J Biol Chem, 2001 Nov 30, 276(48), 45015 - 23 Epub 2001 Sep 19. Thiomandelic acid, a broad spectrum inhibitor of zinc beta-lactamases: kinetic and spectroscopic studies; Mollard C et al.; Resistance to beta-lactam antibiotics mediated by metallo-beta-lactamases is an increasingly worrying clinical problem . Candidate inhibitors include mercaptocarboxylic acids, and we report studies of a simple such compound, thiomandelic acid . A series of 35 analogues were synthesized and examined as metallo-beta-lactamase inhibitors . The K(i) values (Bacillus cereus enzyme) are 0.09 microm for R-thiomandelic acid and 1.28 microm for the S-isomer . Structure-activity relationships show that the thiol is essential for activity and the carboxylate increases potency; the affinity is greatest when these groups are close together . Thioesters of thiomandelic acid are substrates for the enzyme, liberating thiomandelic acid, suggesting a starting point for the design of "pro-drugs." Importantly, thiomandelic acid is a broad spectrum inhibitor of metallo-beta-lactamases, with a submicromolar K(i) value for all nine enzymes tested, except the Aeromonas hydrophila enzyme; such a wide spectrum of activity is unprecedented . The binding of thiomandelic acid to the B . cereus enzyme was studied by NMR; the results are consistent with the idea that the inhibitor thiol binds to both zinc ions, while its carboxylate binds to Arg(91) . Amide chemical shift perturbations for residues 30-40 (the beta(3)-beta(4) loop) suggest that this small inhibitor induces a movement of this loop of the kind seen for other larger inhibitors. J Mol Biol, 2001 Sep 21, 312(3), 569 - 77 On the relationship between protein stability and folding kinetics: a comparative study of the N-terminal domains of RNase HI, E . coli and Bacillus stearothermophilus L9; Sato S et al.; There is currently a great deal of interest in proteins that fold in a single highly cooperative step . Particular attention has been focused on elucidating the factors that govern folding rates of simple proteins . Recently, the topology of the native state has been proposed to be the most important determinant of their folding rates . Here we report a comparative study of the folding of three topologically equivalent proteins that adapt a particularly simple alpha/beta fold . The folding kinetics of the N-terminal domain of RNase HI and the N-terminal domain of the ribosomal protein L9 from Escherichia coli (eNTL9) were compared to the previously characterized N-terminal domain of L9 from Bacillus stearothermophilus (bNTL9) . This 6.2 kDa protein, which is one of simplest examples of the ABCalphaD motif, folds via a two-state mechanism on the millisecond to submillisecond time scale . The RNase HI domain and bNTL9 have very similar tertiary structures but there is little similarity in primary sequence . bNTL9 and eNTL9 share the same biological function and a similar primary sequence but differ significantly in stability . Fluorescence-detected stopped-flow experiments showed that the three proteins fold in a two-state fashion . The folding rates in the absence of denaturant were found to be very different, ranging form 21 s(-1) to 790 s(-1) at 10 degrees C . The diverse folding rates appear to reflect large differences in the stability of the proteins . When compared at an isostability point, the folding rates converged to a similar value and there is a strong linear correlation between the log of the folding rate and stability for this set of proteins . These observations are consistent with the idea that stability can play an important role in dictating relative folding rates among topologically equivalent proteins . Mem Inst Oswaldo Cruz, 2001 Aug, 96(6), 859 - 60 Evaluation of a new tablet formulation based on Bacillus thuringiensis sorovar . israelensis for larvicidal control of Aedes aegypti; de Melo-Santos MA et al.; The effect of sunlight on the efficacy and persistence of an experimental tablet formulation based on Bacillus thuringiensis sorovar . israelensis (C4P1) was evaluated against Aedes aegypti larvae under simulated field conditions . The initial mortality ranged from 93 to 100%, and the residual activity (> or = 70% mortality) recorded in containers exposed to sunlight or shade were, respectively, 13-35 days and 40-54 days . The results suggest that C4P1 can provide long-term larvicidal effect and operational advantages. Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11497 - 502 Epub 2001 Sep 18. Divergent effect of bacillus Calmette-Guérin (BCG) vaccination on Mycobacterium tuberculosis infection in highly related macaque species: implications for primate models in tuberculosis vaccine research; Langermans JA et al.; Despite the widespread use of bacillus Calmette-Guerin vaccination, Mycobacterium tuberculosis infection remains globally the leading cause of death from a single infectious disease . The complicated and often protracted dynamics of infection and disease make clinical trials to test new tuberculosis vaccines extremely complex . Preclinical selection of only the most promising candidates is therefore essential . Because macaque monkeys develop a disease very similar to humans, they have potential to provide important information in addition to small animal models . To assess the relative merits of rhesus and cynomolgus monkeys as screens for tuberculosis vaccines, we compared the efficacy of bacillus Calmette-Guerin vaccination and the course of infection in both species . Unvaccinated rhesus and cynomolgus monkeys both developed progressive disease with high levels of C-reactive protein, M . tuberculosis-specific IgG, and extensive pathology including cavitation and caseous necrosis . Bacillus Calmette-Guerin vaccination protected cynomolgus almost completely toward the development of pathology, reflected in a striking 2-log reduction in viable bacteria in the lungs compared with nonvaccinated animals . Rhesus, on the other hand, were not protected efficiently by the bacillus Calmette-Guerin . The vaccinated animals developed substantial pathology and had negligible reductions of colony-forming units in the lungs . Comparative studies in these closely related species are likely to provide insight into mechanisms involved in protection against tuberculosis. Int Immunopharmacol, 2001 Sep, 1(9-10), 1743 - 51 Mycobacterial antigens attenuate late phase response, airway hyperresponsiveness, and bronchoalveolar lavage eosinophilia in a mouse model of bronchial asthma; Hopfenspirger MT et al.; Allergens, in combination with genetic predisposition, drive undifferentiated T cells towards the type 2 T cells . Some childhood infections may activate the production of a type 1 T cell profile . It is reasonable to speculate that a decrease in childhood infections may increase the incidence of allergy by allowing the immune balance to shift towards the type 2 T cells . We hypothesized that pre-exposure of mycobacterial antigens in sensitized mice would prevent the development of asthma-like conditions . Specifically, we examined the effect of mycobacterial antigens, Bacillus Calmette-Guerin (BCG) vaccine and Mycobacterium vaccae, on antigen-induced bronchoconstriction, airway hyperresponsiveness to methacholine, bronchoalveolar