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1.  Antimicrobial Action of the Cyclic Peptide Bactenecin on Burkholderia pseudomallei Correlates with Efficient Membrane Permeabilization 
Burkholderia pseudomallei is a category B agent that causes Melioidosis, an acute and chronic disease with septicemia. The current treatment regimen is a heavy dose of antibiotics such as ceftazidime (CAZ); however, the risk of a relapse is possible. Peptide antibiotics are an alternative to classical antibiotics as they exhibit rapid action and are less likely to result in the development of resistance. The aim of this study was to determine the bactericidal activity against B. pseudomallei and examine the membrane disrupting abilities of the potent antimicrobial peptides: bactenecin, RTA3, BMAP-18 and CA-MA. All peptides exhibited >97% bactericidal activity at 20 µM, with bactenecin having slightly higher activity. Long term time-kill assays revealed a complete inhibition of cell growth at 50 µM bactenecin and CA-MA. All peptides inhibited biofilm formation comparable to CAZ, but exhibited faster kinetics (within 1 h). Bactenecin exhibited stronger binding to LPS and induced perturbation of the inner membrane of live cells. Interaction of bactenecin with model membranes resulted in changes in membrane fluidity and permeability, leading to leakage of dye across the membrane at levels two-fold greater than that of other peptides. Modeling of peptide binding on the membrane showed stable and deep insertion of bactenecin into the membrane (up to 9 Å). We propose that bactenecin is able to form dimers or large β-sheet structures in a concentration dependent manner and subsequently rapidly permeabilize the membrane, leading to cytosolic leakage and cell death in a shorter period of time compared to CAZ. Bactenecin might be considered as a potent antimicrobial agent for use against B. pseudomallei.
Author Summary
Burkholderia pseudomallei is a category B agent that causes Melioidosis, an acute and chronic disease with septicemia. The current treatment regimen is a heavy dose of antibiotics such as ceftazidime (CAZ), however, the risk of a relapse is possible. In this study we demonstrate that bactenecin, CA-MA, RTA3 and BMAP-18 are able to inhibit the growth and biofilm formation of B. pseudomallei. The strong bactericidal activity of bactenecin is attributed to its greater ability to permeabilize the membrane. Computational modeling of these peptide-membrane interactions provide support for a model in which bactenecin is able to penetrate the membrane most effectively due to its cyclical structure. The peptide, bactenecin has the potential to act as a highly effective alternative to CAZ, or as a combination therapy with CAZ in the treatment of melioidosis. Furthermore, understanding the mechanism of bactenecin may help us better design more effective peptides therapeutics of choice for melioidosis.
PMCID: PMC3681726  PMID: 23785532
2.  Antibacterial activity of plasma from crocodile (Crocodylus siamensis) against pathogenic bacteria 
The Siamese crocodile (Crocodylus siamensis) is a critically endangered species of freshwater crocodiles. Crocodilians live with opportunistic bacterial infection but normally suffer no adverse effects. They are not totally immune to microbial infection, but their resistance thereto is remarkably effective. In this study, crude and purified plasma extracted from the Siamese crocodile were examined for antibacterial activity against clinically isolated, human pathogenic bacterial strains and the related reference strains.
Crude plasma was prepared from whole blood of the Siamese crocodile by differential sedimentation. The crude plasma was examined for antibacterial activity by the liquid growth inhibition assay. The scanning electron microscopy was performed to confirm the effect of crude crocodile plasma on the cells of Salmonella typhi ATCC 11778. Effect of crude crocodile plasma on cell viability was tested by MTT assay. In addition, the plasma was purified by anion exchange column chromatography with DEAE-Toyopearl 650 M and the purified plasma was tested for antibacterial activity.
Crude plasma was prepared from whole blood of the Siamese crocodile and exhibited substantial antibacterial activities of more than 40% growth inhibition against the six reference strains of Staphylococcus aureus, Salmonella typhi, Escherichia coli, Vibrio cholerae, Pseudomonas aeruginosa, and Staphylococcus epidermidis, and the four clinical isolates of Staphylococcus epidermidis, Pseudomonas aeruginosa, Salmonella typhi, and Vibrio cholerae. Especially, more than 80% growth inhibition was found in the reference strains of Salmonella typhi, Vibrio cholerae, and Staphylococcus epidermidis and in the clinical isolates of Salmonella typhi and Vibrio cholerae. The effect of the crude plasma on bacterial cells of Salmonella typhi, a certain antibacterial material probably penetrates progressively into the cytoplasmic space, perturbing and damaging bacterial membranes. The effect of the crude plasma was not toxic by the yellow tetrazolium bromide (MTT) assay using a macrophage-like cell, RAW 264.7. The pooled four fractions, designated as fractions D1-D4, were obtained by column chromatography, and only fraction D1 showed growth inhibition in the reference strains and the clinical, human pathogenic isolates.
The crude and purified plasma from the Siamese crocodile significantly showed antibacterial activity against pathogenic bacteria and reference strains by damage cell membrane of target bacterial cells. From the MTT assay, the Siamese crocodile plasma was not cytotoxic to the cells.
PMCID: PMC3490821  PMID: 22846342
Crocodile (Crocodylus siamensis); Antibacterial activity; Pathogenic bacteria; Cytotoxicity
3.  Biomarker to distinguish hepatocellular carcinoma from cholangiocarcinoma by serum a disintegrin and metalloprotease 12 
Archives of Medical Science : AMS  2011;7(6):1013-1016.
The “a distintegrin and metalloprotease” (ADAM) family contributes to regulation of the cell-cell and cell-matrix interaction that are critical determinants of malignancy. It also plays important roles in the degradation of the basement membrane during tumor invasion. To evaluate a distinguishing biomarker for hepatocellular carcinoma from cholangiocarcinoma, a disintegrin and metalloprotease 12 (ADAM12) level was determined.
Material and methods
The indirect ELISA and Western blot analysis for quantification of ADAM12 level in serum was developed. The subjects were 218 histologically confirmed cases, 128 with intrahepatic cholangiocarcinoma, 30 with hepatocellular carcinoma and 60 healthy people.
The ability of test was verified using an analysis of Receiver Operating Characteristic (ROC) curve. The mean value of serum ADAM 12 in hepatocellular carcinoma was significantly higher than cholangiocarcinoma and healthy people (p = 0.001). The AUC for control vs. HCC was 0.826 while for controls vs. CC was 0.679. The results showed that a disintegrin and metalloprotease 12 for hepatocellular carcinoma had better specificity (77.4%) than for cholangiocarcinoma (64.5%). The serum a disintegrin and metalloprotease 12 level was also found to inversely correlate with overall survival (p = 0.02).
A disintegrin and metalloprotease 12 would be most useful as an adjunct biomarker for distinguishing hepatocellular carcinoma from cholangiocarcinoma.
PMCID: PMC3264993  PMID: 22328884
ADAM12 protein; cholangiocarcinoma; hepatocellular carcinoma; A disintegrin and metalloproteases

Results 1-3 (3)