Bacillus

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 . cereus in the context of intestinal infection.

J Econ Entomol, 2004 Apr, 97(2), 451 - 9
A survey of hymenopteran parasitoids of forest macrolepidoptera in the central Appalachians; Petrice TR et al.; In 1995 and 1996, we conducted a study of the hymenopteran parasitoids of macrolepidopteran larvae in the George Washington National Forest (GWNF), Augusta County, Virginia, and the Monongahela National Forest (MNF), Pocahontas County, West Virginia . Macrolepidopteran larvae were collected from canopy foliage and from under canvas bands placed around tree boles . A total of 115 macrolepidopteran species and 5,235 individual larvae were reared . Forty-two percent (2,221) of the larvae were gypsy moth, Lymantria dispar (L.) (Lymantriidae) . A total of 43 primary and secondary (hyperparasitoid) hymenopteran parasitoid species were reared from 46 macrolepidopteran species . Hymenopteran families represented included Ichneumonidae (23 species), Braconidae (19), Eulophidae (6), Perilampidae (1), and Trigonalidae (1) . We reared 41 and 28 parasitoid species from the GWNF and the MNF, respectively, with 19 species reared from both forests . Many parasitoid species were collected infrequently, suggesting that they are relatively rare on the sampled hosts . The introduced species Cotesia melanoscela (Ratzeburg) (Braconidae), and Euplectrus bicolor (Swederus) (Eulophidae) were among the most commonly reared parasitoids, the latter reared from native hosts . The four most commonly reared native parasitoids were Meteorus hyphantriae, Riley (Braconidae), Microplitis near hyphantriae (Ashmead) (Braconidae), Aleiodes preclarus Marsh & Shaw, and Euplectrus maculiventris (Westwood) (Eulophidae) . A total of 53 new hymenopteran parasitoid-macrolepidopteran host records were documented . Results from this study will be used to evaluate long-term treatment effects of regional applications of Bacillus thuringiensis kurstaki, and the gypsy moth fungus Entomophaga maimaiga Humber, Shimazu & Soper on hymenopteran parasitoids of macrolepidopteran larvae.

J Econ Entomol, 2004 Apr, 97(2), 374 - 82
Damage loss assessment and control of the cereal leaf beetle (Coleoptera: Chrysomelidae) in winter wheat; Buntin GD et al.; Cereal leaf beetle, Oulema melanopus (L.), invaded northern Alabama and Georgia more than a decade ago and since has become an economic pest of winter wheat and other cereal crops in the southeastern United States . A series of trials was conducted beginning in 1995 to determine optimal rate and timing of applications of selected foliar insecticides for managing cereal leaf beetle in soft red winter wheat . These trials, cage studies with larvae, and a manual defoliation experiment were used to provide information on cereal leafbeetle yield loss relationships and to develop economic decision rules for cereal leaf beetle in soft red winter wheat . Malathion, methomyl, carbaryl, and spinosad effectively controlled larval infestations when treatments were applied after most eggs had hatched . Encapsulated endotoxin of Bacillus thuringiensis, methyl parathion, and disulfoton applied at the lowest labeled rates were not effective treatments . Organophosphate insecticides generally were not effective when applied before most eggs had hatched . The most effective treatments were the low rates of lambda cyhalothrin when applied early while adults were still laying eggs and before or near 50% egg hatch . These early applications applied at or before spike emergence virtually eliminated cereal leaf beetle injury . The manual defoliation study demonstrated that defoliation before spike emergence has greater impact on grain yield and yield components than defoliation after spike emergence . Furthermore, flag leaf defoliation causes more damage than injury to lower leaves . Grain test weight and kernel weight were not affected by larval injury in most trials . Regression of larval numbers and yield losses calculated a yield loss of 12.65% or 459 kg/ha per larva per stem, which at current application costs suggested an economic threshold of 0.4 larvae per stem during the spike emergence to anthesis stages.

J Econ Entomol, 2004 Apr, 97(2), 340 - 3
Biological control of Lobesia botrana (Lepidoptera: Tortricidae) larvae by using different formulations of Bacillus thuringiensis in 11 vine cultivars under field conditions; Ifoulis AA et al.; This work describes the results of an experiment that was conducted in the vineyard of the American Farm School, Thermi, Thessaloniki Greece, during 2001 . Its aim was to study the efficacy of two formulations of Bacillus thuringiensis Berliner (wettable powder and dust) to control the larvae of Lobesia botrana Denis & Schiffermueler (Lepidoptera: Tortricidae) . The experimental results showed that the two formulations of B . thuringiensis are significantly more effective than the control, the dusting being more effective in most cultivars (Asyrtico, Sauvignon blanc, Debina, Athiri, Agiorgitico, Limnio, Syrah, and Cabernet sauvignon), and the spraying in a few cultivars (Xinomavro, Roditis, and Chardonnay) . Dusting in the blue-black cultivars was found to be more effective than spraying, this not being the case in the yellow-green cultivars . This fact should be taken into consideration when cultivars of different color were planted in the same field . Single dusting proved to be better in cultivars with loose, average and dense berry cluster compactness, whereas double spraying gave better results in compact cultivars.

J Econ Entomol, 2004 Apr, 97(2), 259 - 64
Assessment of experimental Bt events against fall armyworm and corn earworm in field corn; Buntin GD et al.; Performance of experimental Bacillus thuringiensis (Bt) MON events alone and pyramided with MON810 were evaluated over 3 yr in Georgia and Alabama . Ability of events to prevent whorl defoliation by the fall armyworm, Spodoptera frugiperda (J . E . Smith), and natural ear feeding damage by the corn earworm, Helicoverpa zea (Boddie) was assessed . In each year, near-isogenic hybrids with novel single transformation events and crosses pyramided with the MON810 event were compared with the standard single MON810 event and nontransformed susceptible control . Events were tested for resistance to whorl damage by manual infestations of fall armyworm and ear damage by natural infestations of corn earworm . All Bt events tested reduced fall armyworm whorl damage ratings per plant compared with the susceptible hybrid . All Bt treatments also had considerably less ear infestation and damage by corn earworm compared with the nontransgenic isoline . The MON841, MION849, and MON851 events reduced ear damage by H . zea but were not as effective as other novel events and were not advanced for further testing after the 1999 season . Pyramiding events compared with single events did not improve control of fall armyworm whorl damage, but they generally did prevent more ear damage by corn earworm . The MON84006 event singly and pyramided with MON810 had superior control of whorl-stage damage by S . frugiperda and ear damage by H . zea compared with MON810 . Deployment of new events and genes could provide additional tools for managing the potential for insect resistance to Bt toxins . Furthermore, improved control of whorl and ear infestations by H . zea and S . frugiperda would increase the flexibility of planting corn, Zea mays L., and permit double cropping of corn in areas where these pests perennially reach damaging levels.

J Econ Entomol, 2004 Apr, 97(2), 251 - 8
Screening of the insecticidal activity of Bacillus thuringiensis strains against Lygus hesperus (Hemiptera: Miridae) nymphal population; Wellman-Desbiens E et al.; Lygus hesperus Knight (Hemiptera: Miridae) is an economically important insect pest controlled primarily by chemical pesticides . Bacillus thuringiensis Berliner is a gram-positive bacterium that has been developed for the control of some insect pests in the orders Lepidoptera, Coleoptera, and Diptera . In this study, whole culture extracts of 94 B . thuringiensis strains from 83 serovars were added to an artificial diet and assayed against L . hesperus first and second instars . A total of five B . thuringiensis strains, B . thuringiensis variety thuringiensis, thuringiensis exotoxin +, morrisoni, tolworthi, and darmstadiensis generated > 98% mortality after 7 d of incubation . The screening was repeated with 117 alkali-solubilized trypsin-digested B . thuringiensis cultures and the same five B . thuringiensis strains showed nearly identical results . All five strains produce beta-exotoxin, which exhibits a wide host spectrum activity . No beta-exotoxin-minus B . thuringiensis strains showed significant toxicity against L . hesperus nymphs . The present work is one of the first thorough screenings of the wide diversity of the B . thuringiensis varieties for the control of L . hesperus nymphal populations.

J Food Prot, 2004 May, 67(5), 1036 - 8
Detection of Bacillus spores using PCR and FTA filters; Lampel KA et al.; Emphasis has been placed on developing and implementing rapid detection systems for microbial pathogens . We have explored the utility of expanding FTA filter technology for the preparation of template DNA for PCR from bacterial spores . Isolated spores from several Bacillus spp., B . subtilis, B . cereus, and B . megaterium, were applied to FTA filters, and specific DNA products were amplified by PCR . Spore preparations were examined microscopically to ensure that the presence of vegetative cells, if any, did not yield misleading results . PCR primers SRM86 and SRM87 targeted a conserved region of bacterial rRNA genes, whereas primers Bsub5F and Bsub3R amplified a product from a conserved sequence of the B . subtilis rRNA gene . With the use of the latter set of primers for nested PCR, the sensitivity of the PCR-based assay was increased . Overall, 53 spores could be detected after the first round of PCR, and the sensitivity was increased to five spores by nested PCR . FTA filters are an excellent platform to remove PCR inhibitors and have universal applications for environmental, clinical, and food samples.

J Food Prot, 2004 May, 67(5), 939 - 46
Modeling the prevalence of Bacillus cereus spores during the production of a cooked chilled vegetable product; Malakar PK et al.; In minimally processed vegetable foods, pathogenic spore-forming bacteria pose a significant hazard . As part of a quantitative risk assessment, we used Bayesian belief methods to model the uncertainty and variability of the number of Bacillus cereus spores that can be found in packets of a vegetable puree . The model combines specific information from the manufacturer, experimental data on inactivation of spores, and expert opinion concerning spore concentrations in the raw vegetables and ingredients . Sensitivity analysis revealed that spore contamination of added ingredients contributes most uncertainty to the assessment . The assessment produced a quantitative estimate of the prevalence of B . cereus spores in packets of vegetable puree at the end point of the manufacturing process.

J Food Prot, 2004 May, 67(5), 934 - 8
Variation of the spore population of a natural source strain of Bacillus cereus in the presence of inosine; Collado J et al.; The heat resistance of a wild strain of Bacillus cereus spores isolated from liquid egg was characterized, and the effect of the nutritional germinant inosine on the spore population was then studied, considering different factors such as germination temperature, inosine concentration, and age of spore culture . The heat resistance clearly indicates that these spores can survive mild heat treatments such as those used for cooked refrigerated food of extended durability or liquid egg, posing safety problems for these foods with temperature abuse . The germination study indicates that temperature, spore age, and the interaction between the two were the factors affecting the level of spores remaining after the germination process . No significant differences were found for the three inosine concentrations used in the study (1, 5, and 10 mM) . The highest reduction in the spore concentration was reached at 30 degrees C after 120 min, although the reduction in the spore counts at germination temperatures of 4 and 8 degrees C was also considerable.

Parasitology, 2004 Apr, 128(Pt 4), 433 - 43
Natural variation in the response of Caenorhabditis elegans towards Bacillus thuringiensis; Schulenburg H et al.; Almost nothing is known about the natural ecology of the nematode Caenorhabditis elegans, including its interactions with parasites . To help rectify this discrepancy, we assessed natural variation in the response of C . elegans towards a potential parasite, the soil bacterium Bacillus thuringiensis . Our results show that 10 isolates from across the world differ significantly in survival rate and infection level when confronted with a parasitic strain of B . thuringiensis . Furthermore, behavioural responses are identified as an important component of C . elegans defence, including evasion and possibly reduced ingestion of parasites . Again, the natural isolates show significant differences in these traits . In conclusion, worm defence is indicated to be complex and variable across space, implying that parasites play an important role in the ecology of this species . Based on these results, we expect C . elegans to be a promising model host for future analysis of the evolutionary dynamics of parasite-host interactions.

J Antimicrob Chemother, 2004 Jun, 53(6), 1072 - 5 Epub 2004 May 18.
Role for malonyl coenzyme A:acyl carrier protein transacylase (MCAT) in the growth-inhibitory effect of the calmodulin antagonist trifluoperazine in Mycobacterium bovis BCG; Sinha I et al.; OBJECTIVES: To determine whether the fatty acid synthesis enzyme malonyl coenzyme A:acyl carrier protein transacylase (MCAT) is involved in the growth-inhibitory effect of trifluoperazine in the tubercle bacillus Mycobacterium bovis BCG . METHODS: BCG was grown in liquid culture with various concentrations of trifluoperazine and growth was monitored by OD measurement . To determine the effect of trifluoperazine on MCAT protein level, total protein was extracted from BCG cultures and was analysed by 2D gel electrophoresis and western blot . To confirm trifluoperazine-dependent reduction in the MCAT protein level, two BCG strains overexpressing MCAT at a low and high constitutive level were similarly tested . The synergic effect of trifluoperazine and isoniazid was tested at sub-MIC levels in liquid cultures . RESULTS: Trifluoperazine inhibition of growth correlates with reduction in the steady-state level of MCAT protein . Overexpression of MCAT confers resistance to trifluoperazine . Trifluoperazine acts synergically (albeit weakly) with isoniazid and no resistance towards isoniazid alone was observed due to overexpression of MCAT . This suggests MCAT to be a specific target of trifluoperazine . CONCLUSION: These results indicate MCAT as a target of trifluoperazine and provide an explanation for the inhibitory effect of trifluoperazine on mycobacterial lipid synthesis observed earlier . This makes MCAT a potential target for new antimycobacterials.

Bioconjug Chem, 2004 May-Jun, 15(3), 664 - 71
An S-layer heavy chain camel antibody fusion protein for generation of a nanopatterned sensing layer to detect the prostate-specific antigen by surface plasmon resonance technology; Pleschberger M et al.; The bacterial cell surface layer (S-layer) protein of Bacillus sphaericus CCM 2177 assembles into a square lattice structure and recognizes a distinct type of secondary cell wall polymer (SCWP) as the proper anchoring structure in the rigid cell wall layer . For generating a nanopatterned sensing layer with high density and well defined distance of the ligand on the outermost surface, an S-layer fusion protein incorporating the sequence of a variable domain of a heavy chain camel antibody directed against prostate-specific antigen (PSA) was constructed, produced, and recrystallized on gold chips precoated with thiolated SCWP . The S-layer protein moiety consisted of the N-terminal part which specifically recognized the SCWP as binding site and the self-assembly domain . The PSA-specific variable domain of the camel heavy chain antibody was selected by several rounds of panning from a phage display library of an immunized dromedary, and was produced by heterologous expression in Escherichia coli . For construction of the S-layer fusion protein, the 3'-end of the sequence encoding the C-terminally truncated form rSbpA(31)(-)(1068) was fused via a short linker to the 5'-end of the sequence encoding cAb-PSA-N7 . The S-layer fusion protein had retained the ability to self-assemble into the square lattice structure . According to the selected fusion site in the SbpA sequence, the cAb-PSA-N7 moiety remained located on the outer surface of the protein lattice . After recrystallization of the S-layer fusion protein on gold chips precoated with thiolated SCWP, the monomolecular protein lattice was exploited as sensing layer in surface plasmon resonance biochips to detect PSA.

Int J Med Microbiol, 2004 Apr, 293(7-8), 599 - 607
Pore worms: using Caenorhabditis elegans to study how bacterial toxins interact with their target host; Huffman DL et al.; The interaction of pathogenic bacteria with a target host is regulated both by bacterial virulence factors and by host components that either protect the host or that promote pathogenesis . The soil nematode Caenorhabditis elegans is a host for a number of bacterial pathogens, as briefly reviewed here . Bacillus thuringiensis (Bt) is a pathogenic bacteria that C . elegans is likely to encounter naturally in the soil . The pore-forming Crystal (Cry) toxins made by Bt are recognized as the dominant virulence factor in this host-pathogen interaction . Forward genetic screens for C . elegans mutants resistant to the Cry toxin, Cry5B, have identified a host carbohydrate structure that promotes pathogenesis . Data suggest this structure is likely to be a Cry5B receptor expressed in the host intestine . This finding is discussed in light of other carbohydrate receptors for bacterial toxins . To investigate host-toxin interactions on a global level, the response of C . elegans to the pore-forming Cry5B is also being investigated by gene transcription profiling (microarrays) . These data are beginning to reveal a diverse intracellular response to toxin exposure . To put these investigations in perspective, host responses to other pore-forming toxins are discussed . Investigations with Cry5B in C . elegans show a promising beginning in helping to elucidate host-toxin and host-pathogen interactions.

Int J Med Microbiol, 2004 Apr, 293(7-8), 463 - 70
Host cell modulation by human, animal and plant pathogens; Andersson SG et al.; Members of the alpha-proteobacteria display a broad range of interactions with higher eukaryotes . Some are pathogens of humans, such as Rickettsia and Bartonella that are associated with diseases like epidemic typhus, trench fever, cat scratch disease and bacillary angiomatosis . Others like the Brucella cause abortions in pregnant animals . Yet other species have evolved elaborate interactions with plants; in this group we find both plant symbionts and parasites . Despite radically different host preferences, extreme genome size variations and the absence of toxin genes, similarities in survival strategies and host cell interactions can be recognized among members of the alpha-proteobacteria . Here, we review some of these similarities, with a focus on strategies for modulation of the host target cell.

Methods Mol Biol, 2004, 266, 305 - 22
Discovering new pathogens: culture-resistant bacteria; Lawson AJ; Recent advances in gene-amplification technology and molecular phylogenetics have provided the means of detecting and classifying bacteria directly from their natural habitats without the need for culture . These techniques have revolutionized environmental microbiology, and it is now apparent that the global diversity of microorganisms is much greater than previously thought . In the context of clinical microbiology, this molecular-based approach has facilitated the characterization of culture-resistant bacteria associated with human disease . Examples include Helicobacter heilmannii, a cause of gastritis, Tropheryma whippeli (the agent of Whipple's disease), and the agents of human ehrlichiosis and bacillary angiomatosis . Molecular-based techniques also provide a means of investigating complex bacterial flora within the human ecosystem, such as feces and dental plaque, without the bias of culture-based isolation . This has given a new perspective to the study of polymicrobial infections such as gingivitis, and offers the potential for the detection and identification of novel bacterial pathogens from among complex and numerous endogenous microbial flora.

J Chromatogr A, 2004 May 21, 1036(2), 249 - 53
Gas chromatography using a resistively heated column with mass spectrometric detection for rapid analysis of pyridine released from Bacillus spores; Smith PA et al.; Gas chromatography using a resistively heated analytical column with full scan electron impact mass spectrometry (EI-MS) was used to detect pyridine generated from heating Bacillus spores in a custom designed furnace inlet, along with gasoline range aromatic (GRA) hydrocarbons representing an environmental contaminant that could interfere with detection of the biologically-derived compound . Gas phase materials from the furnace inlet were collected onto a section of cooled open tubular column, and carrier gas flow was then routed through the trapping column onto the analytical column . Both sections of column were contained within low thermal mass tubular metal sheaths, with each independently and resistively heated allowing rapid temperature ramps and cooling . An analysis time of 2 min resolved spore-derived pyridine from the other organics, and allowed identification by mass spectrum match . Throughput of 20 analyses per hour was shown to be possible with a 1-min column cool-down time between analyses.

J Invertebr Pathol, 2004 May-Jun, 86(1-2), 19 - 25
Venom from the pupal endoparasitoid, Pimpla hypochondriaca, increases the susceptibility of larval Lacanobia oleracea to the entomopathogens Bacillus cereus and Beauveria bassiana; Dani MP et al.; Cellular immune responses in insects protect them against parasites and pathogens that enter their hemocoel . Venom from the solitary pupal endoparasitoid, Pimpla hypochondriaca, has previously been shown to suppress certain key, cell-mediated immune responses of Lacanobia oleracea . Experiments were performed to determine if L . oleracea larvae injected with P . hypochondriaca venom would be more susceptible to Bacillus cereus, or Beauveria bassiana, when these microorganisms were subsequently injected . Mortality due to B . cereus (approximately 15 colony-forming units {CFU}/larva) and B . bassiana (approximately 2.4 x 10(3) conidia/larva) was enhanced by prior injection of 4 microg of venom . In addition, injection of venom/Dulbecco's phosphate-buffered saline (DPBS) or DPBS/B . bassiana reduced the rate at which larvae gained weight compared to control larvae . However, the greatest reduction in weight was recorded for larvae that had been injected with venom/B . bassiana conidia.

J Invertebr Pathol, 2004 May-Jun, 86(1-2), 7 - 18
Characterization of Mexican Bacillus thuringiensis strains toxic for lepidopteran and coleopteran larvae; Tamez-Guerra P et al.; Bacillus thuringiensis strains C-4, C-9, GM-7, and GM-10, isolated from northeast Mexico and selected for their high toxicity against lepidopteran and coleopteran pests, were characterized following United States Environmental Protection Agency (EPA)'s guidelines . Flagellar serotyping revealed that GM-7 and GM-10 belonged to serotype aizawai, whereas C-4, C-9 corresponded to the kumamotoensis serotype . GM-10 and C-9 were also shown to be the most effective against lepidoptera and coleoptera larvae, respectively . None of the tested strains produced beta-exotoxin or showed activity against mosquitoes . GM-7 and GM-10 were sensitive to R-41 and CP-51 phages . All strains synthesized crystal proteins of 130-140 kDa . PCR analysis showed that C-4, GM-7, and GM-10 strains expressed cry1 genes, and C-9 expressed cry3 and cry7/8 genes, but not cry1 . However, the C-9 strain had no cross-reaction with antisera raised against Cry3A and Cry7A proteins . GM-7 and GM-10 were sensitive to R-41 and CP-51 phages . When the delta-endotoxin (crystal) from the four strains was subcutaneously injected to Balb/c mice, alone or in combination with spores, only C-4 and C-9 provoked tissue necrosis similar to that caused by the beta-exotoxin producer HD-41 . Tissue necrosis was prevented with the injection of pentoxifylline, an inhibitor of tumor necrosis factor alpha (TNF-alpha) production, suggesting a role of this cytokine in the observed effect . Our results demonstrated that GM-7 and GM-10 strains are effective and suitable for control of lepidopteran pests and safe for mammals under EPA regulations . The potential of the C-9 strain for the control of several coleopteran pests, and the induction of tissue necrosis in mice by C-4 and C-9 strains, are discussed.

Zhonghua Jie He He Hu Xi Za Zhi, 2004 Apr, 27(4), 249 - 52
{Constructing shuttle plasmid fusion expressing Ag85B-ESAT-6 on the surface of Mycobacterium}; Shi CH et al.; OBJECTIVE: To construct the E . coli.-BCG (Bacille Calmette-Guerin) shuttle vector expressing Mycobacterium tuberculosis secreted protein Ag85B-ESAT-6 on the surface of Mycobacterium vaccae . METHODS: The gene fragment containing 19 000 antigen (19-ss) were amplified by polymerase chain reaction (PCR) from the Mycobacterium tuberculosis H(37)Ra . We cloned the 19ss gene into the E . coli.-BCG shuttle vector pOLYG and named the pCW, which can shuttle and express exogenous antigen gene on cell wall of Mycobacterium . Then Mycobacterium tuberculosis secret protein Ag85B and ESAT-6 gene were cloned into the vector and determined by indirect immunofluorescence . RESULTS: The sequence of 19-ss gene was identified with Genbank reported by sequencing . The constructed E . coli.-BCG shuttle vector using 19ss gene had the function of shuttle between E . coli . and Mycobacteria . By indirect immunofluorescence technique the secreted protein Ag85B-ESAT-6 can be fused and expressed on surface of Mycobacterium vaccae . CONCLUSION: The E . coli.-BCG shuttle vector is constructed successfully which could express exogenous antigen gene as a chimeric exported membrane.

Clin Exp Allergy, 2004 May, 34(5), 712 - 9
Effect of Mycobacterium tuberculosis chaperonins on bronchial eosinophilia and hyper-responsiveness in a murine model of allergic inflammation; Riffo-Vasquez Y et al.; BACKGROUND: Epidemiological evidence suggests that infection with Mycobacterium tuberculosis protects children against asthma . Several laboratories have shown that, in mouse models of allergic inflammation, administration of the whole live tuberculosis vaccine, Mycobacterium bovis bacillus Calmette-Guerin (BCG), prevents ovalbumin (OVA)-induced pulmonary eosinophilia . OBJECTIVE: The aim of this study was to characterize specific M . tuberculosis molecules that are known to modulate immune responses to see if they affected pulmonary eosinophilia and bronchial hyper-responsiveness . METHODS: C57Bl/6 mice were sensitized to OVA on days 0 and 7 and subsequently challenged with OVA on day 14 over a 3-day period . Pulmonary eosinophilia and bronchial hyper-responsiveness were measured 24 h following the last antigen challenge . In some groups, mice were pre-treated with M . tuberculosis or M . tuberculosis chaperonins (Cpns)60.1, 60.2 and 10, and the effect of this treatment on the allergic inflammatory response to aerosolized OVA was established . RESULTS: We show that M . tuberculosis Cpns inhibit allergen-induced pulmonary eosinophilia in the mouse . Of the three Cpns produced by M . tuberculosis, Cpn60.1, Cpn10 and Cpn60.2, the first two are effective in preventing eosinophilia when administered by the intra-tracheal route . Furthermore, the increase in airways sensitivity to inhaled methacholine following OVA challenge of immunized mice was suppressed following treatment with Cpn60.1 . The allergic inflammatory response was also characterized by an increase in Th2 cytokines IL-4 and IL-5 in bronchoalveolar lavage fluid, which was also suppressed following treatment with Cpn60.1 . CONCLUSION: These data show that bacterial Cpns can suppress eosinophil recruitment and bronchial hyper-responsiveness in a murine model of allergic inflammation.

Anal Chem, 2004 May 15, 76(10), 2836 - 41
Quantitative determination of heme for forensic characterization of bacillus spores using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Whiteaker JR et al.; A quantitative method was developed for the determination of heme (ferriprotoporphyrin IX) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) . The method was designed for forensic characterization of the use of blood agar in preparation of Bacillus spores . An alkali wash of 0.3 M ammonium hydroxide was used to solubilize heme from spore samples . The wash was concentrated and analyzed by MALDI-TOFMS . Experimental parameters were optimized to obtain the best signal intensity, maximize signal reproducibility, and improve day-to-day repeatability of the measurement . Sinapinic acid was found to be the best matrix . A sandwich sample preparation protocol was determined to increase the shot-to-shot and point-to-point reproducibility of the measurement . Cobalt(III) protoporphyrin was used as an internal standard and the analyte/internal standard ratio responses from solutions of known concentrations were used to construct a calibration curve (R(2) = 0.993) . Limits of detection and quantitation for heme were calculated to be approximately 0.4 (200 fmol) and 0.8 microM (400 fmol), respectively . Spore samples prepared on blood agar and nonblood agar were analyzed using the method . Heme was detected at a concentration of approximately 0.3 ng/mg of spore on samples prepared on blood agar and purified by extensive washing . Heme was not detected on spore samples prepared without blood.

Tuberk Toraks, 2003, 51(2), 163 - 70
{The evaluation of pulmonary tuberculosis patients enrolled to Eskisehir Deliklitas Tuberculosis Control Dispensary}; Kolsuz M et al.; Tuberculosis is an important public health problem in our country . Tuberculosis Control Dispensary has been important role in tuberculosis control programme . In this study we retrospectively evaluate 891 pulmonary tuberculosis patients who registered in Eskisehir Deliklitas Tuberculosis Control Dispensary between January 1990 and June 2000 . The mean age was 38.9 +/- 15.7 years, 159 (18.3%) patients were female, 710 (81.7%) patients were male . The most common symptom was cough (88.7%) . 81 percent of patients have been found by examination of symptomatic people while 6.4 percent of them by examination of close contacts . 26.5 percent of patients had close contacts with tuberculosis patients . The scares of BCG vaccine has been recorded 63.6% of the patients and 80.3% percent of them had at least one scare, tuberculin skin test result has been recorded in 49.6% percent of patients . Cavitary pulmonary tuberculosis was a diagnosed of 27.7% . The sputum examination and culture for acid resistant basil has been performed 89.1% of the patients, 389 (50.2%) of patients had acid fast bacillus in sputum and 548 (70.8%) of patients had culture positivity . 80.3 percent of the patients has completed regularly their treatments (mean duration 10.4 +/- 2.6 months) . The mortality rate was 5.8%.

