• Journal of Microbiology and Biotechnology
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• v.19 no.1
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• pp.108-112
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• 2009

A simplified method for the purification of cholera toxin was developed. The 569B strain of Vibrio cholerae, a recognized hyper-producer of cholera toxin, was propagated in a bioreactor under conditions that promote the production of the toxin. The toxin was separated from the bacterial cells using 0.2-${\mu}m$ crossflow microfiltration, the clarified toxin was passed through the membrane into the permeate, and the bacterial cells were retained in the retentate. The 0.2-${\mu}m$ permeate was then concentrated 3-fold and diafiltered against 10 mM phosphate buffer, pH 7.6, using 30-kDa crossflow ultrafiltration. The concentrated toxin was loaded onto a cation exchange column, the toxin was bound to the column, and most of the impurities were passed unimpeded through the column. The toxin was eluted with a salt gradient of phosphate buffer, pH 7.0, containing 1.0 M NaCl. The peak containing the toxin was assayed for cholera toxin and protein and the purity was determined to be 92%. The toxin peak had a low endotoxin level of $3.1\;EU/{\mu}g$ of toxin. The purified toxin was used to prepare antiserum against whole toxin, which was used in a $G_{M1}$ ganglioside-binding ELISA to determine residual levels of toxin in an oral inactivated whole-cell cholera vaccine. The $G_{M1}$ ganglioside-binding ELISA was shown to be very sensitive and capable of detecting as little as 1 ng/ml of cholera toxin.

• The Korean Journal of Zoology
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• v.29 no.3
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• pp.181-189
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• 1986

The present study was undertaken to investigate whether the known adenylate cyclase activators, forskolin and cholera toxin, would affect the germinal vesicle breakdown (GVBD) and the production of cAMP in mouse oocytes in vitro. To do this, in vitro oocyte culture method and adenylate cyclase assay were employed. In response to different concentrations of forskolin (20 to 80 $\\mu$g/ml) added to a culture medium, the percentage of GVBD significantly decreased (56 to 31%) in a dose-dependent manner as compared to that of control (63%). This inhibitory phenomenon by forskolin was reversible since the rate of GVBD was returned to the control level when the oocytes were transferred to a control medium following exposure to forskolin (80 $\\mu$g/ml). Treatment of cholera toxin (10 to 1, 000 ng/ml) was, however, ineffective in suppressing GVBD. When forskolin (10 to 80 $\\mu$g/ml) was added to the mouse oocyte extracts, cAMP production significantly increased by 5 to 18 fold, whereas cholera toxin (10 to 1, 000 ng/ml) was no longer effective. In addition, treatment of guanidyl-imidodiphosphate (GppNHp, 100 $\\mu$M), which is an activator of the regulatory unit of adenylate cycleas, with forskolin did not exhibit any changes in cAMP production as compared to that induced by forskolin alone. Neither cholera toxin nor cholera toxin plus GppNHp (100 $\\mu$M) exhibited any differences in mouse oocytes. From the above results, the suppression of GVBD by forskolin may be mediated by a high level of intracellular cAMP in mouse oocytes. It appears that the changes in intracellular cAMP level may an important role in the mouse oocyte maturation.

• Asian-Australasian Journal of Animal Sciences
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• v.6 no.4
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• pp.483-489
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• 1993

Bovine fibroblasts were cultured in Dulbecco's Modified Eagle's Medium and then treated with control, insulin (I, $1{\mu}g/ml$), cholera toxin (CT, 0.1-100 ng/ml) or CT (0.1-100 ng/ml) + I ($1{\mu}g/ml$). Cholera toxin, an activator of adenylate cyclase, significantly decreased insulin induced DNA synthesis (p<0.05). The modulation of DNA synthesis apparently involves events occurring in early stage of cell growth, at least between the first 4 and 8 hour of CT treatment. Insulin induced collagen as well as noncollagen synthesis in cell layer, however, these syntheses were reduced by addition of cholera toxin (p<0.05) but were not completely reduced. It is not clear whether the reduction of insulin-induced cell layer collagen or noncollagen proteins by CT is involved in the inhibitory effect on insulin-induced DNA synthesis. However, we could rule out the hypothesis that insulin-induced DNA synthesis is reduced by CT-induced cellular differentiation.

