• Korean Journal of Microbiology
• /
• v.41 no.4
• /
• pp.275-280
• /
• 2005

Anthrax is an infectious disease caused by the gram-positive bacterium, Bacillus anthracis. Anthrax toxin is a tripartite toxin comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the receptor-binding component, which facilitates the entry of LF or EF onto the cytosol. LF is a zinc-dependent metalloprotease, which is a critical virulence factor in cytotoxicity of infected animals. Therefore, it is of interest to develop its potent inhibitors for the neutralization of anthrax toxin. The first step to identify the inhibitors is the development of a rapid, sensitive, and simple assay method with a high-throughput ability. Much efforts have been concentrated on the preparation of powerful assays and on the screening of inhibitors using these system. In the present study, we have tried to construct anthrax lethal factor in yeast expression system to prepare cell-based high-throughput assay system. Here, we have shown the results covering the construction of a new vector system, subcloning of LF gene, and the expression of target gene. Our results are first trial to express LF gene in eukaryote and provide the basic steps in design of cell-based assay system.

• Journal of Oral Medicine and Pain
• /
• v.44 no.1
• /
• pp.11-15
• /
• 2019

Purpose: The aim of this study was to compare the potency-stabilizing effects of two different diluents of botulinum toxin A (10% dextrose solution and 0.9% saline). Methods: A mouse lethality bioassay was undertaken. Ninety mice were divided into experimental and control groups which received varying dosages in subgroups of 10. The experimental group was injected with botulinum toxin A diluted with 10% dextrose solution and the control group was injected with botulinum toxin A diluted with 0.9% saline. A 72 hours after intraperitoneal injection, the number of dead mice was counted to confirm median lethal dose ($LD_{50}$) of each group. Results: The value of $LD_{50}$ in the experimental group was approximately 0.131 mL (1.31 U) and the value of $LD_{50}$ in the control group was approximately 0.107 mL (1.07 U). The potency preservation rate of the experimental group was estimated to be 93.5% and that of the control group was estimated to be 76.3%. Conclusions: Dilution with 10% dextrose solution displayed less potency loss than 0.9% saline.

• Korean Journal of Veterinary Research
• /
• v.36 no.4
• /
• pp.829-837
• /
• 1996

Clostridium perfringens has been identified as a causative organism in necrotic enteritis in chicken. The bacterium has been classified into five toxigenic types (A through E) based on the pattern of the production of major lethal toxins. Seroneutralization with mice or guinea pigs usually has been used to type the organism. Of the types, types A and C of the bacterium had been recognized as the major pathogenic types in chicken. In this experiment, we isolated nine field strains of C perfringens from chicken showing necrotic enteritis in clinical symptoms and pathologic findings and identified by biochemical tests. In order to type the organism, a multiplex polymerase chain reaction (PCR) was used with primers on major lethal toxin genes instead of seroneutralization. Amplification of only a toxin gene with the PCR suggested that the disease in chicken was due to type A of C perfringens in Korea. Furthermore, the PCR method can be replaced with seroneutralization to type C perfringens in future.

• Korean Journal of Veterinary Research
• /
• v.43 no.1
• /
• pp.57-66
• /
• 2003

Single and 28-day repeated dose toxicity studies of botulimnn toxin type A were carried out in ICR mice and Sprague-Dawley rats, respectively. In the single dose toxicity study, botulinwn toxin was injected intraperitoneally to male and female mice at a single dose of 40, 59, 89 133 and 200 ng/10 ml saline/kg. All animals died from 59 ng/kg group. Some clinical signs, such as decrease in locomotor activity, dyspnea, prone position and ptosis, were observed in most of both sexes from 59 ng/kg group, but no signs were seen in all animals at 40 ng/kg group. The results showed that the median lethal dose of botulinum toxin might be in the range of 40-59 ng/kg in both sexes. In the repeated dose toxicity study, the test material was administered intradermally for 28 days at doses of 0 (vehicle-treated control), 1.25, 2.5, 5.0 and $10.0ng/head/50{\mu}{\ell}$ saline in male and female rats. No test material-related changes were noted in survivals, clinical signs, food and water consumptions and gross finding in any group. Botulinum toxin treatment significantly decreased the body weight gain rate in male of 5.0 ng/head group and over and in female of 10.0 ng/head group compared to vehicle-treated control. One or more relative organ weights (i.e., spleen, thymus, liver and kidney) were increased significantly from 5.0 ng/head group compared to vehicle-treated control in both sexes. Serum biochemistry revealed increases in aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine phosphokinase, total protein and albumin in male, and increases in AST and ALT and decreases in $K^+$ and $Cl^-$ in female without dose-pendent manners. In the histopathological study, physical stimulation by needle caused slight inflammations of dennis. In addition, botulinum toxin treatment induced denervation of nerve cell and disuse of muscle, resulting in atrophy of skeletal muscle in both sexes from 2.5 ng/head group. When the antibodies to toxin were determined in all animals, a significant increase in serum antibodies was observed from 5.0 ng/head group. The results showed that the NOAEL of botulinum toxin might be 1.25 ng/head for 28-day repeated dose toxicity in rats.

