• Journal of Veterinary Clinics
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• v.28 no.2
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• pp.196-203
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• 2011

Staphylococcus spp. is one of the most common bacteria isolated from the lesions of atopic dermatitis (AD) in humans, and their colonization is known to be a possible trigger factor of clinical signs. The aim of this study was to determine the prevalence of Staphylococcus spp. in canine AD (CAD), the types of exotoxins present, and their relation with the clinical severity of CAD. From 79 dogs with AD, 72 samples of Staphylococcus spp. were isolated (91.1%), and 65 (90.3%) were confirmed as Staphylococcus pseudintermedius. Concerning the profile of the exotoxin gene, 50 isolates (69.4%) contained at least one exotoxin gene, and 28 isolates (56%) were found to contain more than 2 different exotoxins. There was a significant difference in clinical severity with the presence of staphylococcal exotoxins (P=0.028), whereas no correlation was found with the presence of Staphylococcus spp. (P=0.598). The clinical severity of CAD increased only in relation to staphylococcal enterotoxin D (SED) and exfoliative toxins (P<0.05). Some clinical evaluation criteria (erythema, papule/pustule) were correlated with the presence of the exotoxin gene (P<0.05). This study showed that the high prevalence of Staphylococcus spp. and staphylococcal exotoxins in lesions from dogs with AD may be regarded as an important trigger factor for exacerbation of the clinical signs of CAD.

• Applied Biological Chemistry
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• v.19 no.4
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• pp.189-201
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• 1976

In an effort to develop a microbial in secticide, B. thdringiensis var. thuringiensis was cultured in the medium composed of cocoon-cooked water from a filature. The results obtained are summarized as followss : (1) Bacillus thuringiensis is a bacterium producing a ${\delta}-endotoxin$ especially toxic to lepidopterous insects and a thermostable exotoxin harmful to dipterous insects. (2) With a view to utilizing the cocoon-cooked water discarded from the filature, as a nutrient source in the B. thuingiensis culture, it was analyzed to contain large amounts of various minerals and protein (7.5 mg/ml) believed to be extracted from the pupae. (3) A large amount of the ${\delta}-endotoxin$ can be obtained most cheeply by using cocoon-cooked water instead of distilled water in preparing GYS and citrate salts media. (4) The largest amount of a mixture of the vegetative cells, spores, and crystals was obtained by addition of 8 gr/l of glucose to the GYS medium. (5) The growth of the bacterium was far better, when leucine, isoleucine, and valine were added all together to the citrate salts medium to the concentration of $1.25{\times}10^{-3}M$. (6) The best growth was observed by addition of Na-glutamate to the citrate salts medium to the concentration of $2.5{\times}10^{-3}M$. (7) The optimal culture time ranged from 9 to 15 days. (8) The highest mortality was shown in Pieris rapae Linne with a pH of the total body extract of 8.4, whereas Dendrolimus spectabilis Butler and Bombyx mori Linne with lower pH's were less susceptible to the ${\delta}-endotoxin$. (9) The presence of the thermo stable exotoxin was confirmed by the fact that the supernatant of the culture was very toxic to the Drosophila melanogaster tested.

• BMB Reports
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• v.52 no.8
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• pp.496-501
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• 2019

Conventionally, immunotoxins have been produced as a single polypeptide from fused genes of an antibody fragment and a toxin. In this study, we adopted a unique approach of chemical conjugation of a toxin protein and an antibody fragment. The two genes were separately expressed in Escherichia coli and purified to high levels of purity. The two purified proteins were conjugated using a chemical linker. The advantage of this approach is its ability to overcome the problem of low recombinant immunotoxin production observed in some immunotoxins. Another advantage is that various combinations of immunotoxins can be prepared with fewer efforts, because the chemical conjugation of components is relatively simpler than the processes involved in cloning, expression, and purification of multiple immunotoxins. As a proof of concept, the scFv of trastuzumab and the PE24 fragment of Pseudomonas exotoxin A were separately produced using E. coli and then chemically crosslinked. The new immunotoxin was tested on four breast cancer cell lines variably expressing HER2. The chemically crosslinked immunotoxin exhibited cytotoxicity in proportion to the expression level of HER2. In conclusion, the present study revealed an alternative method of generating an immunotoxin that could effectively reduce the viability of HER2-expressing breast cancer cells. These results suggest the effectiveness of this method of immunotoxin crosslinking as a suitable alternative for producing immunotoxins.

