Apisak Dhiravisit

Complete Amino Acid Sequence of Globin Chains and Biological Activity of Fragmented Crocodile Hemoglobin (Crocodylus siamensis)

Hemoglobin, α-chain, β-chain and fragmented hemoglobin of Crocodylus siamensis demonstrated both antibacterial and antioxidant activities. Antibacterial and antioxidant properties of the hemoglobin did not depend on the heme structure but could result from the compositions of amino acid residues and structures present in their primary structure. Furthermore, thirteen purified active peptides were obtained by RP-HPLC analyses, corresponding to fragments in the α-globin chain and the β-globin chain which are mostly located at the N-terminal and C-terminal parts. These active peptides operate on the bacterial cell membrane. The globin chains of Crocodylus siamensis showed similar amino acids to the sequences of Crocodylus niloticus. The novel amino acid substitutions of α-chain and β-chain are not associated with the heme binding site or the bicarbonate ion binding site, but could be important through their interactions with membranes of bacteria.

Identification of five reptile egg whites protein using MALDI-TOF mass spectrometry and LC/MS-MS analysis

Proteomics of egg white proteins of five reptile species, namely Siamese crocodile (Crocodylus siamensis), soft-shelled turtle (Trionyx sinensis taiwanese), red-eared slider turtle (Trachemys scripta elegans), hawksbill turtle (Eretmochelys imbricate) and green turtle (Chelonia mydas) were studied by 2D-PAGE using IPG strip pH 4-7 size 7 cm and IPG strip pH 3-10 size 24 cm. The protein spots in the egg white of the five reptile species were identified by MALDI-TOF mass spectrometry and LC/MS-MS analysis. Sequence comparison with the database revealed that reptile egg white contained at least seven protein groups, such as serpine, transferrin precursor/iron binding protein, lysozyme C, teneurin-2 (fragment), interferon-induced GTP-binding protein Mx, succinate dehydrogenase iron-sulfur subunit and olfactory receptor 46. This report confirms that transferrin precursor/iron binding protein is the major component in reptile egg white. In egg white of Siamese crocodile, twenty isoforms of transferrin precursor were found. Iron binding protein was found in four species of turtle. In egg white of soft-shelled turtle, ten isoforms of lysozyme were found. Apart from well-known reptile egg white constituents, this study identified some reptile egg white proteins, such as the teneurin-2 (fragment), the interferon-induced GTP-binding protein Mx, the olfactory receptor 46 and the succinate dehydrogenase iron-sulfur subunit.

Antibacterial activity of plasma from crocodile (Crocodylus siamensis) against pathogenic bacteria

BackgroundThe 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.MethodsCrude 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.ResultsCrude 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.ConclusionsThe 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.

Design and synthesis of cationic antibacterial peptide based on Leucrocin I sequence, antibacterial peptide from crocodile (Crocodylus siamensis) white blood cell extracts.

Leucrocin I is an antibacterial peptide isolated from crocodile (Crocodylus siamensis) white blood cell extracts. Based on Leucrocin I sequence, cationic peptide, NY15, was designed, synthesized and evaluated for antibacterial activity against Bacillus sphaericus TISTR 678, Bacillus megaterium (clinical isolate), Vibrio cholerae (clinical isolate), Salmonella typhi (clinical isolate), Salmonella typhi ATCC 5784 and Escherichia coli 0157:H7. The efficacy of the peptide made from all L-amino acids was also compared with all D-amino acids. The peptide made from all D-amino acids was more active than the corresponding L-enantiomer. In our detailed study, the interaction between peptides and the cell membrane of Vibrio cholerae as part of their killing mechanism was studied by fluorescence and electron microscopy. The results show that the membrane was the target of action of the peptides. Finally, the cytotoxicity assays revealed that both L-NY15 and D-NY15 peptides are non-toxic to mammalian cells at bacteriolytic concentrations.

