J Bacteriol, 2004 Jul, 186(13), 4100 - 9 Identification of sigmaB-dependent genes in Bacillus cereus by proteome and in vitro transcription analysis; van Schaik W et al.; The alternative sigma factor sigmaB of the food pathogen Bacillus cereus is activated upon stress exposure and plays a role in the adaptive response of vegetative cells . This study describes the identification of sigmaB-dependent genes in B . cereus . Two-dimensional gel electrophoresis was performed with protein extracts from a sigmaB-overproducing B . cereus strain . Nine protein spots, which were absent from the negative control, were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry or N-terminal sequencing . The sigmaB-dependent expression of the corresponding genes was confirmed by Northern blot analysis with RNA isolated from B . cereus ATCC 14579 and its sigB null mutant . Northern blot analysis also revealed that six other genes were part of sigmaB-dependent operons . The proteins that are predicted to be encoded by the sigmaB-dependent genes include an intracellular protease, a Mg2+ transporter, and a thiamine biosynthesis protein (ThiG) . Highly conserved promoter sites were found to precede all sigmaB-dependent genes, with the exception of thiG . By searching the B . cereus genome for this conserved promoter sequence, five more candidate sigmaB-dependent genes were identified . Northern blot analysis and in vitro transcription experiments with a reconstituted B . cereus sigmaB-RNA polymerase holoenzyme confirmed the sigmaB dependency of two of these genes and strongly suggested that two other genes, encoding an oligopeptide-binding OppA-like protein and subunit II of the cytochrome d ubiquinol oxidase, are also sigmaB dependent . In conclusion, sigmaB of B . cereus not only regulates genes directly involved in the stress response but may also control specific metabolic rearrangements. Drug Metab Dispos, 2004 Jul, 32(7), 707 - 14 Involvement of interleukin-6 and tumor necrosis factor alpha in CYP3A11 and 2C29 down-regulation by Bacillus Calmette-Guerin and lipopolysaccharide in mouse liver; Ashino T et al.; Bacillus Calmette-Guerin (BCG) and lipopolysaccharide (LPS) are well known potent activators of the cell-mediated immune system and thus lead to the decreases in cytochrome P450 (P450) . In this study we used interleukin (IL)-1alpha/beta, IL-6, or tumor necrosis factor alpha (TNFalpha) knockout (KO) mice to investigate how each cytokine is involved in P450 down-regulation, especially CYP3A11 and 2C29 . BCG (40 mg/kg) was found to reduce both CYP3A11 and 2C29 mRNAs at 24 h after treatment in IL-1alpha/beta KO mice in a manner similar to that seen in wild-type mice . CYP3A11 mRNA, but not CYP2C29 mRNA, was significantly decreased by BCG treatment in the TNFalpha KO mice, although the decrease was less than that of wild-type or IL-1alpha/beta KO mice . In contrast, BCG showed no significant effect on CYP3A11 and 2C29 mRNAs in IL-6 KO mice . On the other hand, LPS was able to decrease CYP3A11 and 2C29 mRNA levels in all of the cytokine KO mice and markedly increased systemic levels of TNFalpha; BCG (40 mg/kg) lacked such activity . The present study has shown that IL-6 and TNFalpha are likely to be major factors involved in the down-regulation of CYP3A11 and 2C29 mRNAs in mice . In addition, there exist differences in the amount and/or kind of cytokines released by BCG or LPS, the latter being more potent than the former . This will be a possible reason for differential capability of P450 down-regulation between BCG and LPS. Food Addit Contam, 2004 May, 21(5), 422 - 33 Validation of a microbiological method: the STAR protocol, a five-plate test, for the screening of antibiotic residues in milk; Gaudin V et al.; The results of an in-house laboratory validation of a microbiological method for the screening of antibiotic residues in milk are presented . The sensitivity of this five-plate test, called Screening Test for Antibiotic Residues (STAR), was established by the analysis of milk samples spiked with 66 antibiotics at eight different concentrations . Ten different groups of antibiotics were studied: macrolides, aminoglycosides, cephalosporins, penicillins, quinolones, tetracyclines, sulphonamides, lincosamides, phenicolated and miscellaneous drugs . It was shown that 21 antibiotics were detected by the STAR protocol at or below the maximum residue limit (MRL), and that a further 27 drugs could be detected at levels from the MRL up to four times the MRL . The sensitivity of the STAR protocol was at or below the MRL for three macrolides, one tetracycline, two aminoglycosides, some sulphonamides, half of the beta-lactams, quinolones, lincosamides, trimethoprim and baquiloprim . Moreover, the STAR protocol was at least twice as sensitive as conventional methods for macrolides, quinolones and tetracyclines . The other antibiotics had limits of detection between four and 150 times the MRL . Each plate was preferentially sensitive for one or two families of antibacterials: the plate Bacillus cereus for tetracyclines, the plate Escherichia coli for quinolones, the plate Basillus subtilis for aminoglycosides, the plate Kocuria varians for macrolides, and the plate Bacillus stearothermophilus for sulphonamides and beta-lactams . This method has been used routinely on a day-to-day basis to direct the physicochemical confirmation towards one or two families of antibiotics . Considering the high cost of liquid chromatography coupled with tandem mass spectrometry detection analyses, the reduction of the range of antibiotics to test for confirmation is a significant gain in time and money. Scand J Urol Nephrol, 2004, 38(2), 182 - 3 Vitiligo--an autoimmune side-effect of intravesical bacillus Calmette-Guérin instillation? Beisland C, Holsen DS. We report a case of vitiligo in a 63-year-old man who had undergone intravesical bacillus Calmette-Guerin treatment following removal of a superficial transitional cell carcinoma in the bladder. Mediators Inflamm, 2004 Feb, 13(1), 45 - 9 Secretion of interleukin-8 by human-derived cell lines infected with Mycobacterium bovis; Mendez-Samperio P et al.; BACKGROUND: The variable efficacy of bacillus Calmette-Guerin (Mycobacterium bovis BCG) in protecting humans against tuberculosis has prompted a search for the mechanisms through which BCG induces chemokines . In this study, our experiments were designed to determine the role of the transcription factor nuclear factor-kappaB (NF-kappaB) and intracellular calcium in the production of interleukin (IL)-8, a main chemotactic factor, by human-derived monocytic cell line U937 and by a human epithelial HEp-2 cell line infected with M . bovis BCG . METHODS: The concentrations of IL-8 in culture supernatants of U937 cells or HEp-2 cells infected with M . bovis BCG were determined by enzyme-linked immunosorbent assay . We used sulfasalazine and curcumin, which are well-described inhibitors of NF-kappaB activity, and we used ethylenediamine tetraacetic acid to deplete extracellular Ca2+ or used the cell-permeable agent 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester to chelate releasable intracellular stores of Ca2+ in order to investigate the mechanisms through which M . bovis BCG induces IL-8 secretion in our system . RESULTS: The enzyme-linked immunosorbent assay showed that IL-8 protein secretion was elevated in M . bovis-infected cell lines . This effect was statistically significant (p < 0.01) . When calcium influx was suppressed in M . bovis-infected cell lines, IL-8 secretion was inhibited . Notably, specific inhibitors of NF-kappaB (sulfasalazine and curcumin) inhibited M . bovis-induced IL-8 secretion from U937 cells or HEp-2 cells . CONCLUSIONS: Collectively, these results indicate that activation of NF-kappaB is an important signal transduction pathway in M . bovis-induced IL-8 secretion in monocytic or epithelial cells . Furthermore, the results showed that calcium influx had a direct effect on IL-8 secretion in U937 cells or HEp-2 cells infected with M . bovis. Huan Jing Ke Xue, 2004 Mar, 25(2), 166 - 9 {Biosorption of Pb2+ by Bacillus cereus biomass}; Pan JH et al.; In this paper, the potentiometric titration and the sorption experiments were applied to investigate the biosorption behavior of Pb2+ on Bacillus cereus biomass . It was shown that the biosorption capacities increased with increasing pH from 3 to 7, and the higher concentration of the biomass in solution was favorable to the sorption of Pb2+ . Langmuir model simulated the adsorption data of Pb2+ ion very well, and the saturated sorption capacity for Pb2+ ion achieved 36.7 mg.g-1 dry biomass . According to the result of the potentiometric titration, the Gran plots for the hydroxide back-titration of biomass suspension systems were utilized to derive the specific volume of titrant at the equivalence point (Ve), furthermore, the total surface site concentration was calculated. Biotechnol Lett, 2004 Apr, 26(8), 635 - 9 Molecular cloning and sequence analysis of the chitinase gene from Bacillus thuringiensis serovar alesti; Lin Y et al.; Endogenous chitinase plays a positive role in the pathogenicity of Bacillus thuringiensis to insect pests . The chitinase gene was cloned from B . thuringiensis serovar alesti strain HD-16, and the deduced 676 amino acid sequence showed a high degree of similarity with other Bacillus chitinases . Additionally, the deduced amino acid sequence showed that the protein contained an amino terminus signal peptide and consisted of a catalytic domain, a fibronectin type III domain and a chitin-binding domain . All three domains showed conserved sequences when compared to other bacterial chitinase or cellulase sequences. J Am Chem Soc, 2004 Jun 23, 126(24), 7464 - 75 Alanine racemase free energy profiles from global analyses of progress curves; Spies MA et al.; Free energy profiles for alanine racemase from Bacillus stearothermophilus have been determined at pH 6.9 and 8.9 from global analysis of racemization progress curves . This required a careful statistical design due to the problems in finding the global minimum in mean square for a system with eight adjustable parameters (i.e., the eight rate constants that describe the stepwise chemical mechanism) . The free energy profiles obtained through these procedures are supported by independent experimental evidence: (1) . steady-state kinetic constants, (2) . solvent viscosity dependence, (3) . spectral analysis of reaction intermediates, (4) . equilibrium overshoots for progress curves measured in D(2)O, and (5) . the magnitudes of calculated intrinsic kinetic isotope effects . The free energy profiles for the enzyme are compared to those of the uncatalyzed and the PLP catalyzed reactions . At pH 6.9, PLP lowers the free energy of activation for deprotonation by 8.4 kcal/mol, while the inclusion of apoenzyme along with PLP additionally lowers it by 11 kcal/mol. J Biotechnol, 2004 Jul 1, 111(1), 89 - 96 Pyruvate formation and suppression in recombinant Bacillus megaterium cultivation; Hollmann R et al.; A recombinant Bacillus megaterium strain showed the ability to secrete large amounts of pyruvate (up to 27.8 gl( -1)) for growth rates larger than 0.15 h(-1) . Cultivation below this growth rate avoids pyruvate formation while minimizing acetate and succinate production . Using exponential feeding, final biomass concentrations of up to 80 g l(-1) were achieved . Overall molar yields for the experiments with pyruvate formation were as high as 0.79 mol mol(-1) . Pyruvate formation was caused by the discrepancy between glycolytic and pyruvate dehydrogenase reaction/tricarboxylic acid cycle capacities during glucose excess . High pyruvate resulted in deceleration and subsequent cessation of growth . In addition, this inhibitory effect is likely associated with the phoshoenolpyruvate:glucose phosphotransferase system used by B . megaterium as the main importer for glucose. Clin Exp Immunol, 2004 Jul, 137(1), 24 - 34 Recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) expressing mouse IL-18 augments Th1 immunity and macrophage cytotoxicity; Luo Y et al.; Interleukin-18 (IL-18) has been demonstrated to synergize with BCG for induction of a T-helper-type 1 (Th1) immune response . Since successful treatment of superficial bladder cancer with BCG requires proper induction of Th1 immunity, we have developed a recombinant (r) BCG strain that functionally secretes mouse (m) IL-18 . This rBCG-mIL-18 strain significantly increased production of the major Th1 cytokine IFN-gamma in splenocyte cultures, at levels comparable to that elicited by control BCG plus exogenous rIL-18 . IFN-gamma production by splenocytes was eliminated by addition of neutralizing anti-IL-18 antibody . Endogenous IL-12 played a favourable role whereas IL-10 played an adverse role in rBCG-mIL-18-induced IFN-gamma production . Enhanced host antimycobacterial immunity was observed in mice infected with rBCG-mIL-18 which showed less splenic enlargement and reduced bacterial load compared to control mice infected with BCG . Further, splenocytes from rBCG-mIL-18-infected mice, in response to BCG antigen, displayed increased production of IFN-gamma and GMCSF, decreased production of IL-10, elevated cellular proliferation and higher differentiation of IFN-gamma-secreting cells . rBCG-mIL-18 also enhanced BCG-induced macrophage cytotoxicity against bladder cancer MBT-2 cells in a dose-dependent manner . Neutralizing all endogenous macrophage-derived cytokines tested (IL-12, IL-18 and TNF-alpha) as well as IFN-gamma severely diminished the rBCG-mIL-18-induced macrophage cytolytic activity, indicating a critical role for these cytokines in this process . Cytokine analysis for supernatants of macrophage-BCG mixture cultures manifested higher levels of IFN-gamma and TNF-alpha in rBCG-mIL-18 cultures than in control BCG cultures . Taken together, this rBCG-mIL-18 strain augments BCG's immunostimulatory property and may serve as a better agent for bladder cancer immunotherapy and