Tuberk Toraks, 2004, 52(1), 47 - 51
{BCG vaccination: where are we now?}; Yavuz T et al.; The bacillus Calmette-Guerin (BCG) vaccine is administered and recommended for the prevention and control of tuberculosis in developing countries with high-risk settings . A new general BCG vaccination programme against tuberculosis has been introduced since 1997 in Turkey . The aim of this study was to evaulate the BCG vaccination status in Duzce and to analyze contributing factors for the vaccination programme . BCG screening were performed in 1100 8th class students from nine primary schools of Duzce city centre and seven counties . BCG scar presence was taken as authoritative for vaccination status . BCG vaccination ratio was 0.94 . Of 1100 students, 1030 had BCG scars; 478 had single scar, 536 had two scars and 16 had three scars . The significant negative correlation was observed between the BCG administration ratio and the number of children under age of 15 per health personnel (r= -0.771, p= 0.025) . Furthermore, based on some published studies marked regional variation of BCG vaccination status in Turkey was also discussed in this article.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 941 - 6
Anoxybacillus contaminans sp . nov . and Bacillus gelatini sp . nov., isolated from contaminated gelatin batches; De Clerck E et al.; Aerobic, endospore-forming bacteria that are attributed to the genus Bacillus or related genera constitute a hazard to the quality of gelatin . During repetitive extragenic palindromic DNA (rep)-PCR screening of gelatin isolates, a group of five isolates (group 1) and a group of 66 isolates (group 2) that did not match any pattern in our database were found . On the basis of 16S rDNA sequence analysis, representative strains of the different rep-PCR fingerprint types of group 1 were shown to be related most closely to Anoxybacillus species, but with sequence similarity of <97 % . Likewise, representative strains of group 2 were shown to be related most closely to Bacillus species, with 16S rDNA sequence similarity of <97 % . DNA-DNA reassociation values of isolates that displayed the most divergent rep-PCR profiles revealed that strains within each group belonged to a single species, according to recommendations for species delineation . A mean fatty acid profile could be calculated for each group . Isolates within a single group had similar patterns of results in API and other phenotypic tests; no correlation of patterns of results with rep-PCR groups was seen . Physiological characterization of group 1 isolates allows their distinction from other Anoxybacillus species . Despite the weak reaction of group 2 isolates in API tests, physiological characterization allows distinction between Bacillus species that react weakly in API tests . Two novel species are therefore proposed, with the names Anoxybacillus contaminans sp . nov . (type strain, LMG 21881(T)=DSM 15866(T)) and Bacillus gelatini sp . nov . (type strain, LMG 21880(T)=DSM 15865(T)).

Comp Biochem Physiol B Biochem Mol Biol, 2004 May, 138(1), 65 - 70
Sterols of marine microalgae Pyramimonas cf . cordata (Prasinophyta), Attheya ussurensis sp . nov . (Bacillariophyta) and a spring diatom bloom from Lake Baikal; Ponomarenko LP et al.; The free sterol compositions of two marine microalgal species Pyramimonas cf . cordata (Prasinophyta), Attheya ussurensis sp . nov . (Bacillariophyta), and diatom bloom samples from Lake Baikal were determined by gas chromatography, gas chromatography-mass spectrometry and (for some sterol constituents) using nuclear magnetic resonance spectra . A variety of sterol profiles were found . The principal sterol in the prasinophyte P . cf . cordata, collected in the Sea of Japan near Vladivostok, was 24(R)-ethylcholesta-5,22E-dien-3beta-ol (poriferasterol), but not 24-ethyl-5,24(28)Z-dien-3beta-ol, as reported earlier in the related species Pyramimonas cordata . The principal sterol in the marine diatom A . ussurensis sp . nov . was identified as 24-ethylcholest-5-en-3beta-ol . The sample of diatom bloom caused by Stephanodiscus meyerii with admixtures of several other diatom species, contained cholesterol and 24-methylcholesta-5,24(28)-dien-3beta-ol as main sterol constituents.

BJU Int, 2004 May, 93(7), 980 - 4
Bacillus Calmette-Guérin therapy in stage Ta/T1 bladder cancer: prognostic factors for time to recurrence and progression; Andius P et al.; OBJECTIVE: To report prognostic factors for time to recurrence and progression after bacillus Calmette-Guerin (BCG) prophylaxis in patients with stage Ta/T1 papillary bladder cancer . PATIENTS AND METHODS: The clinical records were assessed retrospectively for 236 patients with papillary stage Ta/T1 bladder cancer treated with BCG between 1986 and 2000 . Patients with known carcinoma in situ were excluded . The median (range) follow-up was 44 (4-155) months . The effect of 13 variables on the time to recurrence and progression was evaluated using multivariate Cox proportional hazard regression and Kaplan-Meier analyses . RESULTS: The recurrence rate was markedly reduced for all grades and stages . Patients with a negative first cystoscopy and maintenance BCG had a significantly longer time to recurrence than those treated with an induction course alone (P < 0.001) . Thirty-seven patients (16%) progressed in stage . The result of the first cystoscopy (P < 0.001), tumour grade (P = 0.003) and six or fewer initial instillations (P = 0.002) had prognostic importance for the time to progression . Twenty-eight patients (12%) had a history of an upper tract tumour, which was 3-10 times the expected rate . Age, number of tumours, number of positive cystoscopies, length of tumour history before BCG, BCG strain and treatment year had no influence on time to recurrence and progression . CONCLUSIONS: Maintenance treatment does not seem to be necessary among patients with TaG1-G2 disease after a negative first cystoscopy, as the progression rate was very low . One new finding was that BCG seemed to be equally effective among patients with or with no history of an upper tract tumour . Another new and surprising finding was that patients treated with fewer than six induction instillations, because of very bothersome side-effects, had an increased risk of tumour progression and of local failure.

Int J Tuberc Lung Dis, 2004 Apr, 8(4), 418 - 23
Estimation of annual risk of tuberculosis infection (ARTI) among children aged 1-9 years in the south zone of India; Kolappan C et al.; OBJECTIVE: To estimate the annual risk of tuberculosis infection (ARTI) among children aged 1-9 years in the south zone of India . SETTING: The survey was carried out in a representative sample of villages and census enumeration blocks of towns in four south Indian states, as a part of a nationwide tuberculin survey . DESIGN: Six districts were selected through systematic random sampling . Four hundred and twenty rural clusters and 180 urban clusters were selected from these districts on the basis of the rural-urban ratio in the entire zone . To obtain the required sample of 12,000 children without bacille Calmette-Guerin (BCG) vaccination, 51,000 had to be covered . Eighty-five children from each cluster were tuberculin tested and read for reaction sizes . The ARTI was computed from the estimated prevalence of TB infection among children without a BCG scar . RESULT: Among 52,951 children registered for the study, 50,846 (96%) had a tuberculin test result . The BCG coverage for the study population was about 65% . Among 17,811 children without a BCG scar, the prevalence of infection was 5.9% (95%CI 4.0-7.7%); the corresponding ARTI was 1.0% (95%CI 0.7-1.4%) {correction} . CONCLUSION: The estimated ARTI for the south zone is 1.0%, as compared to the national average of 1.7% used for programme evaluation . This baseline information should be useful for the assessment of future trends.

J Biol Chem, 2004 Jun 18, 279(25), 26046 - 51 Epub 2004 Mar 31.
Structural determinants of substrate binding to Bacillus cereus metallo-beta-lactamase; Rasia RM et al.; Binding and hydrolysis of the beta-lactams cefotaxime, cephapirin, imipenem, and benzylpenicillin by the metallo-beta-lactamase from Bacillus cereus were studied by presteady state kinetic measurements . In all cases, the substrate was unmodified in the most populated reaction intermediate, and no chemically modified substrate species accumulated to a detectable amount . The cephalosporins tested showed similar formation rate constants for this intermediate, and they differed mostly in their decay rates . Formation of a non-productive enzyme.substrate complex was detected for imipenem . The substrate binding differences can be accounted for by considering the structural features of each substrate . The apoenzyme could not bind any of the substrates, but binding was restored when the apoenzyme was reconstituted with Zn(II), revealing that the metal ions are the main determinants of substrate binding . This evidence is in line with the lack of an optimized substrate recognition patch in B1 and B3 metallo-beta-lactamases that provides a broad substrate spectrum.

J Appl Microbiol, 2004, 96(6), 1317 - 23
Mutation of Gluconobacter oxydans and Bacillus megaterium in a two-step process of l-ascorbic acid manufacture by ion beam; Xu A et al.; AIM: To increase the transformation rate of l-sorbose to 2-keto-l-gulonic (2-KLG) acid in a two-step process of l-ascrobic acid manufacture by ion beam . METHODS AND RESULTS: Gluconobacter oxydans (GO29) and Bacillus megaterium (BM80) were used in the present study . Ion implantation was carried out with the heavy ion implantation facility at the institute of Plasma Physics in China . 2-KLG in whole culture broth was determined by iodometry . Mutants were screened by single-colony isolation and 2-KLG accumulation in broth . GO29 and BM80 were implanted by either hydrogen ions (H(+)) or nitrogen ions (N(+)) with various doses, respectively . The average transformation rate of GM112-302 bred by ion beam in Gram-molecule was increased from 79.3 to 94.5% after eight passages in shaking flasks . Furthermore, in 180-ton fermentors in Jiangsu Jiangshan Pharmaceutical Co . Ltd, the transformation rate was stable at 92.0%, indicating a producer could get 0.99 kg of gulonic acid from 1.0 kg of sorbose . CONCLUSION: Ion beam as a new mutation source had potential advantages in breeding . Comparing with original mixture GO29 and BM80, GM112-302 is more efficient in accumulating 2-KLG, especially at the later phase . SIGNIFICANCE AND IMPACT OF THE STUDY: GM112-302 bred by ion beam implantation dramatically increased the transformation rate by 19.2%, which greatly increased efficiency and reduced the cost of l-ascorbic acid manufacture in a two-step process.

J Appl Microbiol, 2004, 96(6), 1245 - 55
Mechanism of the hydrolysis of 4-methylumbelliferyl-beta-D-glucoside by germinating and outgrowing spores of Bacillus species; Setlow B et al.; AIMS: To determine the mechanism of the hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside (beta-MUG) by germinating and outgrowing spores of Bacillus species . METHODS AND RESULTS: Spores of B . atrophaeus (formerly B . subtilis var . niger, Fritze and Pukall 2001) are used as biological indicators of the efficacy of ethylene oxide sterilization by measurement of beta-MUG hydrolysis during spore germination and outgrowth . It was previously shown that beta-MUG is hydrolysed to 4-methylumbelliferone (MU) during the germination and outgrowth of B . atrophaeus spores (Chandrapati and Woodson 2003), and this was also the case with spores of B . subtilis 168 . Germination of spores of either B . atrophaeus or B . subtilis with chloramphenicol reduced beta-MUG hydrolysis by almost 99%, indicating that proteins needed for rapid beta-MUG hydrolysis are synthesized during spore outgrowth . However, the residual beta-MUG hydrolysis during spore germination with chloramphenicol indicated that dormant spores contain low levels of proteins needed for beta-MUG uptake and hydrolysis . With B . subtilis 168 spores that lacked several general proteins of the phosphotransferase system (PTS) for sugar uptake, beta-MUG hydrolysis during spore germination and outgrowth was decreased >99.9% . This indicated that beta-MUG is taken up by the PTS, resulting in the intracellular accumulation of the phosphorylated form of beta-MUG, beta-MUG-6-phosphate (beta-MUG-P) . This was further demonstrated by the lack of detectable glucosidase activity on beta-MUG in dormant, germinated and outgrowing spore extracts, while phosphoglucosidase active on beta-MUG-P was readily detected . Dormant B . subtilis 168 spores had low levels of at least four phosphoglucosidases active on beta-MUG-P: BglA, BglH, BglC (originally called YckE) and BglD (originally called YdhP) . These enzymes were also detected in spores germinating and outgrowing with beta-MUG, but levels of BglH were the highest, as this enzyme's synthesis was induced ca 100-fold during spore outgrowth in the presence of beta-MUG . Deletion of the genes coding for BglA, BglH, BglC and BglD reduced beta-MUG hydrolysis by germinating and outgrowing spores of B . subtilis 168 at least 99.7% . Assay of glucosidases active on beta-MUG or beta-MUG-P in extracts of dormant and outgrowing spores of B . atrophaeus revealed no enzyme active on beta-MUG and one enzyme that comprised > or =90% of the phosphoglucosidase active on beta-MUG-P . Partial purification and amino-terminal sequence analysis of this phosphoglucosidase identified this enzyme as BglH . CONCLUSIONS: Generation of MU from beta-MUG by germinating and outgrowing spores of B . atrophaeus and B . subtilis is mediated by the PTS-driven uptake and phosphorylation of beta-MUG, followed by phosphoglucosidase action on the intracellular beta-MUG-P . The major phosphoglucosidase catalyzing MU generation from beta-MUG-P in spores of both species is probably BglH . SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the mechanism of uptake and hydrolysis of beta-MUG by germinating and outgrowing spores of Bacillus species, in particular B . atrophaeus . The research reported here provides a biological basis for a Rapid Readout Biological Indicator that is used to monitor the efficacy of ethylene oxide sterilization.

Ying Yong Sheng Tai Xue Bao, 2004 Jan, 15(1), 111 - 5
{Techniques of diseases, insect pests and weeds control and their efficacy in bio-rational rice production}; Li B et al.; Studies on the efficacy of bio-rational pesticides and agricultural methods against the chief diseases, insect pests and weeds of rice showed that the efficacy of the mixtures of jingangmycin and bacillus-cereus, and jingangmycin and polyoxin against rice sheath blight were 75.16%-94.27% after sprayed once at the tiller and boot end stages of rice, respectively, and better than that of chemical fungicide triadimefon . The efficacy of kasugamycin and blasticidin was 50.54%-72.67% on rice leaf blast and 76.66%-87.42% on rice head blast, and equal to the chemical fungicide tricyclazole after sprayed once at the initial stage of rice leaf blast occurrence and the initial and end stages of earing, respectively . The efficacy of bacillus thuringiensis on Chilo suppressalis and Cnaphalocrocis medinalis was better than that of chemical insecticide bisultap, and the efficacy of saponin-nicotine and matrine was equal to that of chemical insecticide bisultap when the three biorational insecticides were sprayed at 1-2 instar larvae of pests . The efficacy of saponin-nicotine and matrine was above 70%, and lower than that of chemical insecticide imidacloprid 3-30 d after sprayed at 1-2 instar larvae of Nilaparvata lugens . The occurrence of weeds could be controlled, and the rice yield could be raised when the suitable non-thorough decomposed organism was applied or weeding was carried after the field had been ploughed twice before rice transplant . The rice yield could be raised by using biorational pesticides and agricultural methods against the chief diseases, insect pests and weeds of rice . The residue of pesticides in rice was lower in the bio-control area than in the chemical control area, according with the demands of health target of green food.

Zhongguo Zhong Yao Za Zhi, 2003 Jul, 28(7), 636 - 9
{Comparative studies on pharmacological effects of angong niuhuang pill with its simplified prescicription}; Ye ZG et al.; OBJECTIVE: Based on the therapeutic claims of Angong Niuhuang pill, a series of pharmacodynamic experiments were designed, where pharmacological effects were investigated comparatively with its simplified prescription(realgar and cinnabar are removed from the original pill) as a parallel control in order to explore possible contribution of cinnabar and realgar to pharmacodynamic activities of the pill as a whole . METHOD: Anti-pyretic, sedative, anti-convulsive, and mice-protected effects of the pill and its simplified prescription as a control were observed, respectively, in rabbits with fever induced by typhoid bacillus, in pentobarbital sodium-induced sleeping mice, in mice with convulsion induced by strychnine, or pentylenetetrazole, and in mice with anoxia induced by NaNO2 . RESULT: Both the pill and its simplified prescription were found to have Anti-pyretic action and protective effect against the mouse death induced by anoxia, and synergistic interaction with pentobarbital sodium in sedative activity, although neither of them was found to have any effects on the convulsion of mice . CONCLUSION: No significant difference between Angong Niuhuang pill and its simplified prescription was found in the above pharmacodynamic experiments.

Internist (Berl), 2004 Aug, 45(8), 935 - 9
{Sepsis and multiple organ failure after BCG-instillation for bladder cancer}; Elmer A et al.; Local Bacillus Calmette-Guerin (BCG) immunotherapy is an effective and widely used treatment for superficial bladder carcinoma . Local side effects are frequent, whereas systemic side effects are rare, but more serious . Systemic BCG infection as a life-threatening complication of intravesical BCG instillation should be suspected in any patient who presents with persistent fever after BCG instillation for bladder cancer . A 62-year-old patient had been treated with 6 intravesical BCG instillations for recurrent, multifocal bladder carcinoma . 4 weeks after the last instillation, he presented with fever, malaise and scleral icterus . Laboratory tests revealed abnormal liver function tests, panzytopenia and signs of coagulation disorder . Bone marrow biopsy and liver biopsy showed noncaseating granulomas . Systemic BCG infection was suspected and antituberculous therapy combined with steroids was started . The patient developed severe sepsis and suffered from multiple organ failure . Despite partial improvement, the course was complicated by intracranial sinus thrombosis, and the patient died two month after admission.

J Wildl Dis, 2004 Jan, 40(1), 66 - 78
Immune responses of white-tailed deer (Odocoileus virginianus) to Mycobacterium bovis BCG vaccination; Waters WR et al.; The objective was to evaluate cellular immune response of captive white-tailed deer (Odocoileus virginianus) to live Mycobacterium bovis bacille Calmette Guerin (BCG) vaccination and to determine diagnostic implications of these responses . In vitro proliferative and interferon-gamma (IFN-gamma) responses to M . bovis purified protein derivative (PPD) were detected beginning 9 days postvaccination . Responses to Mycobacterium avium PPD, however, generally exceeded responses to M . bovis PPD . Interferon-gamma responses to M . avium PPD were not detected prior to vaccination nor in nonvaccinated deer, suggesting that vaccination with BCG boosted prior quiescent M . avium-sensitized cells . Both CD4+ and gammadelta T cells from vaccinated deer proliferated in response to M . bovis PPD stimulation . Intradermal administration of M . bovis PPD resulted in increases in skin thickness of vaccinated deer beginning 24 hr postinjection . Such early reactions were characterized by edema and minimal mononuclear cell infiltration, whereas later reactions (i.e., 72 hr postinjection) were more typical of delayed type hypersensitivity . Upon in vitro activation with pokeweed mitogen, CD44 expression increased and CD62L expression decreased on lymphocytes from deer regardless of vaccination status . Likewise, M . bovis PPD stimulation of lymphocytes from vaccinated deer resulted in increases in CD44 expression and decreases in CD62L expression . These findings demonstrate the potential of BCG vaccination to elicit strong cell-mediated immune responses and appropriate alterations in CD44 and CD62L expression with in vitro stimulation of white-tailed deer lymphocytes . In relation to M . bovis diagnosis, vaccination of white-tailed deer with BCG can induce skin test responses that classify the animal as a tuberculosis reactor . In contrast, BCG vaccination will likely not interfere with tuberculosis testing by the IFN-gamma assay.

Mar Biotechnol (NY) . 2004 May 6; {Epub ahead of print}
Competitive Induction and Enhancement of Indole and a Diketopiperazine in Marine Bacteria; Trischman JA et al.; Thirteen bacterial strains isolated from a sample of Ulva californica were cultured, extracted, and assayed for antibiotic activity . The target strains were 2 gram-positive isolates from the same algal sample . When cultured in monoculture, 2 of the 13 isolates produced extracts with antimicrobial activity . More significantly, one Bacillus isolate (UA-094) produced indole and cyclo(Phe-Pro) at active levels upon challenge with a different Bacillus target strain . These mildly antibiotic compounds were barely detectable in extracts without challenge . This technique of competitive induction and enhancement could be a valuable tool in the search for new antibiotics.

Proc Natl Acad Sci U S A, 2004 May 18, 101(20), 7526 - 9 Epub 2004 May 10.
Contamination of refuges by Bacillus thuringiensis toxin genes from transgenic maize; Chilcutt CF et al.; Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are widely used to control pests, but their benefits will be lost if pests evolve resistance . The mandated high-dose/refuge strategy for delaying pest resistance requires planting refuges of toxin-free crops near Bt crops to promote survival of susceptible pests . We report that pollen-mediated gene flow up to 31 m from Bt maize caused low to moderate Bt toxin levels in kernels of non-Bt maize refuge plants . Immunoassays of non-Bt maize sampled from the field showed that the mean concentration of Bt toxin Cry1Ab in kernels and the percentage of kernels with Cry1Ab decreased with distance from Bt maize . The highest Bt toxin concentration in pooled kernels of non-Bt maize plants was 45% of the mean concentration in kernels from adjacent Bt maize plants . Most previous work on gene flow from transgenic crops has emphasized potential effects of transgene movement on wild relatives of crops, landraces, and organic plantings, whereas implications for pest resistance have been largely ignored . Variable Bt toxin production in seeds of refuge plants undermines the high-dose/refuge strategy and could accelerate pest resistance to Bt crops . Thus, guidelines should be revised to reduce gene flow between Bt crops and refuge plants.

J Mol Biol, 2004 May 28, 339(2), 253 - 8
X-ray crystal structure of a non-crystalline cellulose-specific carbohydrate-binding module: CBM28; Jamal S et al.; Natural cellulose exists as a composite of different forms, which have historically been broadly characterized as "crystalline" or "amorphous" . The recognition of both of these forms of cellulose by the carbohydrate-binding modules (CBM) of microbial glycoside hydrolases is central to natural and efficient biotechnological conversion of plant cell wall biomass . There is increasing evidence that, at least some, individual binding modules target distinct and different regions of non-crystalline "amorphous" cellulose . Competition experiments show that CBM28 modules do not compete with CBM17 modules when binding to non-crystalline cellulose . The structure of the BspCBM28 module from the Bacillus sp . 1139 family GH5 endoglucanase, comprising a 191 amino acid protein, has therefore been determined at 1.4A resolution using single isomorphous replacement with anomalous scattering methods . The structure reveals a "beta-jelly roll" topology, with high degree of similarity to the structure of CBM17 domains . Sequence and structural conservation strongly suggests that these two families of domains have evolved through gene duplication and subsequent divergence . The ligand-binding site "topographies" of CBMs from families 28, 17 and 4 begins to shed light on the differential recognition of non-crystalline cellulose by multi-modular plant cell wall-degrading enzymes.

Protein Expr Purif, 2004 Jun, 35(2), 397 - 403
Optimised expression in Escherichia coli and purification of the functional form of the Bacillus thuringiensis Cry4Aa delta-endotoxin; Boonserm P et al.; Achieving high-level expression of the Bacillus thuringiensis Cry4Aa mosquito-larvicidal protein was demonstrated . The 130-kDa Cry4Aa protoxin was overexpressed as an inclusion body in Escherichia coli under the control of the tac promoter together with the cry4Ba promoter . The solubility of the toxin inclusions in carbonate buffer, pH 10.0, was markedly enhanced at a cultivation temperature of 30 degrees C . Elimination of the tryptic cleavage site at Arg-235 in the loop between helices 5 and 6 still retained the high-level toxicity of E . coli cells expressing the Cry4Aa mutant against Aedes aegypti larvae . Trypsin digestion of the R235Q mutant protoxin produced a protease-resistant fragment of ca . 65kDa . A homogeneous product of the 65-kDa trypsin-treated R203Q protein was obtained after size-exclusion chromatography that would pave the way for the further crystallisation and X-ray crystallographic studies.

J Inorg Biochem, 2004 May, 98(5), 803 - 13
Nickel trafficking: insights into the fold and function of UreE, a urease metallochaperone; Musiani F et al.; UreE is a metallo-chaperone assisting the incorporation of two adjacent Ni(2+) ions in the active site of urease . This study describes an attempt to distill general information on this protein using a computational post-genomic approach for the understanding of the structural details of the molecular function of UreE in nickel trafficking . The two crystal structures recently determined for UreE from Bacillus pasteurii (BpUreE) and Klebsiella aerogenes (KaUreE) were comparatively analyzed . This analysis provided insights into the protein structural and conformational features . A structural database of UreE proteins from a large number of different genomes was built using homology modeling . All available sequences of UreE were retrieved from protein and cDNA databases, and their structures were modeled on the crystal structures of BpUreE and KaUreE . A self-consistent iterative protocol was devised for multiple sequence alignment optimization involving secondary structure prediction and evaluation of the energy features of the obtained modeled structures . The quality of all models was tested using standard assessment procedures . The final optimized structure-based multiple alignment and the derived model structures provided insightful information on the evolutionary conservation of key residues in the protein sequence and surface patches presumably involved in protein recognition during the urease active site assembly.

Biochemistry, 2004 May 18, 43(19), 5912 - 20
Identification of the catalytic residues involved in the carboxyl transfer of pyruvate carboxylase; Yong-Biao J et al.; To clarify the mechanism of carboxyl transfer from carboxylbiotin to pyruvate, the following conserved amino acid residues present in the carboxyl transferase domain of Bacillus thermodenitrificans pyruvate carboxylase were converted to homologous amino acids: Asp543, Glu576, Glu592, Asp649, Lys712, Asp713, and Asp762 . The carboxylase activity of the resulting mutants, D543E, E576D, E576Q, E592Q, D649N, K712R, K712Q, D713E, D713N, D762E, and D762N, was generally less than that of the wild type from mutation, but it decreased the most to 5% or even less than that of the wild type with D543E, D576Q, D649N, K712R, and K712Q . The decrease in activity observed for Asp543, Asp649, and Lys712 mutants was not for structural reasons because their structures seemed to remain intact as assessed by gel filtration and circular dichroism . On the basis of these data, a mechanism is proposed where Lys712 and Asp543 serve as the key acid and base catalyst, respectively.

Biochemistry, 2004 May 18, 43(19), 5820 - 31
Long-range nature of the interactions between titratable groups in Bacillus agaradhaerens family 11 xylanase: pH titration of B . agaradhaerens xylanase; Betz M et al.; Xylanase from Bacillus agaradhaerens belongs to a large group of glycosyl hydrolases which catalyze the degradation of xylan . The protonation behavior of titratable groups of the uniformly (15)N- and (13)C-labeled xylanase was investigated by multinuclear NMR spectroscopy . A total of 224 chemical shift titration curves corresponding to (1)H, (13)C, and (15)N resonances revealed pK(a) values for all aspartic and glutamic acid residues, as well as for the C-terminal carboxylate and histidine residues . Most of the titratable groups exhibit a complex titration behavior, which is most likely due to the mutual interactions with other neighboring groups or due to an unusual local microenvironment . Subsite -1 containing the catalytic dyad shows a long-range interaction over 9 A with Asp21 via two hydrogen bonds with Asn45 as the mediator . This result illuminates the pivotal role of the conserved position 45 among family 11 endoxylanases, determining an alkaline pH optimum by asparagine residues or an acidic pH optimum by an aspartate . The asymmetric interactions of neighboring tryptophan side chains with respect to the catalytic dyad can be comprehended as a result of hydrogen bonding and aromatic stacking . Most of the chemical shift-pH profiles of the backbone amides exhibit biphasic behavior with two distinct inflection points, which correspond to the pK(a) values of the nearby acidic side chains . However, the alternation of both positive and negative slopes of individual amide titration curves is interpreted as a consequence of a simultaneous reorganization of side chain conformational space at pH approximately 6 and/or an overall change in the hydrogen network in the substrate binding cleft.

Theor Appl Genet . 2004 May 5; {Epub ahead of print}
Impact of ecological factors on the initial invasion of Bt transgenes into wild populations of birdseed rape ( Brassica rapa); Vacher C et al.; The inevitable escape of transgenic pollen from cultivated fields will lead to the emergence of transgenic crop-wild plant hybrids in natural patches of wild plants . The fate of these hybrids and that of the transgene depend on their ability to compete with their wild relatives . Here we study ecological factors that may enhance the fitness of genetically modified hybrids relative to wild plants for a Bacillus thuringiensis ( Bt) transgene conferring resistance to insects . Mixed stands of wild plants and first-generation hybrids were grown under different conditions of herbivore pressure and density, with Bt oilseed rape ( Brassica napus) as the crop and B . rapa as the wild recipient . Biomass and fitness components were measured from plant germination to the germination of their offspring . The frequency of transgenic seedlings in the offspring generation was estimated using the green fluorescent protein marker . The biomass of F(1) Bt-transgenic hybrids relative to that of wild-type plants was found to be sensitive to both plant density and herbivore pressure, but herbivore pressure appeared as the major factor enhancing their relative fitnesses . In the absence of herbivore pressure, Bt hybrids produced 6.2-fold fewer seeds than their wild neighbors, and Bt plant frequency fell from 50% to 16% within a single generation . Under high herbivore pressure, Bt hybrids produced 1.4-fold more seeds, and Bt plant frequency was 42% in the offspring generation . We conclude that high-density patches of highly damaged wild plants are the most vulnerable to Bt-transgene invasion . They should be monitored early to detect potential transgene spread.