lavage eosinophilia, and plasma IL-4 and IL-12 levels in ovalbumin (OVA)-sensitized and challenged Balb/c mice . Challenge with OVA produced a 2-3-fold increase in bronchoconstriction within 3-5 min, followed by a delayed response after 60 min, the latter of which was significantly attenuated by both BCG and M . vaccae . Airway hyperresponsiveness to methacholine 24 h after OVA challenge was prevented by BCG and M . vaccae . Airway eosinophilia was also prevented by BCG and M . vaccae . The plasma IL-12 levels were significantly increased and plasma IL-4 levels were significantly decreased following BCG or M . vaccae administration in OVA-sensitized and challenged mice . Interestingly, a significant increase in plasma IL-12 was observed with BCG as compared to M . vaccae administration, suggesting a stronger type 1 response to BCG . These data support our hypothesis and suggest that BCG and M . vaccae may prevent the underlying pathophysiological changes in asthma. J Econ Entomol, 2001 Aug, 94(4), 935 - 41 Overwintering cost associated with resistance to transgenic cotton in the pink bollworm (Lepidoptera: Gelechiidae); Carriere Y et al.; Fitness costs associated with resistance to transgenic crops producing toxins from Bacillus thuringiensis (Bt) may have important effects on the evolution of resistance . We investigated overwintering costs in pink bollworm, Pectinophora gosypiella (Saunders), strains with different degrees of resistance to Bt cotton . Frequency of resistant individuals in a strain was not associated with induction of diapause or emergence from diapause in early winter . Emergence from diapause in the spring was 71% lower in three highly resistant strains than in two heterogeneous strains from which the resistant strains were derived . This underestimates the overwintering cost because the frequency of the resistance allele was relatively high in the heterogeneous strains . Emergence in the spring in hybrid progeny from crosses between the resistant and heterogeneous strains was greater than in resistant strains but did not differ from susceptible strains, showing that the overwintering cost was recessive to some extent. J Econ Entomol, 2001 Aug, 94(4), 920 - 8 Baseline susceptibility to bacterial insecticides in populations of Culex pipiens complex (Diptera: Culicidae) from California and from the Mediterranean Island of Cyprus; Wirth MC et al.; Bacterial insecticides play an increasingly important role in mosquito control . To establish guidelines for detecting resistance at an early stage, information on natural variation in susceptibility of insect populations to these insecticides is needed . Between 1990 and 1993, the susceptibility of Culex pipiens L . complex to Bacillus thuringiensis subsp . israelensis de Barjac and/or Bacillus sphaericus Neide was determined in 31 collections from California . These collections were undertaken before the widespread use of B . thuringiensis subsp . israelensis and before the registration of B . sphaericus in California . Seven collections from the Mediterranean island of Cyprus, where no microbial insecticides have been used, also were tested . The 1990-1991 California collections exhibited limited variation in susceptibility to B . thuringiensis subsp . israelensis . LC50 and LC95 values spanned about a three-fold and four-fold range, respectively . The 1993 Cyprus collections exhibited both higher mean LC values, and greater variability in those values, than the California collections . The LC50s for the Cyprus collections varied over a 10-fold range, whereas the LC50s varied over a 12.5-fold range . Variation in susceptibility to B . sphaericus among the 1991 California collections was about five-fold at the LC50 and LC95 . No significant geographic variation in susceptibility to B . thuringiensis subsp . israelensis was observed among regions within California . Although variation in susceptibility was limited among California collections, the greater variability observed among the Cyprus collections and between the Cyprus and California collections illustrates the importance of establishing regional baselines to monitor accurately for changes in susceptibility. Biochemistry, 2001 Sep 25, 40(38), 11623 - 9 Isolation of an individual allosteric interaction in tetrameric phosphofructokinase from Bacillus stearothermophilus; Kimmel JL et al.; Phosphofructokinase from Bacillus stearothermophilus (BsPFK) is a model allosteric enzyme system in which the interactions between substrates and allosteric effectors have been extensively studied . However, the oligomeric nature of BsPFK has made it difficult to determine the molecular basis of the allosteric regulation because of the multitude of different types of heterotropic and homotropic interactions that are possible between the four active sites and four allosteric sites in the native tetramer . In an attempt to alleviate the complexity of the system and thereby allow the quantitation of a single interaction between one active site and one allosteric site, site-directed mutagenesis has been coupled with a hybrid-forming scheme to create and isolate a tetramer of BsPFK in which only a single active site and a single allosteric site are capable of binding their respective ligands with high (i.e., near wild type) affinity . Characterization of this single allosteric interaction indicates that the free energy involved in the inhibition by the allosteric effector phosphoenolpyruvate (PEP) is 1.48 +/- 0.15 kcal/mol compared to the 3.58 +/- 0.02 kcal/mol measured for the enzyme. Biochemistry, 2001 Sep 25, 40(38), 11525 - 32 Structural and mechanistic insight into the inhibition of aspartic proteases by a slow-tight binding inhibitor from an extremophilic Bacillus sp.: correlation of the kinetic parameters with the inhibitor induced conformational changes; Dash C et al.; We present here the first report of a hydrophilic peptidic inhibitor, ATBI, from an extremophilic Bacillus sp . exhibiting a two-step inhibition mechanism against the aspartic proteases, pepsin and F-prot from Aspergillus saitoi . Kinetic analysis shows that these proteases are competitively inhibited by ATBI . The progress curves are time-dependent and consistent with slow-tight binding inhibition: E + I right arrow over left arrow (k(3), k(4)) EI right arrow over left arrow (k(5), k(6)) EI . The K(i) values for the first reversible complex (EI) of ATBI with pepsin and F-prot were (17 +/- 0.5) x 10(-9) M and (3.2 +/- 0.6) x 10(-6) M, whereas the overall inhibition constant K(i) values were (55 +/- 0.5) x 10(-12) M and (5.2 +/- 0.6) x 10(-8) M, respectively . The rate constant k(5) revealed a faster isomerization of EI for F-prot {(2.3 +/- 0.4) x 10(-3) s(-1)} than pepsin {(7.7 +/- 0.3) x 10(-4) s(-1)} . However, ATBI dissociated from the tight enzyme-inhibitor complex (EI) of F-prot faster {(3.8 +/- 0.5) x 10(-5) s(-1)} than pepsin {(2.5 +/- 0.4) x 10(-6) s(-1)} . Comparative analysis of the kinetic parameters with pepstatin, the known inhibitor of pepsin, revealed a higher value of k(5)/k(6) for ATBI . The binding of the inhibitor with the aspartic proteases and the subsequent conformational changes induced were monitored by exploiting the intrinsic tryptophanyl fluorescence . The rate