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 ß-pore-forming toxins (ß-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.

Identification of a second cytotoxic protein produced by Bacillus thuringiensis A1470.

Bacillus thuringiensis A1470 produces multiple proteins with similar molecular masses (~30 kDa) with cytotoxicity against human cell lines. One that was previously identified, parasporin-4, is a ß-pore-forming toxin. The N-terminal sequence of a second cytotoxic protein was identical to a partial sequence of parasporin-2 produced by B. thuringiensis A1547. PCR was performed on total plasmid DNA from A1470 by using primers for parasporin-2 to amplify a gene which was then cloned. The cloned gene differed from A1547 parasporin-2 by 8 bp and the predicted protein differed by four amino acids. The gene was expressed in Escherichia coli, and the cytotoxic activities of the recombinant protein against four human cell lines (MOLT-4, Jurkat, HeLa, and HepG2) were similar to those of A1547 parasporin-2. We then confirmed that the A1470 strain simultaneously produces parasporin-2 and parasporin-4.

[Clinical consideration of coagulase negative Staphylococci isolated in blood culture].

Despite blood culture's usefulness in antimicrobial therapy, fewer blood cultures and the infrequency of more than 1 set in cultures appear to be problems in Japan. Since June 2007 infection control team (ICT) recommended more than 1 set in blood sampling and intervention in positive blood culture, coagulase negative Staphylococci (CNS) has frequently been isolated from blood culture and its clinical significance is often difficult to judge. To determine the effect of ICT intervention, we evaluated the number of blood culture specimens, the frequency of more than 1 set in all blood culture specimens, and decision-making on antimicrobial treatment for CNS isolated retrospectively from blood. The study was divided into term I in August 2007 to July 2008, term II in August 2008 to July 2009, and term III in August 2009 to February 2010. We also analyzed how physicians treated infection or its suspicion after CNS and its drug susceptibility. The monthly number of blood culture specimens increased from 40.3 to 51.6 between terms I and III. The frequency of more than 1 set in a single blood culture session rose significantly from 67% to 89% between these terms (p < 0.001). The number of indeterminate also dropped cases significantly during these 2 terms from 27% to 6% (p = 0.017). Infection or suspected infection cases--45 of 49--had central vein catheter implantation. Inappropriate treatment by physicians in these cases also dropped significantly from 85% (11/13) to 45% (5/11) (p = 0.043) during the same 2 terms. ICT Intervention may thus increase the number of blood culture specimens, enable more than 1 set in blood sampling, make it easier to judge the presence of infection, and increase appropriate treatment by physicians. We thus believe that the quality of antimicrobial treatment could be improved through education such as ICT action.

Dialkylaluminum hydride-promoted cyclodimerization of silylated 1,3-enynes via skeletal rearrangement.

The dialkylaluminum hydride-promoted reaction of 1-silylalk-3-en-1-ynes gave symmetrical 1,2,3,5-tetrasubstituted benzenes as single regioisomers. The novel cyclodimerization via skeletal rearrangement can be rationalized by an unprecedented mechanism involving sequential hydroalumination, alkene isomerization, carboalumination, carbon-carbon bond cleavage, and retro-hydroalumination.

Mode of action of parasporin-4, a cytocidal protein from Bacillus thuringiensis.

Parasporin-4 (PS4) is a cytotoxic protein produced by Bacillus thuringiensis strain A1470. It exhibits specific cytotoxicity against human cancer cell lines, CACO-2, Sawano, and MOLT-4 cells, in particular. When cells were administrated with PS4, cell swelling and nuclear shrinkage were induced, and, the ballooned cells burst within 24 h. PSI-BLAST search showed that the protein shared homology not only with B. thuringiensis Cry toxins but also with aerolysin-type ß-pore-forming toxins. Circular dichroism measurements suggested that PS4 was a ß-sheet-rich protein. PS4 aggregated into oligomers on the plasma membrane of PS4-susceptible CACO-2 cells, but not on that of PS4-resistant HeLa cells. Leakage of lactate dehydrogenase and influx of extracellular FITC-dextrans were observed only in susceptible cells. The activation of effectors caspase 3 and/or 7 was not observed in PS4-treated CACO-2 cells. It was shown that cytotoxicity of the PS4 against CACO-2 cells was exhibited when treated by cyclodextrin which induces cholesterol depletion. These results suggest that PS4 is a unique ß-pore-forming toxin with a cholesterol-independent activity.

Didodecyldimethylammonium bromide (DDAB) induces caspase-mediated apoptosis in human leukemia HL-60 cells.

