Among the exotoxins produced by V. vulnificus, hemolysin (HS) has been reported to be the most potent one. To investigate the factors up- or down-regulating HS production in the context of pathogenesis, we observed the effects of salinity or/and temperature shifting, glucose, and acidic pH on the production of HS by V. vulnificus C7184 strain in vitro. Significantly more HS was produced when V. vulnificus was cultured in 0.9% salinity and 37 degrees C than in 2.5% and 25 degrees C. When the culture condition reflecting natural habitat of V. vulnificus (2.5% salinity and 25degrees C) was changed into that reflecting human body (0.9% salinity and 37 degrees C), 2.5 fold or more HS was produced than in the V. vulnificus being cultured continuously in 0.9% NaCl at 37 degrees C. This result suggests that V. vulnificus somehow recognizes the shifting in salinity and temperature and stimulate HS production. Glucose addition in the culture medium resulted in a dose- dependent decrease in the HS production. Glucose itself and acidic pH resulting from its metabolism both appeared to inhibit the HS production. Glucose in itself had more dominant role in suppressing the HS production than the lowered pH accompanying the metabolism of glucose. This result suggests that HS production is down-regulated in the presence of glucose and under environmental acidic pH.
Ecosystem ; Exotoxins ; Glucose* ; Human Body ; Hydrogen-Ion Concentration ; Metabolism ; Salinity* ; Vibrio vulnificus* ; Vibrio* ; Virulence

IL-15 and IL-15 receptors (IL-15R) play a crucial role in the pathogenesis of adult T-cell leukemia (ATL), multiple myeloma and inflammatory autoimmune diseases. To develop a novel therapeutic agent capable of eliminating IL-15R-over-expressing abnormal cells, the gene coding for human IL-15 antagonist (IL-15M) was fused with a DNA fragment coding for the mutated form of Pseudomonas exotoxin, PEdelta293. The resulting gene fusion was cloned into pET16b under the control of T7 promoter, giving rise to the expression plasmid pET-IL15M-PEdelta293. Using Ni2+ -NTA affinity chromatography, IL15M-PEdelta293 was purified from E. coli BL21 (DE3) pLysS transformed with pET-IL15M-PEdelta293. The fusion toxin showed cytotoxicity to IL-15R-bearing myelogenous leukemia cell line K562 and K562-derived multidrug resistant cell line K562/AO2. However, IL-15R negative cell line Jurkat was insensitive to IL15M-PEdelta293. In addition, the toxic effect of IL15M-PEdelta293 on K562 was completely blocked by excessive amount of recombinant human IL-15. These results demonstrated that the selective cytotoxicity of IL15M-PEdelta293 correlated with the appropriate IL-15R expression on target cells. The present data suggest that the chimeric toxin constructed in this report may have therapeutic potential in the treatment of diseases associated with abnormal expression of IL-15/IL-15R, even in the treatment of chemotherapy refractory tumors.
Escherichia coli ; genetics ; metabolism ; Exotoxins ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; metabolism ; Humans ; Interleukin-15 ; antagonists & inhibitors ; biosynthesis ; genetics ; K562 Cells ; Pseudomonas aeruginosa ; genetics ; metabolism ; Receptors, Interleukin-15 ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

Exotoxins produced by Actinobacillus (A.) pleuropneumoniae (Apx) play major roles in the pathogenesis of pleuropneumonia in swine. This study investigated the role of ApxI in hemolysis and cellular damage using a novel apxIA mutant, ApxIA336, which was developed from the parental strain A. pleuropneumoniae serotype 10 that produces only ApxI in vitro. The genotype of ApxIA336 was confirmed by PCR, Southern blotting, and gene sequencing. Exotoxin preparation derived from ApxIA336 was analyzed for its bioactivity towards porcine erythrocytes and alveolar macrophages. Analysis results indicated that ApxIA336 contained a kanamycin-resistant cassette inserted immediately after 1005 bp of the apxIA gene. Phenotype analysis of ApxIA336 revealed no difference in the growth rate as compared to the parental strain. Meanwhile, ApxI production was abolished in the bacterial culture supernatant, i.e. exotoxin preparation. The inability of ApxIA336 to produce ApxI corresponded to the loss of hemolytic and cytotoxic bioactivity in exotoxin preparation, as demonstrated by hemolysis, lactate dehydrogenase release, mitochondrial activity, and apoptosis assays. Additionally, the virulence of ApxIA336 appeared to be attenuated by 15-fold in BALB/c mice. Collectively, ApxI, but not other components in the exotoxin preparation of A. pleuropneumoniae serotype 10, was responsible for the hemolytic and cytotoxic effects on porcine erythrocytes and alveolar macrophages.
Actinobacillus pleuropneumoniae/genetics/*pathogenicity/*physiology ; Animals ; *Apoptosis ; Bacterial Proteins/genetics/metabolism ; Blotting, Southern ; Exotoxins/*genetics ; Hemolysin Proteins/genetics/metabolism ; *Hemolysis ; Macrophages, Alveolar/metabolism/*microbiology ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Swine ; Virulence

OBJECTIVETo study inhibitory effect of recombinant transforming growth factor alpha-Pseudomonas exotoxin fusion protein (TP40) on proliferation of the human bladder cancer T24 cells.