constants derived from the fluorescence data were in agreement with those obtained from the kinetic analysis; therefore, the induced conformational changes were correlated to the isomerization of EI to EI . Chemical modification of the Asp or Glu by WRK and Lys residues by TNBS abolished the antiproteolytic activity and revealed the involvement of two carboxyl groups and one amine group of ATBI in the enzymatic inactivation. Proc Natl Acad Sci U S A, 2001 Oct 9, 98(21), 11925 - 30 Epub 2001 Sep 14. Monarch larvae sensitivity to Bacillus thuringiensis- purified proteins and pollen; Hellmich RL et al.; Laboratory tests were conducted to establish the relative toxicity of Bacillus thuringiensis (Bt) toxins and pollen from Bt corn to monarch larvae . Toxins tested included Cry1Ab, Cry1Ac, Cry9C, and Cry1F . Three methods were used: (i) purified toxins incorporated into artificial diet, (ii) pollen collected from Bt corn hybrids applied directly to milkweed leaf discs, and (iii) Bt pollen contaminated with corn tassel material applied directly to milkweed leaf discs . Bioassays of purified Bt toxins indicate that Cry9C and Cry1F proteins are relatively nontoxic to monarch first instars, whereas first instars are sensitive to Cry1Ab and Cry1Ac proteins . Older instars were 12 to 23 times less susceptible to Cry1Ab toxin compared with first instars . Pollen bioassays suggest that pollen contaminants, an artifact of pollen processing, can dramatically influence larval survival and weight gains and produce spurious results . The only transgenic corn pollen that consistently affected monarch larvae was from Cry1Ab event 176 hybrids, currently <2% corn planted and for which re-registration has not been applied . Results from the other types of Bt corn suggest that pollen from the Cry1Ab (events Bt11 and Mon810) and Cry1F, and experimental Cry9C hybrids, will have no acute effects on monarch butterfly larvae in field settings. Proc Natl Acad Sci U S A, 2001 Oct 9, 98(21), 11931 - 6 Epub 2001 Sep 14. Assessing the impact of Cry1Ab-expressing corn pollen on monarch butterfly larvae in field studies; Stanley-Horn DE et al.; Survival and growth of monarch larvae, Danaus plexippus (L.), after exposure to either Cry1Ab-expressing pollen from three Bacillus thuringiensis (Bt) corn (Zea mays L.) events differing in toxin expression or to the insecticide, lambda-cyhalothrin, were examined in field studies . First instars exposed to low doses ( approximately 22 grains per cm(2)) of event-176 pollen gained 18% less weight than those exposed to Bt11 or Mon810 pollen after a 5-day exposure period . Larvae exposed to 67 pollen grains per cm(2) on milkweed leaves from within an event-176 field exhibited 60% lower survivorship and 42% less weight gain compared with those exposed to leaves from outside the field . In contrast, Bt11 pollen had no effect on growth to adulthood or survival of first or third instars exposed for 5 days to approximately 55 and 97 pollen grains per cm(2), respectively . Similarly, no differences in larval survivorship were observed after a 4-day exposure period to leaves with 504-586 (within fields) or 18-22 (outside the field) pollen grains per cm(2) collected from Bt11 and non-Bt sweet-corn fields . However, survivorship and weight gain were drastically reduced in non-Bt fields treated with lambda-cyhalothrin . The effects of Bt11 and Mon810 pollen on the survivorship of larvae feeding 14 to 22 days on milkweeds in fields were negligible . Further studies should examine the lifetime and reproductive impact of Bt11 and Mon810 pollen on monarchs after long-term exposure to naturally deposited pollen. Proc Natl Acad Sci U S A, 2001 Oct 9, 98(21), 11913 - 8 Epub 2001 Sep 14. Temporal and spatial overlap between monarch larvae and corn pollen; Oberhauser KS et al.; To assess the likelihood that monarch larvae will be exposed to Bacillus thuringiensis (Bt) pollen, we studied milkweed and monarch densities in habitats which comprise much of the land available to breeding monarchs, e.g., cornfields, cornfield edges, other agricultural fields, and nonagricultural areas, in four regions of the monarch breeding range . We found that monarchs use milkweed in cornfields throughout their breeding season, and that per plant densities are as high or higher in agricultural habitats as in nonagricultural habitats . As a result of the prevalence of agricultural land, most of the monarchs produced in the upper Midwest are likely to originate in cornfields or other agricultural habitats . There was a greater temporal overlap between susceptible monarchs and corn anthesis in the northern than the southern part of the summer breeding range, because of earlier pollen shed in the south . The importance of agricultural habitats to monarch production suggests that, regardless of the impact of genetically modified crops, agricultural practices such as weed control and foliar insecticide use could have large impacts on monarch populations. Proc Natl Acad Sci U S A, 2001 Oct 9, 98(21), 11908 - 12 Epub 2001 Sep 14. Effects of exposure to event 176 Bacillus thuringiensis corn pollen on monarch and black swallowtail caterpillars under field conditions; Zangerl AR et al.; The widespread planting of corn genetically modified to produce Bacillus thuringiensis endotoxin has led to speculation that pollen from these fields might adversely affect nearby nontarget lepidopterans . A previous study of Bt corn engineered with Monsanto event 810 failed to detect an effect of pollen exposure on the black swallowtail, Papilio polyxenes, in either the field or the laboratory . Here, we report results of a field study investigating the impact of exposure to pollen from a Bt corn hybrid containing Novartis event 176 on two species of Lepidoptera, black swallowtails and monarch butterflies, Danaus plexippus . Nearly half of the 600 monarch larvae died within the first 24 h; this and subsequent mortality was not associated with proximity to Bt corn and may have been due in part to predation . Survivorship of black swallowtails was much higher than that of the monarchs and was also independent of proximity to the transgenic corn . However, despite five rainfall events that removed much of the pollen from the leaves of their host plants during the experiment, we observed a significant reduction in growth rates of black swallowtail larvae that was likely caused by pollen exposure . These results suggest that Bt corn incorporating event 176 can have adverse sublethal effects on black swallowtails in the field and underscore the importance of event selection in reducing environmental impacts of transgenic plants. Eur J Biochem, 2001 Sep, 268(18), 4985 - 91 The effect of changing the hydrophobic S1' subsite of thermolysin-like proteases on substrate specificity; de Kreij A et al.; The hydrophobic S1' subsite is one of the major determinants of the substrate specificity of thermolysin and related M4 family proteases . In the thermolysin-like protease (TLP) produced by Bacillus stearothermophilus (TLP-ste), the hydrophobic S1' subsite is mainly formed by Phe130, Phe133, Val139 and Leu202 . In the present study, we have examined the effects of replacing Leu202 by smaller (Gly, Ala, Val) and larger (Phe, Tyr) hydrophobic residues . The mutational effects showed that the wild-type S1' pocket is optimal for binding leucine side chains . Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P1' position . Rather unexpectedly, the Leu202-->Phe and Leu202-->Tyr mutations, which were expected to decrease the size of the S1' subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P1' position . This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate . To validate these results, we constructed variants of thermolysin with changes in the S1' subsite . Thermolysin and TLP-ste variants with identical S1' subsites were highly similar in terms of their preference for Phe vs . Leu in the P1' position. Mikrobiologiia, 2001 Jul-Aug, 70(4), 552 - 7 {Dynamic of the volume and structure of the soil bacterial complex in the presence of azobenzene}; Koneva ND et al.; Azobenzene exerted no significant effect on the dynamics and the species composition of the saprophytic soil bacterial complex, which remained almost the same as in the control and was characterized by the predominance of Curtobacterium sp., Arthrobacter globiformis, and Bacillus megaterium in all stages of succession . Some heterotrophic bacteria were found to be able to accumulate azobenzene . Bac . cereus and Bac . polymyxa degraded azobenzene during their cultivation in nutrient media. Mikrobiologiia, 2001 Jul-Aug, 70(4), 471 - 6 {The role of cell surface bacterial lectins in the aggregation of Azospirilla}; Nikitina VE et al.; The mutant strain Azospirillum brasilense Sp7.2.3 with impaired lectin activity exhibited poorer cell aggregation than its parent strain A . brasilense Sp7(S) both in the exponential and stationary growth phases . The pretreatment of bacterial cells with the specific haptens (L-fucose and D-galactose) of a lectin located at the cell surface of the mutant strain was found to inhibit the aggregation of azospirilla . The specific binding of the A . brasilense Sp7(S) lectin to the extracellular polysaccharide-containing complexes of this strain was revealed by dot immunoblotting on nitrocellulose membrane filters . The interaction of the lectins of A . brasilense 75, A . brasilense Sp7, and A . lipoferum 59b with the polysaccharide-containing complexes that were isolated from these strains was not specific . No interstrain cross-interaction between the exopolysaccharides and lectins of azospirilla was found . A coflocculation of A . brasilense Sp7 cells with Bacillus polymyxa 1460 cells was shown . The involvement of autogenous lectins in the aggregation of bacterial cells is discussed. Int Endod J, 2001 Sep, 34(6), 435 - 9 Efficacy of chemical sterilization and storage conditions of gutta-percha cones; da Motta PG et al.; AIM: The objective of the present study was to assess the efficacy of 2.5% sodium hypochlorite and 2.2% glutaraldehyde ('Cidex') as sterilizing agents for gutta-percha cones . The efficacy of storage of gutta-percha cones in the presence or absence of paraformaldehyde was also evaluated . METHODOLOGY: Gutta-percha cones artificially contaminated with a suspension of Bacillus stearothermophilus (ATCC/7953) were treated with either 2.2% glutaraldehyde for 10, 15, 30 and 60 min and 10 and 12 h, or 2.5% sodium hypochlorite for 5, 10 and 15 min . The cones were then incubated in thioglycollate medium for the determination of microbial growth . In parallel, additional sterile gutta-percha cones were stored in sealed containers with or without paraformaldehyde tablets for 30 days . The containers were opened 30 min a day and exposed to the environment of a functioning dental clinic . Twelve cones were removed weekly from the containers to determine whether contamination had occurred . RESULTS: The results showed that 2.5% sodium hypochlorite was effective after 5, 10 and 15 min, whereas 10 and 12 h contact with 2.2% glutaraldehyde was necessary to obtain sterilization . There was no contamination of the gutta-percha cones when stored with or without paraformaldehyde . CONCLUSIONS: Sodium hypochlorite (2.5%) and 2.2% glutaraldehyde ('Cidex') proved to be effective as sterilizing agents for gutta-percha cones, with sodium hypochlorite requiring shorter periods of use . No difference was observed between the two methods of cone storage. Rapid Commun Mass Spectrom, 2001, 15(18), 1672 - 80 Optimization of quartz tube pyrolysis atmospheric pressure ionization mass spectrometry for the generation of bacterial biomarkers; Tripathi A et al.; Experimental procedures were investigated to improve the efflux of biomolecule pyrolyzates from quartz tube pyrolysis under atmospheric pressure ionization mass spectrometry conditions . Heating regimes, airflows, and ion focusing parameters were optimized to increase the informative mass spectral signals generated from the pyrolysis of Gram-positive bacterial spores and vegetative cells . Dipicolinic acid (DPA) is found in 5-15% by weight in Gram-positive Bacillus spores, and the parameter optimization procedures provided an intense mass spectral signature of the m/z 168 protonated DPA molecule with a minimization of pyrolytic and ionization fragments . Moreover, mass spectral information from the optimization protocols yielded peaks and mass patterns characteristic of DNA and RNA nitrogen bases, protein diketopiperazines, and amino sugars . J Biol Chem, 2001 Nov 30, 276(48), 44557 - 62 Epub 2001 Sep 12. Hydrophobic amino acid residues in the acceptor binding site are main determinants for reaction mechanism and specificity of cyclodextrin-glycosyltransferase; van der Veen BA et al.; Cyclodextrin-glycosyltransferases (CGTases) (EC ) preferably catalyze transglycosylation reactions with glucosyl residues as acceptor, whereas the homologous alpha-amylases catalyze hydrolysis reactions using water as acceptor . This difference in reaction specificity is most likely caused by the acceptor binding site . To investigate this in detail we altered the acceptor site residues Lys-232, Phe-183, Phe-259, and Glu-264 of Bacillus circulans strain 251 CGTase using site-directed mutagenesis . Lys-232 is of general importance for catalysis, which appears to result mainly from stabilization of the conformation of the loop containing the catalytic nucleophile Asp-229 and His-233, a residue that has been implied in transition state stabilization . Glu-264 contributes to the disproportionation reaction only, where it is involved in initial binding of the (maltose) acceptor . Phe-183 and Phe-259 play important and distinct roles in the transglycosylation reactions catalyzed by CGTase . Mutation of Phe-183 affects especially the cyclization and coupling reactions, whereas Phe-259 is most important for the cyclization and disproportionation reactions . Moreover, the hydrophobisity of Phe-183 and Phe-259 limits the hydrolyzing activity of the enzyme . Hydrolysis can be enhanced by making these residues more polar, which concomitantly results in a lower transglycosylation activity . A double mutant was constructed that yielded an enzyme preferring hydrolysis over cyclization (15:1), whereas the wild type favors cyclization over hydrolysis (90:1). Lett Appl Microbiol, 2001 Sep, 33(3), 241 - 5 Screening for novel cry genes by hybridization; Beard CE et al.; AIMS: To develop an efficient and sensitive