antimycobacterial immunization. Immunology, 2004 Jul, 112(3), 461 - 70 Cellular immune responses induced in cattle by heterologous prime-boost vaccination using recombinant viruses and bacille Calmette-Guérin; Vordermeier HM et al.; The development of novel vaccine strategies to replace or supplement bacille Calmette-Guerin (BCG) is urgently required . Here we study, in cattle, the use of heterologous prime-boost strategies based on vaccination with BCG and the mycobacterial mycolyl transferase Ag85A (Rv3804c) expressed either in recombinant modified vaccinia virus Ankara (MVA85A) or attenuated fowlpox strain FP9 (FP85A) . Five different vaccination schedules were tested in the first experiment: MVA85A followed by BCG (group 1); BCG followed by MVA85A (group 2); BCG followed by FP85A and then MVA85A (group 3); MVA85A followed by MVA85A and then FP85A (group 4); and FP85A followed by FP85A and then MVA85A (group 5) . Vaccine-induced levels of cellular immunity were assessed by determining interferon-gamma (IFN-gamma) responses in vitro . Prime-boost protocols, using recombinant MVA and BCG in combination (groups 1-3), resulted in significantly higher frequencies of Ag85-specific IFN-gamma-secreting cells than the two viral vectors used in combination (P=0.0055), or BCG used alone (groups 2 and 3, P=0.04) . The T-cell repertoires of the calves in all five groups were significantly broader following heterologous booster immunizations than after the primary immunization . In a second experiment, the effects of BCG\MVA85A heterologous prime-boost vaccination were compared with BCG\BCG homologous revaccination . The results suggested a higher Ag85A-specific response with a wider T-cell repertoire in the MVA85A-boosted calves than in the BCG\BCG-vaccinated calves . In conclusion therefore, the present report demonstrates the effectiveness of heterologous prime-boost strategies based on recombinant MVA and BCG to induce strong cellular immune responses in cattle and prioritise such vaccination strategies for rapid assessment of protective efficacy in this natural target species of tuberculosis. J Infect Dis, 2004 Jul 1, 190(1), 123 - 6 Epub 2004 Jun 11. Mycobacterium tuberculosis H37Rv: Delta RD1 is more virulent than M . bovis bacille Calmette-Guérin in long-term murine infection; Sherman DR et al.; Region of difference (RD1) genes are present in virulent Mycobacterium tuberculosis but not the vaccine strain M . bovis bacille Calmette-Guerin (BCG) . The deletion of RD1 from M . tuberculosis produces an attenuation strikingly like that of BCG, which suggests the use of RD1 mutant strains for improvement of the tuberculosis (TB) vaccine . We performed long-term murine infection with M . tuberculosis H37Rv: Delta RD1 and BCG . Mice infected with H37Rv: Delta RD1 gained less weight than did BCG-infected control mice, and, after >1 year, their lungs harbored many more bacteria and displayed significant levels of inflammation . This difference in virulence has important implications for the pursuit of strains lacking RD1 in the development of the TB vaccine. J Infect Dis, 2004 Jul 1, 190(1), 115 - 22 Epub 2004 Jun 04. Enhanced protection against tuberculosis by vaccination with recombinant Mycobacterium microti vaccine that induces T cell immunity against region of difference 1 antigens; Brodin P et al.; Mycobacterium microti, the vole bacillus, which was used as a live vaccine against tuberculosis until the 1970s, confers the same protection in humans as does Mycobacterium bovis bacille Calmette-Guerin (BCG) . However, because the efficacy of the BCG vaccine varies considerably, we have tried to develop a better vaccine by reintroducing into M . microti the complete region of difference 1 (RD1), which is required for secretion of the potent T cell antigens early secreted antigen target (ESAT)-6 and culture filtrate protein (CFP)-10 . The resultant recombinant strain, M . microti OV254::RD1-2F9, induced specific ESAT-6 and CFP-10 immune responses in mice with CD8(+) T lymphocytes that had strong expression of the CD44(hi) activation marker . This vaccine also displayed better efficacy against disseminated disease in the mouse and the guinea pig models of tuberculosis than was seen in animals vaccinated with M . microti alone or with BCG . The M . microti OV254::RD1-2F9 vaccine was less virulent and persistent in mice and than was BCG::RD1-2F9 may represent a safer alternative to BCG::RD1-2F9. Pediatr Infect Dis J, 2004 Jun, 23(6), 544 - 50 Low birth weight infants and Calmette-Guérin bacillus vaccination at birth: community study from Guinea-Bissau; Roth A et al.; BACKGROUND: In developing countries, low birth weight (LBW) children are often not vaccinated with Calmette-Guerin bacillus (BCG) at birth . Recent studies have suggested that BCG may have a nonspecific beneficial effect on infant mortality . We evaluated the consequences of not vaccinating LBW children at birth in Guinea-Bissau . METHODS: Between 1989 and 1999, 7138 children born at the central hospital had a birth weight registered . We assessed BCG coverage until 3 years of age . Data on tuberculin skin test (TST) for 297 children and BCG scar for 1319 children in the study population were reanalyzed for differences between normal birth weight (NBW) children and LBW children . We assessed the effect of early BCG vaccination on mortality to 12 months of age . RESULTS: Among LBW children there were 1.5- to 3-fold more unvaccinated individuals than among NBW children up to 4 months of age . There was no overall difference between LBW and NBW children in TST or BCG scarring; LBW children vaccinated early may have had slightly reduced reactions to tuberculin . Among 845 LBW children, 182 had received BCG within the first week of life . Controlling for background factors and censoring at first diphtheria-tetanuspertussis vaccination, measles vaccination or at 6 months of age (whichever came first), the mortality rate ratio for BCG-vaccinated versus -unvaccinated LBW children was 0.17 (95% confidence interval, 0.06-0.49), with an even stronger effect for LBW children vaccinated in the first week of life (mortality rate ratio, 0.07; 95% confidence interval, 0.01-0.62) . CONCLUSIONS: The policy of not vaccinating with BCG at birth had a negative impact on vaccination coverage for LBW children . Early BCG vaccination had no large negative impact on TST and BCG scarring . Mortality was lower for BCG-vaccinated than for unvaccinated LBW children controlling for available background factors . BCG vaccination of LBW children may have a beneficial effect on survival that cannot be explained by protection against tuberculosis . Future studies should examine possible adverse effects from equalizing BCG policy for LBW and NBW children. Int J Food Microbiol, 2004 Jul 15, 94(2), 175 - 83 Sperm bioassay for rapid detection of cereulide-producing Bacillus cereus in food and related environments; Andersson MA et al.; A novel in vitro method, sperm micro assay for rapidly distinguishing cereulide, the emetic toxin producing Bacillus cereus from non-producers is described and its use for quantitating cereulide and screening large numbers of B . cereus strains/colonies evaluated . The assay is non-laborious and can be executed with equipment present in most laboratories . Boar spermatozoa, purchased as standard semen from artificial insemination suppliers, are used to detect toxicity . Boar sperms respond within 5 min by cessation of motility when exposed at 37 degrees C to heat-treated (100 degrees C) extract prepared from a cereulide containing B . cereus . The assay can be done on individual colonies on the primary plate, with no need for pure culture and the qualitative result is obtained within 30 min . The assay is robust, not sensitive to age or storage of the culture plates . The use of the sperm micro assay for semiquantitative estimation of cereulide in B . cereus was validated with 14 different B . cereus strains using as reference the specific chemical assay for cereulide, based on liquid chromatography-ion trap mass spectrometry (LC-ion trap MS) . The cereulide contents calculated from endpoint dilutions of the sperm micro assay matched the result of the chemical analysis closely . The detection threshold of the sperm micro assay was measured as 0.3 +/- 0.1 ng of cereulide per 5.4 x 10(6) sperm cells in 0.2 ml or 0.9 ng of cereulide per mg of B . cereus biomass (wet wt.) . Food-related B . cereus strains contained 4-400 ng of cereulide per mg (wet wt.) . When a large number of B . cereus of food, non-food, clinical and environmental origins were screened and 107 independent strains/isolates were identified as cereulide producers, it was observed that all of these had low or no haemolytic activity when cultivated on bovine blood agar . None of the strains/isolates with wide, clear zones of haemolysis, considered typical of B . cereus, produced cereulide. J Struct Biol, 2004 Aug, 147(2), 136 - 45 Visualization of alpha-helical features in a density map constructed using 9 molecular images of the 1.8 MDa icosahedral core of pyruvate dehydrogenase; Borgnia MJ et al.; Strategies to achieve the highest resolutions in structures of protein complexes determined by cryo-electron microscopy generally involve averaging information from large numbers of individual molecular images . However, significant limitations are posed by heterogeneity in image quality and in protein conformation that are inherent to large data sets of images . Here, we demonstrate that the combination of iterative refinement and stringent molecular sorting is an effective method to obtain substantial improvements in map quality of the 1.8 MDa icosahedral catalytic core of the pyruvate dehydrogenase complex from Bacillus stearothermophilus . From a starting set of 42,945 images of the core complex, we show that using only the best 139 particles in the data set produces a map that is superior to those constructed with greater numbers of images, and that the location of many of the alpha-helices in the structure can be unambiguously visualized in a map constructed from as few as 9 particles. J Biol Chem, 2004 Aug 27, 279(35), 36771 - 7 Epub 2004 Jun 10. Mycobacterium tuberculosis antigen 85A and 85C structures confirm binding orientation and conserved substrate specificity; Ronning DR et al.; The maintenance of the highly hydrophobic cell wall is central to the survival of Mycobacterium tuberculosis within its host environment . The antigen 85 proteins (85A, 85B, and 85C) of M . tuberculosis help maintain the integrity of the cell wall 1) by catalyzing the transfer of mycolic acids to the cell wall arabinogalactan and 2) through the synthesis of trehalose dimycolate (cord factor) . Additionally, these secreted proteins allow for rapid invasion of alveolar macrophages via direct interactions between the host immune system and the invading bacillus . Here we describe two crystal structures: the structure of antigen 85C co-crystallized with octylthioglucoside as substrate, resolved to 2.0 A, and the crystal structure of antigen 85A, which was solved at a resolution of 2.7 A . The structure of 85C with the substrate analog identifies residues directly involved in substrate binding . Elucidation of the antigen 85A structure, the last of the three antigen 85 homologs to be solved, shows that the active sites of the three antigen 85 proteins are virtually identical, indicating that these share the same substrate . However, in contrast to the high level of conservation within the substrate-binding site and the active site, surface residues disparate from the active site are quite variable, indicating that three antigen 85 enzymes are needed to evade the host immune system. Plant Physiol Biochem, 2004 May, 42(5), 383 - 7 Larvicidal Cry proteins from Bacillus thuringiensis are released in root exudates of transgenic B . thuringiensis corn, potato, and rice but not of B . thuringiensis canola, cotton, and tobacco; Saxena D et al.; Larvicidal proteins encoded by cry genes from Bacillus thuringiensis were released in root exudates from transgenic B . thuringiensis corn, rice, and potato but not from B . thuringiensis canola, cotton, and tobacco . Nonsterile soil and sterile hydroponic solution in which B . thuringiensis corn, rice, or potato had been grown were immunologically positive for the presence of the Cry proteins; from B . thuringiensis corn and rice, the soil and solution were toxic to the larva of the tobacco hornworm (Manduca sexta), and from potato, to the larva of the Colorado potato beetle (Leptinotarsa decemlineata), representative lepidoptera and coleoptera, respectively . No toxin was detected immunologically or by larvicidal assay in soil or hydroponic solution in which B . thuringiensis canola, cotton, or tobacco, as well as all near-isogenic non-B . thuringiensis plant counterparts or no plants, had been grown . All plant species had the cauliflower mosaic virus (CaMV) 35S promoter, except rice, which had the ubiquitin promoter from maize . The reasons for the differences between species in the exudation from roots of the toxins are not known . The released toxins persisted in soil as the result of their binding on surface-active particles (e.g . clay minerals, humic substances), which reduced their biodegradation . The release of the toxins in root exudates could enhance the control of target insect pests, constitute a hazard to nontarget organisms, and/or increase the selection of toxin-resistant target insects . Respir Med, 2004 Jun, 98(6), 509 - 14 Analysis of local T lymphocyte subsets upon stimulation with intravesical BCG: a model to study tuberculosis immunity; Ponticiello A et al.; Cell-mediated immune response can control tuberculosis infection . A significant role for immune cells like CD4, CD8 and gammadelta T lymphocytes have been recognized, but little is known about the kinetics of activation and accumulation of these cells in course of Tuberculosis infection in humans . This is due to both the difficult to access to human lung and the fact that most subjects are examined in different periods of infection which may condition T cell changes . To overcome these problems, we have used intravesical BCG (Bacillus Calmette-Guerin) treatment for preventing the recurrences of bladder cancer as an in vivo experimental model of human tuberculosis infection . 