• Toxicological Research
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• v.4 no.1
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• pp.47-53
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• 1988

Cholera toxin and dibutyryl cyclic adenosine 3', 5'-monophosphate(db-cAMP) increased the rate and number of infections units produced in the in vitro reactivation of latent herpes simplex virus, whereas adenosine diminished them. cAMP concentration in latently infected trigeminal ganglia of mice was greatly increased by cholera toxin but was not affected by adenosine.

• Journal of Microbiology and Biotechnology
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• v.24 no.6
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• pp.725-731
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• 2014

The classical biotype strains of the Vibrio cholerae O1 serogroup harbor the biotype-specific cholera-toxin encoding phage (CTX) $CTX^{cla}$, and the El Tor biotype strains contain CTX-1. Although the classical biotype strains have become extinct, a remnant of classical CTX phage is transferred to the El Tor biotype strains. The prototype El Tor strains, which produce the biotype-specific cholera toxin, are now being replaced by atypical El Tor variant strains producing classical biotype cholera toxin. The genome sequences of the CTX phages in atypical El Tor strains indicate that the CTX phages in atypical El Tor strains are a mosaic of $CTX^{cla}$ and CTX-1. Before the emergence of atypical El Tor stains in the early 1990s, unusual pre-seventh pandemic strains were isolated in the US Gulf Coast between 1973 and 1986. These strains have characteristics of atypical El Tor strains since they are El Tor biotype strains containing $CTX^{cla}$, yet the genome sequence of this CTX phage indicates that it is different from $CTX^{cla}$ and is therefore classified separately as $CTX^{US\;Gulf}$.

• Journal of Nutrition and Health
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• v.34 no.2
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• pp.150-157
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• 2001

Intestinal absorption of dietary taurine is one of the regulatory component maintaining taurine homeostasis along with renal reabsorption, bile acid conjugation and secretion, and de nobo synthesis of taurine in mammals. Recent observations of decreased enterocytic levels of taurine in response to trauma, infection and surgical insults, postulate the possibility that intestinal taurine absorption might be impaired in such stressed conditions. The aim of the present study was to evaluate changes in enterocytic taurine transporter activity using the human intestinal colon carcinoma cell line, HT-29, in various stress-induced conditions. Pretreatment of the HT-29 cells with dexamethasone, a stress hormone(0.1,1,10 or 100$\mu$M) for 3 hrs, or with E coli heat-stable enterotoxin(10, 100, or 200nM) for 30 minutes in order to induce the condition of enterotoxigenic infection did not influence taurine uptake as compared to the value found in control cells. In contrast, pretreatment of the cells with cholera toxin(10, 100, 500, or 1000ng/ml)for 3hr or 24hr significantly decreased taurine uptake by HT-29 cells to 40~50% of the value found in untreated control cells. Kinetic studies of the taurine transporter activity were conducted in control and cholera toxin treated HT-29 cells with varying taurine concentrations(2~60$\mu$M) in the uptake medium. Pretreatment of the cells with cholera toxin(100ng/ml) for 3hr did not influence the Vmax, but resulted in a 55% increase in the Michaelis-Menten constant(Km) of the taurine transporter compared to those in control cells. These results suggest that cholera toxin-induced reduction in taurine transporter activity in HT-29 cells is associated with decreased affinity of the taurine transporter without altering the amount of transporter protein. Intestinal taurine absorption appears to be reduced in the condition of water-borne diseases caused by bacteria such as V. cholerae. This might influence the taurine status of infants and young children more readily, an age group in which the prevalence of intestinal infection is high and the role of intestinal absorption is crucial for maintaining the body taurine pool. (Korean J Nutrition 34(2) : 150-157, 2001)

• Journal of Plant Biology
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• v.37 no.4
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• pp.429-434
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• 1994