Microbiology, 2004 May, 150(Pt 5), 1519 - 27
Genome structure in the vole bacillus, Mycobacterium microti, a member of the Mycobacterium tuberculosis complex with a low virulence for humans; Frota CC et al.; Mycobacterium microti, a member of the Mycobacterium tuberculosis complex, is phylogenetically closely related to M . tuberculosis, differing in a few biochemical properties . However, these species have different levels of virulence in different hosts; most notably M . microti shows lower virulence for humans than M . tuberculosis . This report presents genomic comparisons using DNA microarray analysis for an extensive study of the diversity of M . microti strains . Compared to M . tuberculosis H37Rv, 13 deletions were identified in 12 strains of M . microti, including the regions RD1 to RD10, which are also missing in Mycobacterium bovis BCG . In addition, four new deleted regions, named MiD1, RD1beta, MiD2 and MiD3, were identified . DNA sequencing was used to define the extent of most of the deletions in one strain . Although RD1 of M . bovis BCG and M . microti is thought to be crucial for attenuation, in this study, three of the four M . microti strains that were isolated from immunocompetent patients had the RD1 deletion . In fact, only the RD3 deletion was present in all of the strains examined, although deletions RD7, RD8 and MiD1 were found in almost all the M . microti strains . These deletions might therefore have some relation to the different host range of M . microti . It was also noticeable that of the 12 strains studied, only three were identical; these strains were all isolated from immunocompetent humans, suggesting that they could have arisen from a single source . Thus, this study shows that it is difficult to ascribe virulence to any particular pattern of deletion in M . microti.

Pediatr Infect Dis J, 2004 May, 23(5), 476 - 9
Resistant Mycobacterium bovis Bacillus Calmette-Guérin disease: implications for management of Bacillus Calmette-Guérin Disease in human immunodeficiency virus-infected children; Hesseling AC et al.; Guidelines for the diagnosis and management of Bacillus Calmette-Guerin (BCG) disease in children are lacking, and there are limited data on drug resistance of Mycobacterium bovis BCG . A 6-month-old HIV-infected infant presented with right axillary adenitis ipsilateral to the site of BCG immunization . M . tuberculosis complex was cultured from axillary lymph nodes and gastric aspirates, and M . bovis BCG was isolated . Susceptibility testing before initiation of therapy demonstrated inherent resistance to isoniazid . The organism acquired rifampin resistance during therapy . This was confirmed by the presence of a mutation in codon 531 (Ser531Tyr) of the rpoB gene . Treatment guidelines for BCG disease with consideration of inherent and possible acquired drug resistance should be established in settings with high rates of vertical HIV transmission and routine BCG vaccination.

Lett Appl Microbiol, 2004, 38(6), 447 - 53
Identification, molecular biotyping and ultrastructural studies of bacterial communities isolated from two damaged frescoes of St Damian's Monastery in Assisi; Radaelli A et al.; AIM: To investigate the composition of the microbial community in biodeterioration of two frescoes in St Damian's Monastery in Assisi . METHODS AND RESULTS: A total of 1292 colonies were isolated from the most deteriorated parts, analysed by microbiological, biomolecular and ultrastructural techniques, and taxonomically classified . Molecular biotyping of Staphylococcus cohnii colonies, one of the most prevalent bacterial species, showed a very restricted genome diversity while Bacillus licheniformis were very homogeneous by RFLP, tDNA-PCR and random-amplified polymorphic DNA . Electron microscopy confirmed heterogeneity of the bacterial population in the different sampling areas . CONCLUSIONS: Several of the identified species are widespread in the soil or saprophytes of human skin . Although unable to demonstrate that they are involved in biodeterioration, they may represent trophic elements contributing to fungi-related chromatic alterations when adequate environmental conditions occur . Deterioration may in part be prevented or controlled by adequate air filtering or conditioning of the room.

Org Lett, 2004 May 13, 6(10), 1585 - 8
Synthesis of aminoshikimic acid; Guo J et al.; 5-amino-5-deoxyshikimic acid (aminoshikimic acid) was synthesized from glucose using recombinant Amycolatopsis mediterranei and also synthesized by a tandem, two-microbe route employing Bacillus pumilus and recombinant Escherichia coli.

Biotechnol Lett, 2004 Mar, 26(6), 529 - 32
Newly isolated bacterial strains belonging to Bacillaceae (Bacillus sp.) and Micrococcaceae accelerate death of the honey bee mite, Varroa destructor (V . jacobsoni), in laboratory assays; Tsagou V et al.; Newly isolated bacterial strains belonging to Bacillaceae (Bacillus sp.), Micrococcaceae and three unidentified strains were tested for their pathogenicity against the mite, Varroa destructor . The Bacillus sp . strain and two of the strains belonging to the Micrococcaceae family significantly decreased the time for 50% mortality of the mite population (up to 57%) and hence may be potential control agents . In in vitro bioassay whole cells, extracellular broth and cellular extract of the Bacillus sp . strain effectively killed the mites, suggesting that both endotoxins and exotoxins contributed to the killing.

Nurs Manage, 2000 Sep, 31(9), 47 - 8
Staying on top of TB's rise; Esteves R et al.; Delays in acid-fast bacillis (AFB) emergency department admissions prompted a revised multidisciplinary rounds process for weekends and holidays; AFB isolation cases dropped 10%.

J Bacteriol, 2004 May, 186(10), 3015 - 21
PhaQ, a new class of poly-beta-hydroxybutyrate (phb)-responsive repressor, regulates phaQ and phaP (phasin) expression in Bacillus megaterium through interaction with PHB; Lee TR et al.; Bacillus megaterium can produce poly-beta-hydroxybutyrate (PHB) as carbon and energy storage materials . We now report that the phaQ gene, which is located upstream of the phasin-encoding phaP gene, codes for a new class of transcriptional regulator that negatively controls expression of both phaQ and phaP . A PhaQ binding site that plays a role in this control has been identified by gel mobility shift assays and DNase I footprinting analysis . We have also provided evidence that PhaQ could sense the presence of PHB in vivo and that artificial PHB granules could inhibit the formation of PhaQ-DNA complex in vitro by binding to PhaQ directly . These suggest that PhaQ is a PHB-responsive repressor.

JAMA, 2004 May 5, 291(17), 2086 - 91
Long-term efficacy of BCG vaccine in American Indians and Alaska Natives: A 60-year follow-up study; Aronson NE et al.; CONTEXT: The duration of protection from tuberculosis of BCG vaccines is not known . OBJECTIVE: To determine the long-term duration of protection of a BCG vaccine that was previously found to be efficacious . DESIGN: Retrospective record review using Indian Health Service records, tuberculosis registries, death certificates, and supplemental interviews with trial participants . SETTING AND PARTICIPANTS: Follow-up for the period 1948-1998 among American Indians and Alaska Natives who participated in a placebo-controlled BCG vaccine trial during 1935-1938 and who were still at risk of developing tuberculosis . Data from 1483 participants in the BCG vaccine group and 1309 in the placebo group were analyzed . MAIN OUTCOME MEASURES: Efficacy of BCG vaccine, calculated for each 10-year interval using a Cox regression model with time-dependent variables based on tuberculosis events occurring after December 31, 1947 (end of prospective case finding) . RESULTS: The overall incidence of tuberculosis was 66 and 138 cases per 100 000 person-years in the BCG vaccine and placebo groups, respectively, for an estimate of vaccine efficacy of 52% (95% confidence interval, 27%-69%) . Adjustments for age at vaccination, tribe, subsequent BCG vaccination, chronic medical illness, isoniazid use, and bacille Calmette-Guerin strain did not substantially affect vaccine efficacy . There was slight but not statistically significant waning of the efficacy of BCG vaccination over time, greater among men than women . CONCLUSION: In this trial, BCG vaccine efficacy persisted for 50 to 60 years, suggesting that a single dose of an effective BCG vaccine can have a long duration of protection.

J Pediatr Hematol Oncol, 2004 Mar, 26(3), 194 - 6
Bacillus cereus central line infection in an immunocompetent child with hemophilia; Srivaths PR et al.; Bacillus species are increasingly recognized as pathogens in immunocompromised patients . The authors report a case of Bacillus cereus infection of a central line in an immunocompetent patient with hemophilia, which required line removal for complete cure.

Tunis Med, 2004 Jan, 82(1), 51 - 4
{Cervical-facial skin tuberculosis . Three case reports}; Daabek B et al.; The cervical-face skin tuberculosis is fairly uncommon pathology, it is caused by the Tuberculosis mycobacterium (or Bacille de Koch) . With human or bovine origin this pathology is actually increased as like as AIDS in countries where it was eradicated . Skin lesion may be primitive or secondary to another pre-existing tuberculosis lesions, it may be have many clinical appearance with the same characteristics, as knew as, long evolution and anesthetics sequels . We report in this paper three cases of cervical-face tuberculosis with different age, the diagnosis was given by biology and histology and treated medically.

J Biol Chem, 2004 Jul 2, 279(27), 28051 - 6 Epub 2004 Apr 29.
Bt-R1a extracellular cadherin repeat 12 mediates Bacillus thuringiensis Cry1Ab binding and cytotoxicity; Hua G et al.; The cadherin protein Bt-R(1a) is a receptor for Bacillus thuringiensis Cry1A toxins in Manduca sexta . Cry1Ab toxin is reported to bind specific epitopes located in extracellular cadherin repeat (CR) 7 and CR11 on Bt-R(1) (Gomez, B., Miranda-Rios, J., Riudino-Pinera, E., Oltean, D . I., Gill, S . S., Bravo, A., and Soberon, M . (2002) J . Biol . Chem . 277, 30137-30143; Dorsch, J . A., Candas, M., Griko, N., Maaty, W., Midboe, E., Vadlamudi, R., and Bulla, L . (2002) Insect Biochem . Mol . Biol . 32, 1025-1036) . We transiently expressed CR domains of Bt-R(1a) in Drosophila melanogaster Schneider 2 (S2) cells as fusion peptides between a signal peptide and a terminal region that included membrane-proximal, membrane-spanning, and cytoplasmic domains . A domain consisting of CR11 and 12 was the minimal (125)I-Cry1Ab binding region detected under denaturing conditions . Only CR12 was essential for Cry1Ab binding and cytotoxicity to S2 cells when tested under native conditions . Under these conditions expressed CR12 bound (125)I-Cry1Ab with high affinity (K(com) = 2.9 nm) . Flow cytometry assays showed that expression of CR12 conferred susceptibility to Cry1Ab in S2 cells . Derivatives of Bt-R(1a) with separate deletions of CR7, 11, and 12 were expressed in S2 cells . Only deletion of CR12 caused loss of Cry1Ab binding and cytotoxicity . These results demonstrate that CR12 is the essential Cry1Ab binding component on Bt-R(1) that mediates Cry1Ab-induced cytotoxicity.

Biochemistry, 2004 May 11, 43(18), 5266 - 77
Crystal structure and amide H/D exchange of binary complexes of alcohol dehydrogenase from Bacillus stearothermophilus: insight into thermostability and cofactor binding; Ceccarelli C et al.; The crystal structure of NAD(+)-dependent alcohol dehydrogenase from Bacillus stearothermophilus strain LLD-R (htADH) was determined using X-ray diffraction data at a resolution of 2.35 A . The structure of homotetrameric htADH is highly homologous to those of bacterial and archaeal homotetrameric alcohol dehydrogenases (ADHs) and also to the mammalian dimeric ADHs . There is one catalytic zinc atom and one structural zinc atom per enzyme subunit . The enzyme was crystallized as a binary complex lacking the nicotinamide adenine dinucleotide (NAD(+)) cofactor but including a zinc-coordinated substrate analogue trifluoroethanol . The binary complex structure is in an open conformation similar to ADH structures without the bound cofactor . Features important for the thermostability of htADH are suggested by a comparison with a homologous mesophilic enzyme (55% identity), NAD(+)-dependent alcohol dehydrogenase from Escherichia coli . To gain insight into the conformational change triggered by NAD(+) binding, amide hydrogen-deuterium exchange of htADH, in the presence and absence of NAD(+), was studied by HPLC-coupled electrospray mass spectrometry . When the deuteron incorporation of the protein-derived peptides was analyzed, it was found that 9 of 21 peptides show some decrease in the level of deuteron incorporation upon NAD(+) binding, and another 4 peptides display slower exchange rates . With one exception (peptide number 8), none of the peptides that are altered by bound NAD(+) are in contact with the alcohol-substrate-binding pocket . Furthermore, peptides 5 and 8, which are located outside the NAD(+)-binding pocket, are notable by displaying changes upon NAD(+) binding . This suggests that the transition from the open to the closed conformation caused by cofactor binding has some long-range effects on the protein structure and dynamics.

J Insect Physiol, 2004 May, 50(5), 435 - 43
Effects of Bt plants on the development and survival of the parasitoid Cotesia plutellae (Hymenoptera: Braconidae) in susceptible and Bt-resistant larvae of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae); Schuler TH et al.; A range of crops have been transformed with delta-endotoxin genes from Bacillus thuringiensis (Bt) to produce transgenic plants with high levels of resistance to lepidopteran pests . Parasitoids are important natural enemies of lepidopteran larvae and the effects of Bt plants on these non-target insects have to be investigated to avoid unnecessary disruption of biological control . This study investigated the effects of Cry1Ac-expressing transgenic oilseed rape (Brassica napus) on the solitary braconid endoparasitoid Cotesia plutellae in small-scale laboratory experiments . C . plutellae is an important natural enemy of the diamondback moth (Plutella xylostella), the most important pest of brassica crops world-wide . Bt oilseed rape caused 100% mortality of a Bt-susceptible P . xylostella strain but no mortality of the Bt-resistant P . xylostella strain NO-QA . C . plutellae eggs laid in Bt-susceptible hosts feeding on Bt leaves hatched but premature host mortality did not allow C . plutellae larvae to complete their development . In contrast, C . plutellae developed to maturity in Bt-resistant hosts fed on Bt oilseed rape leaves and there was no effect of Bt plants on percentage parasitism, time to emergence from hosts, time to adult emergence and percentage adult emergence from cocoons . Weights of female progeny after development in Bt-resistant hosts did not differ between plant types but male progeny was significantly heavier on wildtype plants in one of two experiments . The proportion of female progeny was significantly higher on Bt plants in the first experiment with Bt-resistant hosts but this effect was not observed again when the experiment was repeated.

J Vector Borne Dis, 2003 Mar-Jun, 40(1-2), 20 - 32
Biolarvicides in vector control: challenges and prospects; Mittal PK; Biolarvicides, based on mosquitocidal toxins of certain strains of Bacillus sphaericus and Bacillus thuringiensis var israelensis H-14 (Bti) are highly effective against mosquito larvae at very low doses and safe to other non-target organisms . During past two decades various biolarvicide formulations produced in India and abroad have been tested at Malaria Research Centre and some formulations have undergone large-scale operational trials . Biolarvicide formulations of B . sphaericus are useful in the control of Culex and certain Anopheles spp, such as An . stephensi and An . subpictus, but not much effective against An . culicifacies and almost ineffective against Aedes aegypti . Repeated application of B . sphaericus in the same habitat, however, results in the development of resistance in larvae of target mosquitoes . In view of its low specificity for An . culicifacies and the potential for resistance in An . stephensi, B . sphaericus has limited prospects for control of malaria vectors . However, with some resistance management, B . sphaericus can still be used against Culex mosquitoes . On the other hand Bti formulations, which have broader spectrum of activity against Aedes, Culex and Anopheles spp, have not shown significant development of resistance in mosquitoes but their activity in field, particularly against surface feeding anopheline larvae is affected by various bioenvironmental factors, thus requiring weekly application in most habitats . To overcome this problem development of slow release formulations and genetically engineered biolarvicides by transplanting mosquitocidal toxin genes of Bti and B . sphaericus in some other environmentally compatible organisms have been investigated by different scientists.

Crit Rev Microbiol, 2004, 30(1), 33 - 54
Bacterial insecticidal toxins; Chattopadhyay A et al.; Over the years it has been important for humans to control the populations of harmful insects and insecticides have been used for this purpose in agricultural and horticultural sectors . Synthetic insecticides, owing to their various side effects, have been widely replaced by biological insecticides . In this review we attempt to describe three bacterial species that are known to produce insecticidal toxins of tremendous biotechnological, agricultural, and economic importance . Bacillus thuringiensis (BT) accounts for 90% of the bioinsecticide market and it produces insecticidal toxins referred to as delta endotoxins . The other two bacteria belong to the genera Photorhabdus and Xenorhabdus, which are symbiotically associated with entomopathogenic nematodes of the families Heterorhabditidae and Steinernematidae respectively . Whereas, Xenorhabdus and Photorhabdus exist in a mutualistic association with the entomopathogenic nematodes, BT act alone . BT formulations are widely used in the field against insects; however, over the years there has been a gradual development of insect resistance against BT toxins . No resistance against Xenorhabdus or Photorhabdus has been reported to date . More recently BT transgenic crops have been prepared; however, there are growing concerns about the safety of these genetically modified crops . Nematodal formulations are also used in the field to curb harmful insect populations . Resistance development to entomopathogenic nematodes is unlikely due to the physical macroscopic nature of infection . Xenorhabdus and Photorhabdus transgenes have not yet been prepared; but are predicted to be available in the near future . In this review we start with an overview of the synthetic insecticides and then discuss Bacillus thuringiensis, Xenorhabdus nematophilus, and Photorhabdus luminescens in greater detail.

Protein Eng Des Sel, 2004 Mar, 17(3), 223 - 34 Epub 2004 Apr 28.
Cluster analysis of water molecules in alanine racemase and their putative structural role; Mustata G et al.; Conservation of water molecules was identified by a cluster analysis of seven crystal structures of alanine racemase from Bacillus stearothermophilus . A total of 47 clusters of consensus water sites were determined and found to be highly localized, as indicated by their low mobilities . These clusters are located in the region of the active sites as well as at the interface between the N-terminal domain (the alpha/beta-barrel) of the first monomer and the C-terminal domain of the second monomer . The clusters located at the dimer interface form extensive hydrogen-bonding networks linked to the protein backbone . These water-mediated hydrogen bonds, and also all hydrogen-bonding interactions at the dimer interface, were monitored during a 2 ns molecular dynamics simulation and showed that when the inhibitor propionate was bound to the enzyme, some of these interactions were disrupted . The data we present here indicate that the consensus water sites identified at the interface between the two monomers of alanine racemase may play a structural role, which is to maintain and stabilize the alanine racemase dimer . A second role might be to supply the active site continuously with water molecules in order to allow rapid equilibration of active site protons with the solvent.

Folia Microbiol (Praha), 2004, 49(1), 94 - 6
Chitosanase activity in Bacillus thuringiensis; Cruz Camarillo R et al.; The ability to produce extracellular chitosanase (EC 3.2.1.132) was found by plate assays in 18 (23%) out of 77 crystalliferous strains of Bacillus thuringiensis . The best chitosanase producer was selected after the growth chosen in a liquid medium with colloidal chitosan as carbon source . Enzyme production was optimized (a 4-d incubation at 32 degrees C with shaking in a medium of pH 6.5 with 4% colloidal chitosan) and the enzyme was partially characterized . This is the first report on the chitosanase of B . thuringiensis.

J Biochem (Tokyo), 2004 Mar, 135(3), 355 - 63
Two additional carbohydrate-binding sites of beta-amylase from Bacillus cereus var . mycoides are involved in hydrolysis and raw starch-binding; Ye Z et al.; In the previous X-ray crystallographic study, it was found that beta-amylase from Bacillus cereus var . mycoides has three carbohydrate-binding sites aside from the active site: two (Site2 and Site3) in domain B and one (Site1) in domain C . To investigate the roles of these sites in the catalytic reaction and raw starch-binding, Site1 and Site2 were mutated . From analyses of the raw starch-binding of wild-type and mutant enzymes, it was found that Site1 contributes to the binding affinity to raw-starch more than Site2, and that the binding capacity is maintained when either Site1 or Site2 exists . The raw starch-digesting ability of this enzyme was poor . From inhibition studies by maltitol, GGX and alpha-CD for hydrolyses of maltopentaose (G5) and amylose ( (n) = 16) catalyzed by wild-type and mutant enzymes, it was found that alpha-CD is a competitive inhibitor, while, maltitol behaves as a mixed-type or competitive inhibitor depending on the chain length of the substrate and the mutant enzyme . From the analysis of the inhibition mechanism, we conclude that the bindings of maltitol and GGX to Site2 in domain B form an abortive ESI complex when amylose ( (n) = 16) is used as a substrate.

Anal Biochem, 2004 May 15, 328(2), 123 - 30
Stability parameters for one-step mechanism of irreversible protein denaturation: a method based on nonlinear regression of calorimetric peaks with nonzero deltaCp; Arroyo-Reyna A et al.; Thermal transitions of many proteins have been found to be calorimetrically irreversible and scan-rate dependent . Calorimetric determinations of stability parameters of proteins which unfold irreversibly according to a first-order kinetic scheme have been reported . These methods require the approximation that the increase in heat capacity upon denaturation deltaCp is zero . A method to obtain thermodynamic parameters and activation energy for the two-state irreversible process N --> D from nonlinear fitting to calorimetric traces is proposed here . It is based on a molar excess heat capacity function which considers irreversibility and a nonzero constant deltaCp . This function has four parameters: (1) temperature at which the calorimetric profile reaches its maximal value (Tm), (2) calorimetric enthalpy at Tm (deltaHm), (3) deltaCp, and (4) activation energy (E) . The thermal irreversible denaturation of subtilisin BPN' from Bacillus amyloliquefaciens was studied by differential scanning calorimetry at pH 7.5 to test our model . Transitions were found to be strongly scanning-rate dependent with a mean deltaCp value of 5.7 kcal K(-1)mol(-1), in agreement with values estimated by accessible surface area and significantly higher than a previously reported value.

J Agric Food Chem, 2004 May 5, 52(9), 2726 - 34
Compositional equivalency of Cry1F corn event TC6275 and conventional corn (Zea mays L.); Herman RA et al.; Maize (Zea mays L.) plants have been transformed to express a Cry1F insecticidal crystal protein originally isolated from Bacillus thuringiensis Berliner . This protein controls lepidopteran pests of maize, including the European corn borer, Ostrinia nubilalis (Hubner) . As part of the safety assessment for crops containing transgenes, a compositional analysis of the food and feed is conducted . This analysis is designed to detect unintended changes in the nutrient and antinutrient content of the raw commodities produced by the crop due to the insertion of the genes into the genomic DNA of the plant (pleotropic effects) . Samples of transgenic and nontransgenic maize forage and grain were collected from six field sites located in the U.S . and Canada . Forage samples were analyzed for proximates and minerals, and grain was further analyzed for fatty acids, amino acids, vitamins, secondary metabolites, and antinutrients . Results demonstrated that maize expressing the Cry1F protein was equivalent to nontransgenic maize with respect to these important components . Comparison of the variability within the nontransgenic and transgenic hybrid, as compared to composition values reported in the literature, suggest that factors other than transgenes may contribute more substantially to the composition of crops.

J Chem Ecol, 2004 Feb, 30(2), 439 - 52
Casuarina cunninghamiana tissue extracts stimulate the growth of Frankia and differentially alter the growth of other soil microorganisms; Zimpfer JF et al.; Aqueous extracts of host plant Casuarina cunninghamiana tissue altered the in vitro growth of its diazotrophic microsymbiont Frankia and a selection of other soil microorganisms . The growth of actinomycetous Frankia strains, 55005 . AvcI1, CesI5, CjI82 001, and Cj was stimulated by aqueous extracts of C . cunninghamiana tissue . Green cladodes (photosynthetic branches), unsuberized roots, and suberized roots were more stimulatory than dry cladodes and seed tissue . Aqueous extracts of green cladodes of C . cunninghamiana most stimulated the growth of Casuarina-derived Frankia strains CjI82 001 and 55005 . The growth of isolates of soil bacteria Bradyrhizobium japonicum, Arthrobacter globiformis and Bacillus subtillis and of the soil fungi Penicillium oxalicum and Arthroderma cookiellum was either inhibited or not affected by cladode extracts . Cladode extracts stimulated the growth of the actinomycete Streptomyces albus and the fungus Rhizopus homothallicus . The magnitude (as great as 100%) of the increase in growth caused by tissue extracts for the Casuarina-derived Frankia strains relative to other soil microbes suggests a host-specific enhancement of the microsymbiont.

Biotechnol Bioeng, 2004 May 20, 86(4), 377 - 88
Bioprocess design and economic analysis for the commercial production of environmentally friendly bioinsecticides from Bacillus thuringiensis HD-1 kurstaki; Rowe GE et al.; A production process for B . thuringiensis (Bt) bioinsecticides was designed in detail, including alternative batch, low-density fed-batch (LDFB), and high-density fed-batch (HDFB) fermentation configurations . Capital and operating costs, as well as profitability based on simple rate of return, were performed using a purpose-written FORTRAN program, explicitly analyzing production of a water-based flowable product used in forestry applications.The total capital cost was 18 million dollars (Canadian dollars) for a stand-alone plant with base-scale capacity of 3 x 10(7) billion international units (BIU)/year . Raw material costs amounted to 1.5 million dollars yearly, of which approximately half was for formulation ingredients . Per-unit production cost rose sharply for scales of less than 1 x 10(7) BIU/year, but was little affected by scale above 3 x 10(7) BIU/year . Product cost was much lower at all scales for a LDFB as opposed to batch fermentation process, but HDFB gave relatively little additional cost benefit . Profitability analysis performed by co-varying scale and selling price showed that break-even occurred at a price of 0.45 dollars/BIU for a batch process at base scale, while with LDFB fermentation the same production volume sold at 0.35 dollars/BIU gave a 12% rate of return . Since the assumed base scale would represent 8-15% of current world Bt bioinsecticide production, based on value or volume, it was concluded that profitability would require some or all of the following elements: targeting higher-value markets such as disease vector control, in addition to forestry; a potentially lower plant capacity (although at least 1 x 10(7) BIU/year;) and coproduction of other large-volume microbial products to absorb capacity and match bioinsecticide output to market demand .

J Invertebr Pathol, 2004 Mar, 85(3), 182 - 7
Lyophilization of lepidopteran midguts: a preserving method for Bacillus thuringiensis toxin binding studies; Hernandez CS et al.; Binding assays with brush border membrane vesicles (BBMV) from insect midguts are commonly used in the study of the interactions between Bacillus thuringiensis Cry toxins and their receptors . Collaboration between laboratories often require that frozen insect samples are sent in dry ice . Because of customs restrictions and delays, sample thawing is always a risk and often the biological material becomes ruined during shipping . We have tested lyophilization as an alternative method for preserving insect midguts for binding studies with B . thuringiensis Cry toxins . For this purpose, BBMV were prepared from both frozen and lyophilized midguts from three lepidopteran species: Spodoptera exigua, Manduca sexta, and Helicoverpa armigera . Higher membrane protein recovery was always obtained from lyophilized midguts compared to frozen midguts, and similar membrane marker enzyme activities were found in BBMV from either treatment . Comparable equilibrium dissociation constants and binding site concentrations, calculated from binding experiments with labeled (125)I-Cry1Ab toxin, were found using BBMV from either method . In the light of these results, lyophilization is a good preserving method of lepidopteran midguts to study binding of B . thuringiensis Cry toxins.

Int J Food Microbiol, 2004 Apr 15, 92(2), 227 - 34
Inactivation of Bacillus cereus spores in milk by mild pressure and heat treatments; Van Opstal I et al.; The objective of this work was to study the germination and subsequent inactivation of Bacillus cereus spores in milk by mild hydrostatic pressure treatment . In an introductory experiment with strain LMG6910 treated at 40 degrees C for 30 min at 0, 100, 300 and 600 MPa, germination levels were 1.5 to 3 logs higher in milk than in 100 mM potassium phosphate buffer (pH 6.7) . The effects of pressure and germination-inducing components present in the milk on spore germination were synergistic . More detailed experiments were conducted in milk at a range of pressures between 100 and 600 MPa at temperatures between 30 and 60 degrees C to identify treatments that allow a 6 log inactivation of B . cereus spores . The mildest treatment resulting in a 6 log germination was 30 min at 200 MPa/40 degrees C . Lower treatment pressures or temperatures resulted in considerably less germination, and higher pressures and temperatures further increased germination, but a small fraction of spores always remained ungerminated . Further, not all germinated spores were inactivated by the pressure treatment, even under the most severe conditions (600 MPa/60 degrees C) . Two possible approaches to achieve a 6 log spore inactivation were identified, and validated in three additional B . cereus strains . The first is a single step treatment at 500 MPa/60 degrees C for 30 min, the second is a two-step treatment consisting of pressure treatment for 30 min at 200 MPa/45 degrees C to induce spore germination, followed by mild heat treatment at 60 degrees C for 10 min to kill the germinated spores . Reduction of the pressurization time to 15 min still allows a 5 log inactivation . These results illustrate the potential of high-pressure treatment to inactivate bacterial spores in minimally processed foods.