20 male caucasian patients with proven bladder superficial transitional cell carcinoma treated with transurethral resection followed by six weekly intravesical instillations of BCG (T0-T6) were enrolled . Changes in T lymphocyte subsets were assessed by flow cytometry in the bladder wash recovered after each BCG instillation . Our study shows that the action of BCG appears to be T cell dependent . Lymphocytes increase at any new instillation and tend towards the reduction with the suspension of the stimulus . BCG induces a massive increase in the proportion of CD4 Th1 subset followed by an increase in gammadelta T cells, while no significant variation for CD8 and NK cells is found . Our results suggest that BCG infection model represents a valid experimental tool to study the immunological events evoked in vivo by Mycobacterium tuberculosis in humans at the site of infection. Lett Appl Microbiol, 2004, 39(1), 109 - 15 Design of a 5' exonuclease-based real-time PCR assay for simultaneous detection of Bacillus licheniformis, members of the 'B . cereus group' and B . fumarioli in gelatine; De Clerck E et al.; AIMS: The design of a fast, sensitive and specific detection method for Bacillus licheniformis, members of the 'B . cereus group' and B . fumarioli in gelatine . METHODS AND RESULTS: Specific Taqman probes were designed and tested in a real-time PCR setting . A specific fluorescent signal could be obtained for all gelatine isolates attributed to these species in one single real-time PCR reaction . After sample preparation, a gelatine sample spiked with 1 CFU provided enough template DNA for a significant signal . CONCLUSION: The potential of a real-time PCR assay for simultaneous detection of B . licheniformis, members of the 'B . cereus group' and B . fumarioli in gelatine is demonstrated . SIGNIFICANCE AND IMPACT OF THE STUDY: Implementation of the assay in gelatine producing plants may shorten delivery terms and inform on hazards to public health and suitable remediation procedures. Lett Appl Microbiol, 2004, 39(1), 89 - 92 Bacillus thuringiensis serovar shandongiensis strain 89-T-34-22 produces multiple cytotoxic proteins with similar molecular masses against human cancer cells; Okumura S et al.; AIMS: To prove that Bacillus thuringiensis serovar shandongiensis strain 89-T-34-22 produces several novel cytotoxic proteins against human leukaemic T cells . METHODS AND RESULTS: Parasporal inclusion protein was solubilized and processed by proteinase K and was separated by anion-exchange chromatography . Cytopathic effects of each fraction against MOLT-4 and Jurkat cells were monitored . CONCLUSIONS: Existence of at least two novel cytotoxic proteins was suggested and N-terminal sequences of the newly identified proteins were determined to be QSTTDVIREY and X (Y or I) (P or I) NLANELA (X indicates uncertain amino acids) . Molecular masses of the two proteins were approx . 27-28 kDa . SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we demonstrated that the strain 89-T-34-22 produces at least two novel cytotoxic proteins with similar molecular masses against human cancer cells . This is the first strain of B . thuringiensis which produces multiple cytotoxic proteins against human cancer cells. Proteomics, 2004 May, 4(5), 1465 - 90 A proteomic view of cell physiology of Bacillus licheniformis; Voigt B et al.; The still ongoing sequencing of Bacillus licheniformis at the Gottingen Sequencing Laboratory provides the basis for proteome studies of the bacterium . By using two-dimensional (2-D) electrophoresis and protein identification by mass spectrometry, we were able to create master gels for B . licheniformis cells grown either in minimal medium or in complex medium containing about 300 and 180 entries, respectively . With the DECODON Delta 2D software we identified the most abundant protein spots on the gels, which were shown to perform mainly basic metabolic functions in the cell such as translation, amino acid metabolism, glycolysis, and tricarboxylic acid (TCA) cycle . Based on the master gels, we were able to study the regulation of metabolic pathways such as glycolysis and TCA cycle . In cells grown in the presence of glucose a significant increase of the amount of some glycolytic enzymes (TpiA, GapA, Pgk, Pgm, Eno, Pyk) and of the pyruvate dehydrogenase (PdhA-D) was found . At the same time, there is a strong repression of almost all TCA cycle enzymes and of the ATP synthase . Glucose also stimulates the acetate kinase (AckA) and the phosphotransacetylase (Pta) which are known to be involved in the overflow metabolism in B . subtilis . Furthermore, we began developing proteomic signatures for growth of B . licheniformis in complex medium . For this purpose, we compared the proteome pattern of exponentially growing cells with that of cells in different stages during stationary phase . The most obvious proteomic signature indicates that cells during stationary phase are subjected to a severe oxidative stress and a resulting protein stress . Furthermore, the level of many vegetative proteins is strongly reduced when the growth is arrested after entry into stationary phase . The data indicate that proteomics can be a valuable tool to describe the physiological state of B . licheniformis cell populations, e.g., of cells growing in a bioreactor. J Arthroplasty, 2004 Jun, 19(4), 525 - 7 Prosthetic joint infection with Pasturella multocida following cat scratch: a report of 2 cases; Mehta H et al.; Infection is a known cause of failure in a total joint arthroplasty . Secondary or delayed infections are caused by a wider variety of pathogens, including Gram-negative organisms . Pasturella multocida is a Gram-negative bacillus that forms part of the normal nasopharyngeal and gastrointestinal flora of cats and many other animals . Nontrauma-associated infections also have been reported, but these are more often confined to animal handlers . We report 2 patients who had cat scratch and who developed infection of their total hip arthroplasties with P multocida . Both patients were immunocompromised and required revision of their hip arthroplasty . One patient had 1-stage revision, because infective cause was not obvious at the time of surgery . These patients were followed for 18 months to 2 years after surgery, with good results. Protein Eng Des Sel, 2004 May, 17(5), 411 - 6 Epub 2004 Jun 08. Modification of substrate-binding site of glutamyl endopeptidase from Bacillus intermedius; Demidyuk IV et al.; Glutamyl endopeptidases (GEPs) are serine proteases belonging to the chymotrypsin structural family . Although the family as a whole has been described in detail, the molecular mechanism underlying strict substrate specificity of GEPs remains unclear . The most popular hypothesis attributes the key role in recognition of the charged substrates by GEPs to the conserved amino acid His213 (chymotrypsin numbering system) . In order to test the role of this residue in the substrate specificity, we obtained a GEP from Bacillus intermedius with an amino acid substitution (His213-Thr) and studied its catalytic properties . Such modification proved not to affect the primary specificity of the enzyme . The introduced substitution had little effect on the Michaelis constant (Km increased 4.9 times) but considerably affected the catalytic constant (kcat decreased 615 times) . The obtained data suggest that the conserved His213 residue in Bacillus GEPs is not a key element determining their primary substrate specificity. J Immunol, 2004 Jun 15, 172(12), 7618 - 28 Differential immune responses and protective efficacy induced by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA or recombinant protein; Skeiky YA et al.; Key Ags of Mycobacterium tuberculosis initially identified in the context of host responses in healthy purified protein derivative-positive donors and infected C57BL/6 mice were prioritized for the development of a subunit vaccine against tuberculosis . Our lead construct, Mtb72F, codes for a 72-kDa polyprotein genetically linked in tandem in the linear order Mtb32(C)-Mtb39-Mtb32(N) . Immunization of C57BL/6 mice with Mtb72F DNA resulted in the generation of IFN-gamma responses directed against the first two components of the polyprotein and a strong CD8(+) T cell response directed exclusively against Mtb32(C) . In contrast, immunization of mice with Mtb72F protein formulated in the adjuvant AS02A resulted in the elicitation of a moderate IFN-gamma response and a weak CD8(+) T cell response to Mtb32c . However, immunization with a formulation of Mtb72F protein in AS01B adjuvant generated a comprehensive and robust immune response, resulting in the elicitation of strong IFN-gamma and Ab responses encompassing all three components of the polyprotein vaccine and a strong CD8(+) response directed against the same Mtb32(C) epitope identified by DNA immunization . All three forms of Mtb72F immunization resulted in the protection of C57BL/6 mice against aerosol challenge with a virulent strain of M . tuberculosis . Most importantly, immunization of guinea pigs with Mtb72F, delivered either as DNA or as a rAg-based vaccine, resulted in prolonged survival (>1 year) after aerosol challenge with virulent M . tuberculosis comparable to bacillus Calmette-Guerin immunization . Mtb72F in AS02A formulation is currently in phase I clinical trial, making it the first recombinant tuberculosis vaccine to be tested in humans. J Appl Microbiol, 2004, 97(1), 214 - 9 Survival of Bacillus cereus spores and vegetative cells in acid media simulating human stomach; Clavel T et al.; AIMS: To determine the fate of Bacillus cereus spores or vegetative cells in simulated gastric medium . Methods and RESULTS: The effects of acidity on the survival of B . cereus in a medium simulating human stomach content was followed on spores at pH 1.0-5.2, and on vegetative cells at pH 2.5-5.7 . Gastric media (GM) were prepared by mixing equal volumes of a gastric electrolyte solution with J broth (JB), half-skim milk, pea soup and chicken . At pH 1.0 and 1.4, the number of spores slightly decreased in GM-JB and GM-pea soup and remained stable in GM-milk and GM-chicken . A rapid marked decrease (always higher than 2.0 log CFU ml(-1) in 2 h) in vegetative cell counts was observed at pH below 4.2, 4.0, 3.6 and 3.5 in GM-chicken, GM-JB, GM-milk and GM-pea soup, respectively . Between pH 5.0 and 5.3, B . cereus growth was observed in GM-JB (1.2 log CFU ml(-1) increase after 4 h) and in GM-pea soup (1.8 log CFU ml(-1) increase after 4 h) . CONCLUSIONS: Bacillus cereus spores are very much more resistant to gastric acidity than vegetative cells . This resistance strongly depends on the type of food present in the GM . SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that the probability that viable B . cereus cells enter the small intestine, where they can cause diarrhoea, strongly depends on the form of the ingested cells (spores or vegetative cells), on what food they are ingested with, and on the level of stomach acidity. J Appl Microbiol, 2004, 97(1), 158 - 68 The autolytic phenotype of Bacillus thuringiensis; Raddadi N et al.; AIMS: To evaluate the autolytic phenotype of Bacillus thuringiensis . METHODS AND RESULTS: The autolytic rate of 87 strains belonging to different subsp . of B . thuringiensis was examined at pH 6, 6.5 and 8.5 in different buffers under starvation conditions . At pH 6 the extent of autolysis (average in the strain collection 38.3 +/- 21.1) was strain-dependent with wide variability, while at pH 6.5 and 8.5 (averages 72.0 +/- 9.0 and 63.1 +/- 8.2, respectively) it was much more uniform with only a few strains showing low autolytic rates . Forty-one per cent of the strains showed high resistance (>/=80%) to mutanolysin, a commercial muramidase from Streptomyces . The peptidoglycan hydrolase pattern was evaluated by renaturing SDS-PAGE using cells of B . thuringiensis subsp . tolworthi HD125 as indicator . The strain collection showed seven major lytic bands of about 90, 63, 46, 38, 32, 28 and 25 kDa, and in the stationary growth phase (72 h) there was a more intense 25 kDa band in the autolytic pattern . Using Micrococcus lysodeicticus and Listeria monocytogenes as the indicators lytic activity was retained, as seen by the bands of 63, 46, 38, 32 and 25 kDa . Growth in the different media did not affect the autolytic pattern . NaCl abolished the activity of all the peptidoglycan hydrolases in the gel, but in the presence of KCl, MgCl(2), MnCl(2) and EDTA some activity was retained . At basic pH the lytic activity increased . CONCLUSIONS: The autolytic phenotype of B . thuringiensis was found to be strain-dependent, and different proteins exibited peptidoglycan hydrolase activity, particularly at alkaline pH . Several of these proteins retained lytic activity against other bacterial species . SIGNIFICANCE AND IMPACT OF THE STUDY: The characterisation of the autolytic phenotype of B . thuringiensis should expand the prospects of using this species in bacterial bio-control and field applications. J Appl Microbiol, 2004, 97(1), 114 - 23 Isolation of Bacillus pumilus from in vitro grapes as a long-term alcohol-surviving and rhizogenesis inducing covert endophyte; Thomas P; AIMS: To isolate and characterize the alcohol-surviving covert bacterium associated with grape tissue culture . METHODS AND RESULTS: Single colony was isolated by plating the spent rectified spirit used during grape culturing and the organism was identified as Bacillus pumilus using partial 16S rDNA sequence data . Spotting tests (1 microl) using 3-day-old broth culture having a spore content of 20-30% showed similar bacterial survival in 25-90% (v/v) aqueous ethanol for 14 days . Survival in 90% ethanol and 90% rectified spirit appeared affected thereafter with no colony growth from 1 microl samples after 4 months . Plating the samples at this stage gave similar CFU ml(-1) for 25, 50 and 70% ethanol, a significant reduction in 80% ethanol and very few colonies in 90% ethanol and rectified spirit . B . pumilus-inoculated grape microcuttings showed substantial endophytic colonization of original cuttings (7.4 x 10(6) CFU g(-1)) followed by the sprout (5.9 x 10(5)) and roots (2.0 x 10(4)) . The bacterium although a poor root colonizer, induced early rooting and more roots in vitro . Inoculation at ex vitro planting resulted in significantly more roots, root weight and shoot growth . CONCLUSIONS: Bacillus pumilus could remain as a covert endophyte in grape tissue cultures and survive in aqueous ethanol for extended periods . 