Cytotoxicity of the Bacillus thuringiensis Crystal Protein against Mammalian Cells

The crystal proteins produced by Bacillus thuringiensis subsp, israelensis (Bti) and subsp. coreanensis A1519 strain were examined for the cytotoxicity against MOLT-4 and HeLa cells by MTT assay and LDH assay, The A1519 crystal proteins processed by proteinase K exhibited the specific cell-killing activity toward MOLT-4 with little damage to the cell membrane, On the other hand, the Bti crystal proteins processed by proteinase K caused the substantial damage to the cell membrane of both MOLT-4 and HeLa, leading to the cell lysis. The non-digested crystal proteins of both strains exhibited no cytotoxicity, These data suggested that while the Bti crystal proteins caused the colloid-osmotic swelling and cell lysis of MOLT-4 and HeLa, the proteinase K-digested A1519 crystal proteins induced the specific cell death of MOLT-4 through a mechanism other than that of Bti

The cytotoxicity of Bacillus thuringiensis subsp. coreanensis A2316 strain against the human leukemic T cell

Bacillus thuringiensis subsp. coreanensis A2316 is a newly isolated strain from Yonakunijima Island in Japan. It produces the proteinaceous inclusion body (crystal) which has no insecticidal and hemolytic activities. When the crystal proteins were digested by proteinase K, they exhibited the strong cytotoxicity against human leukemic T cell, MOLT-4. The proteinase K-digested A2316 crystal proteins have little damage upon the cell membrane of MOLT-4, suggesting that the cell death of MOLT-4 was induced through a mechanism other than the colloid-osmotic swelling and cell lysis as caused by hitherto known B. thuringiensis crystal proteins. The 29-kDa polypeptide proved to be an active component of the proteinase K-digested A2316 crystal proteins. EC(50) of the purified 29-kDa polypeptide was 0.0579 μg/ml. The N-terminal amino acid sequence of the 29-kDa polypeptide was identical with that of p29 produced by B. thuringiensis A1519 strain and shared no significant homology with all the known proteins, suggesting that this polypeptide belong to a new family of B. thuringiensis crystal proteins

The cytotoxicity of Bacillus thuringiensis subsp. coreanensis A2316 strain against the human leukemic T cell

Bacillus thuringiensis subsp. coreanensis A2316 is a newly isolated strain from Yonakunijima Island in Japan. It produces the proteinaceous inclusion body (crystal) which has no insecticidal and hemolytic activities. When the crystal proteins were digested by proteinase K, they exhibited the strong cytotoxicity against human leukemic T cell, MOLT-4. The proteinase K-digested A2316 crystal proteins have little damage upon the cell membrane of MOLT-4, suggesting that the cell death of MOLT-4 was induced through a mechanism other than the colloid-osmotic swelling and cell lysis as caused by hitherto known B. thuringiensis crystal proteins. The 29-kDa polypeptide proved to be an active component of the proteinase K-digested A2316 crystal proteins. EC(50) of the purified 29-kDa polypeptide was 0.0579 μg/ml. The N-terminal amino acid sequence of the 29-kDa polypeptide was identical with that of p29 produced by B. thuringiensis A1519 strain and shared no significant homology with all the known proteins, suggesting that this polypeptide belong to a new family of B. thuringiensis crystal proteins

Sugar integrated gelators of organic fluids: on their versatility as building blocks and diversity in superstructures

Three 1-O-methyl-4, 6-O-benzylidene derivatives of monosaccharides (D-glucose, D-galactose and D-mannose) were synthesised: they acted as versatile gelators of various organic fluids, indicating that saccharides are useful as potential building-blocks for molecular design of chiral gelators

Response to Leopoldo Palma. Comments on Ekino et al. Cloning and Characterization of a Unique Cytotoxic Protein Parasporin-5 Produced by Bacillus thuringiensis A1100 Strain. Toxins 2014, 6, 1882–1895

I appreciate the thoughtful comments from Dr. Leopoldo Palma [1] on our research about cytotoxic protein parasporin-5 produced by Bacillus thuringiensis (BT) A1100 strain [2]. [...

Cloning and Characterization of a Unique Cytotoxic Protein Parasporin-5 Produced by Bacillus thuringiensis A1100 Strain

Parasporin is the cytocidal protein present in the parasporal inclusion of the non-insecticidal Bacillus thuringiensis strains, which has no hemolytic activity but has cytocidal activities, preferentially killing cancer cells. In this study, we characterized a cytocidal protein that belongs to this category, which was designated parasporin-5 (PS5). PS5 was purified from B. thuringiensis serovar tohokuensis strain A1100 based on its cytocidal activity against human leukemic T cells (MOLT-4). The 50% effective concentration (EC50) of PS5 to MOLT-4 cells was approximately 0.075 μg/mL. PS5 was expressed as a 33.8-kDa inactive precursor protein and exhibited cytocidal activity only when degraded by protease at the C-terminal into smaller molecules of 29.8 kDa. Although PS5 showed no significant homology with other known parasporins, a Position Specific Iterative-Basic Local Alignment Search Tool (PSI-BLAST) search revealed that the protein showed slight homology to, not only some B. thuringiensis Cry toxins, but also to aerolysin-type β-pore-forming toxins (β-PFTs). The recombinant PS5 protein could be obtained as an active protein only when it was expressed in a precursor followed by processing with proteinase K. The cytotoxic activities of the protein against various mammalian cell lines were evaluated. PS5 showed strong cytocidal activity to seven of 18 mammalian cell lines tested, and low to no cytotoxicity to the others