FEMS Microbiol Lett, 2004 May 1, 234(1), 177 - 83
Production of an antifungal protein for control of Colletotrichum lagenarium by Bacillus amyloliquefaciens MET0908; Kim PI et al.; A plant pathogenic fungus, Colletotrichum lagenarium, causing watermelon anthracnose, was isolated from naturally infected leaves, stems, and fruits of watermelon . A bacterial strain, MET0908, showing a potent antifungal activity against C . lagenarium, was isolated from soil . An antifungal protein was purified by 30% ammonium sulfate saturation and concentrated using Centricon 10, DEAE-Sepharose(TM) Fast Flow column and Sephacryl S-100 gel filtration chromatography . The molecular weight of the purified protein was estimated as 40 kDa by SDS-PAGE . The purified protein was stable at 80 degrees C for 20 min and exhibited a broad spectrum of antifungal activity against various plant pathogenic fungi . Confocal microscopy image analysis and scanning electron microscopy showed that the protein acted on the cell wall of C . lagenarium . The purified antifungal protein exhibited beta-1,3-glucanase activity . The N-terminal amino acid sequence of the purified protein was determined as Ser-Lys-Ile-x-Ile-Asn-Ile-Asn-Ile-x-Gln-Ala-Pro-Ala-Pro-x-Ala . A search of the sequence with NCBI BLAST showed no significant homology with any known proteins, suggesting that the purified protein may be novel.

FEMS Microbiol Lett, 2004 May 1, 234(1), 105 - 10
Identification and characterization of a novel inulin binding module (IBM) from the CFTase of Bacillus macerans CFC1; Lee JH et al.; A novel inulin-binding module (IBM), which was identified from the N-terminal region of the cycloinulinooligosaccharide fructanotransferase (CFTase) in Bacillus macerans CFC1, was characterized using the discrete entity of IBM produced by the recombinant Escherichia coli strains . Deletion analyses located the inulin binding activity in the N-terminal region between 241 and 389 amino acid residues, which was removed from the mature enzyme by processing when secreted from the B . macerans CFC1 cells . IBM bound specifically to polyfructans such as inulin and levan but it did not interact with any of the glycan polymers tested in this study including cellulose, xylan, and starch . Binding studies on the IBM revealed that the equilibrium dissociation constant K(d) and the maximum amount of protein bound {(PC)(max)} were 4.7 microM and 22 microM g(-1), respectively . Together, these results indicate that the IBM of CFTase has a relatively high and specific affinity for inulin . Adsorption of the IBM to inulin was highest at pH 7.0 and lowered slowly with decreasing pH down to 3.0 . At pH 7.0, the binding activity was enhanced about twofold by the presence of 1 M MgCl(2) . Chemical modification experiments with the aromatic amino acid-specific modifiers implied that tryptophan and tyrosine residues in the IBM are likely to participate in the interaction with the inulin molecules.

Biochemistry, 2004 May 4, 43(17), 4990 - 7
Investigation of metal ion binding in phosphonoacetaldehyde hydrolase identifies sequence markers for metal-activated enzymes of the HAD enzyme superfamily; Zhang G et al.; The 2-haloalkanoic acid dehalogenase (HAD) family, which contains both carbon and phosphoryl transferases, is one of the largest known enzyme superfamilies . HAD members conserve an alpha,beta-core domain that frames the four-loop active-site platform . Each loop contributes one or more catalytic groups, which function in mediating the core chemistry (i.e., group transfer) . In this paper, we provide evidence that the number of carboxylate residues on loop 4 and their positions (stations) on the loop are determinants, and therefore reliable sequence markers, for metal ion activation among HAD family members . Using this predictor, we conclude that the vast majority of the HAD members utilize a metal cofactor . Analysis of the minimum requirements for metal cofactor binding was carried out using Mg(II)-activated Bacillus cereus phosphonoacetaldehyde hydrolase (phosphonatase) as an experimental model for metal-activated HAD members . Mg(II) binding occurs via ligation to the loop 1 Asp12 carboxylate and Thr14 backbone carbonyl and to the loop 4 Asp186 carboxylate . The loop 4 Asp190 forms a hydrogen bond to the Mg(II) water ligand . X-ray structure determination of the D12A mutant in the presence of the substrate phosphonoacetaldehyde showed that replacement of the loop 1 Asp, common to all HAD family members, with Ala shifts the position of Mg(II), thereby allowing innersphere coordination to Asp190 and causing a shift in the position of the substrate . Kinetic analysis of the loop 4 mutants showed that Asp186 is essential to cofactor binding while Asp190 simply enhances it . Within the phosphonatase subfamily, Asp186 is stringently conserved, while either position 185 or position 190 is used to position the second loop 4 Asp residue . Retention of a high level of catalytic activity in the G185D/D190G phosphonatase mutant demonstrated the plasticity of the metal binding loop, reflected in the variety of combinations in positioning of two or three Asp residues along the seven-residue motif of the 2700 potential HAD sequences that were examined.

Dis Aquat Organ, 2004 Mar 10, 58(2-3), 89 - 97
Pathology and ultrastructure of an intranuclear bacilliform virus (IBV) infecting brown shrimp Crangon crangon (Decapoda: Crangonidae); Stentiford GD et al.; The brown shrimp Crangon crangon supports an important fishery in Europe (over 25000 t, valued at 80 million euros in 2000) . Through the course of histopathological screening of crustaceans from the Clyde estuary, western Scotland, for the biological effect of contaminants, we have discovered a highly prevalent (up to 100%) non-occluded intranuclear bacilliform virus (IBV) infection in the hepatopancreatic tubule epithelia and midgut epithelia of wild C . crangon . This is the first report of an IBV in this family . We have termed this virus Crangon crangon bacilliform virus (CcBV) . Histological and ultrastructural observations suggest that this virus is similar to other IBVs previously described from crabs and penaeid shrimps . The nuclei of virus-infected epithelial cells contained an eosinophilic, hypertrophied viroplasm that marginalised the chromatin of the host nucleus . Infected cells were often separated from their neighbouring cells and their nuclei appeared apoptotic . In heavily infected shrimp, apoptotic cells were expelled into the lumen of the hepatopancreatic tubule or the midgut . Following this stage, some hepatopancreatic tubules became degenerate, with remnants of the basement membrane and myoepithelial lining remaining . Transmission electron microscopy of hypertrophic nuclei revealed the presence of rod-shaped and cylindrical, envelope-bound virions . These virions did not form arrays and were not embedded within occlusion bodies, but did appear to be partially occluded in an amorphous matrix that corresponded to a granular viroplasm . The ultrastructure, morphology and size of the nucleocapsid and the complete virion aligns the virus most closely to the IBVs previously reported from other decapod crustaceans . Due to the pathological manifestation of IBV infection in C . crangon, it appears likely that it can act as a population modulator, particularly at sites where infection prevalence is high, such as that observed in the Clyde estuary.

Appl Microbiol Biotechnol, 2004 Aug, 65(2), 183 - 92 Epub 2004 Apr 24.
Improving the insecticidal activity of Bacillus thuringiensis subsp . aizawai against Spodoptera exigua by chromosomal expression of a chitinase gene; Thamthiankul S et al.; A transcriptionally fused gene comprising the P19 gene from Bacillus thuringiensis subsp . israelensis fused with a chitinase gene (chiBlA) from B . licheniformis was integrated into the B . thuringiensis subsp . aizawai BTA1 genome by homologous recombination . The resulting B . thuringiensis subsp . aizawai strain (INT1) showed growth and sporulation comparable with that of the wild-type strain . INT1 produced four chitinases of different molecular masses (i.e., 66, 55, 39, 36 kDa) . Three of these (66, 55, 36 kDa) were derived from the cloned chiBlA gene, whereas the 39-kDa chitinase originated from BTA1 . Using surface contamination bioassays, the 50% lethal concentration of lyophilized whole culture broth of INT1 against Spodoptera exigua neonate larvae was 12.2 microg/cm2, compared with 30.8 microg/cm2 for BTA1 . Bioassays using filtered culture supernatant of INT1 (110 microg/cm2) together with trypsin-activated purified Cry1C protein of B . thuringiensis (1,280 ng/cm2) showed 75.0% mortality, compared with 56.7% mortality for Cry1C combined with BTA1 at the same concentration . Using scanning electron microscopy, clear perforations were observed in S . exigua fifth instar peritrophic membranes incubated with either crude or purified chitinase, or isolated from fifth instar S . exigua fed purified chitinase since the first instar . These results show that chitinase can increase the activity of B . thuringiensis subsp . aizawai against S . exigua . This is the first documentation of expressing a chimeric chitinase gene on the chromosome of B . thuringiensis; and chromosomal integration might be used as a potential technique for strain improvement.

Microbiol Immunol, 2004, 48(4), 289 - 95
Introduction of 65 kDa antigen of Mycobacterium tuberculosis to cancer cells enhances anti-tumor effect of BCG therapy; Hara I et al.; Bacillus Calmette Guerin (BCG) immunotherapy has anti-tumorigenic effects against bladder cancer . To improve the efficacy of BCG therapy, we introduced the gene encoding the 65 kDa heat shock protein (hsp) of Mycobacterium tuberculosis into a mouse malignant melanoma cell line (B16) . An expression vector harboring the 65 kDa antigen gene was transfected into B16 using Lipofectamine, then expression of the antigen was confirmed by RT-PCR and Western blotting . Several cell lines expressing 65 kDa antigen were established (B16/65 kDa) . We also established a control cell line transfected with the vector alone (B16/con) . All cell lines (B16, B16/con, B16/65 kDa) were injected intraperitoneally into syngeneic mice with or without BCG prior immunization and the development of tumor ascites was examined . To analyze the mechanism of the anti-tumor effect, CD4 T cells or CD8 T cells were depleted in vivo by administering the corresponding monoclonal antibody . B16/65k Da expressed the 65 kDa hsp of M . tuberculosis . The tumor growth of B16/65 kDa was slightly retarded in naive mice, but significantly inhibited by BCG . The anti-tumor effect was totally abrogated in mice deficient in CD4 T cells, suggesting that CD4 T cells are involved in this process . The 65 kDa hsp of M . tuberculosis was expressed after gene transduction in a malignant melanoma cell line and significantly enhanced the anti-tumor effect of BCG immunotherapy . CD4 T cells play an important role in this anti-tumor effect.

Can J Microbiol, 2004 Mar, 50(3), 213 - 9
Weathering of phlogopite by Bacillus cereus and Acidithiobacillus ferrooxidans; Styriakova I et al.; The purpose of this study was to assess the weathering of finely ground phlogopite, a trioctahedral mica, by placing it in contact with heterotrophic (Bacillus cereus) and acidophilic (Acidithiobacillus ferrooxidans) cultures . X-ray diffraction analyses of the phlogopite sample before and after 24 weeks of contact in B . cereus cultures revealed a decrease in the characteristic peak intensities of phlogopite, indicating destruction of individual structural planes of the mica . No new solid phase products or interlayer structures were detected in B . cereus cultures . Acidithiobacillus ferrooxidans cultures enhanced the chemical dissolution of the mineral and formed partially weathered interlayer structures, where interlayer K was expelled and coupled with the precipitation of K-jarosite {KFe3(SO4)2(OH)6}.

J Nat Prod, 2004 Apr, 67(4), 537 - 41
New chromanone acids with antibacterial activity from Calophyllum brasiliense; Cottiglia F et al.; Six novel chromanone acids (1-6) were isolated from the bark of Calophyllum brasiliense Cambess . Their structures were elucidated on the basis of 1D and 2D NMR experiments, as well as mass spectrometry . All compounds showed moderate to strong antibacterial activity against Bacillus cereus and Staphylococcus epidermidis, with 1 and 2 being most active . None of the compounds were cytotoxic against KB, Jurkat T, and myosarcoma cancer cells up to 20 microg/mL.

Antonie Van Leeuwenhoek, 2004 Jul, 86(1), 87 - 92
Molecular characterization of Bacillus thuringiensis strains from Argentina; Franco-Rivera A et al.; Bacillus thuringiensis INTA 7-3, INTA 51-3, INTA Mo9-5 and INTA Mo14-4 strains were obtained from Argentina and characterized by determination of serotype, toxicity, plasmid composition, insecticidal gene content ( cry and vip ) and the cloning of the single- vip3A gene of the INTA Mo9-5 strain . The serotype analysis identified the serovars tohokuensis and darmstadiensis for the INTA 51-3 and INTA Mo14-4 strains, respectively, whereas the INTA Mo9-5 strain was classified as "autoagglutinated" . In contrast to the plasmid patterns of INTA 7-3, INTA 51-3 and INTA Mo9-5 (which were similar to B . thuringiensis HD-1 strain), strain INTA Mo14-4 showed a unique plasmid array . PCR analysis of the four strains revealed the presence of cry genes and vip3A genes . Interestingly, it was found that B . thuringiensis 4Q7 strain, which is a plasmid cured strain, contained vip3A genes indicating the presence of these insecticidal genes in the chromosome . Bioassays towards various lepidopteran species revealed that B . thuringiensis INTA Mo9-5 and INTA 7-3 strains were highly active . In particular, the mean LC(50) obtained against A . gemmatalis larvae with the INTA Mo9-5 and INTA 7-3 strains were 7 (5.7-8.6) and 6.7 (5.6-8.0) ppm, respectively . The INTA Mo14-4 strain was non-toxic and strain INTA 51-3 showed only a weak larvicidal activity.

Acta Crystallogr D Biol Crystallogr, 2004 May, 60(Pt 5), 952 - 3 Epub 2004 Apr 21.
Crystallization of the mosquito-larvicidal binary toxin produced by Bacillus sphaericus; Smith AW et al.; The Bacillus sphaericus binary toxin is expressed during the early stages of sporulation and is composed of two separately encoded polypeptides: BinA (41.9 kDa) and BinB (51.4 kDa) . The binary toxin forms microcrystalline inclusions inside the mother cell that, once ingested, are solubilized in the alkaline pH of the larval gut . B . sphaericus cultures were grown to complete sporulation, harvested and washed . The binary toxin was solubilized with 50 mM NaOH, purified using column chromatography and crystallized . Crystals were grown using the sitting-drop vapor-diffusion method in a glycine-buffered solution containing 0.5 M NaCl and 19.5% PEG 4000 as the precipitant . Native data were collected to a resolution of 2.7 A.

Expert Opin Ther Targets, 2004 Apr, 8(2), 79 - 93
Recent advances towards identification of new drug targets for Mycobacterium tuberculosis; Sharma K et al.; Mycobacterium tuberculosis is a very successful pathogen that remains a leading infectious killer worldwide . The global situation has become precarious due to various factors such as the variable efficacy of the Bacille Calmette-Guerin (BCG) vaccine, drug resistance, delay in diagnosis, association with HIV, and other factors, creating a long-lasting reservoir of impending disease and infection . Surprisingly, no new drugs have been developed in the past 30 years . The release of the complete genome sequence of M . tuberculosis and the availability of advanced genetic tools have provided the powerful repertoire of potential drug targets that are now in hand and can be explored in a more rational and directional manner . In this review, the authors highlight some possible therapeutic targets in M . tuberculosis . The gene products involved in various processes, such as mycobacterial cell wall synthesis, ability to acquire or obtain essential nutrients, persistence, transcription regulation, energy metabolism and others, such as the PE-PGRS family and culture filtrate proteins, would be potential targets for the development of new drugs . Apart from these categories, the importance of signal transduction events in the virulence of mycobacteria is discussed in relation to their potential as therapeutic targets . The potential of all of these therapeutic targets should be investigated together with the potential of being able to synthesise future chemotherapeutic agents.

Protein Eng Des Sel, 2004 Mar, 17(3), 205 - 11 Epub 2004 Apr 19.
Improvement of cyclodextrin glucanotransferase as an antistaling enzyme by error-prone PCR; Shim JH et al.; In an effort to improve the properties of cyclodextrin glucanotransferase (CGTase) as an antistaling enzyme, error-prone PCR was used to introduce random mutations into a CGTase cloned from alkalophilic Bacillus sp . I-5 (CGTase I-5) . A mutant CGTase{3-18} with the three mutations M234T, F259I and V591A was selected by agar plate assay . Sequence alignment of various CGTases indicated that M234 and F259 are located in the vicinity of the catalytic sites of the enzyme and V591 in the starch binding domain E . The cyclization activity of CGTase{3-18} was dramatically decreased by 10-fold, while the hydrolyzing activity was increased by up to 15-fold . These mutations near subsite +1 (M234T) and at subsite +2 (F259I) are likely to alter the enzyme activity in a concerted manner, promoting hydrolysis of substrate while retarding cyclization . The addition of CGTase{3-18} reduced the retrogradation rate of bread by as much as did the commercial antistaling enzyme Novamyl during 7-day storage at 4 degrees C . No cyclodextrin (CD) was detected in bread treated with CGTase{3-18}, whereas 21 mg of CD per 10 g of bread was produced in bread treated with wild-type CGTase.

Immunology, 2004 May, 112(1), 143 - 52
Functional characterization of human natural killer cells responding to Mycobacterium bovis bacille Calmette-Guérin; Esin S et al.; The kinetics of activation and induction of several effector functions of human natural killer (NK) cells in response to Mycobacterium bovis bacille Calmette-Guerin (BCG) were investigated . Owing to the central role of monocytes/macrophages (MM) in the initiation and maintenance of the immune response to pathogens, two different experimental culture conditions were analysed . In the first, monocyte-depleted nylon wool non-adherent (NW) cells from healthy donors were stimulated with autologous MM preinfected with BCG (intracellular BCG) . In the second, the NW cells were directly incubated with BCG, which was therefore extracellular . In the presence of MM, CD4+ T lymphocytes were the cell subset mainly expressing the activation marker, CD25, and proliferating with a peak after 7 days of culture . In contrast, in response to extracellular BCG, the peak of the proliferative response was observed after 6 days of stimulation, and CD56+ CD3- cells (NK cells) were the cell subset preferentially involved . Such proliferation of NK cells did not require a prior sensitization to mycobacterial antigens, and appeared to be dependent upon contact between cell populations and bacteria . Following stimulation with extracellular BCG, the majority of interferon-gamma (IFN-gamma)-producing cells were NK cells, with a peak IFN-gamma production at 24-30 hr . Interleukin (IL)-2 and IL-4 were not detectable in NK cells or in CD3+ T lymphocytes at any time tested . IL-12 was not detectable in the culture supernatant of NW cells stimulated with extracellular BCG . Compared to the non-stimulated NW cells, the NW cells incubated for 16-20 hr with BCG induced the highest levels of expression of apoptotic/death marker on the NK-sensitive K562 cell line . BCG also induced expression of the activation marker, CD25, and proliferation, IFN-gamma production and cytotoxic activity, on negatively selected CD56+ CD3- cells . Altogether, the results of this study demonstrate that extracellular mycobacteria activate several NK-cell functions and suggest a possible alternative mechanism of NK-cell activation as the first line of defence against mycobacterial infections.

Rev Med Suisse Romande, 2003 Feb, 123(2), 85 - 8
{Suspected tuberculosis at a refugee center: 79 referred cases in 2 years at St . Loup Hospital}; Rieder P et al.; The recent opening, close to the hospital of Saint-Loup, of an application centre for foreigners who ask for a status of refugee in Switzerland gave us the opportunity, over the last two years, to evaluate 79 patients with suspected tuberculosis . Of them 67% came from sub-Saharan Africa and 25% from Eastern Europe . A bacillary tuberculosis was found in 19 cases (24%), requiring immediate treatment and a respiratory isolation of a mean duration of 18 days . In addition, 11 cases (14%) had non-bacillary tuberculosis with negative sputum smear but positive culture . This new situation led us to implement a specific strategy of hospital hygiene.

Planta Med, 2004 Apr, 70(4), 359 - 63
Hydroxylation and glucosidation of ent-16beta-Hydroxybeyeran-19-oic acid by Bacillus megaterium and Aspergillus niger; Yang LM et al.; ent-16beta-Hydroxybeyeran-19-oic acid ( 1) has potential antihypertensive activity . To obtain novel and more-effective compounds, 1 was incubated with Bacillus megaterium ATCC 14 581 and Aspergillus niger CCRC 32 720 . The structures of the metabolites were determined by HR-FAB-MS, 1D- and 2D-NMR spectral data, and enzymatic hydrolysis . Bacillus megaterium hydroxylated and glucosidated 1 to yield ent-7alpha,16beta-dihydroxybeyeran-19-oic acid ( 2), ent-16beta-hydroxybeyeran-19-oic acid alpha- D-glucopyranosyl ester ( 3), and ent-7alpha,16beta-dihydroxybeyeran-19-oic acid alpha- D-glucopyranosyl ester ( 4) . Aspergillus niger hydroxylated 1 to yield ent-1beta,7alpha,16beta-trihydroxybeyeran-19-oic acid ( 5) and ent-1beta,7alpha-dihydroxy-16-oxobeyeran-19-oic acid ( 6) . Metabolites 3 - 5 were characterized as new compounds . In addition, 2, 3, 5, and 6 were tested for antihypertensive effects, and we found that 5 and 6 were more potent than the parent compound 1.

Environ Pollut, 1991, 74(2), 89 - 100
The influence of pH on cadmium toxicity to the green alga Stichococcus bacillaris and on the cadmium forms present in the culture medium; Skowronski T et al.; Cadmium toxicity to the green alga Stichococcus bacillaris was investigated in media of pH 3-9 . A significant decrease of cadmium toxicity occurred in both the acidic and alkaline ranges of pH . In media of pH 3 and 9, cadmium did not affect the dry mass content substantially . Maximum toxicity of cadmium was noticed at pH 6-7 . Voltametric investigations showed a significant effect of pH on electrochemically measured cadmium content in the culture media . Hydrolysis of the medium components and formation of cadmium complexes with OH(-) ions caused a considerable decrease in amounts of electrochemically measured cadmium in the alkaline range of pH.

Environ Pollut, 1993, 79(1), 77 - 83
Effects of simetryne on growth of various freshwater algal taxa; Kasai F et al.; The sensitivity of 56 algal strains, representing 7 taxonomic groups to the triazine herbicide, simetryne, was examined using EC50 values for growth . There was a wide range of values from 6.5 to 1500 microg litre(-1) . The Volvocales (Chlorophyceae, Chlorophyta) and Cyanophyceae (Cyanophyta) as a whole were the most sensitive, whereas the Desmidiales (Charophyceae, Chlorophyta) and Bacillariophyceae (Chromophyta) were the most tolerant, although sensitivity differed among strains of a single species . Sensitive and tolerant species were both isolated from samples collected at the same site . The results suggest that changes in species composition and relative abundance will occur when herbicides are applied in natural habitats.

Environ Pollut, 1993, 80(3), 255 - 64
The algal community as an indicator of the trophic status of Lake Patzcuaro, Mexico; Rosas I et al.; An evaluation of water quality and phytoplankton composition position was carried out in order to determine the trophic conditions of Lake Patzcuaro (2035 m above sea level), a high altitude tropical lake . Temperatures ranged from 15 to 23 degrees C . Total phosphorus and inorganic nitrogen showed a seasonal variation; highest values coincided with the rainy season (0.48 and 2.1 mg litre(-1), respectively) . Dissolved oxygen ranged from 2 to 7.9 mg litre(-1) at the surface and from 0.6 to 7.3 mg litre(-1) on the bottom, the lowest values being found in shallow zones . Average transparency varied from 0.62 to 1.4 m Secchi depth . Rainfall was a primary factor in seasonal variability as it influenced both physical and biological conditions by contributing to the transport and deposition of silt, which mixed with sinking algal cells . The composition of the surface phytoplankton segregated along five major divisions comprising a total of 49 species . General seasonal patterns of dominance alternated in a sequence beginning with Bacillariophyta, through Chlorophyta to Cyanophyta . Diatoms, the dominant group from February to early June, included Melosira granulata, Stephanodiscus sp., Synedra sp . and Fragilaria sp . During the rainy season (late June to September), Microcystis aeruginosa, Oscillatoria sp., Anabaena sp., Merismopedia sp., Crucigenia cuadrata, Oocystis lacustris, Selenastrum gracile, Mallomonas sp . and Tetraediella sp . were important . Melosira granulata was present throughout the period of study . Spatial and temporal variability in both physical and biological conditions make it difficult to assign a specific trophic state to Lake Patzcuaro . Nevertheless, analysis of the algal community indicates a generally mesotrophic condition.

Environ Pollut, 1995, 88(3), 275 - 81
Effects of zinc on the structure and growth dynamics of a natural freshwater phytoplankton assemblage reared in the laboratory; Loez CR et al.; The response of a natural phytoplankton assemblage to different concentrations of Zn(2+) was evaluated by means of a static laboratory bioassay . Aliquots of surface water, taken in autumn from a non-polluted point of the Reconquista River (Buenos Aires, Argentia), were incubated in mineral nutrient media containing 2.5, 10 or 25 mg litre(-1) of Zn(2+) (as zinc chloride) . The comparative structure and dynamics of the communities were followed through periodic physico-chemical and biological analyses of samples taken during 24 days of incubation . Under the experimental conditions of the bioassays, the existence of several Zn(2+) tolerant algal species was shown: the most important of them were Chlorella vulgaris Beij . (Chlorophyceae, Chlorococcales), Nitzschia palea (Kutz.) Smith and Gomphonema parvulum (Kutz.) Kutz . (Bacillariophyceae) . It was also demonstrated that the algal responses to Zn(2+) were selectively concentration dependent: at 2.5 and 10 mg litre(-1) a stimulatory effect was observed in the diatoms; at 25 mg litre(-1), diatom toxicity occurred . In contrast, Chlorophyceae growth was stimulated at the maximal Zn level . In general, the diversity, richness and equitability of the community were adversely affected by Zn in a concentration-dependent fashion.

Vet Ophthalmol, 2004 May-Jun, 7(3), 141 - 6
Periocular sarcoid in a horse; Komaromy AM et al.; A periocular nodular sarcoid of the right upper and lower eyelids was diagnosed in an 11-year-old Thoroughbred mare . Computed tomography scan revealed the extent of the tumor . The mass was surgically debulked under general anesthesia, and the affected periocular region was injected intralesionally with Bacillus of Calmette and Guerin (BCG) . An emulsion of cell wall fractions was used, which has been modified to reduce the toxic and allergic effect, but retain the antitumor activity . In total, five injections were performed at 2-week intervals . At follow-up 7 months after the last BCG injection, the tumor was completely resolved . Two years after the last treatment, the horse remains tumor-free.

Microb Cell Fact . 2004 Apr 16;3(1):2.
Identification of pathogenic microbial cells and spores by electrochemical detection on a biochip; Gabig-Ciminska M et al.; BACKGROUND: Bacillus cereus constitutes a significant cause of acute food poisoning in humans . Despite the recent development of different detection methods, new effective control measures and better diagnostic tools are required for quick and reliable detection of pathogenic micro-organisms . Thus, the objective of this study was to determine a simple method for rapid identification of enterotoxic Bacillus strains . Here, a special attention is given to an electrochemical biosensor since it meets the requirements of minimal size, lower costs and decreased power consumption . RESULTS: A bead-based sandwich hybridization system was employed in conjugation with electric chips for detection of vegetative cells and spores of Bacillus strains based on their toxin-encoding genes . The system consists of a silicon chip based potentiometric cell, and utilizes paramagnetic beads as solid carriers of the DNA probes . The specific signals from 20 amol of bacterial cell or spore DNA were achieved in less than 4 h . The method was also successful when applied directly to unpurified spore and cell extract samples . The assay for the haemolytic enterotoxin genes resulted in reproducible signals from B . cereus and B . thuringiensis while haemolysin-negative B . subtilis strain did not yield any signal . CONCLUSIONS: The sensitivity, convenience and specificity of the system have shown its potential . In this respect an electrochemical detection on a chip enabling a fast characterization and monitoring of pathogens in food is of interest . This system can offer a contribution in the rapid identification of bacteria based on the presence of specific genes without preceding nucleic acid amplification.

