• Journal of the Korean Chemical Society
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• v.59 no.1
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• pp.22-30
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• 2015

The effect of diphtheria toxin on cell membrane lipids was studied by examining the phospholipase D (PLD) activity and free fatty acids (FFA) release in HepG2 cells. The diphtheria toxin effects on lipid alteration show apparently maximal at pH 5.1, stimulating PLD activity nearly 3.5 fold and enhancing FFA release approximately 5 fold over the control. These results indicate that the membrane is perturbed and its lipid component is rearranged during the diphtheria toxin translocation. Digitonin, a random membrane perturbing detergent, exhibit about four-fold higher perturbation effect over the diphtheria toxin at neutral pH. This observation suggests that the membrane perturbation induced by diphtheria toxin appears to be rather selective. To investigate the cause of the membrane perturbation, Cibacron blue, an inhibitor of membrane pore formation, and hemagglutinin, an influenza virus with fusion peptide, were tested for their effects on diphtheria toxin action. Cibacron blue decreased the diphtheria toxin effect by almost 50%, but the lipid alteration induced by hemagglutinin was similar to the diphtheria toxin effect. These observations imply that the membrane perturbation induced by diphtheria toxin may be caused by a combination of pore formation and insertion of hydrophobic peptide of toxin to the membrane as well. Additionally, we found that the diphtheria toxin increased the HepG2 cells permeability but the cells viability was maintained at high level at the same time. DNA fragmentation which is related to apoptosis was not induced by the toxin. Under these conditions, we could demonstrate that the lipid alteration of HepG2 cells was brought about by diphtheria toxin at acidic pH.

• KSBB Journal
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• v.14 no.2
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• pp.241-247
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• 1999

Experimental studies were carried out to optimize the culture conditions of Corynebacterium diphtheriae for the production of diphtheria toxin. A new media which does not contain any meat digest products was selected. The main ingredient of new medium was enzymatic digests of casein known as NZ-Case. In fermenter experiments, the toxin production was increased with the increase of cell growth. The optimum initial pH of media, air flow rate and agitation speed were 7.0, 0.22, vvm and 400 rpm, respectively. The contents of iron and calcium-phosphate precipitate were important for maximal cell growth and toxin production. The optimum concentration of iron was 0.3 mg/L and calcium-phosphate precipitate could serve in gradual supply of iron to maintain the optimal culture condition which is required for enhanced yield of toxin production. In potency test, the potency of toxoid from fermentor culture was higher than that from static culture. When diphtheria toxin is produced by fermentor culture, it is possible to produce higher levels of toxin and better toxoid quality in terms of safety, yield, productivity and immunity.

• Microbiology and Biotechnology Letters
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• v.32 no.1
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• pp.11-15
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• 2004

Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst-stage embryos that can be propagated indefinitely and, at the same time, can be differentiated into all the cell types that constitute the body. Current research using ES cells is mainly focused on the efficient generation of specific cell types by employing optimal differentiation conditions, which often requires the genetic manipulation of ES cells. As a way of developing an efficient system to regulate foreign gene expression in ES cells, we have inserted the gene encoding Corynebacterium diphtheria toxin-A (DTA) into an autonomously induced plasmid under positive doxycycline control ('Tet-on' system). In this study, we demonstrate that this system can lead to the cell death of mouse ES cells by the induction of DTA expression when exposed to the tetracycline derivative, doxycycline. MTT assay showed that this induction resulted in the apoptosis of ES cells.

• Development and Reproduction
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• v.16 no.3
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• pp.155-168
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• 2012

Therapeutic cancer is a long lasting and turbulent history accompany with the milestones in surgical intervention, chemotherapy and radiotherapy. In the past decade, however, metastatic cancer still obstinately exists challenging the professional scientist. Beside the major forms of cancer treatment, Diphtheria toxin (DT) which is produced by a pathogenic strain of bacterium Corynebacterium diphtheria to shield themselves against the other dangerous organism, have been researched as a potential candidate to overcome the drawback such as non-specific, non-effect to drug resistant cancer cell and side effects when using chemotherapy and radiotherapy. In the context of suicide gene therapy, the DT expression under controlling of tissue-specific promoter will be targeted in cancer cell but defect in normal cell. The molecular mechanism, characteristic of DT-bases therapy and prominent achievements of preclinical and clinical studies for the past decade are summarized and discussed in this review.

• KSBB Journal
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• v.26 no.6
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• pp.491-504
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• 2011

This review article provides an overview of the evolution of diphtheria vaccine, its value and its future. Diphtheria is an infectious illness caused by diphtheria toxin produced by pathogenic strains of Corynebacterium diphtheriae. It is characterized by a sore throat with membrane formation due to local tissue necrosis, which can lead to fatal airway obstruction; neural and cardiac damage are other common complications. Diphtheria vaccine was first brought to market in the 1920s, following the discovery that diphtheria toxin can be detoxified using formalin. However, conventional formalin-inactivated toxoid vaccines have some fundamental limitations. Innovative technologies and approaches with the potential to overcome these limitations are discussed in this paper. These include genetic inactivation of diphtheria toxoid, innovative vaccine delivery systems, new adjuvants (both TLR-independent and TLR-dependent adjuvants), and heat- and freeze-stable agents, as well as novel platforms for producing improved conventional vaccine, DNA vaccine, transcutaneous (microneedle-mediated) vaccine, oral vaccine and edible vaccine expressed in transgenic plants. These innovations target improvements in vaccine quality (efficacy, safety, stability and consistency), ease of use and/or thermal stability. Their successful development and use should help to increase global diphtheria vaccine coverage.