식물 호르몬의 하나인 앱시스산이 식물의 기공을 닫게 하는 과정 중에 phospholipase C가 활성화되어 inositol 1,4,5-trisphosphate(P3)의 양이 증가함이 보고되었다 (Cot and Crain. 1994). 그러나 아직까지 공변세포에서 phospholipase C의 활성을 조절하는 G-단백질에 대한 보고는 없었다. 그러므로 앱시스산에 의한 기공닫힘과정에 G-단백질이 수반되는지를 조사하고자, G-단백질 활성의 저해제인 pertussis toxin과 촉진제인 cholera toxin을 처리하여 보았다. 닭의장풀(Commelina communis L.)의 잎 뒷면으로부터 얻은 온전한 표피층과 잠두(Vicia faba L)의 잎을 부분 분해하여 공변세포만을 남긴 표피층에 pertussis toxin을 처리하였을 때, 앱시스산에 의한 기공닫힘이 부분적으로 억제됨을 관찰하였다. 그러나 cholera toxin의 경우는 아무런 영향이 없었다. 공변세포만을 지닌 표피층에 pertussis toxin을 전처리한 후 앱시스산을 가했을 때, 앱시스산에 의한 IP3 양의 증가 양상이 억제됨을 확인하였다. 이러한 결과들로부터 앱시스산에 의한 기공닫힘과정에는 pertussis toxin-sensitive, phospholipase C-linked G-protein이 관여하고 있음을 알 수 있었다.

• Journal of Microbiology and Biotechnology
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• v.32 no.11
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• pp.1396-1405
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• 2022

Cholera remains a major global public health problem, for which oral cholera vaccines (OCVs) being a valuable strategy. Patients, who have recovered from cholera, develop antibody responses against LPS, cholera toxin (CT), toxin-coregulated pilus (TCP) major subunit A (TcpA) and other antigens; thus, these responses are potentially important contributors to immunity against Vibrio cholerae infection. However, assessments of the efficacy of current OCVs, especially inactivated OCVs, have focused primarily on O-antigen-specific antibody responses, suggesting that more sophisticated strategies are required for inactivated OCVs to induce immune responses against TCP, CT, and other antigens. Previously, we have shown that the toxT-139F allele enables V. cholerae strains to produce CT and TCP under simple laboratory culture conditions. Thus, we hypothesized that V. cholerae strains that express TCP via the toxT-139F allele induce TCP-specific antibody responses. As anticipated, V. cholerae strains that expressed TCP through the toxT-139F allele elicited antibody responses against TCP when the inactivated bacteria were delivered via a mouse model. We have further developed TCP-expressing V. cholerae strains that have been used in inactivated OCVs and shown that they effect an antibody response against TcpA in vivo, suggesting that V. cholerae strains with the toxT-139F allele are excellent candidates for cholera vaccines.

• Journal of Microbiology and Biotechnology
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• v.33 no.6
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• pp.736-744
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• 2023

The introduction of the toxT-139F allele triggers the expression of TCP (toxin co-regulated pilus) and CT (cholera toxin) under simple laboratory culture conditions in most Vibrio cholerae strains. Such V. cholerae strains, especially strains that have been used in OCVs (oral cholera vaccines), can induce antibody responses against TCP in animal models. However, CT produced in these V. cholerae strains is secreted into the culture medium. In this study, V. cholerae strains that can express intracellular CTB under the control of the toxT-139F allele have been constructed for potential application in OCVs. First, we constructed a recombinant plasmid directly linking the ctxAB promoter to ctxB without ctxA and confirmed CTB expression from the plasmid in V. cholerae containing the toxT-139F allele. We constructed another recombinant plasmid to express NtrCTB, from which 14 internal amino acids-from the 7^th to the 20^th amino acid-of the leader peptide of CTB have been omitted, and we found that NtrCTB remained in the cells. Based on those results, we constructed V. cholerae strains in which chromosomal ctxAB is replaced by ntrctxB or ntrctxB-dimer. Both NtrCTB and NtrCTB-dimer remained in the bacterial cells, and 60% of the NtrCTB-dimer in the bacterial cells was maintained in a soluble form. To develop improved OCVs, these strains could be tested to see whether they induce immune responses against CTB in animal models.