method to facilitate the search for novel Cry toxins . METHODS AND RESULTS: The method uses a cocktail of cry gene sequences as a hybridization probe to screen Bacillus thuringiensis (Bt) strains and gene libraries prepared from them . Under the hybridization and washing conditions used, cross-hybridization between genes from different cry families was not observed . Probes containing either partial or complete cry gene sequences produced similar patterns when hybridized to genomic DNA of several Bt strains although the pattern produced by the probe composed of entire gene coding regions was somewhat more complex . CONCLUSION: As a tool for gene library screening, hybridization with a mixture of cry gene sequences is an efficient means of detecting clones containing a diverse range of cry genes in a single step . SIGNIFICANCE AND IMPACT OF THE STUDY: This technique greatly improves the ease and efficiency of novel toxin gene discovery compared to previous methods. Mol Reprod Dev, 2001 Oct, 60(2), 270 - 6 New DAPI and FISH findings on egg maturation processes in related hybridogenetic and parthenogenetic Bacillus hybrids (Insecta, Phasmatodea); Marescalchi O et al.; Bacillus stick insects have proved adequate for studying a wide array of reproductive modes: sexual, parthenogenetic, hybridogenetic, androgenetic . Hybridogenetic strains (B . rossius-grandii) were thought to discard the paternal "grandii" haploset during first meiotic division and keep the "rossius" hemiclone, whereas the clonal B . whitei (=rossius/grandii) would maintain its hybrid structure by fusing back two nonsister nuclei-each derived from previously segregated heterospecific complements-by the end of the 2(nd) meiotic division . New investigations on laid eggs and ovariole squashes, either DAPI stained or FISH labeled, revealed that in hybridogens the "grandii" set is excluded from the germ line prior to meiosis and that a DNA extra-synthesis should occur to produce hemiclonal eggs after two cytologically normal meiotic divisions . On the other hand, in B . whitei eggs no genome segregation appears to occur and an intrameiotic DNA extra-synthesis must take place to produce 2n tetrachromatidic oocytes I; these divide twice and give unreduced clonal eggs . The new findings bring hybridogenetic oogenesis of Bacillus to be coincident with that of the known hemiclonal organisms and point to an independent onset of B . whitei from hemiclonal strains . In addition, B . whitei gains a closer resemblance to B . lynceorum owing to the sharing of a cytologically identical egg maturation mechanism, of the same maternal ancestor and of peculiar chromosomal features . It is here suggested that B . lynceorum originated from the incorporation of an "atticus" genome into a B . whitei egg, according to a pathway of repeated hybridization often occurred with other polyploid hybrids . Cell Microbiol, 2001 Sep, 3(9), 623 - 32 Evidence of a leading role for VEGF in Bartonella henselae-induced endothelial cell proliferations; Kempf VA et al.; Bartonella henselae causes the vasculoproliferative disorders bacillary angiomatosis (BA) and bacillary peliosis (BP) . The pathomechanisms of these tumorous proliferations are unknown . Our results suggest a novel bacterial two-step pathogenicity strategy, in which the pathogen triggers growth factor production for subsequent proliferation of its own host cells . In fact, B . henselae induces host cell production of the angiogenic factor vascular endothelial growth factor (VEGF), leading to proliferation of endothelial cells . The presence of B . henselae pili was associated with host cell VEGF production, as a Pil- mutant of B . henselae was unable to induce VEGF production . In turn, VEGF-stimulated endothelial cells promoted the growth of B . henselae . Immunohistochemistry for VEGF in specimens from patients with BA or BP revealed increased VEGF expression in vivo . These findings suggest a novel bacteria-dependent mechanism of tumour growth. J Biol Chem, 2001 Nov 30, 276(48), 45065 - 78 Epub 2001 Sep 10. Metal ion binding and coordination geometry for wild type and mutants of metallo-beta -lactamase from Bacillus cereus 569/H/9 (BcII): a combined thermodynamic, kinetic, and spectroscopic approach; de Seny D et al.; One high affinity (nm) and one low affinity (microM) macroscopic dissociation constant for the binding of metal ions were found for the wild-type metallo-beta-lactamase from Bacillus cereus as well as six single-site mutants in which all ligands in the two metal binding sites were altered . Surprisingly, the mutations did not cause a specific alteration of the affinity of metal ions for the sole modified binding site as determined by extended x-ray absorption fine structure (EXAFS) and perturbed angular correlation of gamma-rays spectroscopy, respectively . Also UV-visible absorption spectra for the mono-cobalt enzymes clearly contain contributions from both metal sites . The observations of the very similar microscopic dissociation constants of both binding sites in contrast to the significantly differing macroscopic dissociation constants inevitably led to the conclusion that binding to the two metal sites exhibits negative cooperativity . The slow association rates for forming the binuclear enzyme determined by stopped-flow fluorescence measurements suggested that fast metal exchange between the two sites for the mononuclear enzyme hinders the binding of a second metal ion . EXAFS spectroscopy of the mono- and di-zinc wild type enzymes and two di-zinc mutants provide a definition of the metal ion environments, which is compared with the available x-ray crystallographic data. Urology, 2001 Sep, 58(3), 376 - 9 Risk of continued intravesical therapy and delayed cystectomy in BCG-refractory superficial bladder cancer: an investigational approach; Luciani LG et al.; OBJECTIVES: To assess the risk of continued intravesical therapy and delayed cystectomy in the management of superficial bladder cancer refractory to bacillus Calmette-Guerin (BCG) therapy . METHODS: We retrospectively reviewed the medical records of 24 patients who underwent an experimental intravesical treatment with BCG plus interferon alpha-2b or valrubicin for transitional cell carcinoma of the bladder . All patients had Stage Tis and/or T1 transitional cell carcinoma and had failed multiple prior courses of intravesical therapy, including at least one course of BCG . RESULTS: Patients were followed up for a median of 28.5 months (range 6 to 48) . One patient died of unrelated disease . All other patients were alive at last follow-up . Fourteen patients with preserved bladder were continuing cystoscopic surveillance: four had no recurrence, five had recurrence limited to the mucosa (Ta or Tis) and became free of disease after an additional course of intravesical therapy, and five had recurrent Ta or Tis or positive cytologic findings . The remaining 9 patients underwent radical cystectomy . All pathologic specimens showed no evidence of progression to muscle-invasive disease . Tis of the resected ureters in 6 and involvement of the prostate in 4 of the 9 patients (three in the urethral ducts and glands and one in the prostatic stroma) were noted . CONCLUSIONS: A select group of patients with