J Am Mosq Control Assoc, 2004 Mar, 20(1), 64 - 73
Procedures for the evaluation of field efficacy of slow-release formulations of larvicides against Aedes aegypti in water-storage containers; Mulla MS et al.; In Thailand, water-storage jars, barrels, drums, pails, and tanks constitute vast developmental sites for Aedes aegypti in urban, semiurban, and rural areas . Earthen water jars, cement jars, and concrete tanks constitute the greatest proportion of artificial containers where Ae . aegypti breed . This species is a major vector of the causal agents of dengue and dengue hemorrhagic fever, and vector control by larviciding is one of the main approaches to disease control . At present, temephos sand granules (SG) (1%) are used in large-scale community-based larviciding programs . Because of the use of this larvicide over the past 30 years, the likelihood exists that Ae . aegypti already has become resistant to this larvicide . To develop more options for control and make them available for use, we evaluated VectoBac tablets (Bacillus thuringiensis var . israelensis {Bti} 5%) and a new formulation of zeolite granules (ZG) of temephos (1%) and compared these formulations for efficacy with temephos SG (1%) in water-storage jars . In these tests, we used 48 identical glazed earthen water-storage jars (200-liter capacity) and developed quantitative sampling procedures for larvae, pupae, and pupal skins . Pupal skins were the easiest to count and this technique was used for the 1st time for assessing emergence of adults in water-storage containers . Three water regimens were used: full jars, half-full jars, and full jars emptied half way and refilled weekly . The 3 formulations with 3 regimens of water were assessed over a period of 6 months . VectoBac tablets at the dosage of 1 tablet or 0.37 g per 50 liters of water provided excellent control for about 112 days in full water jars . In the other 2 water regimens, VectoBac gave excellent control for 90 days . The 2 temephos formulations at the operational rate of 5 g per 50 liters of water were equal in efficacy, yielding almost 100% control for more than 6 months . Unlike temephos SG, the temephos ZG had no objectionable odor . Both the temephos ZG and Bti tablets increased clarity of the water, a feature desired by the users . Lack of odor and depression of turbidity are important attributes of Bti tablets and temephos ZG.

Am J Respir Crit Care Med, 2004 Jul 1, 170(1), 65 - 9 Epub 2004 Apr 15.
Comparison of tuberculin skin test and new specific blood test in tuberculosis contacts; Brock I et al.; The tuberculin skin test used to detect latent Mycobacterium tuberculosis infection has many drawbacks, and a new diagnostic test for latent tuberculosis (QuantiFERON-TB {QTF-TB}) has recently been introduced . This test measures the production of IFN-gamma in whole blood upon stimulation with purified protein derivative (PPD) . The QTF-TB test addresses the operational problems with the tuberculin skin test, but, as the test is based on PPD, it still has a low specificity in populations vaccinated with the Bacille Calmette-Guerin (BCG) vaccine . We have modified the test to include the antigens ESAT-6 and CFP-10, which are not present in BCG vaccine strains or the vast majority of nontuberculous mycobacteria . This test was used to detect infection in contacts in a tuberculosis outbreak at a Danish high school . The majority of the contacts were BCG-unvaccinated, which allowed a direct comparison of the skin test and the novel blood test in individuals whose skin test was not confounded by vaccination . An excellent agreement between the two tests was found (94%, kappa value 0.866), and in contrast to the blood test based on PPD, the novel blood test was not influenced by the vaccination status of the subjects tested.

Kidney Int, 2004 May, 65(5), 1826 - 34
Tuberculin skin testing underestimates a high prevalence of latent tuberculosis infection in hemodialysis patients; Sester M et al.; BACKGROUND: Identification of latent Mycobacterium tuberculosis infection in hemodialysis patients is hampered by reduced sensitivity of the established tuberculin skin test . We investigated whether in vitro quantitation of purified protein derivative (PPD)-specific T cells using a rapid 6-hour assay may represent an alternative approach for detecting latent infection . METHODS: One hundred and twenty-seven hemodialysis patients and 218 control patients (blood donors, health care workers, and control patients) were analyzed . Specific T cells toward PPD and early secretory antigenic target-6 (ESAT-6), a protein expressed in Mycobacterium tuberculosis but absent from M . bovis bacillus Calmette-Guerin (BCG) vaccine strains, were flow cytometrically quantified from whole blood, and results were compared with skin testing . RESULTS: Compared to blood donors, a high proportion of both health care workers (48.6%) and hemodialysis patients (53.5%) had PPD-specific Th1-type CD4 T-cell reactivity with similar median frequencies of PPD-specific T cells (0.17%; 0.06-3.75% vs . 0.26%; 0.06-4.12%, respectively) . In contrast, skin test reactivity was significantly reduced in hemodialysis patients . Whereas 85.7% of control patients with PPD reactivity in vitro were skin test-positive, the respective percentage among hemodialysis patients was 51.4% (P= 0.007) . Among individuals with PPD reactivity in vitro, approximately 50% had T cells specific for ESAT-6 . CONCLUSION: Unlike the skin test, measurement of PPD reactivity by in vitro quantitation of PPD-specific T cells was unaffected by uremia-associated immunosuppression . This whole-blood assay may thus be a valuable alternative to skin testing, and detection of ESAT-6-specific T cells could moreover allow distinction of latent M . tuberculosis infection from BCG-induced reactivity to PPD . The assay is well suited for clinical use and may facilitate targeting of preventative therapy in high-risk individuals.

Urol Int, 2004, 72(3), 257 - 60
Isolated renal tuberculosis following intravesical Bacillus Calmette-Guérin therapy for bladder cancer; Wada Y et al.; We present a case of isolated renal tuberculosis following bacillus Calmette-Guerin (BCG) therapy for bladder cancer . In the presurgical radiographic examination, we suspected an atypical renal cell carcinoma . According to the diagnosis of renal cell carcinoma, we performed a radical nephrectomy . The histological findings were tuberculosis-specific inflammatory changes and the patient received an antituberculous multiple drug therapy for a year . It is concluded that we should pay attention to the possibility of a renal tuberculosis granuloma in any patient who presented with subacute formed renal masses following BCG treatment before deciding on the strategy of the treatment of the renal masses, especially in patients who had received such a treatment which induced an immunocompromised state .

Int J Epidemiol, 2004 Apr, 33(2), 367 - 73
Divergent female-male mortality ratios associated with different routine vaccinations among female-male twin pairs; Aaby P et al.; BACKGROUND: Observational studies have suggested that vaccinations have non-specific effects that differ by sex . In the absence of randomized trials, studies of female-male twin pairs would allow us to investigate whether an intervention had sex-specific effects on survival . We therefore examined mortality patterns among female-male twin pairs according to vaccination status . Design We identified female-male twin pairs using the population registers from one urban district and three rural studies from Guinea-Bissau and Senegal and examined the female-male mortality ratio (MR) according to the last vaccine received among pairs in which a death occurred before 18 months of age . As background information, we examined sex- and age-specific mortality patterns in the pre-vaccination era . Subjects In all, 626 female-male twin pairs identified between 1978 and 2000 . RESULTS: There was no sex difference in mortality for boys and girls in the pre-vaccination era . In the combined analysis of all studies, the female-male MR was 0.25 (95% CI: 0.05, 0.93) for pairs having received Bacille Calmette-Guerin (BCG) as the last vaccine, 7.33 (95% CI: 2.20, 38.3) for pairs having received diphtheria, tetanus, pertussis (DTP) as the last vaccine, and 0.40 (95% CI: 0.04, 2.44) for pairs having received measles vaccine as the last vaccine . The female-male MR varied significantly for BCG compared with DTP (exact test of homogeneity, P < 0.001) and for DTP compared with measles vaccine (exact test of homogeneity, P = 0.001) . CONCLUSION: Non-specific effects of routine vaccinations are likely to be important and influence sex-specific mortality patterns in areas with high mortality . The effects of vaccines need to be considered in the planning of immunization programmes for low-income countries.

Free Radic Biol Med, 2004 May 1, 36(9), 1126 - 33
Oxygen tension regulates reactive oxygen generation and mutation of Helicobacter pylori; Park AM et al.; Although both bacillary and coccoid forms of Helicobacter pylori reside in human stomach, the pathophysiological significance of the two forms remains obscure . The present work describes the effect of oxygen tension on the transformation and reactive oxygen species (ROS) metabolism of this pathogen . Most H . pylori cultured under an optimum O2 concentration (7%) were the bacillary form, whereas about 80% of cells cultured under aerobic or anaerobic conditions were the coccoid form . The colony-forming unit of H . pylori decreased significantly under both aerobic and anaerobic culture conditions . The bacillary form of H . pylori generated predominantly superoxide radical, whereas the coccoid form generated preferentially hydroxyl radical . Specific activities of cellular respiration, urease, and superoxide dismatase decreased markedly after transformation of the bacillary form to the coccoid form, with concomitant generation of protein carbonyls and 8-hydroxyguanine . The frequency of mutation of cells increased significantly during culture under nonoptimum O2 conditions . These results indicate that ROS generated by H . pylori catalyze the oxidative modification of cellular DNA, thereby enhancing the transformation from the bacillary to the coccoid form . The enhanced generation of mutagenic hydroxyl radicals in the coccoid form might accelerate mutation and increase the genetic diversity of H . pylori.

Biochem Biophys Res Commun, 2004 May 7, 317(3), 744 - 8
Trp132, Trp154, and Trp157 are essential for folding and activity of a Cyt toxin from Bacillus thuringiensis; Promdonkoy B et al.; Cyt2Aa2 is a cytolytic and mosquito larvicidal toxin produced by Bacillus thuringiensis subsp . darmstadiensis . The toxin contains 3 tryptophan residues at positions 132, 154, and 157 . To study the role of tryptophan on protein structure and functions, each tryptophan residue was substituted by phenylalanine and other different amino acids . Expression test in Escherichia coli showed that all mutant proteins were highly produced as inclusion bodies similar to that of the wild type . The mutant W157F showed haemolytic and mosquito larvicidal activities comparable to the wild type but the mutant W157V and all other mutants at positions 132 and 154 have completely lost these activities . Solubilization and proteinase K activation tests indicated that aromatic residue is required at position 157 and tryptophan residues at positions 132 and 154 are critical residues playing important role to maintain structure and functions of the protein and cannot be changed to any other amino acid.

Waste Manag, 2004, 24(4), 407 - 11
Improving the two-step remediation process for CCA-treated wood: Part II . Evaluating bacterial nutrient sources; Clausen CA; Remediation processes for recovery and reuse of chromated-copper-arsenate- (CCA) treated wood are not gaining wide acceptance because they are more expensive than landfill disposal . One reason is the high cost of the nutrient medium used to culture the metal-tolerant bacterium, Bacillus licheniformis, which removes 70-100% of the copper, chromium, and arsenic from CCA-treated southern yellow pine (CCA-SYP) in a two-step process involving oxalic acid extraction and bacterial culture . To reduce this cost, the nutrient concentration in the culture medium and the ratio of wood to nutrient medium were optimized . Maximum metal removal occurred when B . licheniformis was cultured in 1.0% nutrient medium and at a wood to nutrient medium ratio of 1:10 . Also, malted barley, an abundant by-product of brewing, was evaluated as an alternative nutrient medium . Tests were done to determine absorption of metals by barley, and the results indicate that the barley acted as a biosorbent, removing heavy metals from the liquid culture after their release from CCA to SYP . For comparison, tests were also performed with no nutrient medium . Following bacterial remediation, 17% copper and 15% arsenic were removed from an aqueous slurry of CCA-SYP (no medium) . When oxalic acid extraction preceded the aqueous bacterial culture, 21% copper, 54% chromium, and 63% arsenic were removed . The two-step process (oxalic acid extraction and bacterial culture with nutrient medium) appears to be an effective, yet costly, way to remove metals.

Waste Manag, 2004, 24(4), 401 - 5
Improving the two-step remediation process for CCA-treated wood: Part I . Evaluating oxalic acid extraction; Clausen C; In this study, three possible improvements to a remediation process for chromated-copper-arsenate- (CCA) treated wood were evaluated . The process involves two steps: oxalic acid extraction of wood fiber followed by bacterial culture with Bacillus licheniformis CC01 . The three potential improvements to the oxalic acid extraction step were (1) reusing oxalic acid for multiple extractions, (2) varying the ratio of oxalic acid to wood, and (3) using a noncommercial source of oxalic acid such as Aspergillus niger, which produces oxalic acid as a metabolic byproduct . Reusing oxalic acid for multiple extractions removed significant amounts of copper, chromium, and arsenic . Increasing the ratio of wood to acid caused a steady decline in metal removal . Aspergillus niger removed moderate amounts of copper, chromium, and arsenic from CCA-treated wood . Although A . niger was effective, culture medium costs are likely to offset any benefits . Repeated extraction with commercial oxalic acid appears to be the most cost-effective method tested for the two-step process.

Indian J Pediatr, 2004 Mar, 71(3), 221 - 7
Newer diagnostic modalities for tuberculosis; Lodha R et al.; The gold standard for diagnosis of tuberculosis is demonstration of mycobacteria from various body fluids . This is often not possible in children due to pauci-bacillary nature of illness . Significant improvement in understanding of molecular biology of Mycobacterium tuberculosis has led to development of newer diagnostic techniques of tuberculosis . Polymerase chain reaction (PCR) is an emerging diagnostic tool for diagnosis of TB in children . However, its role in day-to-day clinical practice needs to be defined . A negative PCR never eliminates possibility of tuberculosis, and a positive result is not always confirmatory . The PCR may be useful in evaluating children with significant pulmonary disease when diagnosis is not readily established by other means, and in evaluating immunocompromised children (HIV infection) with pulmonary disease . In the absence of good diagnostic methods for tuberculosis, a lot of interest has been generated in serodiagnosis . ELISA has been used to detect antibodies to various purified or complex antigens of M . tuberculosis in children . Despite a large number of studies published over the past several years, serology has found little place in the routine diagnosis of tuberculosis in children, even though it is rapid and does not require specimen from the site of disease . Sensitivity and specificity depend on the antigen used, gold standard for the diagnosis of tuberculosis and the type of tubercular infection . Though most of these tests have high specificity, their sensitivity is poor . In addition, these tests may be influenced by factors such as age, prior BCG vaccination and exposure to environmental mycobacteria . The serological tests, theoretically, may not be able to differentiate between infection and disease . At present, serodiagnosis does not appear to have any role in diagnosis of childhood pulmonary tuberculosis . A new test (QuantiFERON-TB or QFT) that measures the release of interferon-gamma in whole blood in response to stimulation by purified protein derivative is comparable with the tuberculin skin testing to detect latent tubercular infection, and is less affected by BCG vaccination . It can also discriminate responses due to nontuberculous mycobacteria, and avoids variability and subjectivity associated with placing and reading the tuberculin skin test . Polymerase chain reaction based test for identification of katG and rpoB mutation which are associated with isoniazid and rifampicin resistance may help in early identification of drug resistance in mycobacterium.

Biofouling, 2004 Feb, 20(1), 35 - 42
Biofouling on the walls of a spent nuclear fuel pool with radioactive ultrapure water; Chicote E et al.; Microbial activity in spent nuclear fuel pools which contain ultrapure and radioactive water has been previously observed . The aim of the present research was to isolate and identify the microorganisms attached to the nuclear pool wall of a Spanish nuclear power plant . Amplification of 16S rDNA fragments from the culturable microorganisms by PCR using universal primers for the domain 'Bacteria', followed by Denaturing Gradient Gel Electrophoresis analysis revealed the presence of six different bacteria . The complete gene for 16S rDNA of each one was sequenced and identified as belonging to three different phylogenetic groups, viz . beta-Proteobacteria, Actinomycetales and the Bacillus/Staphylococcus group . A fungus was also found and identified as Aspergillus fumigatus by sequencing the D2 region of the large subunit rDNA gene . The isolation of these microorganisms in oligotrophic and radioactive conditions is of great interest due to the possibility of their use in bioremediation processes of radionuclide-contaminated environments.

Biofouling, 2004 Feb, 20(1), 25 - 33
Influence of surface chemistry on the hygienic status of industrial stainless steel; Boulange-Petermann L et al.; Coupons of fourteen different stainless steels were investigated in terms of surface chemistry and ease of cleaning . Steel surfaces were exposed to Bacillus cereus spores in static saline solution for 2 h . Surfaces were rinsed and then covered with whole milk and allowed to dry . Surfaces were then cleaned in an experimental flow system that mimics an industrial application . After cleaning, remaining spores were released by sonication, spores cultured and colony forming units determined . Surfaces with higher levels of Fe in the outer surface of the passive film cleaned more easily . There was a relation between the polar component and ease of cleaning . The higher the polar component the more easily the surface cleaned . The cleaning mechanism involves dissolution of Fe enriched hydroxide films on the surface.

Mikrobiologiia, 2004 Jan-Feb, 73(1), 25 - 30
{Susceptibility of archaea to the antibiotic effect of the parasporal inclusion proteins from Bacillus thuringiensis subspecies}; Iudina TG et al.; The proteins of parasporal inclusions from three Bacillus thuringiensis subspecies (kurstaki, amagiensis, and monterrey) inhibited growth of methanogenic archaea of two species belonging to two genera, Methanobrevibacter arboriphilus and Methanosarcina barkeri . The minimal inhibitory concentrations of these proteins were 20 to 50 micrograms/ml . Lysozyme exhibited similar bactericidal effect on archaea . The perspective of comparative studies on the effect of polyfunctional proteins on bacteria and archaea is discussed.

Cancer, 2004 Apr 15, 100(8), 1692 - 8
Mature results of a phase III randomized trial of bacillus Calmette-Guerin (BCG) versus observation and BCG plus dacarbazine versus BCG in the adjuvant therapy of American Joint Committee on Cancer Stage I-III melanoma (E1673): a trial of the Eastern Oncology Group; Agarwala SS et al.; BACKGROUND: The local and systemic effects of bacillus Calmette-Guerin (BCG) have been known for decades . To investigate the adjuvant effect of BCG on resected American Joint Committee on Cancer (AJCC) Stage I-III melanoma, the Eastern Cooperative Oncology Group conducted a large trial to study the use of BCG alone or a combination of BCG and dacarbazine between 1974 and 1978 . METHODS: A total of 734 patients were randomized to 4 clinical groups consolidated into 2 cohorts . Cohort I compared BCG with observation and Cohort II compared BCG with a combination of BCG and dacarbazine . The primary end points were survival time and time to disease progression . RESULTS: Within Cohort I, no statistically significant difference in disease-free survival (DFS) (P = 0.84) or overall survival (OS) (5-year survival 67% vs . 62%; P = 0.40) was observed between BCG treatment and observation . Within Cohort II, the addition of dacarbazine to BCG did not improve DFS (P = 0.74) or OS (P = 0.81) compared with BCG alone . Toxicity was mild to moderate in both cohorts . Although toxicity with this agent is mild, the use of BCG is associated with the development of punctate abscesses in greater than two-thirds of patients treated . CONCLUSIONS: In what to our knowledge is the largest ever trial to test the role of BCG as adjuvant therapy for melanoma, no benefit for BCG was observed for patients with AJCC Stage I-III disease . The mature results of the current trial projected to 30 years confirmed the negative results of previous smaller studies utilizing this agent .

Urology, 2004 Apr, 63(4), 682 - 6; discussion 686-7
Intravesical bacille Calmette-Guérin versus mitomycin C in superficial bladder cancer: formal meta-analysis of comparative studies on tumor progression; Bohle A et al.; OBJECTIVES: To compare the therapeutic efficacy of intravesical bacille Calmette-Guerin (BCG) with mitomycin C (MMC) on progression of Stage Ta and T1 bladder carcinoma . METHODS: Combined published and unpublished data from comparative studies on BCG versus MMC in superficial bladder carcinoma were analyzed, considering possible confounding factors . Odds ratios (ORs) and 95% confidence intervals (CIs) were used as the primary effect size estimate . Tumor progression was defined as progression to a higher tumor stage or the development of metastatic disease . RESULTS: In nine eligible clinical trials, 1277 patients were treated with BCG and 1133 with MMC . Within the overall median follow-up of 26 months, 7.67% of the patients in the BCG group and 9.44% of the patients in the MMC group developed tumor progression . In all nine individual studies and in the combined results, no statistically significant difference in the ORs for progression between the BCG and MMC-treated groups was found (combined OR = 0.77; 95% CI 0.57 to 1.03; P = 0.081) . In the subgroup with BCG maintenance, the combined result of the five individual studies showed a statistically significant superiority of BCG over MMC (OR = 0.66; 95% CI 0.47 to 0.94; P = 0.02) . In the four studies without BCG maintenance, the combined result indicated no statistically significant difference between the two treatments (OR = 1.16; 95% CI 0.65 to 2.07; P = 0.612) . Potential confounders, such as tumor risk status, duration of follow-up, BCG strain, BCG and MMC treatment regimen, and year of publication did not significantly influence these results . CONCLUSIONS: The results demonstrated statistically significant superiority for BCG compared with MMC for the prevention of tumor progression only if BCG maintenance therapy was provided.

Toxicol Rev, 2003, 22(2), 83 - 90
Biopesticides; Sudakin DL; The term 'biopesticide' encompasses a broad array of microbial pesticides, biochemicals derived from micro-organisms and other natural sources, and processes involving the genetic incorporation of DNA into agricultural commodities that confer protection against pest damage (plant-incorporated protectants) . Some microbial pesticides, such as Bacillus thuringiensis, have a long history of safe and effective use as a biological insecticide . More recent developments in microbial pest control include the utilisation of other bacterial and fungal species that may competitively inhibit the growth of pathogenic and toxigenic micro-organisms on important agricultural commodities . The use of microbes and their gene products introduces additional considerations to the toxicological dose-response relationship, including a need to determine the plausibility of infectious and immunological effects in association with human exposure to these biopesticides in food or the environment . Studies of substantial equivalence suggest that foods currently derived from plant-incorporated protectants are not likely to differ from conventional foods . However, there is general consensus that the scientific methods to assess risks from genetically modified foods and micro-organisms will continue to evolve in the future.

Curr Opin Oncol, 2004 May, 16(3), 257 - 62
Bladder cancer; Borden LS Jr et al.; PURPOSE OF REVIEW: This article reviews recent advances in the diagnosis and management of bladder cancer . RECENT FINDINGS: Bladder cancer is a significant cause of morbidity and mortality . Recent research has attempted to improve the care of patients with this disease . Evidence suggests that bacillus Calmette-Guerin is the most effective intravesical therapy for the treatment of superficial bladder cancer and that maintenance therapy is superior to an induction course alone . In patients with muscle-invasive disease, nodal status and extent of lymphadenectomy have been shown to correlate with survival after radical cystectomy . The role of chemotherapy in the treatment of bladder cancer continues to evolve as well . Neoadjuvant chemotherapy has recently demonstrated a survival benefit, and trials are ongoing to define the optimal regimen of chemotherapy for urothelial carcinoma . SUMMARY: Improved understanding and advancements in the management of all stages of bladder cancer continue to improve the care of patients with this disease.

J Dairy Res, 2004 Feb, 71(1), 88 - 96
Protease-induced aggregation of bovine alpha-lactalbumin: identification of the primary associating fragment; Otte J et al.; Details in the hydrolysis of alpha-lactalbumin known to result in formation of highly ordered nanotubules, was investigated by incubation of solutions with 10 g alpha-lactalbumin/l with a specific protease from Bacillus licheniformis (BLP) . After 50 min of incubation, soluble aggregates were formed, the concentration of which increased until precipitation occurred after 200 min . The latter aggregates were dissolved in urea or at low pH, like the nanotubules characteristic of gels formed by the action of BLP on alpha-lactalbumin at 100 g/l . On the molecular level, alpha-lactalbumin was initially cleaved into two large hydrophobic fragments with masses of 11.6 and 11.3 kDa, which in turn were cleaved in a stepwise manner into the ultimate fragment of 8.8 kDa . This fragment was the predominating component in the insoluble aggregates, and was identified as the sequences 26-37 and 50-113 of alpha-lactalbumin linked together by a disulphide bond . Cleavage of alpha-lactalbumin into this fragment probably created new hydrophobic surfaces and new calcium binding sites allowing its association into ordered structures.

Appl Environ Microbiol, 2004 Apr, 70(4), 2161 - 71
Characterization of Bacillus probiotics available for human use; Duc le H et al.; Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties . Three B . cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize . Spores of one B . cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids . Spores of all strains were immunogenic when they were given orally to mice, but the B . pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers . Spores of B . pumilus and of a laboratory strain of B . subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B . pumilus and B . subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon . The B . pumilus strain and one B . cereus strain (B . cereus var . vietnami) were found to produce a bacteriocin-like activity against other Bacillus species . The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect . However, the three B . cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.

Appl Environ Microbiol, 2004 Apr, 70(4), 2052 - 60
Identification of the bacterial community of maple sap by using amplified ribosomal DNA (rDNA) restriction analysis and rDNA sequencing; Lagace L et al.; The bacterial community of maple sap was characterized by analysis of samples obtained at the taphole of maple trees for the 2001 and 2002 seasons . Among the 190 bacterial isolates, 32 groups were formed according to the similarity of the banding patterns obtained by amplified ribosomal DNA restriction analysis (ARDRA) . A subset of representative isolates for each ARDRA group was identified by 16S rRNA gene fragment sequencing . Results showed a wide variety of organisms, with 22 different genera encountered . Pseudomonas and Ralstonia, of the gamma- and beta-Proteobacteria, respectively, were the most frequently encountered genera . Gram-positive bacteria were also observed, and Staphylococcus, Plantibacter, and Bacillus were the most highly represented genera . The sampling period corresponding to 50% of the cumulative sap flow percentage presented the greatest bacterial diversity according to its Shannon diversity index value (1.1) . gamma-Proteobacteria were found to be dominant almost from the beginning of the season to the end . These results are providing interesting insights on maple sap microflora that will be useful for further investigation related to microbial contamination and quality of maple products and also for guiding new strategies on taphole contamination control.

J Biotechnol, 2004 Apr 29, 109(3), 255 - 62
Surfactant activity of a naphthalene degrading Bacillus pumilus strain isolated from oil sludge; Calvo C et al.; We studied the growth, biosurfactant activities and petroleum hydrocarbon compounds utilisation of strain 28-11 isolated from a solid waste oil . The isolate was identified as Bacillus pumilus . It grew well in the presence of 0.1% (w/v) of crude oil and naphthalene under aerobic conditions and utilised these substances as carbon and energy source . The capacity of strain 28-11 to emulsify crude oil and its ability to remove hydrocarbons looks promising for its application in environmental technologies.

J Ind Microbiol Biotechnol, 2004 May, 31(4), 161 - 9 Epub 2004 Apr 03.
Detection of Bacillus cereus group bacteria from cardboard and paper with real-time PCR; Priha O et al.; The aim of this study was to develop a PCR-based rapid method to detect Bacillus cereus group cells from paper and cardboard . Primers targeting the 16S rDNA and real-time PCR with SYBR green I detection were used in order to be able to also quantify the target . Both autoclaved cardboard samples spiked with B . cereus vegetative cells or spores and naturally contaminated paper and cardboard samples were studied . Results were compared with culturing verified by commercial (API) tests . Several different methods were tested for DNA isolation from the paper and cardboard samples . Two commercial kits intended for soils, the UltraClean soil DNA kit and the FastDNA spin kit for soil, gave the most reproducible results . In spiked samples, the average yield was 50% of added vegetative cells, but spore yield was only about 10% . PCR results from adding vegetative cells correlated with added colony-forming unit (cfu) values ( r=0.93, P <0.001) in the range 100-10,000 cfu g(-1) . Three out of nine studied paper and cardboard samples contained B . cereus group bacteria, based both on culturing and real-time PCR . The numbers were 10(2)-10(3) bacteria g(-1); and PCR gave somewhat higher results than culturing . Thus, real-time PCR can be used as a rapid semi-quantitative method to screen paper and cardboard samples for contamination with B . cereus group bacteria.

Vaccine, 2004 Mar 29, 22(11-12), 1498 - 508
Immunization with heat-killed Mycobacterium bovis bacille Calmette-Guerin (BCG) in Eurocine L3 adjuvant protects against tuberculosis; Haile M et al.; The current live attenuated vaccine against tuberculosis, BCG, poses a risk of disseminated infections in immunocompromised subjects . Therefore, in this study we compared the protective effect of a heat-killed bacille Calmette-Guerin (H-kBCG) vaccine given in a new adjuvant (Eurocine L3) with the protection provided by the conventional live attenuated BCG vaccine in mice (C57BL/6 and BALB/c) challenged with virulent Mycobacterium tuberculosis (strain Harlingen) . The H-kBCG vaccine alone, in accordance with earlier studies, did not give any or only gave slight protection compared to sham-vaccinated controls . However, the same vaccine given with Eurocine L3 adjuvant, either formulated as a suspension or as an emulsion, afforded significant levels of protection . This protection was at least as good as that of the control live attenuated BCG vaccine . The Eurocine L3 adjuvant is approved for human use as a nasal vaccine adjuvant and a successful phase I trial with nasal immunization with diphtheria vaccine has recently been performed in Sweden . Here we show that, in mice, intranasal priming with H-kBCG in Eurocine L3 adjuvant followed by intranasal booster resulted in the same level of protection as subcutaneous priming followed by intranasal booster . All H-kBCG formulations in the Eurocine L3 adjuvant elicited mycobacterial antigen-specific serum IgG and IFN gamma responses . In general, among the different vaccine formulation(s) in the Eurocine L3 adjuvant those that produced a relatively high Th2 response, as measured by IgG1/IgG2a ratio and IFN gamma production in vitro, were the most protective . In conclusion, H-kBCG in Eurocine L3 adjuvant could represent a safe and a more stable alternative to the conventional live BCG vaccine.