• Journal of environmental and Sanitary engineering
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• v.14 no.2
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• pp.32-39
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• 1999

The following is experimental result of selecting soil bacteria showing toxicity against Root-knot nematode (Meloidogyne hapla). Out of 286 strains isolated from soil, one(NC67) showing toxicity against M.hapla is selected The selected strain(NC67) is identified of B. thuringiensis subsp. indiana. It proved out that the toxic maerial against M. hapla produce by NC67 strain is an exotoxin. The result of examining the existence of the extercellular toxicity product by the toxic strain(NC67) by usign activated carbon column chromatography, Dowex 50W column chromatography and TLC of silical gel etc. proved out that it is a single material.

• Korean Journal of Veterinary Research
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• v.43 no.2
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• pp.247-253
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• 2003

Actinobacillus pleuropneumoniae causes a highly contagious pleuropneumoniae in swine. The bacterium produces several virulence factors such as exotoxin, LPS, capsular polysaccharide, etc. Among them, the exotoxin, called Apx, has been focused as the major virulence factor, and the toxin consists of 4 gene cluster. apx CABD. apxA is the structural gene of toxin and has four different types, I, II, III, and IV. As the first step of development of a new subunit vaccine, the three different types of apxA gene were amplified from A. pleuropneumoniae isolated from Korea by PCR with primer designed based on the N- and C-terminal of the toxin. The sizes of apxIA, IIA and IIIA were 3,073, 2,971 and 3,159bps, respectively. The comparison of whole DNA sequences of apxIA, IIA and IIIA genes with those of the reference strain demonstrated 98%, 99% and 98% homology, respectively. In addition, the phylogenetic analysis was performed based on the amino acid sequences compared with 12 different RTX toxin family using the neighbor-joining method. ApxA proteins of Korean isolates were identical with reference strains in this study. All ApxA proteins were expressed in E. coli with pQE expression vector and identified using Western blot with polyclonal antibodies against culture supernatants of A. pleuropneumoniae serotype 2 or 5. The sizes of each expressed ApxA protein were about 120, 110, 125 kDa (M.W.), respectively. The results obtained in this study could be used for the future study to develop a new vaccine to porcine pleuropneumoniae.

• Journal of Microbiology and Biotechnology
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• v.18 no.8
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• pp.1475-1481
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• 2008

B3 antibody specifically binds the $Lewis^Y$-related carbohydrate antigen of many carcinomas, and it is used as a model antibody in this study. In a previous study, the Fab fragment of the antibody was fused to a 38 kDa truncated form of Pseudomonas exotoxin A, PE38, to make Fab-PE38, where PE38 is fused to the Fd fragment of the Fab domain. This parent monomer molecule, Fab-PE38, had no cysteine in the hinge region, and it could not make a disulfide bond to form a disulfide bond bridged homodimer. In this study, we constructed three different kinds of divalent Fab-toxin fusion homodimers where the toxin is fused to the light chain of Fab, $(Fab-PE38fl)_2$. In addition to the PE38 toxin fused to the light chain, these three molecules have different hinge sequences hi, h2, and h3 making Fabh1-, Fabh2-, and Fabh3-PE38fl monomers, respectively. These hinges contain only one cysteine on different positions of the hinge sequence. The disulfide bond between the hinge region of two monomers forms homodimers $(Fabh1-PE38fl)_2$, $(Fabh2-PE38fl)_2$, and $(Fabh3-PE38fl)_2$. The refolding yields of these dimers were 5-16-fold higher than a previously constructed dimer where the PE38 was fused to the Fd fragment $(Fabh2-PE38)_2$ [8]. Our data suggest that the steric repulsion between the two PE38s in $(Fabh1-PE38)_2$ during disulfide bridge formation is relieved by fusing it at the end of the light chain. The best cytotoxicity value of these dimers showed about 2.5-fold higher on an MCF7 cell line than that of the monovalent reference molecule in ng/ml scale, which is 15-fold higher in pM scale.