90% ethanol was the most effective bactericidal concentration . The bacterium showed endophytic colonization and root and shoot growth promotion . SIGNIFICANCE AND IMPACT OF THE STUDY: The revelation that general recommendation of 70-80% ethanol may not be the most effective bactericidal concentration for all bacteria, elucidation of the possibility of covert bacterial survival in vitro plant cultures and isolation of a potential plant growth promoting endophyte in grape. Immunol Cell Biol, 2004 Jun, 82(3), 253 - 6 Normal levels of immunocompetence in possums (Trichosurus vulpecula) exposed to different laboratory housing conditions post capture; Begg D et al.; Specific and non-specific immunological tests were used to monitor aspects of the immune response in captive possums . The tests included total and differential white blood cell counts, lymphocyte transformation assay, and enzyme linked immunosorbant assay . The level of free cortisol present in possum plasma samples was evaluated as an endocrine marker for stress . Four different housing conditions were used to test whether stress could be managed or avoided in captive animals . Animals were caged individually or as groups in pens . Bacille Calmette-Gurein (BCG) and tetanus toxoid immunization was used to evoke primary cell mediated and antibody responses in test animals . The results indicated that there was no significant difference in immunological responses or endocrine parameters in animals held under any of the housing conditions . The results infer that wild possums adapt quickly post-capture to novel housing conditions and produce representative patterns of immunity when held in housing conditions and fed ad libitum. J Med Entomol, 2004 May, 41(3), 435 - 41 Laboratory selection for resistance to Bacillus thuringiensis subsp . jegathesan or a component toxin, Cry11B, in Culex quinquefasciatus (Diptera: Culicidae); Wirth MC et al.; The bacteria Bacillus thuringiensis subsp . israelensis and Bacillus sphaericus produce insecticidal toxins used to control mosquito larvae throughout the world . Unfortunately, there are few alternative insecticides with similar activity and environmental safety, which may limit the long-term success of these insecticides . Bacillus thuringiensis subsp . jegathesan is another bacterium with toxins that are active against mosquitoes and has potential for development as a commercial product . B . t . subsp . jegathesan would be ineffective if cross-resistance was detected or if treated mosquito populations evolved resistance . B . t . subsp . jegathesan was evaluated for its potential for selecting insecticide resistance in Culex quinquefasciatus Say . Susceptibility changes in mosquitoes selected with the wild-type strain were compared with susceptibility changes in mosquitoes selected with Cry11B, a component toxin of B . t . subsp . jegathesan . Resistance was detected in generation 18 in the Cry11B-selected colony, reached a maximum of 38-fold, and was present through generation 40 . The B . t . subsp . jegathesan-selected colony evolved 13-fold resistance in generation 22, but resistance declined to 2.3-fold in generation 26 and remained low throughout the study . Cry11B-selected mosquitoes showed no significant resistance to the wild-type bacterium, whereas B . t . subsp . jegathesan-selected mosquitoes expressed significant resistance to Cry11B . Both colonies displayed cross-resistance to component toxins of B . t . subsp . israelensis, but they lacked cross-resistance to that wild-type strain . The patterns of resistance and cross-resistance in this study are consistent with the patterns previously observed in mosquitoes selected with B . t . subsp . israelensis and suggest that B . t . subsp . jegathesan might also be at low risk for resistance. J Med Entomol, 2004 May, 41(3), 423 - 9 Laboratory and simulated field evaluation of a new recombinant of Bacillus thuringiensis ssp . israelensis and Bacillus sphaericus against Culex mosquito larvae (Diptera: Culicidae); Zahiri NS et al.; In the laboratory, three microbial mosquito larvicidal products consisting of Bacillus thuringiensis ssp . israelensis de Barjac (Bti), Bacillus sphaericus (Neide) (Bsph) (strain 2362), and the University of California Riverside (UCR) recombinant (producing toxins of both Bacillus sphaericus and Bacillus thuringiensis ssp . israelensis) were bioassayed against larvae of Culex quinequefasciatus Say (susceptible and resistant to Bsph 2362), and Aedes aegypti (L.) . Bti proved highly effective against Cx . Quinequefasciatus susceptible and resistant strains, with LC50 values of 0.009 and 0.011 ppm and LC90 values of 0.057 and 0.026 ppm for Bsph-susceptible and -resistant strains, respectively . Bti was also highly active against Ae . eagypti with LC50 and LC90 values of 0.014 and 0.055 ppm, respectively . The UCR recombinant was equally active against both Bsph-susceptible and -resistant strains of Cx . Quinquefasciatus; LC50 values were 0.005 and 0.009 and LC90 values were 0.030 and 0.043 ppm, respectively . Bti and the UCR recombinant essentially showed similar activity against Bsph-susceptible and -resistant strains . UCR recombinant showed high toxicity against Ae . eagypti with LC50 and LC90 values of 0.023 and 0.064 ppm, respectively . Bsph was highly active against susceptible strain of Cx . quinequefasciatus with LC50 and LC9o values of 0.006 and 0.024 ppm, respectively . Bsph exhibited little toxicity against Ae . eagypti larvae and also no toxicity to Bsph resistance . In the field, we evaluated four experimental corn grit formulations of Bti (VBC 60021), Bsph (VBC 60022), UCR recombinants VBC 60023 (7.89%), and VBC 60024 (1.87%) in simulated field (microcosms) against Bsph-susceptible Culex mosquitoes . Bti and low-concentrate UCR recombinant showed similar initial activity as well as persistence . Both materials provided high-to-moderate level of control for 2-7 d posttreatment at low treatment rates . At low dosages, residual activity of Bti and UCR recombinant lasted for <7 d . Bsph and high-concentrate UCR recombinant (VBC 60023), however, were more effective against natural populations of Cullex and achieved longer control (7-21 d) than the other two materials. J Med Entomol, 2004 May, 41(3), 408 - 13 Insecticide resistance and cross-resistance in Alabama and Florida strains of Culex quinquefasciatus {correction}; Liu H et al.; Insecticide resistance and cross-resistance was determined for three strains of Culex quinquefasciatus Say in the southeastern United States . HAmCq and MAmCq strains were collected in 2002 from Huntsville and Mobile, AL . The VBFmCq strain was collected in 1998 from Vero Beach, FL . VBFmCq, HAmCq, and MAmCq larvae showed resistance to permethrin with resistance ratios of 13, 100, and 940, respectively, compared with the susceptible S-Lab strain . Levels of resistance in HAmCq and MAmCq larvae were 200- and 830-fold to resmethrin and 4- and 70-fold to malathion, respectively . VBFmCq, HAmCq, and MAmCq strains all demonstrated a great ability to develop tolerance and/or cross-resistance to different insecticides, including deltamethrin (50-, 100-, and 300-fold), chlorpyrifos (150-, 33-, and 720-fold), fipronil (10-, 5-, and 15-fold), and imidacloprid (7.5-, 5- and 10-fold, respectively) . Comparison of resistance ratios for pyrethroids, organophosphates, and imidacloprid at LC50 and LC90 and gradual slopes of dose-response curves indicated that VBFmCq, HAmCq, and MAmCq were heterozygous in response to these insecticides . All three strains showed high levels of susceptibility to Bacillus thuringiensis variety israelensis (Bti) and spinosad, although these mosquitoes had been extensively exposed to Bti . Thus, we conclude that Bti and spinosad may be valuable for the management of Cx . quinquefasciatus, especially in situations where local strains are highly resistant to other insecticides. J Clin Microbiol, 2004 Jun, 42(6), 2379 - 87 Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection; Brock I et al.; The currently used method for immunological detection of tuberculosis infection, the tuberculin skin test, has low specificity . Antigens specific for Mycobacterium tuberculosis to replace purified protein derivative are therefore urgently needed . We have performed a rigorous assessment of the diagnostic potential of four recently identified antigens (Rv2653, Rv2654, Rv3873, and Rv3878) from genomic regions that are lacking from the Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine strains as well as from the most common nontuberculous mycobacteria . The fine specificity of potential epitopes in these molecules was evaluated by sensitive testing of the T-cell responses of peripheral blood mononuclear cells derived from M . bovis BCG-vaccinated healthy individuals to synthesized overlapping peptides . Three of the four molecules contained regions with significant specificity problems (Rv2653, Rv3873, and Rv3878) . We selected and combined the specific peptide stretches from the four proteins not recognized by M . bovis BCG-vaccinated individuals . These peptide stretches were tested with peripheral blood mononuclear cells obtained from patients with microscopy- or culture-confirmed tuberculosis and from healthy M . bovis BCG-vaccinated controls . The combination of the most promising stretches from this analysis showed a sensitivity level (57%) comparable to the level found with the two well-known M . tuberculosis-specific proteins ESAT-6 and CFP-10 (75 and 66%, respectively) . The combination of ESAT-6, CFP-10, and the novel specific peptide stretches gave an overall sensitivity of 84% at a specificity of 97% . In a validation experiment with new experimental groups, the sensitivities obtained were 57% for the combination of peptides and 90% for the combination of the peptides, ESAT-6, and CFP-10 . This combination gave a specificity of 95%. Appl Environ Microbiol, 2004 Jun, 70(6), 3769 - 71 Enhancement of Cry19Aa mosquitocidal activity against Aedes aegypti by mutations in the putative loop regions of domain II; Abdullah MA et al.; Improvements in the mosquitocidal activity of Bacillus thuringiensis Cry19Aa were achieved by protein engineering of putative surface loop residues in domain II through rational design . The improvement of Aedes toxicity in Cry19Aa was 42,000-fold and did not affect its toxicity against Anopheles or Culex. Appl Environ Microbiol, 2004 Jun, 70(6), 3329 - 37 Enzyme production-based approach for determining the functions of microorganisms within a community; Nakamura K et al.; The functions of specific microorganisms in a microbial community were investigated during the composting process . Cerasibacillus quisquiliarum strain BLx(T) and Bacillus thermoamylovorans strain BTa were isolated and characterized in our previous studies based on their dominance in the composting system . Strain BLx(T) degrades gelatin, while strain BTa degrades starch . We hypothesized that these strains play roles in gelatinase and amylase production, respectively . The relationship between changes in the abundance ratios of each strain and those of each enzyme activity during the composting process was examined to address this hypothesis . The increase in gelatinase activity in the compost followed a dramatic increase in the abundance ratio of strain BLx(T) . Zymograph analysis demonstrated that the pattern of active gelatinase bands from strain BLx(T) was similar to that from the compost . Gelatinases from both BLx(T) and compost were partially purified and compared . Homologous N-terminal amino acid sequences were found in one of the gelatinases from strain BLx(T) and that of compost . These results indicate strain BLx(T) produces gelatinases during the composting process . Meanwhile, the increase in the abundance ratio of strain BTa was not concurrent with that of amylase activity in the compost . Moreover, the amylase activity pattern of strain BTa on the zymogram was different from that of the compost sample . These results imply that strain BTa may not produce amylases during the composting process . To our knowledge, this is the first report demonstrating that the function of a specific microorganism is directly linked to a function in the community, as determined by culture-independent and enzyme-level approaches. Appl Environ Microbiol, 2004 Jun, 70(6), 3282 - 91 In vivo production of artificial nonribosomal peptide products in the heterologous host Escherichia coli; Gruenewald S et al.; Nonribosomal peptide synthetases represent the enzymatic assembly lines for the biosynthesis of pharmacologically relevant natural peptides, e.g., cyclosporine, vancomycin, and penicillin . Due to their modular organization, in which every module accounts for the incorporation of a single amino acid, artificial assembly lines for the production of novel peptides can be constructed by biocombinatorial approaches . Once transferred into an appropriate host, these hybrid synthetases could facilitate the bioproduction of basically any peptide-based molecule . In the present study, we describe the fermentative production of the cyclic dipeptide D-Phe-Pro-diketopiperazine, as