Biochim Biophys Acta, 2004 Apr 8, 1698(1), 1 - 26
Electron transfer by diflavin reductases; Murataliev MB et al.; Diflavin reductases are enzymes which emerged as a gene fusion of ferredoxin (flavodoxin) reductase and flavodoxin . The enzymes of this family tightly bind two flavin cofactors, FAD and FMN, and catalyze transfer of the reducing equivalents from the two-electron donor NADPH to a variety of one-electron acceptors . Cytochrome P450 reductase (P450R), a flavoprotein subunit of sulfite reductase (SiR), and flavoprotein domains of naturally occurring flavocytochrome fusion enzymes like nitric oxide synthases (NOS) and the fatty acid hydroxylase from Bacillus megaterium are some of the enzymes of this family . In this review the results of the last decade of research are summarized, and some earlier results are reevaluated as well . The kinetic mechanism of cytochrome c reduction is analyzed in light of other results on flavoprotein interactions with nucleotides and cytochromes . The roles of the binding sites of the isoalloxazine rings of the flavin cofactors and conformational changes of the protein in electron transfer are discussed . It is proposed that minor conformational changes during catalysis can potentiate properties of the redox centers during the catalytic turnover . A function of the aromatic residue that shields the isoalloxazine ring of the FAD is also proposed.

Carbohydr Res, 2004 Apr 28, 339(6), 1179 - 84
Purification and characterization of an intracellular cycloalternan-degrading enzyme from Bacillus sp . NRRL B-21195; Kim YK et al.; A novel intracellular cycloalternan-degrading enzyme (CADE) was purified to homogeneity from the cell pellet of Bacillus sp . NRRL B-21195 . The enzyme has a molecular mass of 125 kDa on SDS-PAGE . The pH optimum was 7.0, and the enzyme was stable from pH 6.0 to 9.2 . The temperature optimum was 35 degrees C and the enzyme exhibited stability up to 50 degrees C . The enzyme hydrolyzed cycloalternan {CA; cyclo(-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(-->3)-alpha-d-Glcp-(1-->)} as the best substrate, to produce only isomaltose via an intermediate, alpha-isomaltosyl-(1-->3)-isomaltose . This enzyme also hydrolyzed isomaltosyl substrates, such as panose, alpha-isomaltosyl-(1-->4)-maltooligosaccharides, alpha-isomaltosyl-(1-->3)-glucose, and alpha-isomaltosyl-(1-->3)-isomaltose to liberate isomaltose . Neither maltooligosaccharides nor isomaltooligosaccharides were hydrolyzed by the enzyme, indicating that CADE requires alpha-isomaltosyl residues connected with (1-->4)- or (1-->3)-linkages . The K(m) value of cycloalternan (1.68 mM) was 20% of that of panose (8.23 mM) . The k(cat) value on panose (14.4s(-1)) was not significantly different from that of cycloalternan (10.8 s(-1)) . Judging from its specificity, the systematic name of the enzyme should be cycloalternan isomaltosylhydrolase . This intracellular enzyme is apparently involved in the metabolism of starch via cycloalternan in Bacillus sp . NRRL B-21195, its role being to hydrolyze cycloalternan inside the cells.

J Microbiol Methods, 2004 May, 57(2), 269 - 78
Designing better probes: effect of probe size, mismatch position and number on hybridization in DNA oligonucleotide microarrays; Letowski J et al.; DNA microarrays represent a powerful technology whose use has been hampered by the uncertainty of whether the same principles, established on a scale typical for membrane hybridizations, apply when using the smaller, rigid support of microarrays . Our goal was to understand how the number and position of base pair mismatches, probe length and their G+C content affect the intensity and specificity of the hybridization signal . One set of oligonucleotides (50-mers) based on three regions of the Bacillus thuringiensis cry1Aa1 gene possessing 30%, 42%, and 56% G+C content, a second set with similar G+C content (37% to 40%) but different lengths (30 to 100 bases), and finally amplicon probes (101 to 3000 base pairs) with G+C contents of 37% to 39%, were used . Probes with mismatches distributed over their entire length were the most specific, while those with mismatches grouped at either the 3' or 5'-end were the least specific . Hybridizations done at 8 to 13 degrees C below the calculated T(m) of perfectly matched probes, as compared to the widely used lower temperatures of 20 to 25 degrees C, enhanced probe discrimination . Longer probes produced higher fluorescent hybridization signals than shorter ones . These results should help to optimize the design of oligonucleotide-based DNA microarrays.

Curr Microbiol, 2004 May, 48(5), 321 - 6
Enhanced expression of insecticidal crystal proteins in wild Bacillus thuringiensis strains by a heterogeneous protein P20; Shao Z et al.; P20 is a small chaperone-like protein encoded by a cry11A operon in Bacillus thuringiensis subsp . israelensis (Bti); it is essential to Cyt1Aa expression . In this report, the gene P20 was transformed into wild strains of subsp . kurstaki to raise the yield of crystal proteins . As a result, larger crystals were produced by the transformant than by the wild control, and most were in the form of a big bipyramid (average 2.4 microm long); some were irregular because of too high expression, while the spores turned out to be small spheroids unlike the long rods in the wild strains . SDS-PAGE analysis confirmed that Cry1A protoxin production was doubled by P20, but no increase of Cry2A production was observed . Besides, P20 caused obvious changes not only in bacterial morphology, but in the sporulation process as well . Further investigation discovered that a serious degradation happened to Cry1A in vivo of the wild strains, and reconfirmed that P20 was effective in preventing the degradation . Our results suggest that P20 is useful in engineered strain construction with enhanced protein expression.

Am J Respir Crit Care Med, 2004 Jul 1, 170(1), 59 - 64 Epub 2004 Apr 01.
Specific detection of tuberculosis infection: an interferon-gamma-based assay using new antigens; Mori T et al.; The tuberculin skin test for immunologic diagnosis of Mycobacterium tuberculosis infection has many limitations, including being confounded by bacillus Calmette-Guerin (BCG) vaccination or exposure to nontuberculous mycobacteria . M . tuberculosis-specific antigens that are absent from BCG and most nontuberculous mycobacteria have been identified . We examined the use of two of these antigens, CFP-10 and ESAT-6, in a whole blood IFN-gamma assay as a diagnostic test for tuberculosis in BCG-vaccinated individuals . Because of the lack of an accurate standard with which to compare new tests for M . tuberculosis infection, specificity of the whole blood IFN-gamma assay was estimated on the basis of data from people with no identified risk for M . tuberculosis exposure (216 BCG-vaccinated Japanese adults) and sensitivity was estimated on the basis of data from 118 patients with culture-confirmed M . tuberculosis infection who had received less than 1 week of treatment . Using a combination of CFP-10 and ESAT-6 responses, the specificity of the test for the low-risk group was 98.1% and the sensitivity for patients with M . tuberculosis infection was 89.0% . The results demonstrate that the whole blood IFN-gamma assay using CFP-10 and ESAT-6 was highly specific and sensitive for M . tuberculosis infection and was unaffected by BCG vaccination status.

Lett Appl Microbiol, 2004, 38(5), 393 - 9
Expression and characterization of a recombinant Cry1Ac crystal protein with enhanced green fluorescent protein in acrystalliferous Bacillus thuringiensis; Roh JY et al.; AIMS: To investigate fusion expression between Bacillus thuringiensis crystal protein and a foreign protein, the expression of a fusion protein comprised of Cry1Ac, and enhanced green fluorescent protein (EGFP) in B . thuringiensis Cry(-)B strain was examined . METHODS AND RESULTS: The N-terminal fusion expression of EGFP in Cry1Ac was attempted under the control of the native cry1Ac promoter . The EGFP gene was cloned into pProMu and named pProMu-EGFP . The transformant, ProMu-EGFP/CB produced parasporal inclusions that were of bipyramidal-shaped crystals in size ranging from 200 to 300 nm . The fusion protein was approximately 150 kDa and identified by the immunoblot analysis using a Cry1Ac antibody and also a GFP antibody . The LC(50) of the ProMu-EGFP/CB was twofold higher when compared with that by the ProAc/CB . However, the crystal protein produced by the ProMu-EGFP/CB was effective on Plutella xylostella larvae . CONCLUSIONS: The ProMu-EGFP/CB produced bipyramidal shaped and insecticidal crystals comprising fusion proteins . SIGNIFICANCE AND IMPACT OF THE STUDY: Through the N-terminal fusion expression of EGFP and Cry1Ac, expression and crystallization between the B . thuringiensis crystal protein and a foreign protein were validated.

Pediatr Allergy Immunol, 2004 Apr, 15(2), 148 - 51
Cytokines evaluation in nasal lavage of allergic children after Bacillus clausii administration: a pilot study; Ciprandi G et al.; Respiratory infections are very frequent in children . Bacillus clausii has been demonstrated to exert some immunomodulatory activities and to be safe . We conducted a study to investigate whether B . clausii administration in allergic children with recurrent respiratory infections might modulate cytokine pattern . Ten children (mean age 4.4 yr) attending the nursery school were enrolled at the end of school year (i.e . in the summer) . Bacillus clausii spores (Enterogermina): 2 billion spores per vial) were administered at the dosage schedule of two vials a day for 4 wk . A panel of cytokines, including interleukin (IL)-1, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, interferon (IFN)-gamma, transforming growth factor (TGF)-beta, and tumor necrosis factor (TNF)-alpha, was measured by immunoassay in the fluid recovered from nasal lavage, performed before and after the treatment . Bacillus clausii treatment induced a significant decrease of IL-4 levels (p < 0.01) and a significant increase of IFN-gamma (p < 0.05), IL-12 (p < 0.001), TGF-beta (p < 0.05), and IL-10 (p < 0.05) levels . Other cytokines were not significantly modified . In conclusion, this study shows that the B . clausii may exert immunomodulating activity by affecting cytokine pattern at nasal level in allergic children with recurrent respiratory infections.

Curr Microbiol, 2004 Feb, 48(2), 153 - 8
Endospore degradation in an oligosporogenic, crystalliferous mutant of Bacillus thuringiensis; Sierra-Martinez P et al.; We isolated a new oligosporogenic mutant from Bacillus thuringiensis var . kurstaki HD73 that retains the ability to produce insecticidal crystal inclusions . Sporulation in this mutant initiates in a manner similar to the wild-type strain, and under the electron microscope endospores are seen, but these do not reach maturity (except for 0.2% of them) . At a late stage, the coat surrounding the forespore seems to lack shape and to be empty . Most mutant cells exhibit a well-formed bipyramidal crystal but are completely devoid of the forespore . The mutant has a functional SigK holoenzyme, which is required for the expression of genes involved in the formation of spore coat and cortex and for cry1A transcription from the BtII promoter . Defective maturation of spores could be due to an inadequate forespore coat or cortex structure resulting in the arrest of sporulation at late stage III or early stage IV.

Curr Microbiol, 2004 Mar, 48(3), 196 - 8
Effect of fatty acids on the membrane potential of an alkaliphilic bacillus; Furusawa H et al.; Effect of various fatty acids on the membrane potential of an alkaliphilic Bacillus, YN-2000, was examined . Addition of unsaturated fatty acids such as palmitoleic acid, oleic acid, linoleic acid, and linolenic acid at 30 microM caused the instantaneous depolarization of the membrane potential of the bacterium, which appears to result in the drastic decrease of viability . On the other hand, no depolarization was detected by the addition of saturated acids such as palmitic acid, stearic acid, and 12-hydroxystearic acid even at 1 mM.

Curr Microbiol, 2004 Mar, 48(3), 175 - 81
Expression of chitinase-encoding genes in Bacillus thuringiensis and toxicity of engineered B . thuringiensis subsp . aizawai toward Lymantria dispar larvae; Lertcanawanichakul M et al.; Chitinase genes from Aeromonas hydrophila and Bacillus circulans No.4.1 were cloned into the plasmid pHY300PLK and designated as pHYA2 and pHYB43, respectively . Both plasmids were introduced into various strains of B . thuringiensis by electroporation . Plasmid pHYB43 was generally structurally stable, but showed lower segregrational stability than pHYA2 in B . thuringiensis subsp . aizawai when grown under nonselective conditions . The production of chitinase from B . thuringiensis subsp . aizawai harboring pHYB43 or pHYA2 could be detected after native polyacrylamide gel electrophoresis by using 4-methylumbelliferyl beta-D- N,N'- diacetylchitobioside as the substrate . Moreover, B . thuringiensis subsp . aizawai harboring pHYB43 gave 15 times higher chitinase activity than when harboring pHYA2, as determined by means of a colorimetric method using glycol chitin as the substrate . In addition, B . thuringiensis subsp . aizawai harboring pHYB43 was more toxic to gypsy moth larvae ( Lymantria dispar) than parental B . thuringiensis subsp . aizawai or its clone harboring pHYA2.

Curr Microbiol, 2004 Apr, 48(4), 280 - 4
Site-directed mutagenesis of the conserved threonine, tryptophan, and lysine residues in the starch-binding domain of Bacillus sp . strain TS-23 alpha-amylase; Lo HF et al.; The C-terminal domain of Bacillus sp . strain TS-23 alpha-amylase (BLA) has been known to be involved in the raw starch-binding activity of the enzyme . Sequence comparison revealed that Thr-527, Trp-545, Trp-561, Lys-576, and Trp-588 in this domain are highly conserved in the aligned enzymes . To understand structure-function relationships in the starch-binding domain of BLA, site-directed mutagenesis was conducted to replace these residues with leucine or isoleucine . The overexpressed enzymes have been purified by nickel-chelate chromatography, and the molecular mass of the purified proteins was approximately 64.5 kDa . Starch-binding assay showed that the binding activities of the single-mutated enzymes were significantly reduced, while the combinational mutations did not lead to a complete loss of the activity.

Curr Microbiol, 2004 Apr, 48(4), 270 - 5
Dissection of cry gene profiles of Bacillus thuringiensis isolates in Taiwan; Chen FC et al.; On the basis of the newly revised nomenclature system of cry genes, the PCR amplification method has been adopted to resolve the cry gene combinations of 294 Bacillus thuringiensis isolates from five selected areas of Taiwan . Our results indicate that cry1 (especially cry1A + 1B + 1F) and cry2 were the most abundant cry genes in Taiwan . In contrast, cry3 and cry6 genes were detected only on Yang Ming Mountain, while the cry13 gene was found only on Snow Mountain . In addition, some distinctive combinations of cry genes were detected in distinct areas of Taiwan, such as cry1C, cry1D, cry1C + 1D, cry4, cry1 + 4, cry1 + 11, cry4 + 11, and cry1 + 4 + 11 in the Taipei area; cry1A + 1C + 1F in the Taichung area; cry1E and cry1A + 1B + 1I on Yang Ming Mountain; cry1 + 13, cry1 + 2 + 11, and cry1 + 2 + 13 on Snow Mountain; and cry1 + 5 and cry1 + 2 + 5 on Jade Mountain . These data clearly indicate that the distribution of cry gene combinations of B . thuringiensis isolates seems to be geographically related.

Mem Inst Oswaldo Cruz, 2004 Feb, 99(1), 73 - 9 Epub 2004 Mar 31.
In vivo binding of the Cry11Bb toxin of Bacillus thuringiensis subsp . medellin to the midgut of mosquito larvae (Diptera: Culicidae); Ruiz LM et al.; Bacillus thuringiensis subsp . medellin produces numerous proteins among which 94 kDa known as Cry11Bb, has mosquitocidal activity . The mode of action of the Cry11 proteins has been described as similar to those of the Cry1 toxins, nevertheless, the mechanism of action is still not clear . In this study we investigated the in vivo binding of the Cry11Bb toxin to the midgut of the insect species Anopheles albimanus, Aedes aegypti, and Culex quinquefasciatus by immunohistochemical analysis . Spodoptera frugiperda was included as negative control . The Cry11Bb protein was detected on the apical microvilli of the midgut epithelial cells, mostly on the posterior midgut and gastric caeca of the three mosquito species . Additionally, the toxin was detected in the Malpighian tubules of An . albimanus, Ae . aegypti, Cx . quinquefasciatus, and in the basal membrane of the epithelial cells of Ae . aegypti midgut . No toxin accumulation was observed in the peritrophic membrane of any of the mosquito species studied . These results confirm that the primary site of action of the Cry11 toxins is the apical membrane of the midgut epithelial cells of mosquito larvae.

Mem Inst Oswaldo Cruz, 2004 Feb, 99(1), 57 - 62 Epub 2004 Mar 31.
Therapy of Venezuelan patients with severe mucocutaneous or early lesions of diffuse cutaneous leishmaniasis with a vaccine containing pasteurized Leishmania promastigotes and bacillus Calmette-Guerin: preliminary report; Convit J et al.; Severe mucocutaneous (MCL) and diffuse (DCL) forms of American cutaneous leishmaniasis (ACL) are infrequent in Venezuela . Chemotherapy produces only transitory remission in DCL, and occasional treatment failures are observed in MCL . We have evaluated therapy with an experimental vaccine in patients with severe leishmaniasis . Four patients with MCL and 3 with early DCL were treated with monthly intradermal injections of a vaccine containing promastigotes of Leishmania (Viannia) braziliensis killed by pasteurization and viable Bacillus Calmette- Guerin . Clinical and immunological responses were evaluated . Integrity of protein constituents in extracts of pasteurized promastigotes was evaluated by gel electrophoresis . Complete remission of lesions occurred after 5-9 injections in patients with MCL or 7-10 injections in patients with early DCL . DCL patients developed positive skin reactions, average size 18.7 mm . All have been free of active lesions for at least 10 months . Adverse effects of the vaccine were limited to local reactivity to BCG at the injection sites and fever in 2 patients . Extracts of pasteurized and fresh promastigotes did not reveal differences in the integrity of protein components detectable by gel electrophoresis . Immunotherapy with this modified vaccine offers an effective, safe option for the treatment of patients who do not respond to immunotherapy with vaccine containing autoclaved parasites or to chemotherapy.

Biosci Biotechnol Biochem, 2004 Mar, 68(3), 523 - 8
Functional analysis of two processed fragments of Bacillus thuringiensis Cry11A toxin; Yamagiwa M et al.; The 70-kDa protoxin of Cry11A, a dipteran-specific insecticidal protein, was processed by trypsin into 36- and 32-kDa fragments . To investigate the potent function of the two processed fragments, a GST (Glutathione-S-transferase) fusion protein of each polypeptide was constructed . While neither the 36- nor the 32-kDa fragment was toxic to Culex pipiens larvae, coexpression of the two fragments restored the insecticidal activity . Furthermore, the coprecipitation experiment demonstrated that the 36-kDa fragment was associated with the 32-kDa fragment . It was, therefore, shown that the coexistence of the two processed fragments of Cry11A was essential for the toxicity . The mutant of the 36-kDa fragment lacking the region from Gly(257) to Arg(360) bound to the 32-kDa fragment but the coexpression with the 32-kDa fragment resulted in no toxicity, suggesting that this region was involved in insecticidal activity.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2004, 39(3), 681 - 91
Characterization and nickel sorption kinetics of a new metal hyper-accumulator Bacillus sp; Zaidi S et al.; The heavy metal-resistant bacterial strain SJ-101 has been isolated from fly ash contaminated soil . Based on the morphological and biochemical characteristics, the isolate SJ-101 was presumptively identified as Bacillus sp . The adsorption isotherms revealed the absolute adsorption capacity (Q degrees) of 244 mg Ni g(-1) dry cell mass vis-a-vis 161 mg Ni g(-1) synthetic resin (Amberlite IR-120) . The higher relative adsorption capacity (K(F)) of 7.37, and the intensity of adsorption (1/n) of 0.58 with dry cell biomass suggested higher affinity of Bacillus cells towards nickel ions . The data conform to the Langmuir adsorption model relatively better than the Freundlich model . The thermodynamic parameters indicated the feasibility, endothermic, and interactive nature of nickel adsorption process on the cell surface . Higher Ni tolerance and sorption capacity of Bacillus sp . SJ-101, explicitly signifies its implications in Ni bioremediation process.

J Agric Food Chem, 2004 Apr 7, 52(7), 2097 - 102
Evaluation of Bt (Bacillus thuringiensis) corn on mouse testicular development by dual parameter flow cytometry; Brake DG et al.; The health safety of Bt (Bacillus thuringiensis) corn (Zea mays L.) was studied using mouse testes as a sensitive biomonitor of potential toxic effects . Pregnant mice were fed a Bt corn or a nontransgenic (conventional) diet during gestation and lactation . After they were weaned, young male mice were maintained on the respective diets . At 8, 16, 26, 32, 63, and 87 days after birth, three male mice and an adult reference mouse were killed, the testes were surgically removed, and the percentage of germ cell populations was measured by flow cytometry . Multigenerational studies were conducted in the same manner . There were no apparent differences in percentages of testicular cell populations (haploid, diploid, and tetraploid) between the mice fed the Bt corn diet and those fed the conventional diet . Because of the high rate of cell proliferation and extensive differentiation that makes testicular germ cells highly susceptible to some toxic agents, it was concluded that the Bt corn diet had no measurable or observable effect on fetal, postnatal, pubertal, or adult testicular development . If data from this study were extrapolated to humans, Bt corn is not harmful to human reproductive development.

Syst Appl Microbiol, 2004 Feb, 27(1), 50 - 60
Polyphasic characterization of Bacillus coagulans strains, illustrating heterogeneity within this species, and emended description of the species; De Clerck E et al.; Because of its food spoiling capacity on the one hand and its significant role in the production of industrially valuable products on the other, Bacillus coagulans is of economic concern . Several studies have revealed a great deal of diversity within the species and this has led to a number of taxonomic adjustments . The present study aims to clarify the diversity within Bacillus coagulans sensu stricto and determine the taxonomic status of the species . Therefore, a polyphasic study was performed on a set of B . coagulans strains from diverse habitats . Techniques as ARDRA, SDS-PAGE of whole cell proteins, FAME analysis, routine phenotypic tests and rep-PCR illustrate considerable intra-species heterogeneity, while 16S rDNA sequence comparison and DNA-DNA relatedness support the accommodation of these strains in one species . Although most techniques demonstrate appreciable heterogeneity among the Bacillus coagulans strains, the intraspecies groupings are not consistent throughout all the methods applied and are not supported by any economic, historic or practical traits . Therefore, a division in subspecies seems inappropriate . In attempt to achieve a better species delineation, an emended description of Bacillus coagulans is included.

PDA J Pharm Sci Technol, 2004 Jan-Feb, 58(1), 6 - 14
Effect of terminal sterilization by irradiation on amber Type I and Type III glass containers containing veterinary oxytetracycline suspension; Wong J et al.; The purpose of this study is to determine the effect of terminal sterilization by gamma irradiation on 500-mL amber Type I and Type III glass containers (bottles) containing oxytetracycline (OTC) (25% W/V) suspension formulated using 20% (W/V) phospholipids syrup . The formulation was developed for veterinary parenteral administration . The results of terminal sterilization were used to assess the acceptability for this suspension product . OTC is light-sensitive and needs to be stored in amber glass containers . Amber Type I and Type III glass containers were considered for this formulation during development . Type I is a highly resistant, borosilicate glass (oxidized glass) . Type III, which is soda-lime glass (reduced glass, see Materials and Methods), may also be used for parenteral products . The amber Type I and Type III glass containers, containing the suspension, were irradiated at 20-40 kGy for 150 min . In order to determine the sterility of the suspension, ampoules of the biological indicator, Bacillus pumilus, were placed in a number of bottles of the suspension . The bottles with the biological indicators were then positioned among the rest of the production bottles as per a radiation dose-mapping study conducted at the sterilization facility . Potency determination and stability evaluations for OTC were performed using high pressure liquid chromatography (HPLC) . The results indicate that the gamma irradiation dose of 20-40 kGy for 150 min was able to inactivate 10(6) Bacillus pumilus spores in ampoules . After gamma sterilization, OTC concentration, pH, particle size, endotoxins, and sterility were evaluated . The assay results were comparable for the suspension in amber Type I and Type III glass containers . Sterility and pyrogenicity were measured by the USP Membrane Filtration Method and USP Bacterial Endotoxins Test, respectively . The suspension in the amber Type III glass container was also chemically (HPLC) and physically (suspension mean particle size, viscosity, density, and syringeability) stable for at least 12 months at room temperature . However, amber Type I glass darkened following irradiation, leading to a substantial reduction in glass transparency . The appearance of amber Type III glass was acceptable . Thus, the "reduced" glass, such as soda-lime, was found to be much less susceptible to darkening than the highly oxidized borosilicate amber . Due to the potential aesthetic concerns with Type I glass, the amber Type III glass container was selected for this suspension formulation.

Rocz Panstw Zakl Hig, 2003, 54(4), 345 - 53
{Detection of genetic modification in maize and maize products by ELISA-test}; Urbanek-Karlowska B et al.; Enzyme immunoassay methods--TRAIT Test--was applied for detection of genetic modification in maize seeds and foodstuffs, which have been produced from this crop . TRAIT Test is based on the identification GMO protein Cry 1Ab produced by a gene derived from Bacillus thuringiensis (Bt) incorporated into insect resistant corn grain . The experiment was carried out on maize standards and foodstuffs from Warsaw market . The positive result was obtained for one maize product, which was not labelled as GMO . The presence of GMO material was approximately equal to 1% . In conclusion, this test is proper for fast routine qualitative (yes/no) determination GMO material in maize seeds and unprocessed food products.

Biotechnol Bioeng, 2004 Apr 20, 86(2), 174 - 87
New-generation multicistronic expression platform: pTRIDENT vectors containing size-optimized IRES elements enable homing endonuclease-based cistron swapping into lentiviral expression vectors; Fux C et al.; Capitalizing on a proven multicistronic expression vector platform we have designed novel pTRIDENT vectors which (1) . enable coordinated expression of three desired transgenes, (2) . are size-optimized, (3) . take advantage of small highly efficient internal ribosome entry sites of the GTX or Rbm3 type, (4) . harbor various sites specific for homing endonucleases facilitating promoter/multicistronic expression unit/polyadenylation site swapping as well as (5) . straightforward integration into human HIV-l-based lentiviral expression vectors tailored to contain compatible homing endonucleases . Multicistronic expression profiles of novel pTRIDENT vectors engineered for different tricistronic expression configurations encoding human low-molecular-weight urokinase-type plasminogen activator (u-PA(LMW)) or Bacillus stearothermophilus-derived alpha-amylase (SAMY), human vascular endothelial growth factor (hVEGF), and human placental secreted alkaline phosphatase (SEAP) have been quantified in Chinese hamster ovary cells (CHO-K1), mouse fibroblasts (NIH/3T3), and/or human fibrosarcoma (HT-1080) cells . In addition, a pTRIDENT-derived SAMY-VEGF-SEAP expression cassette transferred into a compatible lentiviral expression vector enabled simultaneous high-level transgene expression following transduction of transgenic lentiviral particles into primary human chondrocytes .

Anal Biochem, 2004 Apr 15, 327(2), 278 - 83
Tight attachment of chitin-binding-domain-tagged proteins to surfaces coated with acetylated chitosan; Bernard MP et al.; Several excellent procedures for trapping tagged proteins have been devised, but many of these are expensive, cannot be used outside a limited pH range, fail to work in the presence of chaotropic agents, or are difficult to use . The chitin binding domain (CBD) of Bacillus circulans chitinase, which binds to chitin matrices prepared from inexpensive reagents isolated from crab shells, is an alternative tag that can be used under a variety of pH and denaturing conditions . Kits based on the interaction between the CBD and the chitin beads are available commercially . Here, we show that simultaneous treatment of microtiter plates with chitosan, a deacetylated form of chitin, and acetic anhydride produces a surface-bound film of chitin that also interacts tightly with the CBD . Chitin-coated microtiter well plates captured a CBD-tagged heterodimeric human glycoprotein hormone analog directly from mammalian cell culture media, even when present in trace amounts . Binding to the surface was stable in sodium dodecylsulfate and reversed only partially at low pH or in 8M urea at 37 degrees C . This technique appears well suited to surface attachment and permits biochemical or other analyses of molecules that can be tagged with a CBD.