• KSBB Journal
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• v.14 no.2
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• pp.248-254
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• 1999

Adverse reactions after injection of diphtheria vaccine are induced by impurities present in crude toxoids that cannot be removed completely by purification of toxoids after formalization. To increase toxoid purity, toxin purification was tried before formalization. Crude toxin was purified with ultrafiltration and ion-exchange chromatography. Purified toxin purity was improved 2.9 times higher than crude toxin, and purity was 2,560 Lf/mg PN. Purified toxin was detoxified with formalin and lysine, and potency test were performed. Toxoid, prepared from toxin treated with formalin and lysine, did not show reversion to toxin and purity was higher than the toxoid purified after formalization. Therefore, we concluded that the use of toxoid vaccine prepared from toxin purified is a useful method of minimize adverse reaction after injection of diphtheria vaccine.

• BMB Reports
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• v.30 no.2
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• pp.144-149
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• 1997

An expression vector was constructed containing the gene encoding diphtheria toxin A (DTA) which was placed after a T7 promoter. Cytoplasmic expression of the DTA gene resulted in the formation of an insoluble inclusion body. The inclusion body was collected after the complete lysis of the cell, and subsequent washing with 0.1% Triton X-100 released 16~30% of DTA protein from the inclusion body along with other contaminating proteins. The released DTA protein was purified by dialysis. The remaining pellet was dissolved in 8 M urea containing 5% ${\beta}-mercaptoethanol$, and the denatured DTA was renatured by the dilution-dialysis method. The total yield was 35%, and about 5 mg DTA was obtained from 1 L culture. The DTA protein has a free sulfhydryl group exposed to the protein surface, and was shown to have a tendency to dimerize through disulfide formation in the absence of ${\beta}-mercaptoethanol$. The utility of the sulfhydryl group was tested for the construction of recombinant toxins.

• Clinical and Experimental Vaccine Research
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• v.11 no.1
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• pp.12-29
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• 2022

Purpose: In the present study, whole diphtheria toxin (dt) and fragment B (dtb) genes from Corynebacterium diphtheriae Park William were cloned into Escherichia coli, the purified expressed proteins were evaluated for ultimately using as a candidate vaccine. Materials and Methods: The dt and dtb genes were isolated from bacterial strain ATCC (American Type Culture Collection) no. 13812. Plasmid pET29a+ was extracted by DNA-spin TM plasmid purification kit where genes were inserted using BamHI and HindIII-HF. Cloned pET29a+dt and pET29a+dtb plasmids were transformed into E. coli BL21(DE3)PlysS as expression host. The identity of the sequences was validated by blasting the sequence (BLASTn) against all the reported nucleotide sequences in the NCBI (National Center for Biotechnology Information) GenBank. Production of proteins in high yield by different types and parameters of fermentation to determine optimal conditions. Lastly, the purified concentrated rdtx and rdtb were injected to BALB/c mice and antibody titers were detected. Results: The genetic transformation of E. coli DH5α and E. coli BL21 with the pET-29a(+) carrying the dt and dtb genes was confirmed by colony polymerase chain reaction assay and were positive to grow on Luria-Bertani/kanamycin medium. The open reading frame of dt and dtb sequences consisted of 1,600 bp and 1,000 bp, were found to be 100% identical to dt and dtb sequence of C. diphtheriae (accession number KX702999.1 and KX702993.1) respectively. The optimal condition for high cell density is fed-batch fermentation production to express the rdtx and rdtb at 280 and 240 Lf/mL, dissolved oxygen was about 24% and 22% and the dry cell weight of bacteria was 2.41 g/L and 2.18 g/L, respectively. Conclusion: This study concluded with success in preparing genetically modified two strains for the production of a diphtheria vaccine, and to reach ideal production conditions to achieve the highest productivity.

• Korean Journal of Animal Reproduction
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• v.27 no.3
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• pp.225-231
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• 2003

Transgenic mice containing Diphtheria Toxin-A (DT-A) gene fused to proximal lck promoter sequences was used for analysis of DT-A gene expression and thymocyte development. The diphtheria toxin gene was expressed in thymus, spleen and liver of transgenic mice confirmed by RT-PCR and Northern blotting. A FACS analysis with thymocyte cell surface antigens antibodies (CD4 and CD8) showed that the number of peripheral mature single positive thymocytes ($CD4^{+}\;and\;CD8^{+}$ cells) T-cells was severely reduced in transgenic mice compared to that in the non-transgenic littermates. A relative portion of $CD8^{+}$ single positive thymocytes was about 33.2% in transgenic peripheral T-cells while 50.6% in wild type. Reduction of $CD4^{+}$ cell numbers in transgenic mice was observed (5.9% in transgenic versus 10.3% in non-transgenic). The data from analysis of these transgenic mice indicate that the proximal lck promoter regulated the expression of DT-A gene at high level in developing thymocytes and the DT-A disrupted developing thymocytes in transgenic mice.

• Horticultural Science & Technology
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• v.17 no.4
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• pp.473-475
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• 1999

This study was carried out to restore the fertility by suppression of male sterility-induced gene using an antisense construct. Tobacco (cv. Petit Havana SR1) was transformed with the binary vector containing a GBAN215-6 promoter, an antisense diphtheria toxin (DTx-A) gene (pKDA215b) and a hygromycin resistant gene. Seventy-six confirmed transgenic plants regenerated from leaf disks were designated as the $R_0$ generation and selfed to produce the $R_1$ generation. From the inheritance study, five $R_1$ lines with multiple copies of the antisense construct were selected and selfed to identify homozygosity for the antisense construct. In order to restore fertility and finally to select restore lines, five $R_2$ lines with multiple copies of the antisense construct were crossed with male sterile plants. From these crosses, three different phenotypes have been observed: completely restored, partially restored, and not restored pollens, and otherwise tobacco plants were phenotypically same as normal plants. These plants were scored for the degree of restoration and selected for further study.