a prototype for the exploitation of the heterologous host Escherichia coli, and the use of artificial nonribosomal peptide synthetases . E . coli provides a tremendous potential for genetic engineering and was manipulated in our study by stable chromosomal integration of the 4'-phosphopantetheine transferase gene sfp to ensure heterologous production of fully active holoenzmyes . D-Phe-Pro-diketopiperazine is formed by the TycA/TycB1 system, whose components represent the first two modules for tyrocidine biosynthesis in Bacillus brevis . Coexpression of the corresponding genes in E . coli gave rise to the production of the expected diketopiperazine product, demonstrating the functional interaction of both modules in the heterologous environment . Furthermore, the cyclic dipeptide is stable and not toxic to E . coli and is secreted into the culture medium without the need for any additional factors . Parameters affecting the productivity were comprehensively investigated, including various genetic setups, as well as variation of medium composition and temperature . By these means, the overall productivity of the artificial system could be enhanced by over 400% to yield about 9 mg of D-Phe-Pro-diketopiperazine/liter . As a general tool, this approach could allow the sustainable bioproduction of peptides, e.g., those used as pharmaceuticals or fine chemicals. Biochem Biophys Res Commun, 2004 Jul 2, 319(3), 1017 - 25 Cloning and characterization of two thermostable xylanases from an alkaliphilic Bacillus firmus; Chang P et al.; Two genes encoding thermostable xylanases, named xyn10A and xyn11A, from an alkaliphilic Bacillus firmus were cloned and expressed in Escherichia coli . The E . coli harboring either gene showed clear zone with Congo red clearance assay on xylan plate . The Xyn10A and Xyn11A have molecular weights of 45 and 23kDa, respectively, and both show activities on xylan-zymogram . The xyn10A encodes 396 amino acid residues and is very similar to an alkaliphilic xylanase A from alkaliphilic Bacillus halodurans . The Xyn11A contains 210 amino acid residues and only one amino acid different from an endo-beta-1,4-xylanase from B . halodurans . From alignment of the amino acid sequences with other xylanases, Xyn10A and Xyn11A belong to family 10 and 11 glycosyl hydrolases, respectively . Both show activities over the pH range of 4-11 at 37 degrees C and over 80% activities at 70 degrees C . Interestingly both still retain over 70% activities after 16h preincubation at 62 degrees C. Carbohydr Res, 2004 Jun 22, 339(9), 1643 - 8 Characterisation of the core part of the lipopolysaccharide O-antigen of Francisella novicida (U112); Vinogradov E et al.; Francisella novicida (U112), a close relative of the highly virulent bacterium F . tularensis, is known to produce a lipopolysaccharide that is significantly different in biological properties from the LPS of F . tularensis . Here we present the results of the structural analysis of the F . novicida LPS core part, which is found to be similar to that of F . tularensis, differing only by one additional alpha-Glc residue:where R is an O-chain, linked via a beta-bacillosamine (2,4-diamino-2,4,6-trideoxyglucose) residue . The lipid part of F . novicida LPS contains no phosphate substituent and apparently has a free reducing end, a feature also noted in F . tularensis LPS. Carbohydr Res, 2004 Jun 22, 339(9), 1603 - 8 Enzymatic synthesis of a beta-D-galactopyranosyl cyclic tetrasaccharide by beta-galactosidases; Higashiyama T et al.; The galactosyl transfer reaction to cyclo-{-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->} (CTS) was examined using lactose as a donor and beta-galactosidases from Aspergillus oryzae and Bacillus circulans . The A . oryzae beta-galactosidase produced three galactosyl derivatives of CTS . The main galactosyl derivative produced by the A . oryzae enzyme was identified as 6-O-beta-D-galactopyranosyl-CTS, cyclo-{-->6)-alpha-D-Glcp-(1-->3)-{beta-D-Galp-(1-->6)}-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->} . The B . circulans beta-galactosidase also synthesized three galactosyl-transfer products to CTS . The structure of main transgalactosylation product was 3-O-beta-D-galactopyranosyl-CTS, cyclo-{-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-{beta-D-Galp-(1-->3)}-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->} . These results showed that beta-galactosidase transferred galactose directly to the ring glucose residue of CTS. J Immunol Methods, 2004 May, 288(1-2), 81 - 9 Monitoring leukocyte traffic in vivo into human delayed-type hypersensitivity reaction; Savilahti E et al.; Leukocyte traffic from blood to sites of inflammation has been elaborately studied in animal models and in vitro . However, to date, little understanding has accumulated on the process in humans in vivo . A noninvasive light confocal microscopy technique has enabled us to image leukocyte rolling, arrest and transmigrated cells in vivo in human tissues . In the current study, a delayed-type hypersensitivity (DTH) reaction was elicited in the lower lip of healthy, Bacille Calmette-Guerin (BCG)-vaccinated volunteers by injection of respective purified protein derivate (PPD), mimicking the classic cutaneous Mantoux reaction . Subjects were imaged with real-time confocal microscopy at baseline and 2, 4, 24 and 48 h after the injection . The number of rolling leukocytes did not increase significantly until at 48 h . Even then, rolling cells were seen in only a minority (23%) of blood vessels . The frequency of engaged blood vessels was, nevertheless, significantly greater than at baseline . As the inflammation generated with this challenge was mild, transmigrated leukocytes could be detected in the confocal microscopy only occasionally . Histology of biopsies taken immediately after imaging at 48 h showed a T cell-dominated leukocyte population at the site of the DTH reaction . We have thus developed a method to monitor noninvasively leukocyte traffic in vivo in human subjects during the classic inflammatory model of delayed-type hypersensitivity reaction . Biochemistry, 2004 Jun 15, 43(23), 7413 - 20 Manipulation of the active site loops of D-hydantoinase, a (beta/alpha)8-barrel protein, for modulation of the substrate specificity; Cheon YH et al.; We previously proposed that the stereochemistry gate loops (SGLs) constituting the substrate binding pocket of D-hydantoinase, a (beta/alpha)(8)-barrel enzyme, might be major structural determinants of the substrate specificity {Cheon, Y . H., et al . (2002) Biochemistry 41, 9410-9417} . To construct a mutant D-hydantoinase with favorable substrate specificity for the synthesis of commercially important non-natural amino acids, the SGL loops of the enzyme were rationally manipulated on the basis of the structural analysis and sequence alignment of three hydantoinases with distinct substrate specificities . In the SGLs of D-hydantoinase from Bacillus stearothermophilus SD1, mutations of hydrophobic and bulky residues Met 63, Leu 65, Phe 152, and Phe 159, which interact with the exocyclic substituent of the substrate, induced remarkable changes in the substrate specificities . In particular, the substrate specificity of mutant F159A toward aromatic substrate hydroxyphenylhydantoin (HPH) was enhanced by approximately 200-fold compared with that of the wild-type enzyme . Saturation mutagenesis at position 159 revealed that k(cat) for aromatic substrates increased gradually as the size of the amino acid side chain decreased, and this seems to be due to reduced steric hindrance between the bulky exocyclic group of the substrate and the amino acid side chains . When site-directed random mutagenesis of residues 63 and 65 was conducted with the wild type and mutant F159A, the selected enzymes (M63F/L65V and L65F/F159A) exhibited approximately 10-fold higher k(cat) values for HPH than the wild-type counterpart, which is likely to result from reorganization of the active site for efficient turnover . These results indicate that the amino acid residues of SGLs forming the substrate binding pocket are critical for the substrate specificity of D-hydantoinase, and the results also imply that substrate specificities of cyclic amidohydrolase family enzymes can be modulated by rational design of these SGLs. Int J Tuberc Lung Dis, 2004 Jun, 8(6), 718 - 23 Risk of infection with Mycobacterium tuberculosis in Malawi: national tuberculin survey 1994; Salaniponi FM et al.; OBJECTIVE: To estimate the annual risk of tuberculosis infection among schoolchildren in Malawi . METHODS: A school survey was conducted in twelve randomly selected districts in Malawi . Children in standard 1-4 and aged 6-11 years were eligible . Tuberculin skin testing was performed according to World Health Organization/International Union Against Tuberculosis and Lung Disease guidelines . RESULTS: Of the 17123 eligible children, 80% were tested . Of those tested 79% were read . The prevalence of infection according to various criteria was 9-12% in children without bacille Calmette-Guerin (BCG) scar . The prevalence of reactions of 10 mm or more was lower in girls than in boys, increased with age, and was higher in those with than in those without BCG scar . The annual risk of infection was estimated to be within the range 0.6-1.4% . CONCLUSION: Annual risk of infection in Malawi was in the order of 1% . This study is expected to provide valuable baseline information for an assessment of the impact of human immunodeficiency virus (HIV) on tuberculosis transmission in Malawi. Environ Toxicol Chem, 2004 May, 23(5), 1297 - 304 Responses of nontarget Lepidoptera to Foray 48B Bacillus thuringiensis var . kurstaki on Vancouver Island, British Columbia, Canada; Boulton TJ; Impacts of a gypsy moth (Lymantria dispar) eradication program on native, nontarget Lepidoptera were assessed in 1999, on southeastern Vancouver Island (BC, Canada) . The microbial insecticide Bacillus thuringiensis var . kurstaki (Btk) was applied aerially over two areas totalling 12,805 ha on May 8, May 19, and June 8, 1999, at a dosage of 50 billion international units in 4.0 L/ha . Lepidoptera were collected from two host plant species: Garry oak (Quercus garryana) and common snowberry (Symphoricarpos albus) . Lepidopteran larvae were collected from common snowberry foliage at 24 urban parks and from Garry oak foliage at 28 oak-dominated habitats, representing 12 and 14 replicates, respectively, of two treatments: unsprayed (reference) and sprayed (treatment) . Prespray data were collected from March 25 to May 6 for S . albus, and from April 26 to May 6 for Q . garryana . Postspray data were collected from May 10 through June 15 for S . albus and from May 10 to July 6 for Q . garryana . The 15 most abundant lepidopteran species were analyzed statistically . However, the majority of species were collected infrequently, and, therefore were pooled for statistical analysis . After the Btk spray applications, 11 of the individual species and groups of uncommon species were found to be significantly less abundant in the treatment sites than in the reference sites . The effects of sample date were statistically significant on almost all groups of Lepidoptera analyzed, both before and after Btk spray applications, indicating temporal variation in lepidopteran abundance . Significant variation in diversity of members of the Lepidoptera, as a result of Btk spray application, was not detected on S . albus or Q . garryana . However, results showed significant variation in lepidoptera richness and abundance on both host plant species. Hum Reprod, 2004 Aug, 19(8), 1886 - 93 Epub 2004 Jun 03. Increase in peripheral blood mononuclear cell (PBMC)- and CD56+ cell-mediated killing of endometrial stromal cells by mycobacteria; a possible role in endometriosis immunotherapy? Clayton RD, Duffy SR, Wilkinson N, Garry R, Jackson AM. BACKGROUND: Immunological therapies have shown promising results in the treatment of endometriosis . Mycobacteria are one of the most common immune therapies used in other diseases . We have assessed the effects of mycobacteria in altering the ability of peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells to kill endometrial stromal cells using an in vitro model . This may have implications in the immunotherapy of endometriosis . METHODS: Primary cultures of endometrial stromal cells were grown from female patients and PBMCs were extracted from healthy female volunteers . Effector cells (PBMCs or NK cells) were exposed to varying concentrations of mycobacteria before their ability to kill cultured endometrial cells was tested using a 51Cr-release assay . RESULTS: Treatment of effector cells with the Connaught Substrain Bacillus of Calmette and Guerin (BCG) led to increased killing of target cells by PBMCs and NK cells . The optimal concentration for treatment of effector cells with Connaught BCG was approximately 0.1 multiplicities of infection (m.o.i.) . There was a trend towards increased killing after treatment with Pasteur BCG . CD56+ (NK) cells treated with BCG at 0.1 m.o.i . showed increased killing of target cells compared with untreated effector cells . CONCLUSIONS:Endometrial stromal cells are susceptible to killer cells activated by mycobacteria . This in vitro work suggests a possible role for mycobacteria in the immunotherapy of endometriosis . Biochem Biophys Res Commun, 2004 Jun 25, 319(2), 677 - 82 Identification of monohydroxy progesterones produced by CYP106A2 using comparative HPLC and electrospray ionisation collision-induced dissociation mass spectrometry; Lisurek M et al.; Two previously uncharacterised products, produced by recombinant CYP106A2 of Bacillus megaterium ATCC 13368 using progesterone as substrate, were identified . For this purpose a combination of comparative HPLC and electrospray ionisation collision induced dissociation mass spectrometry (ESI CID MS) was established and applied for rapid identification of the steroids, which were identified as 