• Korean Journal of Microbiology
• /
• v.41 no.4
• /
• pp.262-268
• /
• 2005

Intoxication of murine macrophages (RAW 264.7) with the anthrax lethal toxin (LeTx 100 ng/ml) results in profound alterations in the host cell gene expression. The role of LeTx in mediating these effects is unknown, largely due to the difficulty in identifying and assigning function to individual proteins. In this study, we have used two-dimensional polyacrylamide gel electrophoresis to analyze the protein profile of murine macrophages treated with the LeTx, and have coupled this to protein identification using MALDI-TOF mass spectrometry. Interpretation of the peptide mass fingerprint data has relied primarily on the ProFound database. Among the differentially expressed spots, cleaved mitogen-activated protein kinase kinase (Mek1) and glucose-6-phosphate dehydrogenase were increased in the LeTx treated macrophages. Mek1 acts as a negative element in the signal transduction pathway, and G6PD plays the role for the protection of the cells from the hyper-production of active oxygen. Our results suggest that this proteomic approach is a useful tool to study protein expression in intoxicated macrophages and will contribute to the identification of a putative substrate for LeTx.

• Archives of Pharmacal Research
• /
• v.16 no.1
• /
• pp.36-42
• /
• 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
• /
• v.16 no.11
• /
• pp.1784-1790
• /
• 2006

Bacillus anthracis is a causative agent of anthrax. Anthrax toxins are composed of a protective antigen (PA), lethal factor (LF), and edema factor (EF), in which the PA is a central mediator for the delivery of the two enzymatic moieties LF and EF. Therefore, the PA has been an attractive target in the prevention and vaccinization for anthrax toxin. Recently, it has been reported that the molecule consisting of multiple copies of PA-binding peptide, covalently linked to a flexible polymer backbone, blocked intoxification of anthrax toxin in an animal model. In the present study, we have screened novel diverse peptides that bind to PA with a high affinity (picomolar range) from an M13 peptide display library and characterized the binding regions of the peptides. Our works provide a basis to develop novel potent inhibitors or diagnostic probes with a diverse polyvalence.

• Journal of Life Science
• /
• v.27 no.1
• /
• pp.67-71
• /
• 2017

Foulbrood disease is infected by Paenibacillus larvae on larval stage of honeybee, and is lethal disease to result in population death. This disease was manifested in 2008 in Korea, is still suffered by the secondary damages. In this study, we are to examine diagnostic PCR approaches to manage the Foulbrood disease. PCR amplification of 16S rRNA is generally using for microbial infection, but the specificity is little poor for the correct diagnosis. Therefore, we are to identify specific genes expressed in Paenibacillus larvae, and perform PCR analysis. We selected five distinct genes from literature references. Those genes are commonly known as toxic genes for host infection, and include Toxin1, Toxin2A & 2B, SplA, CBP49, and SevA&SevB. PCR amplification for these genes is difficult to detect at the first time. So, we performed the second PCR using the first PCR product as a template. This approach using the nested PCR was very useful for detecting large marker genes. When Paenibacillus larvae was cultured in the medium containing plant extracts, PCR amplification of the identified genes is correlated with the microbial growth inhibition. Therefore, these results suggest that the identified genes might be useful to study diagnostic PCR markers for honeybee Foulbrood disease.

• Microbiology and Biotechnology Letters
• /
• v.16 no.5
• /
• pp.389-392
• /
• 1988

A clone pSL 2-1, which is a recombinant plasmid believed to contain the mosquitocidal crystal-line protein gene of the Bacillus sphaericus 1593, was expressed in Escherichia coli JM83 and the product of the clone was purified and identified. The unsolubilized mosquitocidal crystal proteins from the B. sphaericus had formed 43, 58, 64, 100, 113, and 130 Kd bands in the SDS-polyacrylamide gel, but the NaOH-solublized proteins at pH 12 formed 2 protein bands of 43- and 64Kd in the gel because the larger protein (precursor) bands were cleaved. The products of the pSL 2-1 clone was purified by Sephadex G-200 and only the fractions having lethal activity to the 3rd in-star larvae of mosquito Culex pipiens were analyzed by the gel. The only single protein band of 42 Kd toxic to the larvae was formed. The major toxic protein being produced from the B. sphaericus 1593 and the pSL 2-1 clone was found to be the 42 Kd.

• The Korean Journal of Mycology
• /
• v.12 no.3
• /
• pp.117-119
• /
• 1984

To find toxic components in Korean higher fungi, the carpophores of Naematoloma fasciculare which had caused several cases of lethal intoxication were examined for toxicity. The components of high molecular weight were separated by ethanol precipitation and dialysis from the aqueous extract of the carpophores. After the components were freeze-dried, a brown powder was obtained. When a dose of 60mg/kg of this macromolecular fraction was intraperitoneally injected into mice, the mice began to die in six days and a half of them died within seven days. This toxic component was named nematoxin after the genus name of the mushroom.