OBJECTIVETo clone and produce ribosome inactivating protein MAP30 from the seeds of Momordica charantia L(bitter melon), and to evaluate the biological activity of the recombinant protein.

METHODSThe DNA sequence encoding MAP30 was cloned from the fresh seeds of Momordica charantia by PCR, the target DNA fragments were sequenced after T-A cloning. The expression plasmid was constructed by inserting the MAP30 fragment into vector pET30a. MAP30 was expressed in E.coli by addition of IPTG into final concentration of 1.0 mmol/L. The recombinant MAP30 was identified by SDS-PAGE, and the biological activity of MAP30 protein was evaluated by using MTT assay in cancer cells and normal cells following fluid-phase endocytosis.

RESULTThe nucleotide and amino acid sequences of the cloned MAP30 were identical with those of reported MAP30. The solubility of recombinant protein was analyzed by SDS-PAGE, and the MAP30 was mainly produced in soluble form. The recombinant MAP30 showed a greater cytotoxicity to cancer cells than that to normal cells.

CONCLUSIONThe gene of MAP30 has been successfully cloned.The recombinant MAP30 protein expressed by E.coli is bioactive.

Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Momordica charantia ; chemistry ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Ribosome Inactivating Proteins, Type 2 ; biosynthesis ; genetics ; metabolism ; Seeds ; chemistry ; Transformation, Bacterial

This study was conducted to investigate the presence of Escherichia (E.) coli O157 and E. coli O157:H7 and stx1 and stx2 genes on cattle carcasses and in rectal samples collected from Samsun Province of Turkey. A total of 200 samples collected from cattle carcasses and the rectal contents of 100 slaughtered cattle from two commercial abattoirs were tested using the immunomagnetic separation technique and multiplex PCR methods. E. coli O157 and E. coli O157:H7 were detected in 52 of the 200 samples (26%) tested. Of the positive samples, 49 were E. coli O157 and three were E. coli O157:H7. The E. coli O157 strain was isolated from 24 carcasses and 25 rectal samples, while E. coli O157:H7 was isolated from two carcasses and one rectal sample. Of the 49 samples positive for E. coli O157, 32 were from the rectal and carcass samples of the same animal, while two E. coli O157:H7 isolates were obtained from rectal swabs and carcasses of the same animal. The stx1 and stx2 genes were both detected in 35 E. coli O157 isolates and one E. coli O157:H7 isolate, but the stx2 gene was only detected alone in two E. coli O157 isolates. Overall, 16 carcasses tested positive for E. coli O157 and one carcass tested positive for E. coli O157:H7 based on both carcass and rectal samples. Overall, the results of this study indicate that cattle carcasses pose a potential risk to human health due to contamination by E. coli O157 and E. coli O157:H7 in the feces.
Abattoirs ; Animals ; Cattle ; Escherichia coli O157/*genetics/isolation & purification ; *Immunomagnetic Separation ; Meat/microbiology ; *Polymerase Chain Reaction ; Rectum/microbiology ; Shiga Toxin 1/*genetics ; Shiga Toxin 2/*genetics ; Turkey

China ; epidemiology ; Escherichia coli Infections ; epidemiology ; microbiology ; Escherichia coli O157 ; classification ; isolation & purification ; metabolism ; Humans ; Shiga Toxin 1 ; biosynthesis ; isolation & purification ; Shiga Toxin 2 ; biosynthesis ; isolation & purification ; Sorbitol

We evaluated the effect of the conjugate of basic fibroblast growth factor (FGF2) and saporin (FGF2-SAP) on proliferation of cultured keratocytes. Cultured rabbit and human keratocytes were incubated in medium containing 0.01 to 100 nM of chemical conjugate of EGF2 conjugated by disulfide bond to saporin (CCFS1), FGF2 genetically fused to saporin (rFGF2-SAP), FGF2, or saporin for three hours or four days and cell proliferation was quantified four days after the drug treatment. Proliferation of rabbit and human keratocytes was effectively inhibited by three hour and by four day exposure to CCFS1 and rFGF2-SAP in a dose-dependent manner, whereas it was affected minimally by four day exposure to saporin. Their inhibitory effects were detected at concentrations above 0.1 or 1 nM, and were most prominent in serum-stimulated rabbit keratocytes. These results suggest a potential role for FGF2-SAP in limiting proliferation of keratocytes during corneal wound healing.
Animals ; Antineoplastic Agents, Phytogenic/*pharmacology ; Cell Division/drug effects ; Cell Line ; Cells, Cultured ; Corneal Stroma/*cytology/drug effects ; Dose-Response Relationship, Drug ; Fibroblast Growth Factor 2/*pharmacology ; Humans ; Immunotoxins/pharmacology ; *N-Glycosyl Hydrolases ; Plant Proteins/*pharmacology ; Rabbits ; Ribosome Inactivating Proteins, Type 1