J Invertebr Pathol, 2004 Feb, 85(2), 120 - 7
The Bacillus thuringiensis Cry1Aa toxin: effects of trypsin and chymotrypsin site mutations on toxicity and stability; Bah A et al.; The objective of the present work was to create an active Cry1Aa toxin showing enhanced resistance to degradation by spruce budworm (Choristoneura fumiferana) midgut proteases by mutating potential chymotrypsin and trypsin sites . Fourteen Cry1Aa mutants were created in an Escherichia coli-Bacillus shuttle vector and expressed in a crystal minus Bacillus thuringiensis host . Using spruce budworm gut juice, commercial bovine trypsin and chymotrypsin we performed protease resistance assays with Cry1Aa wild type and mutant toxins . Although many mutants showed little or no change, several mutants showed a > 2-fold increase (R543S, R566G, and F570S) up to a > 4-fold increase in toxicity (F576S), in bioassay studies against C . fumiferana . The in vitro protease resistance assay results indicated a possible involvement of other gut juice components in toxin overdigestion.

Brain Behav Immun, 2004 May, 18(3), 223 - 30
Interleukin-1beta mediates the memory impairment associated with a delayed type hypersensitivity response to bacillus Calmette-Guérin in the rat hippocampus; Palin K et al.; Interleukin-1beta (IL-1beta) plays a major role in the initiation and exacerbation of brain inflammation, and its action is limited by the natural antagonist of IL-1 receptors, IL-1Ra . The aim of the present study was to test the hypothesis that IL-1beta mediates the functional consequences of inflammation during the course of delayed-type hypersensitivity response to bacillus Calmette-Guerin (BCG) in the hippocampus of Lewis rats . Animals were primed with an injection of BCG in the right hippocampus and challenged 4 weeks later with BCG administered subcutaneously . Concentrations of IL-1beta and IL-1Ra were measured by ELISA in the BCG injected hippocampus and compared to those measured in the contralateral hippocampus during the first 2 weeks post-challenge . IL-1beta levels increased in response to BCG challenge and peaked 12 days after challenge . The same variations appeared in the contralateral hippocampus but to a lesser extent . Hippocampal IL-1Ra levels increased in response to intrahippocampal injection of BCG . They further increased at days 6 and 9 post-challenge and decreased from day 12 back to baseline values on day 16 . The increase in IL-1beta levels and the decline in IL-1Ra levels were associated with an impairment in spatial memory in a Y-maze on day 16 post-challenge, that was abrogated by chronic administration of IL-1Ra via a subcutaneously implanted osmotic minipump geared to deliver 7 mg IL-1Ra/day . These results show that overexpression of IL-1beta in the brain during the course of a chronic inflammation has deleterious consequences on cognitive processes, that are reversed by blockade of IL-1 receptors.

Joint Bone Spine, 2004 Mar, 71(2), 150 - 3
Tuberculous sacroiliitis . Four cases; Benchakroun M et al.; Tuberculous sacroiliitis with no local abscesses or tuberculosis at other sites occurred in four patients, three men and one woman with a mean age of 42 years . Slow progression characterized this uncommon variant of joint tuberculosis . Two patients reported contact with family members known to have tuberculosis . Mean time from symptom onset to presentation was 14 months . Surgical biopsy showed epithelioid and giant-cell granulomas with caseous necrosis; culturing on Lowenstein-Jensen medium recovered the tubercle bacillus . Treatment was with antitubercular drugs, and protection from weight bearing for 4 weeks . These four cases illustrate the slowly progressive course of the clinical and radiological manifestations and underline the diagnostic difficulties met in the early stages of tuberculous sacroiliitis without tuberculosis at other sites . Antitubercular treatment can restore joint function provided the diagnosis is made before radiological destruction occurs.

Mol Microbiol, 2004 Apr, 52(1), 81 - 92
The VirB type IV secretion system of Bartonella henselae mediates invasion, proinflammatory activation and antiapoptotic protection of endothelial cells; Schmid MC et al.; Bartonella henselae is an arthropod-borne zoonotic pathogen causing intraerythrocytic bacteraemia in the feline reservoir host and a broad range of clinical manifestations in incidentally infected humans . Remarkably, B . henselae can specifically colonize the human vascular endothelium, resulting in inflammation and the formation of vasoproliferative lesions known as bacillary angiomatosis and bacillary peliosis . Cultured human endothelial cells provide an in vitro system to study this intimate interaction of B . henselae with the vascular endothelium . However, little is known about the bacterial virulence factors required for this pathogenic process . Recently, we identified the type IV secretion system (T4SS) VirB as an essential pathogenicity factor in Bartonella, required to establish intraerythrocytic infection in the mammalian reservoir . Here, we demonstrate that the VirB T4SS also mediates most of the virulence attributes associated with the interaction of B . henselae during the interaction with human endothelial cells . These include: (i) massive rearrangements of the actin cytoskeleton, resulting in the formation of bacterial aggregates and their internalization by the invasome structure; (ii) nuclear factor kappaB-dependent proinflammatory activation, leading to cell adhesion molecule expression and chemokine secretion, and (iii) inhibition of apoptotic cell death, resulting in enhanced endothelial cell survival . Moreover, we show that the VirB system mediates cytostatic and cytotoxic effects at high bacterial titres, which interfere with a potent VirB-independent mitogenic activity . We conclude that the VirB T4SS is a major virulence determinant of B . henselae, required for targeting multiple endothelial cell functions exploited by this vasculotropic pathogen.

Curr HIV Res, 2003 Oct, 1(4), 441 - 6
Use of fusion proteins and procaryotic display systems for delivery of HIV-1 antigens: development of novel vaccines for HIV-1 infection; De Berardinis P et al.; Two non-pathogenic scaffolds (represented by the filamentous bacteriophage fd and the dihydrolipoyl acetyltransferase E2 protein of the Bacillus stearothermophilus pyruvate dehydrogenase (PDH) complex) able to deliver human immunodeficiency virus (HIV)-1 antigenic determinants, were designed in our laboratories and investigated in controlled assay conditions . Based on a modification of the phage display technology, we developed an innovative concept for a safe and inexpensive vaccine in which conserved antigenic determinants of HIV-1 reverse transcriptase (RTase) were inserted into the N-terminal region of the major pVIII coat protein of bacteriophagefd virions . Analogously, we developed another antigen delivery system based on the E2 component from the PDH complex and capable of displaying large intact proteins on the surface of an icosahedral lattice . Our data show that both of these systems can deliver B and T epitopes to their respective presentation compartments in target cells and trigger a humoral response as well as a potent helper and cytolytic response in vitro and in vivo.

Protein Eng Des Sel, 2004 Feb, 17(2), 149 - 56 Epub 2004 Feb 03.
Engineering a substrate-specific cold-adapted subtilisin; Tindbaek N et al.; One region predicted to be highly flexible for a psychrophilic enzyme, TA39 subtilisin (S39), was transferred in silico to the mesophilic subtilisin, savinase (EC 3.4.21.62), from Bacillus lentus (clausii) . The engineered hybrid and savinase were initially investigated by molecular dynamic simulations at 300 K to show binding region and global flexibility . The predicted S39 region consists of 12 residues, which due to homology between the subtilisins, results in a total change of eight residues . By site-directed modifications, the region was transferred to the binding region of savinase, thus a savinase-S39 hybrid, named H5, was constructed . The designed hybrid showed the same temperature optimum and pH profile as savinase, but H5 had higher specific activity on the synthetic substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF) at all temperatures measured and, at the same time, H5 showed a decrease in thermostability . The H5 hybrid showed broader substrate specificity, measured at room temperature, due to an increase in catalytic efficiency on AAPF, AAPA and FAAF compared with savinase (N-succinyl-XXXX-pNA; XXXX = AAPF, AAPA and FAAF) . The H5 hybrid showed increased activity at low temperature, increased binding region and global flexibility, as investigated by molecular dynamic simulations, and global destabilization from differential scanning calorimetry measurements . These psychrophilic characteristics indicated an increase in binding site flexibility, probably due to the modifications P129S, S130G, P131E, and thus we show that it is possible to increase low temperature activity and global flexibility by engineered flexibility in the binding region.

Arch Biochem Biophys, 2004 Apr 15, 424(2), 201 - 9
Novel inhibition mechanism of Bacillus cereus sphingomyelinase by beryllium fluoride; Fujii S et al.; Phosphate analogs have been known to inhibit competitively various phosphatases and phospholipase C and D . We found for the first time that only beryllium fluoride (BeF(x)) among the phosphate analogs studied inhibits Bacillus cereus sphingomyelinase (SMase) activity . The active inhibitory species proved to be not BeF(3)(-) but BeF(2) by the measurement of SMase activity and of (19)F NMR spectroscopy in the presence of a fixed concentration of BeCl(2) and different concentrations of NaF, although both the species have been reported for other kinds of enzymes . The result of kinetic experiment also indicated that the BeF(x) binds in the vicinity of the essential binding site for the substrate and that the Mg(2+) binding to SMase is essential for the binding of BeF(x) to the enzyme.

Arch Virol, 2004 Apr, 149(4), 791 - 8 Epub 2004 Jan 05.
Sugarcane bacilliform virus encapsidates genome concatamers and does not appear to integrate into the Saccharum officinarum genome; Geijskes RJ et al.; Sugarcane bacilliform virus (SCBV) DNA molecules larger than the complete genome length of 7.6 kbp were detected in infected plants and in virions . We have confirmed that these high molecular weight nucleic acids were open circular DNA and viral in origin . Due to their open circular conformation, accurate size determination of the DNA molecules was not possible using conventional electrophoresis . Using field inversion gel electrophoresis (FIGE), however, the DNA appeared to increase in genome size increments, with sizes ranging from 1 to 4 genomes (31 kbp) detected . The DNA was packaged into virions, which may explain the observation of purified virions with lengths corresponding to one, two or three times the modal length of 130 nm . The DNA products were possibly concatamers formed during replication as a result of a terminal overlap on the sense strand, and were shown to be overlapped individual genome-length molecules and not covalently-bonded continuous DNA strands . Southern analysis indicated that SCBV sequences are not integrated into the sugarcane genome and that the high molecular weight DNA observed in the sugarcane accessions analysed represents SCBV concatamers.

Amino Acids, 2004 Mar, 26(2), 209 - 14 Epub 2003 Dec 22.
N-Acyl derivatives of Asn, new bacterial N-acyl D-amino acids with surfactant activity; Peypoux F et al.; New N-acyl D-amino acids were isolated from Bacillus pumilus IM 1801 . Their structures were determined by chemical analysis and mass spectrometry . The lipid part was identified as a mixture of fatty acids with 11, 12, 13, 15, and 16 carbon atoms in the iso, anteiso or n configuration linked by an amide bond with a D-asparagine . They exhibited surfactant properties.

Eur Urol, 2004 Apr, 45(4), 475 - 82
Pretreatment p53 nuclear overexpression as a prognostic marker in superficial bladder cancer treated with Bacillus Calmette-Guérin (BCG); Saint F et al.; INTRODUCTION: Altered p53 gene product correlates with the stage and grade of bladder tumor, but its value as a predictor of BCG response has been disappointing . In order to revisit the prognostic value of pretreatment p53 nuclear overexpression for the BCG response, we studied a large cohort of consecutive patients with superficial bladder cancer treated with BCG . METHODS: From 1988 to 2001, 102 patients with a history of multifocal, recurrent, and/or high-risk papillary transitional cell carcinoma or carcinoma in situ, were treated for the first time with BCG . p53 immunostaining was performed on paraffin-embedded tissues using monoclonal antibody DO7 and an automated immunostainer . Special attention was paid to the conditions of tumor fixation . p53 overexpression was defined as more than 20% tumor cells with p53-stained nuclei . RESULTS: Immunostaining was significantly higher for Ta/T1 G3 +/- Cis (p < 0.001), tumoral substage T1b (p = 0.001), grade 3 (p = 0.0001), and Cis (p = 0.002) . Times to recurrence, progression and cancer death were shorter among patients with p53 overexpression (p = 0.03; p < 0.0001; p = 0.0003) . In multivariate analysis, p53 overexpression was an independent predictor of recurrence (p = 0.0003) {RR = 0.15; 95%CI, 0.06 to 0.42} . CONCLUSION: Pretreatment p53 nuclear overexpression in superficial bladder tumors is associated with a high risk of disease recurrence, progression and cancer death after BCG therapy . Applying antibody DO7 with an automated immunostainer and stringent fixative conditions, p53 nuclear immunostaining yields clinically relevant information and may be a useful tool for selecting patients with superficial bladder cancer who might be resistant to BCG.

Acta Leprol, 2003, 12(3), 123 - 8
Pattern of bacillary clearance in multibacillary leprosy patients with multidrug therapy; Kumar A et al.; The bacteriological index (BI) of the skin smears is traditionally one of the important parameters of assessment of severity and of progress of leprosy under multidrug therapy . The present study reports on BI clearance among 578 multibacillary treated leprosy patients and the factors that influence this clearance . The patients were treated till smear negativity or for 2 years fixed duration and their skin smears periodically examined every 6 to 12 months till negativity (and even afterwards) . We confirm that bacterial clearance is a slow process . The time taken for each log-unit decline in BI is between 13.6 to 24 months probably depending on initial BI level . The rate of smear negativity appears to be dependent on immune competence of the patients as reflected by a rapid BI decline in borderline BT-BB patients vis-a-vis BL-LL lepromatous patients both in the low and high BI group . Patients who had several episodes of ENL, took significantly longer time (63.7 months versus 53.5 months, p<0.0001) to become smear negative than those without ENL.

Biochem J, 2004 Jun 15, 380(Pt 3), 635 - 41
In vitro synthesis of a crystalline (1-->3,1-->4)-beta-D-glucan by a mutated (1-->3,1-->4)-beta-D-glucanase from Bacillus; Faijes M et al.; Oligo- and poly-saccharides have a large number of important biological functions, and they occur in natural composite materials, such as plant cell walls, where they self-assemble during biosynthesis in a poorly understood manner . They can also be used for the formation of artificial composite materials with industrial applications . Fundamental and applied research in biology and nanobiotechnology would benefit from the possibility of synthesizing tailor-made oligo-/poly-saccharides . In the present paper, we demonstrate that such syntheses are possible using genetically modified glycoside hydrolases, i.e . glycosynthases . The ability of the endoglycosynthase derived from Bacillus (1-->3,1-->4)-beta-D-glucanase to catalyse self-condensation of sugar donors was exploited for the in vitro synthesis of a regular polysaccharide . The specificity of the enzyme allowed the polymerization of alpha-laminaribiosyl fluoride via the formation of (1-->4)-beta-linkages to yield a new linear crystalline (1-->3,1-->4)-beta-D-glucan with a repeating 4betaG3betaG unit . MS and methylation analyses indicated that the in vitro product consisted of a mixture of oligosaccharides, the one having a degree of polymerization of 12 being the most abundant . Morphological characterization revealed that the (1-->3,1-->4)-beta-D-glucan forms spherulites which are composed of platelet crystals . X-ray and electron diffraction analyses allowed the proposition of a putative crystallographic structure which corresponds to a monoclinic unit cell with a =0.834 nm, b =0.825 nm, c =2.04 nm and gamma=90.5 degrees . The dimensions of the ab plane are similar to those of cellulose I(beta), but the length of the c -axis is nearly twice that of cellulose I . It is proposed that four glucose residues are present in an extended conformation along the c -axis of the unit cell . The data presented show that glycosynthases represent promising enzymic systems for the synthesis of novel polysaccharides with specific and controlled structures, and for the analysis in vitro of the mechanisms of polymerization and crystallization of polysaccharides.

Microb Ecol, 2004 Jul, 48(1), 111 - 9 Epub 2004 Mar 25.
Isolation and biodiversity of hitherto undescribed soil bacteria related to Bacillus niacini; Felske AD et al.; The hitherto largely not described phylogenetic neighborhood of Bacillus niacini has been explored by a comprehensive cultivation experiment and genomic variety studies . Previous culture-independent studies demonstrated that approximately 15% of all Bacillus 16S rDNA directly extracted from soils worldwide was affiliated to B . niacini . Seven different media were inoculated with soil suspensions in serial dilutions and incubated at different temperatures . Then, bacterial colonies were picked and analyzed by sequencing . A mineral medium with acetate as carbon source yielded a B . niacini rate of >3% of all picked colonies . Other media were less efficient but also successful . Applying this culturing approach, we succeeded in obtaining 64 isolates from different Dutch soils . The isolates turned out to be diverse, although closely related to B . niacini as revealed by 16S rDNA sequencing . Close matches with environmental clones were also found, thus demonstrating much more diversity beyond previously known 16S rDNA sequences . The rep-PCR fingerprinting method revealed a high genomic variety, redundancy could not be observed among our isolates . Hence, the hitherto neglected B . niacini lineage, apparently among the most abundant soil Bacillus, was accessible to our cultivation approach.

Nihon Hansenbyo Gakkai Zasshi, 2004 Feb, 73(1), 37 - 46
{Global situation of leprosy and recent progress in molecular epidemiology of the disease}; Izumi S et al.; Recent discovery of genetic diversity of Mycobacterium leprae such as variable number of tandem repeats opened a new era in molecular epidemiology of leprosy infection . It was revealed that the leprosy bacillus in residential environment of endemic villages is an important source of infection . The global elimination strategy will be revised taking new molecular epidemiological knowledge into account . Responsibility of leprosy specialist is to propose feasible control program to local administration based on the epidemiological analysis on transmission of the disease.

Nat Rev Microbiol, 2003 Nov, 1(2), 97 - 105
Tuberculosis: a problem with persistence; Stewart GR et al.; Mycobacterium tuberculosis is one of most successful pathogens of mankind, infecting one-third of the global population and claiming two million lives every year . The ability of the bacteria to persist in the form of a long-term asymptomatic infection, referred to as latent tuberculosis, is central to the biology of the disease . The persistence of bacteria in superficially normal tissue was recognized soon after the discovery of the tubercle bacillus, and much of our knowledge about persistent populations of M . tuberculosis dates back to the first half of the last century . Recent advances in microbial genetics and host immunity provide an opportunity for renewed investigation of this persistent threat to human health.

Skeletal Radiol, 2004 May, 33(5), 249 - 59 Epub 2004 Mar 18.
Musculoskeletal disorders associated with HIV infection and AIDS . Part I: infectious musculoskeletal conditions; Tehranzadeh J et al.; The musculoskeletal system can be affected by a variety of abnormalities in association with human immunodeficiency virus (HIV) infection . Although not as common as complications involving other organ systems, such as the pulmonary and the central nervous systems, HIV-associated musculoskeletal disorders are sometimes the initial presentation of the viral illness . Knowledge of the existence and the characteristic appearance of the conditions affecting bone, joint, and muscle in HIV-infected patients is valuable to radiologists for diagnosis and to clinicians for detection and appropriate treatment . We reviewed recent literature to provide a comprehensive assessment of the HIV-associated musculoskeletal disorders, and present radiologic examples from our own collection . This article is divided into two parts . In the first part we review the infectious musculoskeletal disorders associated with HIV illness and AIDS, including cellulitis, abscesses, pyomyositis, septic bursitis, septic arthritis, osteomyelitis, and bacillary angiomatosis . We also present a comprehensive spectrum of mycobacterial infections, consisting of tuberculous spondylitis and spondylodiskitis, arthritis, osteomyelitis, and tenosynovitis, as well as infections caused by atypical mycobacteria . Part II of this review will concentrate on non-infectious musculoskeletal conditions, including rheumatic disorders and neoplasms.

Org Biomol Chem, 2004 Apr 7, 2(7), 961 - 2 Epub 2004 Feb 26.
Formation of LacNAc mimetics employing novel donor substrates for enzymatic beta 1-->4 galactosylation; Weingarten S et al.; In examining C-6 modified 4-nitrophenyl beta-D-galactopyranosides as donor structures the beta-galactosidase (Bacillus circulans) revealed an unexpectedly broad substrate specificity which allowed successful syntheses of various disaccharide components.

J Immunol, 2004 Apr 1, 172(7), 4425 - 34
Toll-like receptor 2 (TLR2)-dependent-positive and TLR2-independent-negative regulation of proinflammatory cytokines by mycobacterial lipomannans; Quesniaux VJ et al.; Lipoarabinomannans (LAM) and lipomannans (LM) are integral parts of the mycobacterial cell wall recognized by cells involved in the innate immune response and have been found to modulate the cytokine response . Typically, mannosylated LAM from pathogenic mycobacteria have been reported to be anti-inflammatory, whereas phosphoinositol-substituted LAM from nonpathogenic species are proinflammatory molecules . In this study, we show that LM from several mycobacterial species, including Mycobacterium chelonae, Mycobacterium kansasii, and Mycobacterium bovis bacillus Calmette-Guerin, display a dual function by stimulating or inhibiting proinflammatory cytokine synthesis through different pathways in murine primary macrophages . LM, but none of the corresponding LAM, induce macrophage activation characterized by cell surface expression of CD40 and CD86 and by TNF and NO secretion . This activation is dependent on the presence of Toll-like receptor (TLR) 2 and mediated through the adaptor protein myeloid differentiation factor 88 (MyD88), but independent of either TLR4 or TLR6 recognition . Surprisingly, LM exerted also a potent inhibitory effect on TNF, IL-12p40, and NO production by LPS-activated macrophages . This TLR2-, TLR6-, and MyD88-independent inhibitory effect is also mediated by LAM from M . bovis bacillus Calmette-Guerin but not by LAM derived from M . chelonae and M . kansasii . This study provides evidence that mycobacterial LM bear structural motifs susceptible to interact with different pattern recognition receptors with pro- or anti-inflammatory effects . Thus, the ultimate response of the host may therefore depend on the prevailing LM or LAM in the mycobacterial envelope and the local host cell receptor availability.

J Chromatogr A, 2004 Mar 12, 1029(1-2), 87 - 95
Liquid chromatography-mass spectrometry analysis of enzyme-hydrolysed carboxymethylcellulose for investigation of enzyme selectivity and substituent pattern; Cohen A et al.; A series of celloendoglucanases: Bacillus agaradhaerens Cel 5a, Humicola insolens Cel 5a, H . insolens Cel 7b, H . insolens Cel 45a, Trichoderma reesei Cel 7b, and T . reesei Cel 45a were used to hydrolyse carboxymethylcellulose (CMC) and the hydrolysis products were investigated with a novel liquid chromatography-mass spectrometry (LC-MS) method . Separation was achieved using a graphitised carbon chromatographic column which allowed the use of electrospay compatible eluents . Analysis of the compounds produced during enzyme hydrolysis of CMC is used to understand enzyme selectivities and substitution pattern of CMC . Conventional high-performance anion-exchange chromatography (HPAEC)-pulsed amperometric detection (PAD), size-exclusion chromatography (SEC)-refractive index (RI) detection, and reducing end analysis are also used to analyse enzyme-hydrolysed CMC . The LC-MS method presented allows for a more detailed investigation of hydrolysis products, which facilitates characterisation of both enzymes and substrates.

Eur J Biochem, 2004 Apr, 271(7), 1391 - 400
Protein engineering of pyruvate carboxylase: investigation on the function of acetyl-CoA and the quaternary structure; Sueda S et al.; Pyruvate carboxylase (PC) from Bacillus thermodenitrificans was engineered in such a way that the polypeptide chain was divided into two, between the biotin carboxylase (BC) and carboxyl transferase (CT) domains . The two proteins thus formed, PC-(BC) and PC-(CT+BCCP), retained their catalytic activity as assayed by biotin-dependent ATPase and oxamate-dependent oxalacetate decarboxylation, for the former and the latter, respectively . Neither activity was dependent on acetyl-CoA, in sharp contrast to the complete reaction of intact PC . When assessed by gel filtration chromatography, PC-(BC) was found to exist either in dimers or monomers, depending on the protein concentration, while PC-(CT + BCCP) occurred in dimers for the most part . The two proteins do not associate spontaneously or in the presence of acetyl-CoA . Based on these observations, this paper discusses how the tetrameric structure of PC is built up and how acetyl-CoA modulates the protein structure.

Eur J Biochem, 2004 Apr, 271(7), 1357 - 63
Jackbean, soybean and Bacillus pasteurii ureases: biological effects unrelated to ureolytic activity; Follmer C et al.; In this work we compared two plant ureases, jackbean urease (JBU) and embryo-specific soybean urease (SBU) and a bacterial (Bacillus pasteurii) urease, for kinetic parameters and other biological properties described recently for ureases that are independent of the ureolytic activity . The insecticidal effect of ureases was investigated in feeding trials with the cotton sucker bug, Dysdercus peruvianus (Hemiptera) as an insect model . Contrasting with B . pasteurii urease (PBU), both plant ureases presented potent insecticidal activity, with LD(50) values of 0.017% (w/w) and 0.052% (w/w) for JBU and SBU, respectively . The insecticidal property of JBU or SBU was not affected by treatment with p-hydroxymercuribenzoate, an irreversible inhibitor of ureolytic activity of both proteins . Also, contrasting with canatoxin - a urease isoform from jackbean seeds that displays a toxic effect in mice (LD(50) = 2 mg x kg(-1)) - no lethality was seen in mice injected intraperitoneally with JBU or SBU (20 mg x kg(-1)) . Similarly to canatoxin, the three enzymes promoted aggregation of blood platelets (EC(50) = 400.0 micro g x mL(-1), 22.2 micro g x mL(-1), 15.8 micro g x mL(-1) for BPU, SBU and JBU, respectively) . This platelet activating property was also independent of urease activity . Comparison of the kinetic properties indicated that SBU is fivefold less susceptible than JBU to inhibition by acetohydroxamic acid, a chelator of Ni(+2) and Zn(+2) ions . The ureases also showed different susceptibility to agents that modify cysteine residues, such as p-hydroxymercuribenzoate and p-benzoquinone . Altogether, these data emphasize that biological properties that are independent of ureolytic activity are not restricted to jackbean ureases and that these proteins may have a role in plant defense against insect predators.

Prikl Biokhim Mikrobiol, 2004 Jan-Feb, 40(1), 70 - 3
{Microorganisms--degraders of polychlorinated biphenyls}; Kim AA et al.; Four strains belonging to the genus Bacillus, capable of degrading polychlorinated biphenyls (PCB), were isolated by screening the collection strains of soil bacteria, degrading a organochlorine pesticide, hexachlorocyclohexane (HCCH) . A method for production of tritium-labeled PCB was developed . Consumption and degradation of PCB by the soil bacterial strains selected were studied using tritium-labeled PCB and GLC . It was demonstrated that PCB are degradable both in culture media and under in model soil samples.

Prikl Biokhim Mikrobiol, 2004 Jan-Feb, 40(1), 28 - 31
{Isolation of conjugates of albumin and ribonuclease}; Zelepuga EA et al.; A method for separation of albumin-ribonuclease (RNase) conjugates has been proposed, based on the use of macroporous silicates . It was established that about 76% of ligand-free human serum albumin (LFHSA) formed complexes with enzymes . It was shown that most of the conjugates of albumin and pancreatic RNase contained up to 2 mol enzyme per 1 mol LFHSA . The conjugates of albumin and bacterial RNase, isolated from the cells of the strain Bacillus intermedius 7P, displayed higher specific activities, containing, on average, 2.3 mol RNase per 1 mol LFHSA (for the conjugates with molecular weights below 92 kDa) or 3.3 mol RNase per 1 mol protein carrier (for the conjugates with higher molecular weight).

J Fr Ophtalmol, 2004 Feb, 27(2), 179 - 83
{Cat's cratch disease and Parinaud's oculoglandular syndrome}; Escarmelle A et al.; By presenting this case report describing Parinaud's oculoglandular syndrome, we review the medical literature on its most frequent etiology: catscratch disease, a self-limited, systemic illness caused by a Gram-negative bacillus, Bartonella henselae, principally affecting children under 15 years of age . Typical symptoms include regional lymphadenopathy, fever, malaise, and fatigue, possibly with more severe complications such as splenomegaly, granulomatous hepatitis, and encephalopathy . Ocular manifestations may include follicular conjunctivitis, Parinaud's oculoglandular syndrome, neuroretinitis, optic neuritis, and chorioretinitis . Diagnosis is based on serologic tests, and when necessary, antimicrobial treatment can be considered.

Urology, 2004 Mar, 63(3), 596 - 601
Antitumor effects of recombinant BCG and interleukin-12 DNA vaccines on xenografted murine bladder cancer; Yu DS et al.; OBJECTIVES: To evaluate the antitumor effects of recombinant bacille Calmette-Guerin (BCG) DNA (multi-rBCG) and murine interleukin-12 DNA (mIL-12) vaccines on xenografted MBT-2 murine bladder tumors . METHODS: Treatment with combined multi-rBCG and mIL-12 was examined in syngeneic C3H/HeN mice and athymic nude mice . The delivery efficiency of multi-rBCG expression was detected by flow cytometry . Inhibition of tumor growth was monitored, and antitumor effects were evaluated after one dose of electroporation immunogenetherapy, with measurement of cytokines and phenotyping of infiltrating lymphocytes in tumors . RESULTS: In vivo expression of multi-rBCG was efficient and reached a maximum on day 7 after electroporation . Treatment with multi-rBCG plus mIL-12 significantly inhibited tumor growth in C3H/HeN mice, with increased production of Th1-type cytokines, including interferon-gamma and IL-12 . Treatment with multi-rBCG and/or mIL-12 in C3H/HeN mice induced infiltration of CD4+/CD8+ T cells and expansion of natural killer cells within tumors . By contrast, however, athymic nude mice treated in the same way showed no significant immune cells within tumors and died of the fast growing tumors . CONCLUSIONS: Electroporation using multi-rBCG plus mIL-12 could be effective immunotherapy for existing bladder cancer . The antitumor effects correlated with the elicitation of Th1 lymphocytes and natural killer cell-mediated cytotoxic immune responses.