Comparative proteomic analysis of leaves, leaf sheaths, and roots of drought-contrasting sugarcane cultivars in response to drought stress

A better understanding of drought response proteins may improve our understanding of the mechanisms underlying drought tolerance in sugarcane. In this research, drought-tolerant (K86-161) and drought-sensitive (B34-164) sugarcane cultivars were grown and exposed to drought stress. The changes in protein expression in leafs, leaf sheaths and roots were analyzed using proteomics techniques. Proteins that responded to drought in both cultivars could be classified into four major categories, including energy and metabolism, photosynthesis, antioxidant, and defense protein. Interestingly, an increased abundance of fructose-bisphosphate aldolase under drought was observed in all three organs of K86-161. Elevated expression of oxygen-evolving enhancer protein was also found in leaves and leaf sheaths of K86-161, when compared with their controls. Additionally, SOD was abundant in the leaves and roots of K86-161. Importantly, the expression level of these proteins decreased in B34-164 under drought stress. These contrasting results suggest that these proteins were inhibited by drought stress in the drought-sensitive cultivar. This proteomic research is the first to combine analyses of leaves, leaf sheaths and roots in sugarcane, which may enhance our understanding of drought responses at the molecular level and lead to selective breeding for enhanced drought tolerance.

Improving the antibacterial activity and selectivity of an ultra short peptide by hydrophobic and hydrophilic amino acid stretches

The principle of amino acid stretches tagged at the C terminal of Luecrocin I, which is an ultra-short antibacterial peptide, by tryptophan and arginine or lysine has been reported. The choice of amino acid type at each stretch position depends on the hydrophobic and hydrophilic regions visualized in the helical wheel pattern of Luecrocin I. Oligopeptide tagging should also consider the properties such as positive charge, hydrophobicity, the content of hydrophobic amino acids, polar angle, the properly hydrophilic and hydrophobic facets. Amidation at C terminal and lysine substitute for arginine can increase selectivity between mammalian cells (hemolytic and MTT assay) and bacterial cells tested. KT2 and RT2 which have 53% hydrophobic residues, 7 positive charges, 160° polar angle, -0.02 (KT2) and -0.04 (RT2) hydrophobicity were effective against S. typhi DMST 22842, S. epidermidis ATCC 12228, E. coli ATCC 25922 and V. cholerae non-O1, non-O139. The SEM images implied that the antibacterial mechanism of RT2 and KT2 may depend on concentration rather than time. Finally, RT2 and KT2 can be new antibacterial agents or may be further developed for alternative antibiotics.

Purification and characterization of antimicrobial substances from Bacillus licheniformis BFP011

Bacillus licheniformis BFP011 isolated from papaya (Thailand) could produce extracellular antimicrobial substances which were active against some important phytopathogens, pathogenics and spoilage microorganisms such as Colletotrichum capsici, Escherichia coli O157: H7 and Salmonella typhi ATCC 5784. The antimicrobial substances of this bacterium showed resistance to pronase enzyme and high temperature at 100 and 121°C for 15 min. They were purified by TLC on silica gel plates F254 using the different solvent mixtures. The best solvent mixture was revealed as n-butanol: ethanol: acetic acid: water (30: 60: 5: 30, v/v). The spots F4, F5 and F6 from TLC were able to inhibit growth of S. typhi ATCC 5784 assayed in vitro by the disc diffusion method. The characterization of the active fractions F4, F5 and F6 from TLC and reversedphase HPLC indicated that the antimicrobial substances of B. licheniformis BFP011 contain peptides and unsaturated fatty acids.

Purification and characterization of macrolactins and amicoumacins from Bacillus licheniformis BFP011: a new source of food antimicrobial substances

ABSTRACT In this study, Bacillus licheniformis BFP011 has been cultivated for producing antimicrobial compounds, which were concentrated from the crude supernatant and purified using thin-layer chromatography. The antimicrobial activity was investigated in the presence of several organic solvents and detergents, and inhibiting effects to various Gram-negative and Gram-positive human pathogenic and food spoilage bacteria were observed. Three bands (F4, F5 and F6) showing antimicrobial activity against Salmonella typhi ATCC 5784 were subjected to reversed-phase high-performance liquid chromatography purification. Two peaks of fraction F5, F5-P3 and F5-P4, showed 100% inhibition against S. typhi ATCC 5784. The contained mixture of antimicrobial compounds consists of macrolactins and amicoumacins, which induced the collapse and breaking of S. typhi ATCC 5784 cell membranes in a time-dependent manner. Taken collectively, the results indicate that these compounds may represent promising candidates for the development of food preservative agents of natural origin, as well as novel antimicrobial drug candidates against multiresistant bacterial strains.