METHODSExpression of epidermal growth factor receptor (EGFR) in cultured T24 cells was analyzed with Western blot assay. Human bladder cancer T24 cells were exposed to TP40 at 5 - 1 000 microg/L. Methyl thiazolyl tetrazolium assay was applied to evaluate the cell proliferation by measuring the absorbance (A) at 570 nm with a microplate reader. Tritium labeled thymine deoxyriboside ([(3)H]-TdR) uptake was measured to observe DNA synthesis. Competition assays were performed by the EGF at 1 - 7 500 microg/L.

RESULTSExpression of EGFR was high in human bladder cancer T24 cells. Cell growth was suppressed by 10%, 19%, 27%, 41%, 47%, 53% and 61% after 96 h treatment with TP40 at 5, 50, 100, 250, 500, 750 and 1 000 microg/L, respectively. [(3)H]-TdR incorporation was 80%, 69%, 48% and 51% after 24 h, 48 h, 72 h, 96 h treatment with TP40 at 750 microg/L, respectively. When the concentration was 1 - 7 500 microg/L, EGF could block the inhibitory effect of TP40 to some extent.

CONCLUSIONSHuman bladder cancer T24 cells express EGFR at a high level. TP40 could inhibit the growth of T24 cells effectively in a dose- and time-dependent manner. The cytotoxic effects of TP40 were specifically mediated by EGFR.

Cell Proliferation ; drug effects ; Exotoxins ; pharmacology ; Humans ; Receptor, Epidermal Growth Factor ; metabolism ; Recombinant Fusion Proteins ; pharmacology ; Transforming Growth Factor alpha ; pharmacology ; Tumor Cells, Cultured ; Urinary Bladder Neoplasms ; metabolism ; pathology

OBJECTIVEThe aim of this study was to explore the possibility of creating a toxin, C-CPE-ETA', by fusing C-terminal high affinity binding domain of CPE (C-CPE) with a truncated form of Pseudomonas aeruginosa exotoxin A (ETA') and to examine whether C-CPE-ETA' could specifically target CLDN-3, 4 molecule and the targeted toxin was cytotoxic against CLDN-3,4-overexpressing ovarian cancer.

METHODSCLDN-3 and CLDN-4 expressions were analyzed at the mRNA level in three ovarian cancer cell lines and epithelial ovarian cancer tissues from 20 patients. After transforming an expression plasmid of C-CPE-ETA' into E. coli BL21 (DE3) plysS strain, the recombinant protein was purified using His-Bind resin chromatography column and analyzed by Western blot and Coomassie blue staining. The specific binding, proapoptotic and cytolytic activities were evaluated by flow cytometry, fluorescence microscopy with the JC-1 probe and MTT assay in CLDN-3,4-overexpressing ovarian cancer cells.

RESULTSQuantitive RT-PCR results showed there existed high levels of CLDN-3 and CLDN-4 in ovarian cancer cells, CAOV3, OVCAR3 and SKOV3. Moreover, high expressions of CLDN-3 and CLDN-4 were observed in 90.0% (18/20) and 60.0% (12/20) of ovarian cancer tissues, with an expression level 10-fold higher than that in the normal ovarian tissue. A 58 000 recombinant protein C-CPE-ETA' was demonstrated by Western blot and Coomassie blue staining. Purified and recombinant C-CPE-ETA' was bound with high affinity to CLDN-3,4-overexpressing ovarian cancer cells, CAOV3, OVCAR3 and SKOV3 cells. C-CPE-ETA' was strongly proapoptotic and cytotoxic towards the CLDN-3,4-overexpressing ovarian cancer cells. The concentration of IC(50) was 7.364 ng/ml for CAOV3 cells, 8.110 ng/ml for OVCAR3 cells and 22.340 ng/ml for SKOV3 cells, respectively. However, control CLDN-3,4-deficient cell line HUVEC was not susceptible to the recombinant C-CPE-ETA' at a concentration up to 10 µg/ml.

CONCLUSIONSThe C-CPE-ETA' protein exhibits remarkably specific cytotoxicity for CLDN-3,4-overexpressing ovarian cancer cells. Its therapeutic potential warrants further development for ovarian cancer molecular targeted therapy.