11alpha-hydroxyprogesterone and 9alpha-hydroxyprogesterone . The pharmaceutical relevance of these steroids is discussed . Furthermore, the hydroxylation activity was quantified for all monohydroxylation products (15beta-hydroxyprogesterone, 6beta-hydroxyprogesterone, 11alpha-hydroxyprogesterone, and 9alpha-hydroxyprogesterone) . The V(max) values for 15beta-hydroxyprogesterone, 6beta-hydroxyprogesterone, 11alpha-hydroxyprogesterone, and 9alpha-hydroxyprogesterone were determined as 337.3+/-43.7, 22.3+/-0.9, 17.5+/-0.9, and 6.5+/-0.3nmol product/min/nmol CYP106A2, respectively. J Microbiol Methods, 2004 Jul, 58(1), 1 - 12 MALDI-TOFMS compared with other polyphasic taxonomy approaches for the identification and classification of Bacillus pumilus spores; Dickinson DN et al.; To verify the efficacy of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) protein profiling for identifying and differentiating bacterial species, several strains of Bacillus pumilus were examined in a thorough taxonomic study incorporating a polyphasic approach . Sixteen isolates of putative B . pumilus isolated from spacecraft assembly facilities, the Mars Odyssey spacecraft, and the International Space Station, were characterized for their biochemical and molecular profiles using the Biolog system, DNA techniques, and MALDI-TOFMS protein profiling . MALDI-TOFMS protein profiling was more accurate than Biolog metabolic profiling, more discriminating than 16S rDNA sequence analysis, and complemented the results of gyrB sequence analysis and DNA-DNA hybridization for the identification of the B . pumilus spores . This is the first report whereby MALDI-TOFMS generated protein profiles from a set of microbes is compared directly with DNA-DNA hybridization yielding a positive correlation . Unique, cluster-specific biomarker peaks have been identified in the spores of the B . pumilus examined in this study . MALDI-TOFMS protein profiling is a rapid and simple analysis and has been demonstrated as a useful taxonomic tool for differentiating spores of the genus Bacillus . For practical purposes, it would be ideal (and necessary) to have a publicly available, standardized MALDI profile database, to facilitate the use of the technique as a diagnostic method to differentiate bacterial species . J Chromatogr B Analyt Technol Biomed Life Sci, 2004 Jul 25, 807(1), 25 - 31 N-terminal tagged lactate dehydrogenase proteins: evaluation of relative hydrophobicity by hydrophobic interaction chromatography and aqueous two-phase system partition; Fexby S et al.; The hydrophobic contributions of 17 individual peptides, fused to the N-terminal of Bacillus stearothermophilus lactate dehydrogenase (LDH) were studied by hydrophobic interaction chromatography (HIC) and aqueous two-phase system (ATPS) . The constructs were sequenced from a protein library designed with a five-amino acid randomised region in the N-terminal of an LDH protein . The 17 LDH variants and an LDH control lacking the randomised region were expressed in Escherichia coli . HIC and ATPS behaviour of the proteins indicated significant differences in protein hydrophobicity, even though the modifications caused only 1% increase in protein molecular weight and 2% variation in isoelectric points . HIC and ATPS results correlated well (R(2) = 0.89) . Protein expression was clearly affected by N-terminal modification, but there was no evidence that the modification affected protein activity . A GluAsnAlaAspVal modification resulted in increased protein expression . In most cases, HIC and ATPS results compared favourably with those predicted on the basis of 34 amino acid residue hydrophobicity scales; assuming exposure of tag residues to solution . Exceptions included LeuAlaGlyValIle and LeuTyrGlyCysIle modifications, which were predicted, assuming full solution exposure, to be more hydrophobic than observed. Expert Rev Vaccines, 2004 Jun, 3(3), 299 - 306 Developing an improved vaccine against tuberculosis; McShane H; Despite the availability of a vaccine for over 80 years, the tuberculosis epidemic continues to be a major cause of mortality and morbidity throughout the world . The factors contributing to the resurgence of tuberculosis and the possible explanations for the failure of the current vaccine, bacille Calmette-Guerin, are discussed . The nature of protective immunity to Mycobacterium tuberculosis and how this relates to the development of new candidate vaccines is then considered . The issues surrounding the progression of the most promising candidates into Phase I clinical trials are also discussed. J Biol Inorg Chem, 2004 Jul, 9(5), 600 - 8 Epub 2004 Jun 03. Protein stability and mutations in the axial methionine loop of a minimal cytochrome c; Bartalesi I et al.; The minimal mono-heme ferricytochrome c from Bacillus pasteurii, containing 71 amino acids, has been further investigated through mutagenesis of different positions in the loop containing the iron ligand Met71 . These mutations have been designed to sample different aspects of the loop structure, in order to obtain insights into the determinants of the stability of the iron(III) environment . In particular, positions 68, 72 and 75 have been essayed . Gln68 has been mutated to Lys to provide a suitable alternate ligand that can displace Met71 under denaturing conditions . Pro72 has been mutated to Gly and Ala to modify the range of allowed backbone conformations . Ile75, which is in van der Waals contact with Met71 and partly shields a long-lived water molecule in a protein cavity, has been substituted by Val and Ala to affect the network of inter-residue interactions around the metal site . The different contributions of the above amino acids to protein parameters such as structure, redox potential and the overall stability against unfolding with guanidinium hydrochloride are analyzed . While the structure remains essentially the same, the stability decreases with mutations . The comparison with mitochondrial c-type cytochromes is instructive. Cancer Immunol Immunother, 2004 Jul, 53(7), 617 - 24 Epub 2004 Feb 07. WT1 peptide vaccination combined with BCG-CWS is more efficient for tumor eradication than WT1 peptide vaccination alone; Nakajima H et al.; A Wilms' tumor gene WT1 is expressed at high levels not only in most types of leukemia but also in various types of solid tumors, including lung and breast cancer . WT1 protein has been reported to serve as a target antigen for tumor-specific immunotherapy both in vitro in human systems and in vivo in murine models . We have shown that mice immunized with WT1 peptide or WT1 cDNA could reject a challenge from WT1-expressing tumor cells (a "prophylactic" model) . However, it was not examined whether WT1 peptide vaccination had the potency to reject tumor cells in a "therapeutic" setting . In the present study, we demonstrated for the first time that WT1 peptide vaccination combined with Mycobacterium bovis bacillus Calmette-Guerin cell wall skeleton (BCG-CWS) was more effective for eradication of WT1-expressing tumor cells that had been implanted into mice before vaccination (a "therapeutic" model) compared with WT1 peptide vaccination alone . An intradermal injection of BCG-CWS into mice, followed by that of WT1 peptide at the same site on the next day, generated WT1-specific cytotoxic T lymphocytes (CTLs) and led to rejection of WT1-expressing leukemia or lung cancer cells . These results showed that BCG-CWS, which was well known to enhance innate immunity, could enhance WT1-specific immune responses (acquired immunity) in combination with WT1 peptide vaccination . Therefore, WT1 peptide vaccination combined with BCG-CWS may be applied to cancer immunotherapy in clinical settings . Rapid Commun Mass Spectrom, 2004, 18(12), 1277 - 85 Fragmentation of biotinylated cyclic peptides; Lassman ME et al.; Electrospray ionization coupled with tandem mass spectrometry (MS/MS) was used to determine the preferred binding site(s) of biotin NHS ester with a series of cyclic peptides with antibiotic properties . The peptides investigated are polymyxins, cyclic peptides produced by Bacillus polymyxa . In spite of the 1:1 stoichiometry used in the labeling reaction, multiple biotin molecules were incorporated into intact polymyxin peptides . Given the amine specificity of the activated biotin and the large number of amino acids with primary amines in the polymyxins, it was not clear by inspection which binding sites were more reactive than others . MS/MS was used to characterize the structure of the biotinylated peptides . MS/MS spectra of cyclic peptides often lead to ambiguous structure determinations due to the potential for multiple ring openings which result in the generation of multiple ion series . The MS/MS spectra of polymyxin peptides are especially difficult to characterize due to the lack of variety in their amino acids; however, the added complexity of the biotin aided the elucidation of the fragmentation pathways . MS/MS spectra of the species with biotin additions were used to rationalize the preferential binding sites of these molecules. Nucleic Acids Res, 2004 Jun 01, 32(10), 2977 - 86 Print 2004. DnaG interacts with a linker region that joins the N- and C-domains of DnaB and induces the formation of 3-fold symmetric rings; Thirlway J et al.; Loading of the replicative ring helicase onto the origin of replication (oriC) is the final outcome of a well coordinated series of events that collectively constitute a primosomal cascade . Once the ring helicase is loaded, it recruits the primase and signals the switch to the polymerization mode . The transient nature of the helicase-primase (DnaB-DnaG) interaction in the Escherichia coli system has hindered our efforts to elucidate its structure and function . Taking advantage of the stable DnaB-DnaG complex in Bacillus stearothermophilus, we have reviewed conflicting mutagenic data from other bacterial systems and shown that DnaG interacts with the flexible linker that connects the N- and C-terminal domains of DnaB . Furthermore, atomic force microscopy (AFM) imaging experiments show that binding of the primase to the helicase induces predominantly a 3-fold symmetric morphology to the hexameric ring . Overall, three DnaG molecules appear to interact with the hexameric ring helicase but a small number of complexes with two and even one DnaG molecule bound to DnaB were also detected . The structural/functional significance of these data is discussed and a speculative structural model for this complex is suggested. Chem Phys Lipids, 2004 Jul, 130(2), 127 - 34 Cholesterol modulation of sphingomyelinase activity at physiological temperatures; Contreras FX et al.; Bacillus cereus sphingomyelinase activity was assayed on large unilamellar vesicles composed of sphingomyelin (SM)/cholesterol (Ch) mixtures at varying proportions . Natural (egg) SM was used with a gel-fluid transition temperature at ca . 40 degrees C . When the enzyme was assayed at 37 degrees C, the activity on pure SM was exceedingly low, but a small increase was observed as soon as some Ch was added, and a large enhancement of activity occurred with Ch proportions above 25 mol% . The data were interpreted in terms of sphingomyelinase activity being higher in the cholesterol-induced liquid-ordered phase than in the gel phase . The abrupt increase in activity above 25 mol% Ch would occur as a result of a change in domain connectivity, when the Ch-rich liquid-ordered domains coalesced . In equimolar SM/Ch mixtures, that were in the liquid-ordered state in a wide range of temperatures, sphingomyelinase activity was virtually constant in the 30-70 degrees C range . The results demonstrate that at the mammalian and bird physiological temperatures Ch modulates sphingomyelinase activity, and that this can occur precisely because most SM have a gel-fluid transition temperature above the physiological temperature range . In addition, Ch activation of sphingomyelinase and the strong affinity of Ch for SM allow the rapid, localised and self-contained production of the metabolic signal ceramide in specific microdomains (rafts). Pathophysiol Haemost Thromb, 2003 May-Jun, 33(3), 138 - 43 The effect of dietary bacillus natto productive protein on in vivo endogenous thrombolysis; Yamashita T et al.; The influence of dietary bacillus natto productive protein (BNPP) on endogenous thrombolysis was investigated in the rat . Animals were given a standard feed for 14 weeks, to which 0.2 or 1% BNPP was added . Thrombolysis was evaluated using an He-Ne laser-induced thrombosis model in mesenteric microvessels . Changes in thrombus volume, reflecting thrombolysis, decreased to 82% of the initial value in the control group . In contrast, the thrombus volume decreased to 67% in the animals fed 0.2% BNPP, and decreased to 51% in the group given 1% BNPP . The extent of thrombolysis in the 1% BNPP group was equivalent to that seen in animals treated with a bolus intravenous infusion of 0.2 mg/kg tissue plasminogen activator . The results demonstrated that the dietary administration of BNPP enhanced endogenous thrombolysis in a dose-dependent manner . Argatroban (2 mg/kg/h) enhanced endogenous fibrinolysis only in control animals, but not in the BNPP groups . The results support the suggestion that dietary supplementation with BNPP may provide a simple means to promote fibrinolysis not only in the treatment of thromboembolism but also in the prevention of venous occlusion . Biochemistry (Mosc), 2004 Apr, 69(4), 420 - 6 Purification and characterization of serine proteinase 2 from Bacillus intermedius 3-19; Balaban NP et al.; A proteinase secreted in the late stationary phase was isolated from the culture fluid of Bacillus intermedius 3-19 by ion-exchange chromatography on CM-cellulose followed by FPLC on a Mono S column . The enzyme was completely inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride . The maximum proteolytic activity against the synthetic chromogenic substrate Z-Ala-Ala-Leu-pNA was observed at pH 9.0 . The molecular weight