Zhonghua Nan Ke Xue, 2004 Feb, 10(2), 117 - 8, 121
{Efficacy of local injection of bacillus calmette-guerin polysaccharide nucleic acid following CO2 laser resection on condyloma acuminatum}; Yu X et al.; OBJECTIVE: To observe the efficacy of local injection of bacillus calmette-guerin polysaccharide-nuclear acid (BCG-PSN) following CO2 laser resection on condyloma acuminatum (CA) . METHODS: One hundred and six patients with CA were randomized into BCG-PSN group (60 cases) and interferon group (46 cases), treated respectively with BCG-PSN local injection and interferon subcutaneous injection following CO2 laser resection and followed up for 3 months . RESULTS: The healing rate of the BCG-PSN group was 88.3% and that of the interferon group was 71.7% . CONCLUSION: The BCG-PSN local injection following CO2 laser resection is effective for CA patients.

Arch Insect Biochem Physiol, 2004 Apr, 55(4), 169 - 77
Bacillus thuringiensis delta-endotoxin binding to brush border membrane vesicles of rice stem borers; Alcantara EP et al.; The receptor binding step in the molecular mode of action of five delta-endotoxins (Cry1Ab, Cry1Ac, Cry1C, Cry2A, and Cry9C) from Bacillus thuringiensis was examined to find toxins with different receptor sites in the midgut of the striped stem borer (SSB) Chilo suppressalis (Walker) and yellow stem borer (YSB) Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae) . Homologous competition assays were used to estimate binding affinities (K(com)) of (125)I-labelled toxins to brush border membrane vesicles (BBMV) . The SSB BBMV affinities in decreasing order was: Cry1Ab = Cry1Ac > Cry9C > Cry2A > Cry1C . In YSB, the order of decreasing affinities was: Cry1Ac > Cry1Ab > Cry9C = Cry2A > Cry1C . The number of binding sites (B(max)) estimated by homologous competition binding among the Cry toxins did not affect toxin binding affinity (K(com)) to both insect midgut BBMVs . Results of the heterologous competition binding assays suggest that Cry1Ab and Cry1Ac compete for the same binding sites in SSB and YSB . Other toxins bind with weak (Cry1C, Cry2A) or no affinity (Cry9C) to Cry1Ab and Cry1Ac binding sites in both species . Cry2A had the lowest toxicity to 10-day-old SSB and Cry1Ab and Cry1Ac were the most toxic . Taken together, the results of this study show that Cry1Ab or Cry1Ac could be combined with either Cry1C, Cry2A, or Cry9C for more durable resistance in transgenic rice . Cry1Ab should not be used together with Cry1Ac because a mutation in one receptor site could diminish binding of both toxins .

J Biol Chem, 2004 May 14, 279(20), 21282 - 6 Epub 2004 Mar 16.
A Bacillus thuringiensis crystal protein with selective cytocidal action to human cells; Ito A et al.; Bacillus thuringiensis crystal proteins, well known to be toxic to certain insects but not pathogenic to mammals, are used as insecticidal proteins in agriculture and forest management . We here identified a crystal protein that is non-insecticidal and non-hemolytic but has strong cytocidal activity against various human cells with a markedly divergent target specificity, e.g . highly cytotoxic to HepG2 and Jurkat and less cytotoxic to the normal hepatocyte (HC) and HeLa . In slices of liver and colon cancer tissues, the toxin protein preferentially killed the cancer cells, leaving other cells unaffected . The cytocidal effect of the protein is non-apoptotic with swelling and fragmentation of the susceptible cells, although the apoptotic process does occur when the cell damage proceeded slowly . The amino acid sequence deduced from the nucleotide sequence of the cloned gene of the protein has little sequence homology with the insecticidal crystal proteins of B . thuringiensis . These observations raise the presence of a new group of the B . thuringiensis toxin and the possibility of new applications for the protein in the medical field.

J Biol Chem, 2004 Jun 18, 279(25), 26546 - 54 Epub 2004 Mar 15.
Replacement of amino acid sequence features of a- and c-subunits of ATP synthases of Alkaliphilic Bacillus with the Bacillus consensus sequence results in defective oxidative phosphorylation and non-fermentative growth at pH 10.5; Wang Z et al.; Mitchell's (Mitchell, P . (1961) Nature 191, 144-148) chemiosmotic model of energy coupling posits a bulk electrochemical proton gradient (Deltap) as the sole driving force for proton-coupled ATP synthesis via oxidative phosphorylation (OXPHOS) and for other bioenergetic work . Two properties of proton-coupled OXPHOS by alkaliphilic Bacillus species pose a challenge to this tenet: robust ATP synthesis at pH 10.5 that does not correlate with the magnitude of the Deltap and the failure of artificially imposed potentials to substitute for respiration-generated potentials in energizing ATP synthesis at high pH (Krulwich, T . (1995) Mol . Microbiol . 15, 403-410) . Here we show that these properties, in alkaliphilic Bacillus pseudofirmus OF4, depend upon alkaliphile-specific features in the proton pathway through the a- and c-subunits of ATP synthase . Site-directed changes were made in six such features to the corresponding sequence in Bacillus megaterium, which reflects the consensus sequence for non-alkaliphilic Bacillus . Five of the six single mutants assembled an active ATPase/ATP synthase, and four of these mutants exhibited a specific defect in non-fermentative growth at high pH . Most of these mutants lost the ability to generate the high phosphorylation potentials at low bulk Deltap that are characteristic of alkaliphiles . The aLys(180) and aGly(212) residues that are predicted to be in the proton uptake pathway of the a-subunit were specifically implicated in pH-dependent restriction of proton flux through the ATP synthase to and from the bulk phase . The evidence included greatly enhanced ATP synthesis in response to an artificially imposed potential at high pH . The findings demonstrate that the ATP synthase of extreme alkaliphiles has special features that are required for non-fermentative growth and OXPHOS at high pH.

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 617 - 21
Bacillus galactosidilyticus sp . nov., an alkali-tolerant beta-galactosidase producer; Heyndrickx M et al.; A novel Bacillus isolate from raw milk and four strains from diverse origins that were identified previously as Bacillus lentus, Bacillus firmus and Bacillus circulans showed a high degree of similarity in amplified rDNA restriction analysis, SDS-PAGE and routine phenotypic tests, whilst 16S rDNA sequence comparisons and DNA relatedness data showed that this taxon was different from related Bacillus species . On the basis of these data, Bacillus galactosidilyticus sp . nov . is proposed, with the type strain LMG 17892(T) (=DSM 15595(T)=Logan B2188(T)=MB 800(T)).

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 373 - 6
Bacillus shackletonii sp . nov., from volcanic soil on Candlemas Island, South Sandwich archipelago; Logan NA et al.; A sample of mossy soil taken from the eastern lava flow of northern Candlemas Island, South Sandwich archipelago, yielded six isolates of aerobic, endospore-forming bacteria . Miniaturized routine phenotypic tests and other observations, amplified rDNA restriction analysis and SDS-PAGE analysis suggested that the strains represent a novel taxon . 16S rDNA sequence comparisons support the proposal of a novel species, Bacillus shackletonii sp . nov., the type strain of which is LMG 18435(T) (=CIP 107762(T)).

J Control Release, 2004 Mar 24, 95(3), 455 - 62
Floating pellets containing bacterial antagonist for control sheath blight of rice: formulations, viability and bacterial release studies; Wiwattanapatapee R et al.; Floating pellets containing spores of bacterial biological control agent, Bacillus megaterium were prepared by extrusion-spheronization process . The formulations composed of hydrogenated vegetable oil (HVO), lactose, microcrystalline cellulose (Avicel(R) PH101), and a disintegrant; cross-linked sodium carboxymethylcellulose (Ac-Di-Sol(R)) . The finishing pellets contained bacteria ranging from 10(7) to 10(8) CFU/g and the viability of bacteria in all formulations remained high after 6 months storage . The scanning electron microscope (SEM) was used to observe endospores of B . megaterium on both the surface and the inside of the pellets . The formulations were tested for their physical properties, floating ability and bacterial release . The level of disintegrant in the formulations influenced the floating ability and the liberation of antagonistic bacteria from pellets . The bacterial pellets showed promising result in suppression of the development of sheath blight lesions in greenhouse experiment.

Am J Clin Pathol, 2004 Mar, 121(3), 335 - 42
Immunostaining for human herpesvirus 8 latent nuclear antigen-1 helps distinguish Kaposi sarcoma from its mimickers; Cheuk W et al.; We assessed the usefulness of a mouse monoclonal antibody (13B10) against human herpesvirus 8 (HHV-8) latent nuclear antigen-1 (LNA-1) in diagnosis of Kaposi sarcoma (KS) and for distinguishing it from various mimickers by studying 50 cases of KS and 53 mimickers (angiosarcoma, 15; kaposiform hemangioendothelioma, 6; spindle cell hemangioma, 3; reactive angioendotheliomatosis, 3; bacillary angiomatosis, 4; acroangiomatous dematitis, 2; microvenular hemangioma, 2; hobnail hemangioma, 2; pyogenic granuloma, 5; dermatofibroma, 8; arteriovenous hemangioma, 1; verrucous hemangioma, 1; nonspecific vascular proliferation, 1) from patients with or without acquired HIV infection . Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue sections . All 50 cases of KS were positive for HHV-8 LNA-1, with immunolocalization in the nuclei of the spindle cells and cells lining the primitive and thin-walled vascular channels, whereas all 53 mimickers (including 4 lesions from HIV-positive patients) tested negative . The results idicate that positive immunostaining for HHV-8 LNA- 1 exhibits high sensitivity and specificity for the diagnosis of KS and is, thus, useful for distinguishing it from the mimickers.

Extremophiles, 2004 Jun, 8(3), 229 - 35 Epub 2004 Mar 12.
A new subtilisin family: nucleotide and deduced amino acid sequences of new high-molecular-mass alkaline proteases from Bacillus spp; Okuda M et al.; Six genes encoding high-molecular-mass subtilisins (HMSs) of alkaliphilic Bacillus spp . were cloned and sequenced . Their open reading frames of 2,394-2,424 bp encoded prosubtilisins of 798-808 amino acids (aa) consisting of the prepropeptides of 151-158 aa and the mature enzymes of 640-656 aa . The deduced aa sequences of the mature enzymes exhibited 60-95% identity to those of FT protease of Bacillus sp . strain KSM-KP43, a subtilisin-like serine protease, and a minor serine protease, Vpr, of Bacillus strains . Three of the six recombinant enzymes were susceptible to proteolysis, but the others were autodigestion resistant . All enzymes had optimal pH values of 10.5-11.0, optimal temperatures of 40-45 degrees C for hydrolysis of a synthetic substrate, and were heat labile . These alkaline proteases seem to form a new subtilisin family, as judged by their aa sequences and phylogenetic analysis .

J Biol Chem, 2004 May 28, 279(22), 23274 - 86 Epub 2004 Mar 12.
Flavocytochrome P450 BM3 mutant A264E undergoes substrate-dependent formation of a novel heme iron ligand set; Girvan HM et al.; A conserved glutamate covalently attaches the heme to the protein backbone of eukaryotic CYP4 P450 enzymes . In the related Bacillus megaterium P450 BM3, the corresponding residue is Ala264 . The A264E mutant was generated and characterized by kinetic and spectroscopic methods . A264E has an altered absorption spectrum compared with the wild-type enzyme (Soret maximum at approximately 420.5 nm) . Fatty acid substrates produced an inhibitor-like spectral change, with the Soret band shifting to 426 nm . Optical titrations with long-chain fatty acids indicated higher affinity for A264E over the wild-type enzyme . The heme iron midpoint reduction potential in substrate-free A264E is more positive than that in wild-type P450 BM3 and was not changed upon substrate binding . EPR, resonance Raman, and magnetic CD spectroscopies indicated that A264E remains in the low-spin state upon substrate binding, unlike wild-type P450 BM3 . EPR spectroscopy showed two major species in substrate-free A264E . The first has normal Cys-aqua iron ligation . The second resembles formate-ligated P450cam . Saturation with fatty acid increased the population of the latter species, suggesting that substrate forces on the glutamate to promote a Cys-Glu ligand set, present in lower amounts in the substrate-free enzyme . A novel charge-transfer transition in the near-infrared magnetic CD spectrum provides a spectroscopic signature characteristic of the new A264E heme iron ligation state . A264E retains oxygenase activity, despite glutamate coordination of the iron, indicating that structural rearrangements occur following heme iron reduction to allow dioxygen binding . Glutamate coordination of the heme iron is confirmed by structural studies of the A264E mutant (Joyce, M . G., Girvan, H . M., Munro, A . W., and Leys, D . (2004) J . Biol . Chem . 279, 23287-23293).

J Neuroimmunol, 2004 Apr, 149(1-2), 22 - 30
Influence of the course of brain inflammation on the endogenous IL-1beta/IL-1Ra balance in the model of brain delayed-type hypersensitivity response to bacillus Calmette-Guérin in Lewis rats; Palin K et al.; Interleukin-1beta (IL-1beta) is a key player in the pathogenesis of acute and chronic inflammatory diseases at the periphery and in the brain . Its action is regulated by interleukin-1 receptor antagonist (IL-1Ra), the specific endogenous antagonist of IL-1 receptors . The ratio between local concentrations of IL-1Ra and IL-1beta is known to influence the initiation and progression of many inflammatory and autoimmune diseases at the periphery . In order to determine whether this is also the case in the brain, brain and plasma concentrations of IL-1beta and IL-1Ra were measured by ELISA in a model of chronic brain inflammation in Lewis rats, the hippocampal delayed-type hypersensitivity (DTH) response to bacillus Calmette-Guerin (BCG) . Brain IL-1beta increased rapidly after intracerebral (i.c.) injection of BCG and came back to baseline concentrations 1 week later, whereas IL-1Ra increased gradually over time and remained elevated during the last 2 weeks post-BCG intracerebral injection . Following peripheral BCG challenge, brain IL-1beta increased at the site of the brain BCG and peaked 12 days later before decreasing on day 16 post-challenge . Brain IL-1Ra remained elevated during the first days post-challenge and then decreased from the 12th day post-challenge . The same temporal variations were observed in the plasma concentrations of IL-1beta and IL-1Ra . The increase in the IL-1beta/IL-1Ra ratio that was apparent from day 3 to day 12 post-challenge might be correlated with the invasion of peripheral inflammatory cells at the site of intracerebral injection . Besides showing that the course of inflammation alters the brain IL-1beta/IL-1Ra ratio, these findings point to the importance of monitoring plasma IL-1beta/IL-1Ra ratio to predict the course of brain inflammation.

J Insect Physiol, 2004 Feb-Mar, 50(2-3), 175 - 83
Bacillus thuringiensis toxin (Cry1Ab) has no direct effect on larvae of the green lacewing Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae); Romeis J et al.; Earlier studies have shown that larvae of the green lacewing predator Chrysoperla carnea are negatively affected when preying on lepidopteran larvae that had been fed with transgenic maize expressing the cry1Ab gene from Bacillus thuringiensis . To test whether the observed effects were directly caused by the Cry1Ab toxin, we have developed a bioassay which allows us to feed high concentrations of the toxin directly to the predator . The results of these feeding studies show no direct toxic effect of Cry1Ab on C . carnea larvae . The amount of toxin ingested by first instar C . carnea in the present study was found to be a factor 10,000 higher than the concentration ingested when feeding on Bt-reared lepidopteran larvae, a treatment that was previously shown to have a negative impact on the predator . In addition, feeding first instar C . carnea with the Cry1Ab toxin did not affect the utilisation of subsequently provided prey . Furthermore, the quality of the prey provided to first instars did not affect the sensitivity of second and third instar C . carnea to the Bt-toxin . The presented results strongly suggest that C . carnea larvae are not sensitive to Cry1Ab and that earlier reported negative effects of Bt-maize were prey-quality mediated rather than direct toxic effects . These results, together with the fact that lepidopteran larvae are not regarded as an important prey for C . carnea in the field, led us to conclude that transgenic maize expressing Cry1Ab poses a negligible risk for this predator.

Curr Microbiol, 2004 Jan, 48(1), 47 - 50
Isolation and characterization of a strain of Bacillus thuringiensis subsp . morrisoni PG-14 encoding delta-endotoxin Cry1Ac; Choi YS et al.; Bacillus thuringiensis 656-3, isolated from a soil sample collected at mushroom houses, showed high toxicity to mushroom flies, Lycoriella mali and Coboldia fuscipes . B . thuringiensis 656-3 produced bipyramidal inclusions and reacted with the H antiserum of B . thuringiensis subsp . morrisoni (H8a8b) . The plasmid and protein profiles of B . thuringiensis 656-3 were similar to those of its reference strain, subsp . morrisoni PG-14 . However, PCR analysis using cry gene primers showed that B . thuringiensis 656-3, unlike its reference strain, had cry4A, cry4B, cry10A, cry11A, and cry1Ac genes, suggesting that B . thuringiensis 656-3 was a unique strain with respect to gene type . In addition, B . thuringiensis 656-3 showed a high level of toxicity against mushroom flies, L . mali and C . fuscipes.

Curr Microbiol, 2004 Jan, 48(1), 39 - 46
Comparative genomic analysis of the sigB operon in Listeria monocytogenes and in other Gram-positive bacteria; Ferreira A et al.; The stress-responsive, alternative sigma factor sigmaB has been described in members of three Gram-positive genera, Bacillus, Listeria, and Staphylococcus . In these bacteria, sigmaB appears to play an important role in facilitating rapid adaptation to and survival in stressful environments . sigmaB activity is regulated through a complex system of phosphatases and kinases encoded by rsb (regulator of sigma B) genes . We describe the sigB operon structure for the facultative intracellular pathogen Listeria monocytogenes and apply this sequence as well as other previously described sigB operon sequences to probe the evolution and functional conservation of the sigmaB stress response system among different Gram-positive bacteria . While sigmaB as well as two Rsbs (RsbS and RsbT) are highly conserved (73%, 84%, and 79% average amino acid {aa} identities, respectively), the predicted aa sequences of the other Rsb proteins showed less conservation (62-71% aa identities) . Furthermore, the sigB operon structure varies among bacterial species . Bacterial species differ in the numbers and identities of rsb genes encoded in their genomes . We thus conclude that the sigmaB stress-response system as represented by the sigB operon has diverged in both its overall components as well as in the sequences of its individual proteins, even among closely related bacterial species . Differential evolution of this stress response system among various genera may represent a strategy that enables bacteria to adapt cellular response and survival systems to a variety of stress conditions.

Curr Microbiol, 2004 Jan, 48(1), 1 - 9
Interaction of two Bacillus thuringiensis delta-endotoxins with the digestive system of Lygus hesperus; Brandt SL et al.; The active-toxin form of CrylAc (65 kDa) or Cry2Ab was fed to a non-susceptible insect, Lygus hesperus, in an artificial diet . Biochemical and immunocytochemical methods were used to determine the distribution of ingested toxin . The toxins did not elicit a feeding deterrent response . CrylAc and Cry2Ab were ingested; small amounts were absorbed into the hemolymph as holoproteins, but most was excreted . SDS-PAGE analysis of CrylAc and Cry2Ab incubations with salivary gland homogenate showed a small decrease in the molecular weight of the active toxins . Proteolytic processing of the toxins also occurred in vivo, within the digestive system of L . hesperus . Excreted CrylAc and Cry2Ab retained activity toward lepidopteran larvae . Immunocytochemical in vivo localization studies showed negligible association of CrylAc with L . hesperus tissues . In contrast, strong extracellular association of Cry2Ab was observed with L . hesperus midgut brush border microvilli and basement membrane, as well as with cellular outlines within the hemolymph and fat body.

J Huazhong Univ Sci Technolog Med Sci, 2003, 23(4), 339 - 43, 347
A comparative study on the effect of BCG-PSN and thymopeptides on T-lymphocyte subsets of normal and immunosuppressed mice; Deng Y et al.; To compare the effects of polysaccharide nucleic acid fraction of bacillus calmette guerin (BCG-PSN) and thymopeptides on T-lymphocytes of normal and immunosuppressed mice, CD4+ and CD8+ T-lymphocyte subsets of single nucleic cell in thymus, spleen and peripheral blood were detected successively by flow cytometry after application of BCG-PSN and thymopeptides . Meanwhile, CD4+/CD8+ ratio was also calculated . The results showed that both BCG-PSN and thymopeptides could decrease the proportion of CD4+ CD8+ T-lymphocyte subsets in the thymus, at the same time increase CD4+ T-lymphocyte, CD8+ T-lymphocyte proportion in the three tissues . The fluctuation in amplitude was greater in thymopeptides group than that in BCG-PSN group . It is concluded that acting location of thymopeptides is in thymus, its stimulating action is stronger than that of BCG-PSN, while BCG-PSN not only accelerates the differentiation in thymus, but also has some direct stimulation to peripheral CD4+ T-lymphocytes, and can maintain CD4+/CD8+ ratio within normal range . So, BCG-PSN is safer.

Zhongguo Zhong Yao Za Zhi, 2003 Aug, 28(8), 756 - 8, 793
{Protective effect of curcumin on experimental liver injury in mice}; Liu YG et al.; OBJECTIVE: To Study the protective effect of curcumin on three models of experimental liver injury in mice . METHOD: The experimental models of live injury were induced by carbon tetrachloride (CCl4), D-galactosamine (D-Gal N), and Bacillus Calmette-Guerin (BCG) Plus lipolysaccharides (LPS), respectively, in mice . The serum ALT, AST, NO and liver MDA were measured to evaluate the protective effect of curcumin on experimental injury in mice . RESULT: Curcumin (50 mg.kg-1, 100 mg.kg-1, 150 mg.kg-1), like biophenyldicarboxylate, were shown to significantly inhibit the increase of serum ALT, AST, NO and liver molondialdehyde (MDA) content induced by CCl4, D-Gal N, BCG + LPS . CONCLUSION: Curcumin showed protective effect against liver injury induced by CCl4, D-Gal N, BCG plus LPS.

Arch Microbiol, 2004 Apr, 181(4), 314 - 23 Epub 2004 Mar 11.
Bacillus amyloliquefaciens strains isolated from moisture-damaged buildings produced surfactin and a substance toxic to mammalian cells; Mikkola R et al.; Fungicidic Bacillus amyloliquefaciens strains isolated from the indoor environment of moisture-damaged buildings contained heat-stable, methanol-soluble substances that inhibited motility of boar spermatozoa within 15 min of exposure and killed feline lung cells in high dilution in 1 day . Boar sperm cells lost motility, cellular ATP, and NADH upon contact to the bacterial extract (0.2 microg dry wt/ml) . Two bioactive substances were purified from biomass of the fungicidal isolates . One partially characterized substance, 1,197 Da, was moderately hydrophobic and contained leucine, proline, serine, aspartic acid, glutamic acid and tyrosine, in addition to chromophore(s) absorbing at 365 nm . In boar sperm and human neural cells (Paju), the compound depolarized the transmembrane potentials of mitochondria (Delta Psi(m)) and the plasma membrane (Delta Psi(p)) after a 20-min exposure and formed cation-selective channels in lipid membranes, with a selectivity K(+):Na(+):Ca(2+) of 26:15:3.5 . The other substance was identified as a plasma-membrane-damaging lipopeptide surfactin . Plate-grown biomass of indoor Bacillus amyloliquefaciens contained ca . 7% of dry weight of the two substances, 1,197 Da and surfactin, in a ratio of 1:6 (w:w) . The in vitro observed simultaneous collapse of both cytosolic and mitochondrial ATP in the affected mammalian cell, induced by the 1,197-Da cation channel, suggests potential health risks for occupants of buildings contaminated with such toxins.

J Appl Microbiol, 2004, 96(4), 725 - 41
Analysis of the action of compounds that inhibit the germination of spores of Bacillus species; Cortezzo DE et al.; AIMS: To determine the mechanism of action of inhibitors of the germination of spores of Bacillus species, and where these inhibitors act in the germination process . METHODS AND RESULTS: Spores of various Bacillus species are significant agents of food spoilage and food-borne disease, and inhibition of spore germination is a potential means of reducing such problems . Germination of the following spores was studied: (i) wild-type B . subtilis spores; (ii) B . subtilis spores with a nutrient receptor variant allowing recognition of a novel germinant; (iii) B . subtilis spores with elevated levels of either the variant nutrient receptor or its wild-type allele; (iv) B . subtilis spores lacking all nutrient receptors and (v) wild-type B . megaterium spores . Spores were germinated with a variety of nutrient germinants, Ca2+-dipicolinic acid (DPA) and dodecylamine for B . subtilis spores, and KBr for B . megaterium spores . Compounds tested as inhibitors of germination included alkyl alcohols, a phenol derivative, a fatty acid, ion channel blockers, enzyme inhibitors and several other compounds . Assays used to assess rates of spore germination monitored: (i) the fall in optical density at 600 nm of spore suspensions; (ii) the release of the dormant spore's large depot of DPA; (iii) hydrolysis of the dormant spore's peptidoglycan cortex and (iv) generation of CFU from spores that lacked all nutrient receptors . The results with B . subtilis spores allowed the assignment of inhibitory compounds into two general groups: (i) those that inhibited the action of, or response to, one nutrient receptor and (ii) those that blocked the action of, or response to, several or all of the nutrient receptors . Some of the compounds in groups 1 and 2 also blocked action of at least one cortex lytic enzyme, however, this does not appear to be the primary site of their action in inhibiting spore germination . The inhibitors had rather different effects on germination of B . subtilis spores with nutrients or non-nutrients, consistent with previous work indicating that germination of B . subtilis spores by non-nutrients does not involve the spore's nutrient receptors . In particular, none of the compounds tested inhibited spore germination with dodecylamine, and only three compounds inhibited Ca2+-DPA germination . In contrast, all compounds had very similar effects on the germination of B . megaterium spores with either glucose or KBr . The effects of the inhibitors tested on spores of both Bacillus species were largely reversible . CONCLUSIONS: This work indicates that inhibitors of B . subtilis spore germination fall into two classes: (i) compounds (most alkyl alcohols, N-ethylmaleimide, nifedipine, phenols, potassium sorbate) that inhibit the action of, or response to, primarily one nutrient receptor and (ii) compounds {amiloride, HgCl2, octanoic acid, octanol, phenylmethylsulphonylfluoride (PMSF), quinine, tetracaine, tosyl-l-arginine methyl ester, trifluoperazine} that inhibit the action of, or response to, several nutrient receptors . Action of these inhibitors, is reversible . The similar effects of inhibitors on B . megaterium spore germination by glucose or KBr indicate that inorganic salts likely trigger germination by activating one or more nutrient receptors . The lack of effect of all inhibitors on dodecylamine germination suggests that this compound stimulates germination by creating channels in the spore's inner membrane allowing DPA release . SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the steps in spore germination that are inhibited by various chemicals, and the mechanism of action of these inhibitors . The work also provides new insights into the process of spore germination itself.

J Appl Microbiol, 2004, 96(4), 684 - 92
Assessment of bacterial endospore viability with fluorescent dyes; Laflamme C et al.; AIM: To validate three fluorescence viability assays designed primarily for vegetative cells on pure Bacillus endospores . METHODS AND RESULTS: Purified fresh and gamma-irradiated Bacillus endospores (Bacillus cereus, B . coagulans and two strains of B . subtilis) were used . The viability assays were: 5-cyano-2,3-diotolyl tetrazolium chloride (CTC) to test respiratory activity and early germination, DiBAC4(3) and Live/Dead BacLight to measure membrane energization and permeabilization, respectively . Gamma irradiation treatment completely eliminated spore culturability and was used as negative control . The untreated spores showed respiratory activity after 1 h of incubation and this was characteristic of almost 100% of spores after 24 h . The membrane potential assessment gave no answer about spore viability . A lower proportion of untreated spores had permeabilized membrane compared with gamma-irradiated spores using Live/Dead BacLight (P < 0.02) . CONCLUSION: It is possible to use CTC and Live/Dead BacLight to rapidly test endospore viability and evaluate the proportion of spores in a preparation that could not be recovered with plate count . SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that fluorescence tests could be applied to assess viability in potentially pathogenic Bacillus spore preparations within 1 h.

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