Didodecyldimethylammonium bromide (DDAB) is widely used for an efficient delivery system into mammalian cells. However, the biological activities of DDAB nanoparticles in mammalian cells are insufficiently understood. The purpose of this study was to establish a critical role of DDAB in cellular response. Here, we demonstrate that DDAB is a potent inducer of cell death in a wide range of tumor cell lines, wherein leukemia cells (HL-60 and U937) and neuroblastoma cells (Neuro2a) were more sensitive to DDAB than carcinoma cells such as HepG2 and Caco-2 cells. Moreover, in HL-60 cells, treatment with DDAB led to increased numbers of apoptotic cells with fragmented DNA (99.6%) and high levels of caspase-3 activation in comparison to that with actinomycin D. Cotreatment with a caspase-8 inhibitor (Z-IETD-FMK) or a polyethylene glycol (Mr 2000) (PEG 2000) effectively prevented the activation of caspase-3 induced by DDAB. These results suggest that DDAB can trigger caspase-3-mediated apoptosis through the extrinsic caspase-8 pathway and cytotoxic pore formation in cell membrane. Therefore, the present findings provide new insight into the biological activity of DDAB to induce caspase-mediated apoptosis.

Identification and characterization of a novel cytotoxic protein, parasporin-4, produced by Bacillus thuringiensis A1470 strain.

In 1901, a unique bacterium was isolated as a pathogen of the sotto disease of the silkmoth larvae, and later in 1915, the organism was described as Bacillus thuringiensis. Since the discovery, this bacterium has widely attracted attention of not only insect pathologists but many other scientists who are interested in strong and specific insecticidal activity associated with inclusion bodies of B. thuringiensis. This has led to the recent worldwide development of B. thuringiensis-based microbial insecticides and insect-resistant transgenic plants, as well as the epoch-making discovery of parasporin, a cancer cell-specific cytotoxin. In the review, we introduce a detection study of interaction between inclusion proteins of B. thuringiensis and brush border membrane of insects using surface plasmon resonance-based biosensor, and then identification and cloning of parasporin-4, a latest cancer cell-killing protein produced by B. thuringiensis A1470 strain. Inclusion bodies of the parasporin-4 produced by recombinant Escherichia coli were solubilized and activated with a new method and purified by an anion-exchange chromatography. At last the characterization of the recombinant parasporin-4 was shown.

Rapid and reversible shape changes of molecular crystals on photoirradiation.

The development of actuators based on materials that reversibly change shape and/or size in response to external stimuli has attracted interest for some time. A particularly intriguing possibility is offered by light-responsive materials, which allow remote operation without the need for direct contact to the actuator. The photo-response of these materials is based on the photoisomerization of constituent molecules (typically trans-cis isomerization of azobenzene chromophores), which gives rise to molecular motions and thereby deforms the bulk material. This effect has been used to create light-deformable polymer films and gels, but the response of these systems is relatively slow. Here we report that molecular crystals based on diarylethene chromophores and with sizes ranging from 10 to 100 micrometres exhibit rapid and reversible macroscopic changes in shape and size induced by ultraviolet and visible light. We find that on exposure to ultraviolet light, a single crystal of 1,2-bis(2-ethyl-5-phenyl-3-thienyl)perfluorocyclopentene changes from a square shape to a lozenge shape, whereas a rectangular single crystal of 1,2-bis(5-methyl-2-phenyl-4-thiazolyl)perfluorocyclopentene contracts by about 5-7 per cent. The deformed crystals are thermally stable, and switch back to their original state on irradiation with visible light. We find that our crystals respond in about 25 microseconds (that is, about five orders of magnitude faster than the response time of the azobenzene-based polymer systems) and that they can move microscopic objects, making them promising materials for possible light-driven actuator applications.

Investigation of a novel Bacillus thuringiensis gene encoding a parasporal protein, parasporin-4, that preferentially kills human leukemic T cells.

A novel gene encoding a leukemic cell-killing parasporal protein, designated parasporin-4, was cloned from an isolate of Bacillus thuringiensis serovar shandongiensis. The amino acid sequence of the parasporin-4, as deduced from the gene sequence, had low-level homologies of <30% with the established B. thuringiensis Cry proteins including the three known parasporins. When the gene was expressed in a recombinant of Escherichia coli BL21(DE3), the parasporin-4 formed intracellular inclusion bodies. Alkali-solubilized and proteinase K-activated inclusion protein exhibited strong cytotoxic activity against human leukemic T cells (MOLT-4) and weak for normal T cells, but no adverse effect on human uterus cervix cancer cells (HeLa).

Identification of a novel cytotoxic protein, Cry45Aa, from Bacillus thuringiensis A1470 and its selective cytotoxic activity against various mammalian cell lines.

Parasporal inclusion proteins produced by Bacillus thuringiensis strain A1470 exhibit strong cytotoxicity against human leukemic T cells when activated by protease treatment. One of the cytotoxic proteins was separated by anion exchange chromatography and gel filtration chromatography and designated Cry45Aa. Its gene was then expressed in recombinant Escherichia coli, in which the Cry45Aa precursor was accumulated in an inclusion body. It was solubilized in sodium carbonate buffer and processed with proteinase K, and cytotoxic activities of the protein against various mammalian cell lines were evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assay. The protein exhibited high cytotoxic activity against CACO-2, Sawano, MOLT-4, TCS, and HL60 cells and moderate activity against U-937 DE-4, PC12, and HepG2 cells. On the other hand, the EC50 values against Jurkat, K562, HeLa, A549, Vero, COS-7, NIH3T3, CHO, and four normal tissue cells (human primary hepatocyte cells, UtSMC, MRC-5, and normal T cells) were >2 microg/mL.