A multiplex loop-mediated isothermal amplification (mLAMP) assay was developed for simultaneous detection of the stx1 and stx2 genes and applied for detection of shiga toxin-producing Escherichia coli (STEC) in cattle farm samples. Two target genes were distinguished based on T m values of 85.03 +/- 0.54degrees C for stx1 and 87.47 +/- 0.35degrees C for stx2. The mLAMP assay was specific (100% inclusivity and exclusivity), sensitive (with a detection limit as low as 10 fg/microL), and quantifiable (R 2 = 0.9313). The efficacy and sensitivity were measured to evaluate applicability of the mLAMP assay to cattle farm samples. A total of 12 (12/253; 4.7%) and 17 (17/253; 6.7%) STEC O157, and 11 (11/236; 4.7%) non-O157 STEC strains were isolated from cattle farm samples by conventional selective culture, immunomagnetic separation, and PCR-based culture methods, respectively. The coinciding multiplex PCR and mLAMP results for the types of shiga toxin revealed the value of the mLAMP assay in terms of accuracy and rapidity for characterizing shiga toxin genes. Furthermore, the high detection rate of specific genes from enrichment broth samples indicates the potential utility of this assay as a screening method for detecting STEC in cattle farm samples.
Animals ; Cattle ; Cattle Diseases/epidemiology/microbiology ; Escherichia coli Infections/epidemiology/microbiology/*veterinary ; Feces/microbiology ; Multiplex Polymerase Chain Reaction/veterinary ; Nucleic Acid Amplification Techniques/*veterinary ; Shiga Toxin 1/*genetics/isolation & purification ; Shiga Toxin 2/*genetics/isolation & purification ; Shiga-Toxigenic Escherichia coli/*genetics/isolation & purification

A total of 156 Shiga-like toxin producing Escherichia coli (STEC) were isolated from fecal samples of Korean native (100/568, 18%) and Holstein dairy cattle (56/524, 11%) in Korea between September 2010 and July 2011. Fifty-two STEC isolates (33%) harbored both of shiga toxin1 (stx1) and shiga toxin2 (stx2) genes encoding enterohemolysin (EhxA) and autoagglutinating adhesion (Saa) were detected by PCR in 83 (53%) and 65 (42%) isolates, respectively. By serotyping, six STEC from native cattle and four STEC from dairy cattle were identified as O-serotypes (O26, O111, O104, and O157) that can cause human disease. Multilocus sequence typing and pulsed-field gel electrophoresis patterns highlighted the genetic diversity of the STEC strains and difference between strains collected during different years. Antimicrobial susceptibility tests showed that the multidrug resistance rate increased from 12% in 2010 to 42% in 2011. Differences between isolates collected in 2010 and 2011 may have resulted from seasonal variations or large-scale slaughtering in Korea performed to control a foot and mouth disease outbreak that occurred in early 2011. However, continuous epidemiologic studies will be needed to understand mechanisms. More public health efforts are required to minimize STEC infection transmitted via dairy products and the prevalence of these bacteria in dairy cattle.
Animals ; Anti-Bacterial Agents/pharmacology ; Cattle/microbiology ; Drug Resistance, Multiple, Bacterial ; Electrophoresis, Gel, Pulsed-Field/veterinary ; Escherichia coli Infections/epidemiology/microbiology/*veterinary ; Female ; Genes, Bacterial/genetics ; Latex Fixation Tests/veterinary ; Microbial Sensitivity Tests/veterinary ; Multilocus Sequence Typing/veterinary ; Prevalence ; Republic of Korea/epidemiology ; Shiga Toxin 1/genetics ; Shiga Toxin 2/genetics ; *Shiga-Toxigenic Escherichia coli/drug effects/genetics

Escherichia (E.) coli serotype O157:H7 is a globally distributed human enteropathogen and is comprised of microorganisms with closely related genotypes. The main reservoir for this group is bovine bowels, and infection mainly occurs after ingestion of contaminated water and food. Virulence genetic markers of 28 O157:H7 strains were investigated and multilocus enzyme electrophoresis (MLEE) was used to evaluate the clonal structure. O157:H7 strains from several countries were isolated from food, human and bovine feces. According to MLEE, O157:H7 strains clustered into two main clonal groups designated A and B. Subcluster A1 included 82% of the O157:H7 strains exhibiting identical MLEE pattern. Most enterohemorrhagic E. coli (EHEC) O157:H7 strains from Brazil and Argentina were in the same MLEE subgroup. Bovine and food strains carried virulence genes associated with EHEC pathogenicity in humans.
Animals ; Argentina/epidemiology ; Brazil/epidemiology ; Cattle ; Cattle Diseases/epidemiology/*microbiology ; Enterohemorrhagic Escherichia coli/genetics/*isolation & purification/pathogenicity ; Escherichia coli O157/*genetics/*isolation & purification/pathogenicity ; Food Microbiology ; Gene Expression Regulation, Bacterial/physiology ; Genetic Markers ; Humans ; Polymerase Chain Reaction/veterinary ; Shiga Toxin 1/genetics/metabolism ; Shiga Toxin 2/genetics/metabolism ; Virulence