BCG-refractory transitional cell carcinoma and a poor surgical risk for cystectomy may benefit from continued intravesical therapy without a significant risk of progression . However, a cautious approach to this treatment modality is recommended, and very close follow-up is necessary to detect bladder recurrences and involvement of the upper tract and prostatic urethra. Appl Microbiol Biotechnol, 2001 Aug, 56(3-4), 431 - 4 Heterologous expression of the Bacillus pumilus endo-beta-xylanase (xynA) gene in the yeast Saccharomyces cerevisiae; Nuyens F et al.; The endo-beta-xylanase-encoding gene (xynA) of Bacillus pumilus PLS was isolated from a genomic DNA library and the open reading frame (ORF) was inserted in expression vectors for the yeast Saccharomyces cerevisiae . Plasmid pFN3 harboured the xynA ORF fused to the yeast mating pheromone alpha-factor signal sequence (MFalpha1s) under the control of the alcohol dehydrogenase II gene promotor (ADH2P) and terminator (ADH2T) sequences . In plasmid pFN4, the MFalpha1S-xynA ORF was brought under the control of the phosphoglycerate kinase I gene promotor (PGK1p) and terminator (PGK1T) sequences . Autoselective, recombinant S . cerevisiae {fur1::LEU2} strains bearing pFN3 or pFN4 secreted functional endo-beta-xylanase when grown in complex medium . Enzymatic activities in the culture supernatants reached maximum levels of 8.5 nkat/ml and 4.5 nkat/ml, respectively . The temperature and pH optimum for both the bacterial and the recombinant xylanase were 58 degrees C and pH 6.2. Appl Microbiol Biotechnol, 2001 Aug, 56(3-4), 402 - 5 Isolation and characterization of a cell-associated protein of Bacillus pumilus PH-01; Hong HB et al.; A cell-associated protein released from Bacillus pumilus PH-01 showed an affinity for some dioxins, like 1,2,3,4-tetrachlorodibenzo-p-dioxin (TCDD) and 1,2,3,4-tetrachlorodibenzofuran (TCDF), and the concentration of the protein increased when B . pumilus PH-01 was boiled in minimal salts medium . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-mass spectrometry revealed that the boiled culture supernatant contained a major protein with a molecular mass of 5,313.4 Da . The adsorption behavior of the protein for 1,2,3,4-TCDD and 1,2,3,4-TCDF was examined by digesting it with proteinase K and trypsin, showing that the proteolyzed protein lost the ability to adsorb the compounds . The amino acid sequence of the protein was determined by automated Edman degradation and tandem mass spectrometry . A search of the protein databases showed no existence of proteins with an homologous sequence. Appl Microbiol Biotechnol, 2001 Aug, 56(3-4), 395 - 401 Chitinase from Bacillus thuringiensis subsp . pakistani; Thamthiankul S et al.; The chitinase gene (chiA71) from Bacillus thuringiensis subsp . pakistani consists of an open reading frame of 1,905 nucleotides encoding 635 amino acid residues with an estimated molecular mass of 71 kDa . Comparison of the deduced amino acid sequence of the mature enzyme to other microbial chitinases shows a putative catalytic domain and a region with conserved amino acids similar to that of the type III module of fibronectin and a chitin-binding domain . By activity detection of chitinase on SDS-PAGE after renaturation, the molecular mass of protein bands with chitinase activity were 66, 60, 47, and 32 kDa . The N-terminal amino acid sequence of each chitinase activity band was the same (Asp-Ser-Pro-Lys-Gln), suggesting that the 60-, 47-, and 32-kDa chitinases were derived from the 66-kDa chitinase by processing step(s) at the C-terminus . The enzyme was identified as an exochitinase, since it generated N-acetylglucosamine from early stage of colloidal chitin hydrolysis . The crude protein (2.3-18.4 mg/ml), containing chitinase at final activities of 8, 16, 32, and 64 mU/ml, was toxic to Aedes aegypti larvae and caused mortalities of 7.5, 15.0, 51.3, and 70.0% respectively, but the same amount of crude protein from a B . thuringiensis subsp . pakistani mutant lacking chitinase was not toxic. Microbios, 2001, 106 Suppl 2, 143 - 54 Detection of antigenic surface proteins of Actinobacillus actinomycetemcomitans using the immunoblotting method; Mineyama R; Antigenic surface proteins of Actinobacillus actinomycetemcomitans (three strains), which can be recognized by antibodies in human serum, were examined using the Western blot method . By comparing the immunoblotting profiles between protease-treated cells and untreated cells, IgG-antigenic and IgM-antigenic surface proteins were found . The IgG-antigenic proteins revealed the following molecular weights: strain ATCC 29522, 52 and 49 kD; strain ATCC 29523, 45, 49, 52 and 70 kD; strain Y4, 36, 38, 44, 53 and 58 kD . Molecular weights of the IgM-antigenic proteins ranged from 50 to 92 kD: strain ATCC 29522, 68, 80, 90 and 92 kD; strain ATCC 29523, 62, 68 and 80 kD; strain Y4, 50, 64, 73, 81 and 86 kD . The IgG-antigenic proteins were very sensitive to trypsin and Bacillus licheniformis protease, but were resistant to V8 protease, while the IgM-antigenic proteins were sensitive to various proteases . These results suggested that IgG-antigenic and IgM-antigenic components were different from the serotype-specific antigen or species-specific antigen associated with polysaccharides or lipopolysaccharides with respect to molecular weights and that they were proteins. J Urol, 2001 Oct, 166(4), 1300 - 4, discussion 1304-5 Salvage intravesical therapy with interferon-alpha 2b plus low dose bacillus Calmette-Guerin is effective in patients with superficial bladder cancer in whom bacillus Calmette-Guerin alone previously failed; O'Donnell MA et al.; PURPOSE: We determined whether combining low dose bacillus Calmette-Guerin (BCG) interferon-alpha 2B would be effective for patients in whom previous BCG failed . MATERIALS AND METHODS: A total of 40 patients in whom 1 (19) or more (21) previous induction courses of BCG failed received 6 to 8 weekly treatments of 1/3 dose (27 mg.) BCG plus 50 million units interferon-alpha 2B . Additional 3 week miniseries of further decreased BCG (1/10, 1/30 or 1/100) titrated to symptoms without changing the interferon-alpha 2B dose were given at 5, 11 and 17 months . In 12 patients a second induction course was given with 1/10 BCG plus 100 million units interferon-alpha 2B . There was multifocal disease in 39 patients, previous BCG had failed within 6 months in 34, disease was aggressive (stage T1, grade 3 or carcinoma in situ in 31, there had been 2 or more previous recurrences in 25 and disease history was greater than 4 years in 13 . RESULTS: At a median followup of 30 months 63% and 53% of patients were disease-free at 12 and 24 months, respectively . Patients in whom 2 or more previous BCG courses had failed fared as well as those with 1 failure . Of the 18 failures 14 occurred at the initial cystoscopy evaluation . Of 22 patients initially counseled to undergo cystectomy 12 (55%) are disease-free with a functioning bladder . Combination therapy was well tolerated . CONCLUSIONS: While longer followup and larger multicenter studies are required to validate these encouraging findings, intravesical low dose BCG plus interferon-alpha 2B appears to be effective in many cases of high risk disease previously