Production of secretory immunoglobulin A against Shiga toxin-binding subunits in mice by mucosal immunization.

The toxicity of Shiga toxins (Stx) depends on the binding of their B subunits to carbohydrate ligands on host cells. The production of antibodies against B subunits, especially immunoglobulin A (IgA) secreted on the mucosal surface, should contribute to host defense. One of the major problems in attempts to produce IgA against Stx was the poor immunogenicity of B subunits. We were able to produce serum IgA as well as IgG against Stx1B in mice of the H-2d haplotype by means of intranasal immunization with recombinant B subunits of Stx (Stx1B) together with cholera toxin as a mucosal adjuvant. Secretory IgA (S-IgA) was detected in nasal washes but not in feces. We prepared chemically cross-linked Stx1B for use as an immunogen, and the formation of stable oligomers was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. When the cross-linked Stx1B was used together with cholera toxin for the intranasal immunization of BALB/c mice, strong enhancement of the immune response was observed. The S-IgA titers in nasal washes were 16- to more than 64-fold higher than those in mice immunized with native Stx1B plus cholera toxin. Furthermore, fecal IgA was detectable when the cross-linked Stx1B was used. The use of cholera toxin was necessary for the induction of high titers of S-IgA in the nasal washes. However, the effect of cross-linking was dependent on the major histocompatibility complex haplotype; that is, no enhancement of IgA production was observed in C57BL/6 mice. The present results provide a practical means of producing IgA against Stx1B in BALB/c mice.

Six novel P gene mutations and oculocutaneous albinism type 2 frequency in Japanese albino patients.

Type 2 oculocutaneous albinism (OCA2) is an autosomal recessive disorder that results from mutations in the P gene that codes one of the melanosomal proteins, the function of which remains unknown. In this paper, we report the frequency of OCA2, 8%, among the Japanese albino population, six novel mutations containing four missense substitutions (P198L, P211L, R10W, M398I), and two splice site mutations (IVS15+1 G>A, IVS24-1 G>C). One of them, R10W, was within the putative signal peptide at the N-terminal of the P protein. This is the first report on the frequency of OCA2 in the Japanese albino population.

Restricted expression of shiga toxin binding sites on mucosal epithelium of mouse distal colon.

Shiga toxins (Stx) are some of the major virulence factors of enterohemorrhagic Escherichia coli strains such as serotype O157:H7. To explore how Stx might initially gain access to the bloodstream from sites of infection, frozen sections of mouse colon were immunohistochemically examined for binding sites for recombinant binding subunits (Stx1B). Binding sites were selectively expressed on the epithelium in the distal half of the mouse colon, whereas the proximal half did not exhibit any binding sites. In agreement with this observation, we also demonstrated the distal-part-restricted distribution of glycolipids that bind to Stx1B in the mouse colon. For comparison, the binding sites of several control lectins were also examined. Selective binding to the distal part of the colon was not seen with any other control lectins, including Griffonia simplicifolia lectin-I isolectin B4 (GS-I-B4), which shares alpha-galactose specificity with Stx1B. Partial overlapping of the specificities of Stx1B and GS-I-B4 was seen by assay with globotriose-conjugated multivalent ligands. The results indicate that Stx1B is stricter in the recognition of carbohydrate determinants than GS-I-B4 when examined with biological ligands.

Lack of Shiga-like toxin binding sites in germinal centres of mouse lymphoid tissues.

B cells in germinal centres are known to express carbohydrate antigen CD77 in human lymphoid tissues. The CD77 antigen is specifically recognized by Shiga-like toxins (SLTs) that are produced by enterohaemorrhagic Escherichia coli O157:H7. To determine whether the binding subunits of Shiga-like toxin-1 (SLT-1B) could have adverse effects on the murine immune system when used as an immunogen, we investigated whether SLT-1B could bind to germinal centres of mouse lymphoid tissues. Frozen sections of peripheral lymph nodes and Peyer's patches from immunized mice were tested for the presence of SLT-1B-binding sites by immunohistological methods. Germinal centres were not stained with SLT-1B, while they were intensely stained with peanut agglutinin (PNA), another marker of germinal centres. On the other hand, SLT-1B specifically bound to renal tubules and collecting ducts in frozen sections of mouse kidney. This is consistent with results from human tissues. We also demonstrated that B220/PNA double-positive populations in lymph nodes from immunized mice exhibited only marginal staining with SLT-1B. The present results suggest that SLTs would not impede germinal centre functions of the murine immune system.