of the enzyme is 28 kD and its isoelectric point is 9.2 . We have also determined pH- and thermostability and Km and kcat of this proteinase . The enzyme has been classified as a thiol-dependent serine proteinase . N-Terminal amino acid sequence (10 residues) and amino acid composition of the protein were also determined . By the mode of hydrolysis of peptide bonds in the oxidized B-chain of insulin, this enzyme is similar to the thiol-dependent serine proteinase 1 from B . intermedius 3-19 secreted during vegetative growth. Biosci Biotechnol Biochem, 2004 May, 68(5), 1059 - 66 Characterization of endo-beta-N-acetylglucosaminidase from alkaliphilic Bacillus halodurans C-125; Fujita K et al.; The genome sequencing project on alkaliphilic Bacillus halodurans C-125 revealed a putative endo-beta-N-acetylglucosaminidase (Endo-BH), which consists of a signal peptide of 24 amino acids, a catalytic region of 634 amino acids exhibiting 50.1% identity with the endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A), and a C-terminal tail of 220 amino acids . Transformed Escherichia coli cells carrying the Endo-BH gene exhibited endo-beta-N-acetylglucosaminidase activity . Recombinant Endo-BH hydrolyzed high-mannose type oligosaccharides and hybrid type oligosaccharides, and showed transglycosylation activity . On deletion of 219 C-terminal amino acid residues of Endo-BH, the wild type level of activity was retained, whereas with deletions of the Endo-A homolog domain, the proteins were expressed as inclusion bodies and these activities were reduced . These results suggest that the enzymatic properties of Endo-BH are similar to those of Endo-A, and that the C-terminal tail does not affect the enzyme activity . Although the C-terminal tail region is not essential for enzyme activity, the sequence is also conserved among endo-beta-N-acetylglucosaminidases of various origins. Biotechnol Lett, 2004 Apr, 26(7), 603 - 5 Evaluation of cellulose-binding domain fused to a lipase for the lipase immobilization; Hwang S et al.; A cellulose-binding domain (CBD) fragment of a cellulase gene of Trichoderma hazianum was fused to a lipase gene of Bacillus stearothermophilus L1 to make a gene cluster for CBD-BSL lipase . The specific activity of CBD-BSL lipase for oil hydrolysis increased by 33% after being immobilized on Avicel (microcrystalline cellulose), whereas those of CBD-BSL lipase and BSL lipase decreased by 16% and 54%, respectively, after being immobilized on silica gel . Although the loss of activity of an enzyme immobilized by adsorption has been reported previously, the loss of activity of the CBD-BSL lipase immobilized on Avicel was less than 3% after 12 h due to the irreversible binding of CBD to Avicel. Curr Opin Otolaryngol Head Neck Surg, 2004 Jun, 12(3), 223 - 31 Vaccination and allergy; Rottem M et al.; PURPOSE OF REVIEW: Vaccines have had a major effect on controlling the spread of infectious diseases, but use of certain vaccines was linked to potential allergic and autoimmune side effects in healthy and often in certain high-risk populations . In this review the authors summarize the current knowledge of such risks . RECENT FINDINGS: Immediate systemic allergic reactions after vaccination with commonly used vaccines are extremely rare . Use of certain vaccines was linked to potential allergic side effects in healthy and often in certain high-risk populations . The authors review the data on the risk associated with important vaccines including influenza, smallpox, pneumococcus, Japanese encephalitis, Bacille Calmette-Guerin, pertussis, and measles, mumps, and rubella . Two main components were identified as a source for allergic reactions in vaccines: gelatin and egg protein . There is growing interest in the potential interactions between infant vaccination and risk for development of atopic disease . In addition, there is concern that genetic risk for atopy influences capacity to respond to vaccination during infancy . There is no evidence that vaccines such as Bacille Calmette-Guerin; pertussis; influenza; measles, mumps, and rubella; or smallpox have an effect on the risk of the development of atopy later in life . Immunotherapy provides an efficacious and safe method for the treatment of allergic conditions by immunomodulation of the immune system . The possibility of vaccination triggering or unmasking autoimmunity in genetically susceptible individuals cannot be ruled out, but for the general population the risk-to-benefit ratio is overwhelmingly in favor of vaccinations . SUMMARY: Childhood vaccination remains an essential part of child health programs and should not be withheld, even from children predisposed to allergy . Vaccinations are safe, but special attention should be taken in high-risk individuals with anaphylactic reactions to foods, and in patients with autoimmune diseases. J Biochem Biophys Methods, 2004 Jun 30, 59(3), 267 - 74 Microcalorimetric investigation on the growth model and the protein yield of Bacillus thuringiensis; Xiaoyan L et al.; A novel microcalorimetric technique based on the bacterial heat output was applied to evaluate the special growth model, the protein expression and the generation time of Bacillus thuringiensis for the first time . The thermogenic curves of the aerobic metabolism of B . thuringiensis strains YBT-833, YBT-1520 and YBT-833-2-1 were determined by using an LKB-2277 BioActivity Monitor . The analysis of the thermogenic curves indicated both the mutant strain and the wild-type strains followed the same linear growth model during sporulation . The metabolism heat output revealed heat output was correlated to the yield of the insecticidal crystal proteins (ICPs) very well, the more protein product, and the less heat output . Based on the data acquired, we proposed that this method could be a useful tool in monitoring the fermentation of B . thuringiensis. Comp Biochem Physiol A Mol Integr Physiol, 2004 May, 138(1), 45 - 51 Haematological parameters in Umbrina cirrosa (Teleostei, Sciaenidae): a comparison between diploid and triploid specimens; Ballarin L et al.; Haematological features were compared between diploid and triploid specimens of the ray-finned fish Umbrina cirrosa . No significant differences between diploids and triploids were reported in haematocrit and total haemoglobin concentration, but erythrocytes and thrombocytes were significantly greater in size in triploids . Glycaemia was significantly lower in diploids, whereas triploid erythrocytes were more resistant to osmotic stress . In triploids, a greater fraction of leukocytes was positive for alkaline phosphatase activity, when stimulated with Bacillus clausii spores, otherwise no significant increase of oxygen consumption was observed in triploid leukocytes after stimulation, based on assays for superoxide anions . Triploids were characterized by a lower concentration of circulating blood cells with a lower surface/volume ratio when compared with diploids . These features may lead to a general disadvantage of triploids in withstanding stress conditions: a situation that needs to be taken into account in aquaculture practice. J Biotechnol, 2004 Jun 10, 110(3), 273 - 85 Synthesis of alkylgalactosides using whole cells of Bacillus pseudofirmus species as catalysts; Das-Bradoo S et al.; Whole cells of alkaliphilic Bacillus pseudofirmus AR-199, induced for beta-galactosidase activity, were used for the synthesis of 1-hexyl-beta-d-galactoside and 1-octyl-beta-d-galactoside, respectively, by transglycosylation reaction between lactose and the corresponding alcohol acceptor . The product yield was strongly influenced by the initial water content in the reaction mixture . Water content of 10% (v/v) was optimal providing 3.6-36 mM hexyl galactoside from 10 to 150 mM lactose, and no secondary product hydrolysis . Product yield could be enhanced by supplementing the reaction mixture with more cells or partly replacing the product with fresh substrate, but was decreased with time to the initial equilibrium level . Cell permeabilisation or disruption resulted in increased reaction rate and higher product yield but was followed by product hydrolysis . Octyl galactoside synthesis using whole cells was optimal at water content of 2% (v/v) with a yield of 26% . The cells were immobilised in cryogels of polyvinyl alcohol for use in continuous process, where hexyl galactoside was produced with a constant yield of 50% from 50mM lactose for at least a week . J Basic Microbiol, 2004, 44(3), 241 - 52 Isolation, characterization and beneficial effects of rice associated plant growth promoting bacteria from Zanzibar soils; Yasmin S et al.; This study was undertaken to isolate and characterize plant growth promoting bacteria (PGPB) occurring in four soils of Zanzibar, Tanzania as well as to evaluate their potential use as biofertilizers for rice . A total of 12 PGPB strains were isolated from rice and studied for growth characteristics, carbon/nitrogen source utilization patterns using QTS-24 kits, phosphate solubilization, indole acetic acid (IAA) production, antibiotic resistance patterns and growth at different pH, temperature and salt concentrations . All the isolates were motile and gram negative except Z3-4 . Acetylene reduction activity was detected in all isolates ranging from 5.9-76.4 nmole C2H2 reduced/h x mg protein while 9 isolates produced IAA ranged from 20-90.8 mg/l . Most of the isolates showed resistance against different environmental stresses like 10-40 degrees C temperature, 0.2-1 M salt concentration and 4-8.5 pH range . Only one isolate Z2-7 formed clear zones on Pikovskaia's medium showing its ability to solubilize phosphates . Z3-2 was used to develop fluorescent antibodies to check the cross reactivity of the isolates . Inoculation of these bacterial isolates resulted in higher plant biomass, root area, and total N and P contents on Tanzanian rice variety BKN PRAT3036B under controlled conditions . Bacillus sp . Z3-4 and Azospirillum sp . Z3-1 are effective strains and, after further testing under field conditions, can be used for inoculum production of rice in Tanzania . The plant growth promoting effects of these PGPRs suggest that these can be exploited to improve crop productivity of rice in Tanzania . J Agric Food Chem, 2004 Jun 2, 52(11), 3356 - 9 Studies on cultivation kinetics for elastase production by Bacillus sp . EL31410; Chen QH et al.; It was the first time to study elastase batch cultivation kinetics . This paper discusses the growth kinetics, elastase production, and substrate consumption kinetics model of Bacillus sp . EL31410 in batch cultivation . A simple model was proposed using a logistic equation for growth, the Luedeking-Piret equation for elastase production, and the Luedeking-Piret-like equation for glucose consumption . The model appeared to provide a reasonable description for each parameter during the growth phase . This study could provide some support for studying elastase fermentation kinetics, especially for studying its singular growth phenomenon . However, the model for elastase production is not good for explaining the real process and is still up to research. Vaccine, 2004 Feb 25, 22(8), 1063 - 71 A single dose of killed Mycobacterium bovis BCG in a novel class of adjuvant (Novasome) protects guinea pigs from lethal tuberculosis; Chambers MA et al.; The only vaccine currently available for the prevention of tuberculosis in man is a live attenuated vaccine, bacille Calmette-Guerin (BCG), derived from Mycobacterium bovis . Concerns over the lack of the universal efficacy and safety of BCG have resulted in efforts to develop a new generation of TB vaccines . Historically, killed whole-cell preparations of mycobacteria have been ineffective vaccines . We revisited the potential of killed whole-cell vaccines by comparing their efficacy with live BCG Pasteur in a guinea pig challenge model . BCG Pasteur was inactivated with a low concentration of formalin and showed to be non-viable in culture or severe combined immunodeficient mice . Formalin-inactivated BCG was mixed with non-phospholipid liposome adjuvants (Novasomes) and administered to guinea pigs as a single subcutaneous inoculation . All formulations were well tolerated and one conferred a significant survival advantage against lethal aerogenic challenge with M . bovis. Microbiol Res, 2004, 159(1), 59 - 71 Isolation, geographical diversity and insecticidal activity of Bacillus thuringiensis from soils in Spain; Quesada-Moraga E et al.; Bacillus thuringiensis is a spore-forming bacterium showing the unusual ability to produce endogenous crystals during sporulation that are toxic for some pest insects . This work was performed to study the composition, ecological distribution and insecticidal activity of isolates of this entomopathogenic bacterium from the Spanish territory . Using a standard isolation method, B . thuringiensis was isolated from 115 out of 493 soil samples collected in the Iberian Peninsula and the Canary and Balearic Archipelagos . The percentages of samples with B . thuringiensis were 31.7, 27.6 and 18.5 and the B . thuringiensis index 0.065, 0.067 and 0.11 for the Iberian Peninsula, Canary and Balearic Archipelagos, respectively . The prairies were shown to be the worst source of B . thuringiensis while forests, urban and agricultural habitats showed similar percentages . Strain classification based on H-antigen agglutination showed a great diversity among the Spanish isolates, which were distributed among 24 subspecies, including three new ones andaluciensis, asturiensis and palmanyolensis . We differentiated 65 different protein profiles of spore-crystal mixtures by sulfate-polyacrylamide gel electrophoresis and we selected 109 isolates representative of these profiles to evaluate their insecticidal activity against insects from the Orders Orthoptera, Dictyoptera, Coleoptera, Lepidoptera and Diptera . We found variable percentages of isolates active against Coleoptera and Lepidoptera, one isolate highly active