was purified by ammonium sulfate fractionation. Amicon PM I0 concentration. Sephadex G-200 and Scphadcx G-75 column chromatography. The purified killer toxin showed a single band by SIX-polyacvlamide gel electrophoresis. The protein part of killer toxin was active site. which was found by treating the proteolytic enzyme such as pronase E and pepsin to killer toxin. The killer toxin was stable at pH 2.0-5.0 and 20$^{\circ}$C. but inactivated with increasing temperature. The molecular weight was determined to be approximately 13.000 according to the results obtained from the SDS-polyacrylamide gel electrophoresis. It was confirmed that the purified killer toxin is glycoprotein by showing a red single band after st'tining with Schiffs reagent.

• Journal of the Korean Society of Laryngology, Phoniatrics and Logopedics
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• v.23 no.2
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• pp.93-98
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• 2012

Botulinum toxins are the most potent toxins known to mankind. Botulinum toxin acts by blocking the cholinergic neuromuscular or the cholinergic autonomic innervation of exocrine glands and smooth muscles. Seven distinct antigenic botulinum toxins (A, B, C, D, E, F and G) produced by different strains of Clostridium botulinum have been described and only A and B type of botulinum toxins were clinically used. Toxins were consisted of a heavy chain with a molecular weight of 100 kD and a light chain with a molecular weight of 50 kD. Toxins are bound with an astounding selectivity to glycoprotein structures located on the cholinergic nerve terminal. Subsequently light chain of toxin is internalized and cleaves different proteins of the acetylcholine transport protein cascade transporting the acetylcholine vesicle from the intracellular space into the synaptic cleft. After a decade of therapeutic application of the toxin, no anaphylaxis or deaths have been reported and systemic adverse effects have not been reported so far. However the toxin's immunologic properties can lead to the stimulation of antibody production, potentially rendering further treatments ineffective. Botulinum toxin is a safe and effective treatment. Use of botulinum toxin in clinical medicine has grown exponentially in recent years, and many parts of the human body are now being targeted for therapeutic purposes.

• Archives of Pharmacal Research
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• v.16 no.1
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• pp.36-42
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• 1993

To find biologically active components of the higher fungi of Korea, the carpophores of Auricularia polytricha, a well-known edible mushroom, were extracted with 0.14 M NaCl solution. The extract was successively fractionated by adding ammonium sulfate at various concentrations, and the respective precipitates were separated by centrifugation, then dialyzed and freeze-dried. When a does of 60 mg/kg of each was injected i.p. into ICR mice, the fraction which precipitated at 20% ammonium sulfate showed the highest toxicity, killing seven out of seven mice within two days. The fraction obtained at 40% ammonium sulfate showed the second highest toxicity. The two fractions were named auritoxin I and II after the genus name. However, they Nere shown to have nearly identical composition by physicochemical and 6.8% protein. The polysaccharide moiety was found to have 12.3% $\alpha$-linkage and 87.7% $\beta$-linkage and to be a heteromannoglucan consisting of 45.1% glucose, 435 mannose and 11.0% xylose. The protein moiety contained ten amino adids. The molecular weight of the toxin was $1.5\times10^6$ dalton by Sepharose CL-4B gel filtration. The modian lethal doses of auritoxin in mice were 56.4, 157.2 and 454.6 mg/kg by i.p., s.c. and p.o.administrations, respectively. The signs of intrxication were convulsion during the first 30 minutes after the injection, coma or sleeping within an hour, termor, lacrimation, nasal bleeding congestion, and death in 24 hours. Smong the various organs, the spleen was found to be enlarged remarkably. Human platelet aggregation was inhibited by the addition of auritoxin. The activity of malic dehydrogenase in vitro was inhibited by the toxin.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.490-497
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• 2004

Although the E. coli heat-labile enterotoxin B subunit (LTB) is known to be a potent mucosal adjuvant towards co-administrated unrelated antigens and immunoregulator in T-helper 1-type-mediated autoimmune diseases, a more efficient and useful LTB is still required for prospective vaccine adjuvants. To determine whether a novel chimeric LTB subunit would produce an enhanced mucosal adjuvant activity and immune response, a number of LTB subunits were genetically fused with chimeric proteins using the epitope genes of the envelope glycoprotein E2 (gp51-54) from the classical swine fever virus (CSFV). It was found that the total serum immunoglobulin (Ig) levels of BALB/c mice orally immunized with chimeric proteins containing an N-terminal linked LTB subunit (LE1, LE2, and LE3) were higher than those of mice immunized with LTB, E2 epitope, and chimeric proteins that contained a C-terminal linked LTB subunit. In particular, immunization with LE1 markedly increased both the total serum Ig and fecal IgA level compared to immunization with LTB or the E2 epitope. Accordingly, the current results demonstrated that the LTB subunit in a chimeric protein exhibited a strong mucosal adjuvant effect as a carrier molecule, while the chimeric protein containing the LTB subunit stimulated the mucosal immune system by mediating the induction of antigen-specific serum Ig and mucosal IgA. Consequently, an LE1-mediated mucosal response may contribute to the development of effective antidiarrhea vaccine adjuvants.

