Anti-cancer Parasporin Toxins of New Bacillus thuringiensis Against Human Colon (HCT-116) and Blood (CCRF-CEM) Cancer Cell Lines.

Bacillus thuringiensis is one of the most important microorganisms used against cancer cell lines in latest studies all over the world. This study aims to perform the isolation, molecular identification, and to identify novel B. thuringiensis strains that specifically targeting human cancer cell-killing activities in Iran. A total of 88 B. thuringiensis isolates were recovered from Iran. Upon the treatment of the non-hemolytic crystal proteins by proteinase K, five isolates belonging to three biotypes, thuringiensis, kurstaki and sotto of B. thuringiensis are found to have different cytotoxicity toward HCT-116 and CCRF-CEM cell lines. Digested inclusions of the isolates consisted of one major poly peptide of 34-kDa, as estimated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The structure, molecular identification, and functionality of five isolates inclusion proteins have shown to be closely like to parasporin-2 but their size of activated protein is not similar to this parasporin. It is unclear that discovered damaging proteins are parasporin-2. This 34-kD protein exhibited varying degrees of cytocidal activity toward human colon and blood cancer cells and caused cell swelling and the formation of blebs in the surface of the cells or alteration in cytoskeleton. The soil in the humid and temperate climates of Iran is a good reservoir for parasporin producing B. thuringiensis. The isolated B. thuringiensis strains exhibit specific and different cytocidal activities against human colon and blood cancer cells. Parasporin is a novel cytotoxic protein to human cancer cells produced by B. thuringiensis and these toxins appeared to attack an identical target on human cancer cells.

Bacillus thuringiensis toxins: an overview of their biocidal activity.

Bacillus thuringiensis (Bt) is a Gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing Cry and Cyt proteins, some of which are toxic against a wide range of insect orders, nematodes and human-cancer cells. These toxins have been successfully used as bioinsecticides against caterpillars, beetles, and flies, including mosquitoes and blackflies. Bt also synthesizes insecticidal proteins during the vegetative growth phase, which are subsequently secreted into the growth medium. These proteins are commonly known as vegetative insecticidal proteins (Vips) and hold insecticidal activity against lepidopteran, coleopteran and some homopteran pests. A less well characterized secretory protein with no amino acid similarity to Vip proteins has shown insecticidal activity against coleopteran pests and is termed Sip (secreted insecticidal protein). Bin-like and ETX_MTX2-family proteins (Pfam PF03318), which share amino acid similarities with mosquitocidal binary (Bin) and Mtx2 toxins, respectively, from Lysinibacillus sphaericus, are also produced by some Bt strains. In addition, vast numbers of Bt isolates naturally present in the soil and the phylloplane also synthesize crystal proteins whose biological activity is still unknown. In this review, we provide an updated overview of the known active Bt toxins to date and discuss their activities.

Parasporin 1Ac2, a novel cytotoxic crystal protein isolated from Bacillus thuringiensis B0462 strain.

Two novel parasporin (PS) genes were cloned from Bacillus thuringiensis B0462 strain. One was 100 % identical even in nucleotide sequence level with that of parasporin-1Aa (PS1Aa1) from B. thuringiensis A1190 strain. The other (PS1Ac2) showed significant homology (99 % identity) to that of PS1Ac1 from B. thuringiensis 87-29 strain. The 15 kDa (S(113)-R(250)) and 60 kDa (I(251)-S(777)) fragments consisting of an active form of PS1Ac2 were expressed as His-tag fusion. Upon purification under denaturing condition and refolding, the recombinant polypeptides were applied to cancer cells to analyze their cytotoxicities. 3-(4,5-Dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay revealed that either of 15 or 60 kDa polypeptide exhibited no cytotoxicity to HeLa cells, but they became cytotoxic upon mixed together. Our results suggested that PS1Ac2 was responsible for the cytotoxicity of B. thuringiensis B0462 strain, and that the formation of hetero-dimer of 15 and 60 kDa polypeptide was required for their cytotoxicity.

Evaluation of quantification methods of occupational endotoxin exposure.