• Journal of Life Science
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• v.9 no.1
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• pp.106-110
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• 1999

Lipopolysaccharide(LPS) from Vibrio vulnificus was purified, the fatty acid composition was analyzed, and Limulus gelation activity and lethal toxic activity were tested in order to investigate the cause of cytotoxicity by V. vutnificus. These results were compared to those of Escherichia coli LPS and Salmonella typhimurium LPS. LPS from V. vulnificus had a different fatty acid composition from those of E coli and S. typhimurium. The major fatty arid from each LPS was lauric acid for E. coli, rapric acid for S. typhimurium, and myristic acid for V. vulnificus. The Limulus gelation activities of three LPSs were the same(0.1ng/ml) and the lethal toxicity in BALB/c mouse of V vulnificus LPS was similar to those of E. coli LPS and S. typhimurium LPS. Such factor as exotoxin need to be considered to be the cause of cytotoxicity by V. vulnificus LPS.

• Journal of Life Science
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• v.18 no.3
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• pp.298-303
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• 2008

Using a phage peptide library approach, we have isolated a peptide ligand that binds to transferrin receptor on the surface of human melanoma cell, B16F10. The library was first screened twice by recovering internalized phages and was further screened three times by competitively eluting transferrin receptor-specific phages with human transferrin among the phages bound to the cell surface. The peptides displayed by the selected phages were fused to translocation and catalytic domain of Pseudomonas exotoxin to prepare recombinant toxins. After estimating cytotoxicity of each recombinant toxin toward B16F10 cell, seven clones were selected. Sequence analysis revealed that one of the clones displayed a peptide which had a significant sequence homology with human transferrin. The peptide was chemically synthesized and was shown to be functional in delivering cytotoxic agents into B16F10 cell via interaction with transferrin receptor.

• Bulletin of the Korean Chemical Society
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• v.22 no.12
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• pp.1361-1365
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• 2001

Recombinant immunotoxins are hybrid cytotoxic proteins designed to selectively kill cancer cells. A divalent immunotoxins, [B3(FabH1)-PE38]2, was constructed by recombining Fab domain of B3 antibody as a cell-targeting domain and Pseudo monas exotoxin A (PE) as a cytotoxic domain. Monoclonal antibody, B3, is the murine antibody (IgG1k) directed against Lewis Y-related carbohydrate antigens, which are abundant on the surface of many carcinomas. Fab fragment of this antibody was used in this study with the modified hinge sequence where last two cysteines out of three were mutated to serine. PE is a 66 kDa bacterial toxin that kills eukaryotic cells by inhibiting protein synthesis with ADP ribosylation of ribosomal elongation factor 2 (EF2). Fc region of B3 antibody was substituted with the truncated form of PE (38 kDa, PE38) on DNA level. [B3(FabH1)-PE38]2 was formed by disulfide bond between cysteines in the modified hinge region of B3(FabH1)-PE38. Each polypeptide for recombinant immunotoxins was overexpressed in Escherichia coli and collected as inclusion bodies. Each inclusion body was solubilized and refolded, and cytotoxic effects were measured. Divalent immunotoxins, [B3(FabH1)-PE38]2, had ID50 values of about 10 ng/mL on A431 cell lines and about 4 ng/mL on CRL1739 cell lines. Control immunotoxins, B3(scFv)-PE40, had ID50 values of about 28 ng/mL on A431 cell lines and about 41 ng/mL on CRL1739 cell lines. Divalent immunotoxins, [B3(FabH1)-PE38]2, had higher cytotoxic effects than B3(scFv)-PE40 control immunotoxins.

• 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.