The investigation of antibacterial properties of peptides and protein hydrolysates derived from serum of Asian water monitor (Varanus salvator).

It is well known that the Asian water monitors or Varanus salvator are both scavengers and predators. They can live and survive in the place that exposed to harmful microorganisms. Most people believe that they have some protected mechanisms to confront those infections. The aim of this study is to determine the antibacterial activities of crude peptides and protein hydrolysates extracted from serum of the Varanus salvator. Ten types of bacteria were cultured with crude peptides and protein hydrolysates which were isolated from 21 Varanus salvator's serum. The crude peptides showed some interested inhibition percentages against Enterobacter aerogenes ATCC13048 = 25.6%, Acinetobacter baumannii ATCC19606 = 33.4%, Burkholderia cepacia ATCC25416 = 35.3% and Pseudomonas aeruginosa ATCC27853 = 25.8%, whereas the protein hydrolysates had some inhibition potential on Burkholderia cepacia ATCC25416 = 24.3%. For the rest results of other tests were below 20% of inhibition. In addition, the evidences show that crude peptides have better antibacterial performances significantly than protein hydrolysates on most tested bacteria. Furthermore, antimicrobial peptides prediction shows about 10 percent hit (41/432 sequences). The interpretation shows that the best hit sequence is highly hydrophobic. It may destroy outer membrane of Gram-negative hence prevents the invasion of those bacteria. Altogether, bioinformatics and experiments show similar trends of antimicrobial peptide efficacy from Varanus salvator. Further studies need to be conducted on peptide purification and antimicrobial peptide candidate should be identified.

Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel enterotoxin.

Unusual outbreaks of food poisoning in Japan were reported in which Clostridium perfringens was strongly suspected to be the cause based on epidemiological information and fingerprinting of isolates. The isolated strains lack the typical C. perfringens enterotoxin (CPE) but secrete a new enterotoxin consisting of two components: C. perfringens iota-like enterotoxin-a (CPILE-a), which acts as an enzymatic ADP-ribosyltransferase, and CPILE-b, a membrane binding component. Here we present the crystal structures of apo-CPILE-a, NAD+-CPILE-a and NADH-CPILE-a. Though CPILE-a structure has high similarity with known iota toxin-a (Ia) with NAD+, it possesses two extra-long protruding loops from G262-S269 and E402-K408 that are distinct from Ia. Based on the Ia-actin complex structure, we focused on actin-binding interface regions (I-V) including two protruding loops (PT) and examined how mutations in these regions affect the ADP-ribosylation activity of CPILE-a. Though some site-directed mutagenesis studies have already been conducted on the actin binding site of Ia, in the present study, mutagenesis studies were conducted against both α- and ß/γ-actin in CPILE-a and Ia. Interestingly, CPILE-a ADP-ribosylates both α- and ß/γ-actin, but its sensitivity towards ß/γ-actin is 36% compared with α-actin. Our results contrast to that only C2-I ADP-ribosylates ß/γ-actin. We also showed that PT-I and two convex-concave interactions in CPILE-a are important for actin binding. The current study is the first detailed analysis of site-directed mutagenesis in the actin binding region of Ia and CPILE-a against both α- and ß/γ-actin.

Comparative Studies of Actin- and Rho-Specific ADP-Ribosylating Toxins: Insight from Structural Biology.

Mono-ADP-ribosylation is a major post-translational modification performed by bacterial toxins, which transfer an ADP-ribose moiety to a substrate acceptor residue. Actin- and Rho-specific ADP-ribosylating toxins (ARTs) are typical ARTs known to have very similar tertiary structures but totally different targets. Actin-specific ARTs are the A components of binary toxins, ADP-ribosylate actin at Arg177, leading to the depolymerization of the actin cytoskeleton. On the other hand, C3-like exoenzymes are Rho-specific ARTs, ADP-ribosylate Rho GTPases at Asn41, exerting an indirect effect on the actin cytoskeleton. This review focuses on the differences and similarities of actin- and Rho-specific ARTs, especially with respect to their substrate recognition and cell entry mechanisms, based on structural studies.

Sequence to structure approach of estrogen receptor alpha and ligand interactions.