ADP Ribose Transferases ; metabolism ; physiology ; Apoptosis ; Bacterial Toxins ; metabolism ; Cell Line, Tumor ; Claudin-3 ; Claudin-4 ; Claudins ; genetics ; metabolism ; Enterotoxins ; metabolism ; physiology ; Exotoxins ; metabolism ; physiology ; Female ; Humans ; Immunotoxins ; metabolism ; Ovarian Neoplasms ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Recombinant Fusion Proteins ; metabolism ; physiology ; Virulence Factors ; metabolism ; physiology

The objective was to identify some biochemical and physical properties for fusion protein IL6D24-PE40KDEL. Edman degradation, SDS-PAGE, peptide mass fingerprinting, Western blot and MTT were used for identification of the protein. The results showed that the sequence of N-terminus is Met-Ile-Asp-Lys-Gln-Ile, Met was added because of prokaryotic expression system; Western blot revealed that the purified protein could react with IL6 and PEA antibody. The purified protein IL6D24-PE40KDEL could kill the multiple myeloma cell lines U266 expressing high affinity IL6R, but it could not kill the cell lines CEM which not expressed IL6R; The molecular weight was 58.7 kD measuring by SDS-PAGE; peptide mass fingerprinting (PMF) confirmed that the construction of IL6D24-PE40KDEL was correct. A novel protein by Peptident database in EXPASY web site was identified. In conclusion, IL6D24-PE40KDEL is a new targeting protein with bioactivity of specific killing effect.
ADP Ribose Transferases ; chemistry ; metabolism ; pharmacology ; Amino Acid Sequence ; Blotting, Western ; Cell Line, Tumor ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Exotoxins ; chemistry ; metabolism ; pharmacology ; Humans ; Interleukin-6 ; chemistry ; metabolism ; pharmacology ; Molecular Sequence Data ; Pseudomonas aeruginosa ; genetics ; metabolism ; Recombinant Fusion Proteins ; chemistry ; metabolism ; pharmacology ; Sequence Analysis, Protein ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

OBJECTIVETo investigate the pro-apoptotic effect of Her-2 targeted recombinant caspase-6 fusion protein on osteosarcoma SOSP-9607 cells.

METHODSRecombinant immunocasp-6 was generated by sequential fusion of the genes of a signal peptide, a single-chain Her-2 antibody (e23sFv), a PEA translocation domain (PEA aa253-364) and an active caspase-6. The immunocasp-6 gene was cloned into pCMV plasmid to construct a kind of eukaryotic expression vector, i.e. pCMV-e23sfv-PE II-caspase-6 (abbr. pCMV-6) and transfected into SOSP-9607 cells. Murine xenograft models were randomly divided into two groups that received i.m. injections of liposome encapsulated pCMV-6 or pCMV alone. The tumor volume and weight of the nude mice and the tumor weight of the cured mice were observed and statistically analyzed. The morphological changes of the tumors were examined with HE staining, apoptotic morphology of the tumor was observed by TUNEL staining and the gene expression was analyzed by immunohistochemical staining.

RESULTSThe tumor growth of the mice in the treatment group was significantly slower than that of the control group (P = 0.001). The weight of the nude mice in the treatment group was significantly higher than that of the control group (P = 0.0002). The tumor weight of the mice in the treatment group was significantly lower than that of the control group (P = 0.0006). HE and TUNEL staining of the tumor of nude mice in the treatment groups showed typical characteristics of apoptosis, while normal structure was found in the control group. Furthermore, caspase-6 was not found in the tumor and muscle tissues in the control group, but only in the treatment group by immunohistochemistry.

CONCLUSIONImmunocasp-6 can selectively recognize and bind to and kill HER-2 positive osteosarcoma cells, therefore, to offer some foundation for the clinical treatment of osteosarcoma.

ADP Ribose Transferases ; genetics ; Animals ; Apoptosis ; Bacterial Toxins ; genetics ; Bone Neoplasms ; metabolism ; pathology ; Caspase 6 ; genetics ; metabolism ; Cell Line, Tumor ; Exotoxins ; genetics ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Osteosarcoma ; metabolism ; pathology ; Plasmids ; Random Allocation ; Receptor, ErbB-2 ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Tumor Burden ; Virulence Factors ; genetics

A recombinant immunotoxin named CEA/PE38/KDEL was constructed, which was composed of anti-CEA single-chain Fv and the truncated and modified form of Pseudomonas exotoxin (PE38/KDEL). The CEA/PE38/KDEL immunotoxin was expressed in the E. coli strain BL21 (DE3)-star as inclusion bodies. The denatured inclusion bodies were purified with Ni-NTA chelate agarose, then the constant gradient dialysis was used to perform the refolding of the CEA/PE38/KDEL immunotoxin. Results of FACS and MTT assay indicate that the refolded immunotoxins keep potent and specific cytotoxicity to tumor cells bearing CEA antigens.
ADP Ribose Transferases ; biosynthesis ; genetics ; pharmacology ; Antibodies ; genetics ; metabolism ; pharmacology ; Antineoplastic Agents ; metabolism ; pharmacology ; Bacterial Toxins ; biosynthesis ; genetics ; pharmacology ; Carcinoembryonic Antigen ; immunology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Exotoxins ; biosynthesis ; genetics ; pharmacology ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; Immunotoxins ; genetics ; isolation & purification ; metabolism ; pharmacology ; Protein Renaturation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Virulence Factors ; biosynthesis ; genetics ; pharmacology