against mosquito larvae and for the first time, three isolates active against cockroaches and locusts. Acta Crystallogr D Biol Crystallogr, 2004 Jun, 60(Pt 6), 1149 - 51 Epub 2004 May 21. Crystallization and preliminary X-ray diffraction analysis of a thermostable endo-1,5-alpha-L-arabinanase from Bacillus thermodenitrificans TS-3; Yamaguchi A et al.; A thermostable endo-1,5-alpha-L-arabinanase ABN-TS from Bacillus thermodenitrificans TS-3 with a molecular weight of 35 kDa was crystallized by the hanging-drop vapour-diffusion method using sodium citrate as a precipitant . The crystals were loop-mounted in a cryoprotectant solution containing 28%(w/v) sucrose and 1 M sodium citrate pH 6.0 and flash-cooled . Sucrose was selected as the most suitable cryoprotectant . The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 40.3, b = 77.8, c = 89.7 angstroms . The calculated VM based on one molecule per asymmetric unit was 2.0 angstroms3 Da(-1) . A complete data set from a frozen crystal was collected to 1.9 angstroms resolution using synchrotron radiation at SPring-8 . A molecular-replacement solution was obtained using the structure of alpha-arabinanase 43A from Cellvibrio japonicus . Biochim Biophys Acta, 2004 Jun 1, 1699(1-2), 123 - 30 Effect of pH and ionic strength on the cytolytic toxin Cyt1A: a fluorescence spectroscopy study; Manceva SD et al.; Cyt1A is a cytolytic toxin produced by Bacillus thuringiensis var . israelensis . Due to its toxicity in vivo against mosquitoes and black flies, it is used as an environmentally friendly insecticide, although its mode of action is not completely understood . The toxin is membrane-active, but its membrane-bound conformation is unknown . In the absence of direct structural data, fluorescence spectroscopy was used to obtain indirect information on Cyt1A conformation changes in the environment mimicking the vicinity of the lipid membrane (lower pH and increased ionic strength) . With decreasing pH, Cyt1A's surface hydrophobicity increased, which is consistent with an increased interaction with model membranes at low pH values, as observed previously . The pK(a) value of this conformation change is 4.4+/-0.1 . Intrinsic tryptophan fluorescence decreased with decreasing pH, and the pK(a) value was the same as the one determined with synthetic probes . The protein has two types of hydrophobic binding sites, and at low pH these sites bind more probe molecules (bis-ANS) with a higher affinity than at pH 7.4 . When bound to the lipid, the toxin exhibited conformation similar to the molten-globule state and showed some characteristics also observed at low pH . However, the conformation of the lipid-bound toxin did not depend on pH . Neutral salts like NaCl and KCl induced conformational changes at neutral pH, but not at low pH . These changes were most probably due to specific interactions of the salt ions with the charged amino acids on the protein surface rather than due to general effects such as Hofmeister and Debye-Huckel . Our results might contribute to elucidating the mode of action of Cyt1A, and perhaps also to improving the formulation of the insecticidal preparations. Arch Biochem Biophys, 2004 Jun 15, 426(2), 286 - 97 Mutation of active site residues in the chitin-binding domain ChBDChiA1 from chitinase A1 of Bacillus circulans alters substrate specificity: use of a green fluorescent protein binding assay; Hardt M et al.; A fluorescent binding assay was developed to investigate the effects of mutagenesis on the binding affinity and substrate specificity of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12 . The chitin-binding domain was genetically fused to the N-terminus of a green fluorescent protein, and the polyhistidine-tagged hybrid protein was expressed in Escherichia coli . Residues likely to be involved in the binding site were mutated and their contributions to binding and substrate specificity were evaluated by affinity electrophoresis and depletion assays . The experimental binding isotherms were analyzed by non-linear regression using a modified Langmuir equation . Non-conservative substitution of tryptophan residue (W687) nearly abolished chitin-binding affinity and dramatically lowered chitosan binding while retaining the original level of curdlan binding . Double mutation E668K/P689A had altered specificity for several substrates and also impaired chitin binding significantly . Other substitutions in the binding site altered substrate specificity but had little effect on overall affinity for chitin . Interestingly, mutation T682A led to a higher specificity towards chitinous substrates than the wildtype . Furthermore, the ChBD-GFP hybrid protein was tested for use in diagnostic staining of cell walls of fungi and yeast and for the detection of fungal infections in tissue samples. Biochim Biophys Acta, 2004 May 27, 1663(1-2), 178 - 87 Electron holography of non-stained bacterial surface layer proteins; Simon P et al.; We report transmission electron microscopy (TEM) investigations on bacterial surface layers (S-layers) which belong to the simplest biomembranes existing in nature . S-layers are regular 2D protein crystals composed of single protein or glycoprotein species . In their native form, S-layers are weak phase objects giving only poor contrast in conventional TEM . Therefore, they are usually examined negatively stained . However, staining with heavy metal compounds may cause the formation of structural artefacts . In this work, electron microscopy studies of non-stained S-layers of Bacillus sphaericus NCTC 9602 were performed . Compared to other proteins, these S-layers are found relatively stable against radiation damage . Electron holography was applied where information about phase and amplitude of the diffracted electron wave is simultaneously obtained . In spite of small phase shifts observed, the phase image reconstructed from the hologram of the non-stained S-layer is found to be sensitive to rather slight structure and thickness variations . The lateral resolution, obtained so far, is less than that of conventional electron microscopy of negatively stained S-layers . It corresponds to the main lattice planes of 12.4 nm observed in the reconstructed electron phase image . In addition, as a unique feature of electron holography the phase image provides thickness information . Thus, the existence of double layers of the protein crystals could be easily visualized by the height profile of the specimen. Proc R Soc Lond B Biol Sci, 2004 Mar 22, 271(1539), 617 - 23 The reinfection threshold promotes variability in tuberculosis epidemiology and vaccine efficacy; Gomes MG et al.; Population patterns of infection are determined largely by susceptibility to infection . Infection and vaccination induce an immune response that, typically, reduces susceptibility to subsequent infections . With a general epidemic model, we detect a 'reinfection threshold', above which reinfection is the principal type of transmission and, consequently, infection levels are much higher and vaccination fails . The model is further developed to address human tuberculosis (TB) and the impact of vaccination . The bacille Calmette-Guerin (BCG) is the only vaccine in current use against TB, and there is no consensus about its usefulness . Estimates of protection range from 0 to 80%, and this variability is aggravated by an association between low vaccine efficacy and high prevalence of the disease . We propose an explanation based on three postulates: (i) the potential for transmission varies between populations, owing to differences in socio-economic and environmental factors; (ii) exposure to mycobacteria induces an immune response that is partially protective against reinfection; and (iii) this protection is not significantly improved by BCG vaccination . These postulates combine to reproduce the observed trends, and this is attributed to a reinfection threshold intrinsic to the transmission dynamics . Finally, we demonstrate how reinfection thresholds can be manipulated by vaccination programmes, suggesting that they have a potentially powerful role in global control. Clin Infect Dis, 2004 May 15, 38(10), 1495 - 7 Epub 2004 Apr 29. Adverse reactions to accidental forearm injection of Bacille Calmette-Guerin vaccine in schoolchildren: 12-month cohort follow-up; Gross S et al.; This study examined the natural history of reaction after accidental intradermal administration of bacille Calmette-Guerin (BCG) vaccine instead of purified protein derivative (PPD) in 226 schoolchildren . At 18 days after vaccination, a local reaction with a diameter of 4.5-14 mm was found in 62% of the students, and ulceration with discharge was found in 26.6%; corresponding rates at 120 days were 72.3% and 38% and at 281 days were 73% and 6% . At 345 days, 85% of the students had a dry scar measuring 5-14 mm in diameter, and none had ulceration or discharge. Methods Mol Biol, 2004, 268, 207 - 11 Interaction between lactic acid bacteria and gastrointestinal nematodes of caprine origin; Draksler D et al.; To compare the level of parasitism with gastrointestinal nematodes in sheep and goats, several studies have been conducted . They have generally shown that goats were more infected than sheep, as they exhibited higher worm burdens and egg excretion . This difference between two host species has been attributed not only to a difference in feeding behavior, but also to a lesser ability of goats to develop resistance to trichostrongylate infection (In kids and lambs the greatest damage is observed from weaning until 1 yr of age; mature mothers, before and after parturition and during suckling, are affected).In the last few decades, the most common tool (and frequently the only one) used for controlling internal parasites in livestock was the anthelmintic drugs . The application of anthelmintic treatments; must be accompanied by epidemic data and determinations supporting the appropriate timing and frequency of animal treatment . This view has not always been respected . In our country, because of a decrease in price, the anthelmintic drugs were used indiscriminately, causing the resistance we see today . This serious problem, added to the objective of producing organic foods without drug residuals, calls for better use of the antiparasitic drugs and for the developmentment of alternative methods that are ecologically viable and without risks for human health.Little information is available on the interactions between bacteria and intestinal nematodes of caprine origin . Some reports note ovicidal activity of different strains of Bacillus thuringiensis on the eggs of zooparasitic nematodes . Recent work found inhibitory actions of lactic bacteria on gastrointestinal nematodes (both of caprine origin) . In the present chapter we describe the methods used for the determination of interactions between lactic acid bacteria and nematodes. J Biol Chem, 2004 Jul 30, 279(31), 31995 - 2000 Epub 2004 May 23. The catalytic role of aspartate in a short strong hydrogen bond of the Asp274-His32 catalytic dyad in phosphatidylinositol-specific phospholipase C can be substituted by a chloride ion; Zhao L et al.; Phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis catalyzes the cleavage of the phosphorus-oxygen bond in phosphatidylinositol . The focus of this work is to dissect the roles of the carboxylate side chain of Asp(274) in the Asp(274)-His(32) dyad, where a short strong hydrogen bond (SSHB) was shown to exist based on NMR criteria . A regular hydrogen bond (HB) was observed in D274N, and no low field proton resonance was detected for D274E and D274A . Comparison of the activity of wild type, D274N, and D274A suggested that the regular HB contributes significantly (approximately 4 kcal/mol) to catalysis, whereas the SSHB contributes only an additional 2 kcal/mol . The mutant D274E displays high activity similar to wild type, suggesting that the negative charge is sufficient for the catalytic role of Asp(274) . To further support this interpretation and rule out possible contribution of regular HB or SSHB in D274E, we showed that the activity of D274G can be rescued by exogenous chloride ions to a level comparable with that of D274E . Comparison between different anions suggested that the ability of an anion to rescue the activity is due to the size and the charge of the anion not the property as a HB acceptor . In conclusion, a major fraction of the functional role of Asp(274) in the Asp(274)-His(32) dyad can be attributed to a negative charge (as in D274E and D274G-Cl(-)), and the SSHB in the wild type enzyme provides minimal contribution to catalysis . These results represent novel insight for an Asp-His catalytic dyad and for the mechanism of phosphatidylinositol-specific phospholipase C. Infect Immun, 2004 Jun, 72(6), 3106 - 12 Disassembly of F-actin cytoskeleton after interaction of Bacillus cereus with fully differentiated human intestinal Caco-2 cells; Minnaard J et al.; In the present study, the role of direct procaryote-eucaryote interactions in the virulence of Bacillus cereus was investigated . As a model of human enterocytes, differentiated Caco-2 cells were used . Infection of fully differentiated Caco-2 cells with B . cereus in the exponential phase of growth, in order to minimize the concentration of spores or sporulating microorganisms, shows that a strain-dependent cytopathic effect develops . Interestingly, addition of 3-h-old cultures of some strains resulted in complete detachment of the cultured cells after a 3-h infection whereas no such effect was found after a 3-h infection with 16-h-old cultures . Infection of enterocyte-like cells with B . cereus leads to disruption of the F-actin network and necrosis . Even though the effect of secreted factors cannot be ruled out, direct eucaryote-procaryote interaction seems to be necessary . In addition, we observed that some B . cereus strains were able to be internalized in Caco-2 cells . Our findings add a new insight into the mechanisms of virulence of B . cer