deemed BCG refractory . However, early failure while on this regimen should be aggressively pursued with more radical treatment options. J Urol, 2001 Oct, 166(4), 1296 - 9 Does early cystectomy improve the survival of patients with high risk superficial bladder tumors? Herr HW, Sogani PC. PURPOSE: We compared survival after early versus delayed cystectomy in patients with high risk superficial bladder tumors . MATERIALS AND METHODS: Of 307 patients with high risk superficial bladder tumors who were treated initially with transurethral resection and bacillus Calmette-Guerin (BCG) therapy 90 (29%) underwent cystectomy for recurrent tumor during a followup of 15 to 20 years . Disease specific survival distribution of these 90 patients was determined relative to the indications for and time of cystectomy . RESULTS: Of the 90 patients who underwent cystectomy 44 (49%) survived a median of 96 months . Of 35 patients with recurrent superficial bladder tumors 92% and 56% survived who underwent cystectomy less than 2 years after initial BCG therapy and after 2 years of followup, respectively . Of 55 patients with recurrent muscle invasive bladder disease 41% and 18% survived when cystectomy was performed within and after 2 years, respectively . Multivariate analysis showed that survival was improved in patients who underwent earlier rather than delayed cystectomy for nonmuscle invasive tumor relapse . CONCLUSIONS: Earlier cystectomy improves the long-term survival of patients with high risk superficial bladder tumors in whom BCG therapy fails. Int J Food Microbiol, 2001 Aug 15, 68(1-2), 141 - 8 Combined effect of nisin and carvacrol at different pH and temperature levels on the viability of different strains of Bacillus cereus; Periago PM et al.; The influence of pH and temperature on the bactericidal action of nisin and carvacrol on vegetative cells of different Bacillus cereus strains was studied . The five strains tested showed significant differences in sensitivity towards nisin, at pH 7.0 and 30 degrees C . Carvacrol concentrations of 0.3 mmol l(-1) had no effect on viability of B . cereus cells . When the same carvacrol concentration was combined with nisin, however, it resulted in a greater loss of viability of cells than when nisin was applied alone . The concentration of carvacrol played an important role on the bactericidal effect of nisin and, therefore, on the synergistic action of both compounds combined . At lower pH values (6.30 and 5.75), nisin was more active against B . cereus cells than at pH 7.0 at 30 degrees C, with a different sensitivity of the strains tested . The combined effect of nisin and carvacrol was found to be significantly different at pH 7.0 and 5.75 . When the temperature was 8 degrees C, nisin was significantly less active against B . cereus IFR-NL 94-25 than at 30 degrees C, both at pH 7.0 and 6.30 . At 8 degrees C, there was a significant increased effect of nisin at lower pH values . Also at this low temperature, a synergistic effect between nisin and carvacrol on B . cereus cells was observed at the pHs tested . This study indicates the potential of nisin and carvacrol at lower pHs to be used for preservation of minimally processed foods. J Immunol, 2001 Sep 15, 167(6), 3300 - 7 ATP-mediated killing of intracellular mycobacteria by macrophages is a P2X(7)-dependent process inducing bacterial death by phagosome-lysosome fusion; Fairbairn IP et al.; Mycobacterium tuberculosis survives within host macrophages by actively inhibiting phagosome fusion with lysosomes . Treatment of infected macrophages with ATP induces both cell apoptosis and rapid killing of intracellular mycobacteria . The following studies were undertaken to characterize the effector pathway(s) involved . Macrophages were obtained from p47(phox) and inducible NO synthase gene-disrupted mice (which are unable to produce reactive oxygen and nitrogen radicals, respectively) and P2X(7) gene-disrupted mice . RAW murine macrophages transfected with either the natural resistance-associated macrophage protein gene 1 (Nramp1)-resistant or Nramp1-susceptible gene were also used . The cells were infected with bacille Calmette-Guerin (BCG), and intracellular mycobacterial trafficking was analyzed using confocal and electron microscopy . P2X(7) receptor activation was essential for effective ATP-induced mycobacterial killing, as its bactericidal activity was radically diminished in P2X(7)(-/-) macrophages . ATP-mediated killing of BCG within p47(phox-/-), inducible NO synthase(-/-), and Nramp(s) cells was unaffected, demonstrating that none of these mechanisms have a role in the ATP/P2X(7) effector pathway . Following ATP stimulation, BCG-containing phagosomes rapidly coalesce and fuse with lysosomes . Blocking of macrophage phospholipase D activity with butan-1-ol blocked BCG killing, but not macrophage death . ATP stimulates phagosome-lysosome fusion with concomitant mycobacterial death via P2X(7) receptor activation . Macrophage death and mycobacterial killing induced by the ATP/P2X(7) signaling pathway can be uncoupled, and diverge proximal to phospholipase D activation. Adv Space Res, 1997, 20(10), 1945 - 8 Catalase activity as a potential indicator of the reducer component of small closed ecosystems; Sarangova AB et al.; Dynamics of catalase activity has been shown to reflect the growth curve of microorganisms in batch cultivation (celluloselythic bacteria Bacillus acidocaldarius and bacteria of the associated microflora Chlorella vulgaris) . Gas and substrate closure of the three component ecosystems with spatially separated components "producer-consumer-reducer" (Chl . vulgaris-Paramecium caudatum-B . acidocaldarius, two bacterial strains isolated from the associated microflora Chl . vulgaris) demonstrated that the functioning of the reducer component can be estimated by the catalase activity of mciroorganisms of this component. Icarus, 1992 Aug, 98(2), 233 - 9 Survival of microorganisms in smectite clays: implications for Martian exobiology; Moll DM et al.; Manned exploration of Mars may result in the contamination of that planet with terrestrial microbes, a situation requiring assessment of the survival potential of possible contaminating organisms . In this study, the survival of Bacillius subtilis, Azotobacter chroococcum, and the enteric bacteriophage MS2 was examined in clays representing terrestrial (Wyoming type montmorillonite) or Martian (Fe(3+)-montmorillonite) soils exposed to terrestrial and Martian environmental conditions of temperature and atmospheric pressure and composition, but not to UV flux or oxidizing conditions . Survival of bacteria was determined by standard plate counts and biochemical and physiological measurements over 112 days . Extractable lipid phosphate was used to measure microbial biomass, and the rate of 14C-acetate incorporation into microbial lipids was used to determine physiological activity . MS2 survival was assayed by plaque counts . Both bacterial types survived terrestrial or Martian conditions in Wyoming montmorillonite better than Martian conditions in Fe(3+)-montmorillonite . Decreased survival may have been caused by the lower pH of the Fe(3+)-montmorillonite compared to Wyoming montmorillonite . MS2 survived simulated Mars