Although pandemic community-associated (CA-) methicillin-resistant Staphylococcus aureus (MRSA) ST30 clone has successfully spread into many Asian countries, there has been no case in Korea. We report the first imported case of infection caused by this clone in a Korean traveler returning from the Philippines. A previously healthy 30-yr-old Korean woman developed a buttock carbuncle while traveling in the Philippines. After coming back to Korea, oral cephalosporin was given by a primary physician without any improvement. Abscess was drained and MRSA strain isolated from her carbuncle was molecularly characterized and it was confirmed as ST30-MRSA-IV. She was successfully treated with vancomycin and surgery. Frequent international travel and migration have increased the risk of international spread of CA-MRSA clones. The efforts to understand the changing epidemiology of CA-MRSA should be continued, and we should raise suspicion of CA-MRSA infection in travelers with skin infections returning from CA-MRSA-endemic countries.
Adult ; Bacterial Toxins/metabolism ; Carbuncle/microbiology ; Cephalosporins/therapeutic use ; Community-Acquired Infections/drug therapy/microbiology ; Exotoxins/metabolism ; Female ; Humans ; Leukocidins/metabolism ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus/*isolation & purification ; Philippines ; Republic of Korea ; Staphylococcal Skin Infections/*drug therapy/surgery ; Travel ; Vancomycin/therapeutic use

OBJECTIVETo explore the pathogenesis of the damage to peripheral nerves induced by Campylobacter jejuni exotoxin (CJT).

METHODS(1) Animal models: (1) The CJT was extracted from PEN 19-CJ and injected perineurally and intravenously to Wistar rats. (2) The sera and the supernatants of peripheral blood mononuclear cells (PBMCs), taken from the rats immunized with the CJT, were injected perineurally at sciatic nerves of experimental rats and intravenously, respectively. (2) Histopathologic study of sciatic nerves: the animals were sacrificed and their sciatic nerves were examined for tease fibers, transverse section with toluidine blues staining and electron microscopy. (3) Immunohistochemistry: sections of sciatic nerves of either normal rats or human which were incubated with CJT and the sciatic nerves with pathological changes induced by CJT were obtained for observation of the binding capability of CJT with peripheral nerves by SABC and FITC-immunofluorescence methods, and nucleic acid hybridization techniques for detection of TNF-alpha mRNA expression in pathological sciatic nerves samples.

RESULTS(1) Remarkable peripheral neuropathies with axon degeneration and/or demyelination were found in the nerves induced by both CJT injection perineurally and intravenously. The axon degeneration was more obvious. Pathological changes were identified in 76.8% (2,763/3,600) of teasing fibers after perineural injection, but only 9.6% (230/2,400) of fibers were damaged in control group (P < 0.01). The peak severity of fiber damage was found on the 3rd day after CJT intravenous injection with the incidence of abnormal fibers was 19.5% (390/2,000), and abnormalities of 15.5% (310/2000) on the 14th day. However, no abnormal changes were demonstrated in control group (P < 0.01). So was in the groups injected with anti-CJT sera and the supernatants of PBMCs compared with control (P > 0.05). (2) Binding of CJT to the nerve was found dominant in the sciatic nerves taken from normal rats or human either incubated with CJT or in the pathological sciatic nerves induced by CJT to various degrees. The binding of CJT to all these nerves was determined. (3) After intravenous injection with CJT, no histopathologic change could be found in the other viscera of the rats, with the exception of remarkable pathological change in peripheral nerves.

CONCLUSIONS(1) CJT could remarkably damage the peripheral nerves in rats. Specific pathogenicity of CJT to peripheral nerves was well shown, because no histopathologic abnormalities could be found in the other viscera, such as brain, liver and kidney etc. although there was remarkable pathological change along the peripheral nerve in the animals. (2) No immunological pathogenicity of CJT could be demonstrated in the nerves of rats after immunization with CJT.

Animals ; Antibodies, Anti-Idiotypic ; blood ; Bacterial Toxins ; immunology ; toxicity ; Campylobacter jejuni ; immunology ; Exotoxins ; immunology ; toxicity ; Gene Expression ; drug effects ; Peripheral Nerves ; drug effects ; metabolism ; pathology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Sciatic Nerve ; drug effects ; metabolism ; pathology ; Tumor Necrosis Factor-alpha ; genetics