Endotoxin has been identified as important component of organic-dust exposure and is suspected as main cause of work-related adverse health effects in dusty areas. Although the determination of endotoxin levels by using the Limulus amoebocyte lysate (LAL) assay is internationally accepted, reliability and variation of values measured with this test remain a point of discussion. Therefore, the purpose of the study was to determine the influence of different parameters on endotoxin activity measured in airborne samples. This study thus analyzed: (a) dust filter extraction procedures, (b) storage of samples, (c) usage of different commercially available LAL assays, and (d) results of the whole blood assay (WBA) compared to the LAL test. Using a parallel sampler, 120 filters were loaded with dust at 4 different occupational settings and extracted in 2 labs using a standardized protocol. Parameters like Tween in the extraction medium, extraction volume, centrifugation speed, and material of tubes used for extraction were tested. The LAL test and the WBA were able to determine the differences in dust load of filters obtained from the settings investigated. In addition, results varied significantly with modifications in extraction procedures. Using Tween for filter extraction mainly influenced the resulting endotoxin activity. In addition, LAL test differences according to manufacturer of LAL test, extraction volume, and whether the samples are freshly processed or frozen also resulted in significant variations in the endotoxin levels. In conclusion, a reliable assessment of exposure to endotoxin activity is only possible if standard operation procedures (SOPs) for sampling and determination are established.

[Use of reactions with Limulus amoebocyte lysate (LAL) to determine biological activity of lipopolysaccharides from reference and clinical strains of the Bacteroides fragilis group]. / Zastosowanie reakcji z lizatem amebocytów Limulus (LAL) do oznaczania biologicznej aktywnosci lipopolisacharydów referencyjnych i klinicznych szczepów z grupy Bacteroides fragilis.

The aim of this study was to determine and compare a biological activity of lipopolysaccharides (LPS) from reference and clinical strains of strictly anaerobic bacteria belonging to the Bacteroides fragilis group (BFG) by means of quantitative, photometric BET (LAL) method with Limulus polyphemus amoebocyte lysate and chromogenic substrate S-2423. Lipopolysaccharides of five BFG species were extracted by Westphal and Jann method (1965) from eight reference and two clinical strains of B. fragilis group. Crude LPS preparations were purified according to the procedure described by Gmeiner (1975) with ultracentrifugation and nuclease treatment. Biological activities of bacterial endotoxins were determined by quantitative BET method with chromogenic substrate S-2423 (ENDOCHROME kit, Charles River Endosafe Ltd., USA). Tests were performed according to the producer's recommendations. E. coli O55:B5 LPS was applied to compare its activity in reaction with LAL reagent with activities of LPS preparations from rods of the Bacteroides genus. Among examined bacterial compounds the most active in BET method was E. coli O55:B5 LPS. Activities of lipopolysaccharides from five species of BFG rods in reaction with Limulus amoebocyte lysate were differentiated. Greater ability to activate LAL proenzyme revealed lipopolysaccharides of these species of the Bacteroides genus, which are important from the clinical point of view--B. fragilis and B. thetaiotaomicron.

Nucleotide sequence and analysis of the N-terminal coding region of the Spodoptera-active delta-endotoxin gene of Bacillus thuringiensis aizawai 7.29.

The nucleotide sequence of a 2711bp DNA segment which contains the N-terminal coding sequence and the 5' flanking region of a crystal protein gene (bta) from Bacillus thuringiensis subsp. aizawai 7.29 has been determined. The coding region encodes an 824 amino-acid polypeptide corresponding to a carboxy-terminally truncated delta-endotoxin specifically active against the cotton leaf worm Spodoptera littoralis. Comparison of the deduced amino acid sequence of the bta gene with that of the 4.5, 5.3 and 6.6 kb classes of lepidopteran-active delta-endotoxins revealed that the Bta sequence contains a very high level of amino acid substitutions in the N-terminal part of the protoxin molecule. The substitutions are grouped in several highly variable segments separated by highly conserved regions. These conserved domains are also present in the dipteran- and coleopteran-active delta-endotoxins. The control region of the bta gene shows considerable DNA identity with the control regions of the other lepidopteran-active genes. Deletions of the 3' region of the gene were carried out and the toxic fraction of the bta delta-endotoxin was identified with the N-terminal half of the molecule.