OBJECTIVES: Two related cases of Hemolytic-Uremic Syndrome (HUS) were reported to the Korea National Institute of Health in May, 2001. Shiga toxin 2 genes were detected in both stool samples. We suspected an enterohemorrhagic Escherichia coli (EHEC) infection as the cause of the HUS, and conducted an investigation to find the source of the infection and its route of transmission. METHODS: We performed case investigations on these two related HUS cases, and obtained interviews and rectal swabs form the family members and other close contacts. Additionally, we performed rectal swabs on the cattle raised by the household of the index patient. RESULTS: We found a 20 month old index patient and a 6 year-old cousin had developed HUS, where there had been a 2 day history of contact with the index, and bacteriological examinations for these two patients revealed, indistinguishably, the same E. coli O171. The grandmother of the index patient was found to be asymptomatic, but E. coli O26 was isolated. We also found a probable case in the mother of the cousin. She reported a history of contact with the index, and developed bloody diarrhea of 3 days duration. The test results for the cattle revealed E. coli O26 in one cow, and E. coli O26 and O55 in another. E. coli O26, which was isolated in both cows and the grandmother of the index, were indistinguishably the same. CONCLUSIONS: We found that the E. coli O26 in the grandmother had originated from the cows, and that the E. coli O171 found in the index patient had been transmitted to the cousin through person-to-person contact.
Animals ; Cattle ; Diarrhea ; Enterohemorrhagic Escherichia coli* ; Family Characteristics ; Grandparents ; Hemolytic-Uremic Syndrome ; Humans ; Korea* ; Mothers ; Shiga Toxin 2 ; Zoonoses

OBJECTIVETo develop toxin targeting vascular endothelial growth factor receptor II (VEGF-II/KDR) fused with a KDR-binling peptide screened from peptide library.

METHODSBy affinity to KDR molecular which expressed specifically by new born vascular endothelial cell, peptides to KDR were screened from C7 peptide library by phage display. Among them, a peptide binding to KDR with high affinity termed as P5 was selected and fused to the N-terminal of Shiga toxin subunit A (StxA). The protein (P5-StxA) was expressed in E. coli.

RESULTSELISA and Western blot were applied to characterize the binding interaction between the fusion protein, P5-StxA and KDR. Cytotoxicity assay showed that P5-StxA maintained similar toxicity to cell as StxA. In the model of angiogenesis, P5-StxA inhibited selectively VEGF-induced growth of preexisting vessels of the chick chorioallantoic membrane.

CONCLUSIONThese studies demonstrate the small peptide, P5, maybe be used as carrier of toxin targeting to KDR.

Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Humans ; Peptide Library ; Protein Subunits ; Recombinant Fusion Proteins ; metabolism ; Shiga Toxin ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism

OBJECTIVEVerotoxin-producing Escherichia coli (VTEC) strains of serotype O157 : H7 have been implicated in a wide spectrum of diseases, including blood diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). To further explore the pathological role of verotoxin (VT) in HUS and other VTEC associated diseases, we investigated the effects of recombinant verotoxin 2 (rVT2) on the biological activity of neutrophils.

METHODSThe technique of flow cytometry, a fluorescent probe 2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF/AM), and the assay of reduced cytochrome c to detect superoxide production were used in this study.

RESULTSgammaVT2 significantly inhibited spontaneous apoptosis in neutrophils. Neutrophils with prolonged survival due to gammaVT2 maintained various biological functions, such as the expression of adhesion molecules (shading CD62L and raising CD11b/CD18), adherence to human umbilical vein endothelial cells (HUVECs), and generation of superoxide (O(2)(-)).

CONCLUSIONProlongation of the functional life-span of neutrophils by gammaVT2 may accelerate inflammatory responses at sites of inflammation. This may play a crucial role in neutrophil-mediated tissue injury in HUS and other VTEC-associated diseases.

Apoptosis ; drug effects ; Cell Adhesion ; Endothelium, Vascular ; cytology ; Humans ; Neutrophils ; drug effects ; physiology ; Recombinant Proteins ; toxicity ; Shiga Toxin 2 ; toxicity ; Superoxides ; metabolism