• IMMUNE NETWORK
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• v.12 no.6
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• pp.261-268
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• 2012

Respiratory syncytial virus (RSV) and influenza virus are the most significant pathogens causing respiratory tract diseases. Composite vaccines are useful in reducing the number of vaccination and confer protection against multiple infectious agents. In this study, we generated fusion of RSV G protein core fragment (amino acid residues 131 to 230) and influenza HA1 globular head domain (amino acid residues 62 to 284) as a dual vaccine candidate. This fusion protein, Gcf-HA1, was bacterially expressed, purified by metal resin affinity chromatography, and refolded in PBS. BALB/c mice were intranasally immunized with Gcf-HA1 in combination with a mucosal adjuvant, cholera toxin (CT). Both serum IgG and mucosal IgA responses specific to Gcf and HA1 were significantly increased in Gcf-HA1/CT-vaccinated mice. To determine the protective efficacy of Gcf-HA1/CT vaccine, immunized mice were challenged with RSV (A2 strain) or influenza virus (A/PR/8/34). Neither detectable viral replication nor pathology was observed in the lungs of the immune mice. These results demonstrate that immunity induced by intranasal Gcf-HA1/CT immunization confers complete protection against both RSV and homologous influenza virus infection, suggesting our Gcf-HA1 vaccine candidate could be further developed as a dual subunit vaccine against RSV and influenza virus.

• Biomolecules & Therapeutics
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• v.27 no.2
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• pp.152-159
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• 2019

Multiple sclerosis (MS) is an autoimmune disease characterized by progressive neuronal loss, neuroinflammation, axonal degeneration, and demyelination. Previous studies have reported that 6-shogaol, a major constituent of ginger (Zingiber officinale rhizome), and its biological metabolite, 6-paradol, have anti-inflammatory and anti-oxidative properties in the central nervous system (CNS). In the present study, we investigated whether 6-shogaol and 6-paradol could ameliorate against experimental autoimmune encephalomyelitis (EAE), a mouse model of MS elicited by myelin oligodendrocyte glycoprotein ($MOG_{35-55}$) peptide immunization with injection of pertussis toxin. Once-daily administration of 6-shogaol and 6-paradol (5 mg/kg/day, p.o.) to symptomatic EAE mice significantly alleviated clinical signs of the disease along with remyelination and reduced cell accumulation in the white matter of spinal cord. Administration of 6-shogaol and 6-paradol into EAE mice markedly reduced astrogliosis and microglial activation as key features of immune responses inside the CNS. Furthermore, administration of these two molecules significantly suppressed expression level of tumor necrosis $factor-{\alpha}$, a major proinflammatory cytokine, in EAE spinal cord. Collectively, these results demonstrate therapeutic efficacy of 6-shogaol or 6-paradol for EAE by reducing neuroinflammatory responses, further indicating the therapeutic potential of these two active ingredients of ginger for MS.

• Journal of Microbiology and Biotechnology
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• v.24 no.7
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• pp.905-913
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• 2014

Lj-RGD3, an RGD (Arg-Gly-Asp) toxin protein from the salivary gland of Lampetra japonica, exhibits antifungal activity against Candida albicans. Lj-RGD3 has three RGD motifs and shows homology to histidine-rich glycoprotein. We synthesised two mutant derivatives of Lj-RGD3: Lj-26, which lacks all three RGD motifs and contains no His residues; and Lj-112, which lacks only the three RGD motifs. We investigated the effects of the wild-type and mutated toxins on a gram-positive bacterium (Escherichia coli), a gram-negative bacterium (Staphylococcus aureus), and a fungus (C. albicans). rLj-RGD3 and its mutants exhibited antifungal but not antibacterial activity, as measured by a radial diffusion assay. The C. albicans inhibition zone induced by rLj-112 was larger than that induced by the other proteins, and its inhibitory effect on C. albicans was dose-dependent. In viable-count assays, the rLj-112 MIC was $7.7{\mu}M$, whereas the MIC of the positive control (ketoconazole) was $15{\mu}M$. Time-kill kinetics demonstrated that rLj-112 effectively killed C. albicans at $1{\times}$ and $2{\times}$ MIC within 12 and 6 h, respectively. Electron microscopy analysis showed that rLj-RGD3 and rLj-112 induced C. albicans lysis. Our results demonstrate a novel anticandidal activity for rLj-RGD3 and its mutant derivatives.

• Molecules and Cells
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• v.46 no.12
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• pp.764-777
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• 2023

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated Pseudomonas exotoxin (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in Escherichia coli, achieving high solubility and purity post-purification. In vitro cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC₅₀ values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.

• Development and Reproduction
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• v.11 no.1
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• pp.49-54
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• 2007

Ethane 1,2-dimethane sulfonate(EDS), a toxin which specifically kills Leydig cells(LC), has been widely used to prepare the reversible testosterone(T) depletion rat model. In the present study, we monitored the gene expression profiles of pituitary gonadotropins, LH and FSH, up to 7 weeks after EDS injection. Adult male Sprague-Dawley rats($300{\sim}350\;g$ B.W.) were injected with a single dose of EDS(75 mg/kg i.p.) and sacrificed on weeks 0, 1, 2, 3, 4, 5, 6 and 7. Total RNAs were purified from each pituitary, and the message levels of common alpha subunit($C{\alpha}$) of pituitary glycoprotein hormones, LH beta subunit($LH{\beta}$), FSH beta subunit($FSH{\beta}$) and GnRH receptor(GnRH-R) were evaluated by semi-quantitative RT-PCRs. The message levels of $C{\alpha}$ increased sharply during weeks 1-4, then return to the control level on week 5. The mRNA levels of $LH{\beta}$ were elevated after week 2, reached the peak at week 4, then declined to the control level after week 5. The message levels of $FSH{\beta}$ were elevated after week 2, reached the peak at week 3, then declined to the nadir at week 5. Similarly, the mRNA levels of GnRH-R were elevated after week 2, reached the peak at week 3, then gradually declined to the control level after week 5. The present study indicated that EDS treatment could induce reversible alterations in the transcriptional activities of gonadotropin subunits and GnRH-R in the anterior pituitary from male rats. EDS injection model might be useful to understand the mechanism of hormonal regulation of hypothalamus- pituitary neuroendocrine axis in male rats.