Estrogen receptors (ERs) are steroid receptors located in the cytoplasm and on the nuclear membrane. The sequence similarities of human ERα, mouse ERα, rat ERα, dog ERα, and cat ERα are above 90%, but structures of ERα may different among species. Estrogen can be agonist and antagonist depending on its target organs. This hormone play roles in several diseases including breast cancer. There are variety of the relative binding affinity (RBA) of ER and estrogen species in comparison to 17ß-estradiol (E2), which is a natural ligand of both ERα and ERß. The RBA of the estrogen species are as following: diethyl stilbestrol (DES)>hexestrol>dienestrol>17ß-estradiol (E2)>17-estradiol>moxestrol>estriol (E3)>4-OH estradiol>estrone-3-sulfate. Estrogen mimetic drugs, selective estrogen receptor modulators (SERMs), have been used as hormonal therapy for ER positive breast cancer and postmenopausal osteoporosis. In the postgenomic era, in silico models have become effective tools for modern drug discovery. These provide three dimensional structures of many transmembrane receptors and enzymes, which are important targets of de novo drug development. The estimated inhibition constants (Ki) from computational model have been used as a screening procedure before in vitro and in vivo studies.

Prediction of cadmium (Cd) toxicity in cattle.

Hepato- and nephrotoxicity can be induced by the exposure to cadmium (Cd). This toxicity can be detected by the elevation of blood biomarkers such as ALT AST, ALP BUN and creatinine. These elevations are found in small animals, e.g. mice and rats. However the alteration of biomarkers did not investigate in large animals, e.g. cattle. Hepato- and nephrotoxicity induced by cadmium can also be examined by the alterations of metallothionein (MT) and metal transcription factor-1 (MTF-1). To present study the expressions of these markers, the cattle were classified into five groups according to the levels of cadmium in the kidneys. ALT, AST andALP were analyzed to determine liver damage whereas BUN and creatinine were examined for kidney damage. The results showed that blood biomarkers were not sensitive enough to be correlated markers to cadmium induced hepato- and nephrotoxicity in cattle. The expressions of MT and MTF-1 protein were investigated by immunofluorescence method. The expressions of MT and MTF-1 proteins were firstly found in the cattle group which had low cadmium concentration in tissues (< 0.5 mg/kg). Thus, these proteins could be used as the sensitive markers to determine the cadmium exposure. The MT and MTF-1 gene expressions were also studied. However, there was no correlation between the level of RNA and the protein expressions due to the concentration of protein levels bearing unclear relationship with the mRNA level. The investigation of these protein expressions is very useful because the result can be used as a protective method to prevent consumption of cadmium-contaminated beef.

Binding capacity of ER-α ligands and SERMs: comparison of the human, dog and cat.

The estrogen molecule is the major risk factor related to mammary gland tumors, with estrogen receptor alpha (ER- α) as the important target stimulating growth. Therefore one alternative approach to treatment of breast cancer is to use selective estrogen receptor modulator (SERM), hormonal therapy. In this study, the structures of ER- α in humans, dogs and cats were predicted using the amino acid sequencing data bank and corrected for general protein structures, receptor sites and docking by adding 2,344 ligands with 15 SERMs into the database and calculating estimated inhibition constants (Ki). Thereby, ranking of best ligands of SERMs in humans, dogs and cats could be achieved. The results show that the shapes of ER- α differ between species but the major pocket sites are the same. Bazedoxifene, a new SERM proved to be the best estrogen antagonist and ER- α inhibitor in all species (human, dog, cat) with the lowest Ki. The other good ligands for dogs and cats are Neohesperidin, Dihydrochalcone, and Schreiber2. The differences in these protein structures may explain why there are only a few SERMs or other ligands which can be used as anti-cancer drugs.

Immunohistochemical determination of estrogen and progesterone receptors in canine mammary tumors.

Mammary gland tumors are by far the most commonly found tumors in domestic dogs. Effective therapeutic procedures with prompt accurate diagnoses are of prime importance for this life threatening neoplasm. Although immunohistochemical methods provide valuable information such as the location and semi-quantitative data of the interested antigens in particular tumors, conventional methods like histopathological diagnosis remain useful and necessary for identification and classification of tumors. In the present study, we combined histopathology with immunohistochemical staining of estrogen receptors (ER) and progesterone receptors (PR) in canine mammary gland tumors. Fifty dogs with primary mammary tumors underwent surgery at the Veterinary Teaching Hospital of Mahidol University during 2005 to 2007. Three of them were diagnosed with precancerous lesions and negatively stained for ER or PR antibody. Twenty-one were diagnosed with benign tumors classified as adenomas and benign mixed mammary gland tumors. Nearly 60% of thelesions were negatively stained for ER or PR. PR positively stained, both PR and ER stained and ER stained tumors accounted for 19%, 19% and 5%, respectively. Of the malignant tumors, eighty-six percent were adenocarcinomas and 14% were malignant mixed mammary gland tumors. Nearly 70% were negatively stained for ER or PR, 14% were PR positively stained, 14% were both PR and ER stained and 5% were ER stained. Four dogs had unidentified lesions. In summary, more than half of of our benign and malignant canine mammary tumors were negatively stained for ER and PR. This indicates a lack of correlation with estrogen and/or progesterone receptor expression.