conditions better than the terrestrial environment, likely due to stabilization of the virus caused by the cold and dry conditions of the simulated Martian environment . The survival of MS2 in the simulated Martian environment is the first published indication that viruses may be able to survive in Martian type soils . This work may have implications for planetary protection for future Mars missions. Curr Microbiol, 1990, 21, 325 - 7 PCR amplification of 16S rDNA from lyophilized cell cultures facilitates studies in molecular systematics; Wisotzkey JD et al.; The sequence of the major portion of a Bacillus cycloheptanicus strain SCH(T) 16S rRNA gene is reported . This sequence suggests that B . cycloheptanicus is genetically quite distinct from traditional Bacillus strains (e.g., B . subtilis) and may be properly regarded as belonging to a different genus . The sequence was determined from DNA that was produced by direct amplification of ribosomal DNA from a lyophilized cell pellet with straightforward polymerase chain reaction (PCR) procedures . By obviating the need to revive cell cultures from the lyophile pellet, this approach facilitates rapid 16S rDNA sequencing and thereby advances studies in molecular systematics. Eur J Oral Sci, 2001 Aug, 109(4), 249 - 59 Ultrastructural analysis of structural framework in dental plaque developing on synthetic carbonate apatite applied to human tooth surfaces; Takeuchi H et al.; This study focused on determining the structural framework by oral microbiota in supragingival plaque on a carbonate apatite film applied to human tooth surfaces . The sequential phases of plaque formation over a 3-wk period were found to be equivalent to those previously reported for natural tooth surfaces . Scanning electron microscopy of specimens prepared by vertical sectioning demonstrated the organization of two types of framework between certain genera of initial and secondary colonizers in the pre- and post-organization phases, respectively . The initial colonizers in the pre-organization phase were of a coccoid type, while colonizers in the post-organization phase were of a bacillary type . Secondary colonizers, filamentous cells, were common to both frameworks . Transmission electron microscopy using freeze-substitution and immunohistochemistry demonstrated two types of coaggregation, fibril- and saliva-mediated modes, among the plaque microbiota . Coaggregation between microbiota, which organized the framework, showed a tendency to occur in the fibril-mediated mode, and the filamentous secondary colonizers were characterized by inducing multigeneric coaggregation . The present findings indicate that a structural framework and specific cells to form this framework are essential for plaque formation. J Biochem (Tokyo), 2001 Sep, 130(3), 417 - 23 Changing the amino acid specificity of yeast tyrosyl-tRNA synthetase by genetic engineering; Ohno S et al.; In an attempt to generate mutant aminoacyl-tRNA synthetases capable of charging non-canonical amino acids, a series of yeast tyrosyl-tRNA synthetase (TyrRS) mutants was constructed by site-specific mutagenesis of putative active site residues, which were deduced by analogy with those of Bacillus stearothermophilus TyrRS . Among these mutants, one with the replacement of tyrosine at position 43 by glycine, "Y43G," was found to be able to utilize several 3-substituted tyrosine analogues as substrates for aminoacylation . The catalytic efficiency (k(cat)/K(m)) of mutant Y43G for aminoacylation with L-tyrosine was about 400-fold decreased as compared to that of the wild-type TyrRS . On the other hand, the ability to utilize 3-iodo-L-tyrosine was newly generated in this mutant TyrRS, since the wild-type TyrRS could not accept 3-iodo-L-tyrosine at all under physiological conditions . This mutant TyrRS should serve as a new tool for site-specific incorporation of non-canonical amino acids, such as those in 3-substituted tyrosine analogues, into proteins in an appropriate translation system in vivo or in vitro. J Med Assoc Thai, 2001 Jun, 84 Suppl 1, S127 - 30 Primary conjunctival tuberculoma: a case report; Tulvatana W et al.; Conjunctival tuberculoma is very rare in modern medicine . We report a case of a 34-year-old Thai female patient presenting with multiple peculiar conjunctival nodules . Two small nodules were excised and submitted for histopathological study, which showed discrete granulomatous inflammation with caseating necrosis . The acid-fast bacilli's staining for tuberculous bacteria was positive . There was no other organ involvement of active tuberculosis . The patient was then treated with systemic anti-tuberculous regimen for 6 months . The remaining lesions disappeared within the first few months of treatment . Conjunctival tuberculoma should always be considered in cases of conjunctival nodules. Eur Urol, 2001 Aug, 40(2), 169 - 75 Prospective evaluation of Ki-67 labeling in predicting the recurrence and progression of superficial bladder transitional cell carcinoma; Blanchet P et al.; PURPOSE: We studied a series of superficial transitional cell carcinoma of the bladder to assess whether the Ki-67 labeling index predicts recurrence and progression in a cohort of patients treated by transurethral resection alone or receiving adjuvant intravesical bacillus Calmette-Guerin therapy (BCG) . MATERIALS AND METHODS: From 1989 to 1990, we prospectively studied 70 consecutive cases of superficial transitional cell carcinoma of the bladder using Ki-67 immunostaining . The tumors were 43 pTa and 27 pTl . Thirteen were treated with transurethral resection only and 57 received adjuvant intravesical BCG . The median follow-up times was 64 months . The threshold index values of Ki-67 for recurrence and progression were determined using ROC curves . The relative predictive values of the Ki-67 labeling index and tumor characteristics for recurrence and progression were evaluated using Cox's proportional hazards model . RESULTS: A cutoff value of 13% was determined . The recurrence free survival rate at 5 years was 68% for cases with a Ki-67 labeling index of 13 or higher and 71% for those with an index of less than 13 (NS) . The progression-free survival rate at 5 years was 43% in cases with an index of 13 or higher and 89% in those with an index of less than 13 (p<0.0001) . Using multivariate analysis the Ki-67 labeling index is an independent risk factor for tumor progression with a relative risk of 4.61 (p<0.05) . CONCLUSION: When BCG is used for high and intermediate risk superficial bladder cancers, the Ki-67 labeling index is an independent predictive factor of progression but not of recurrence. Acta Crystallogr D Biol Crystallogr, 2001 Sep, 57(Pt 9), 1344 - 7 Epub 2001 Aug 23. Oligosaccharide binding to family 11 xylanases: both covalent intermediate and mutant product complexes display (2,5)B conformations at the active centre; Sabini E et al.; The glycoside hydrolase sequence-based classification reveals two families of enzymes which hydrolyse the beta-1,4-linked backbone of xylan, xylanases, termed families GH-10 and GH-11 . Family GH-11 xylanases are intriguing in that catalysis is performed via a covalent intermediate adopting an unusual (2,5)B (boat) conformation, a conformation which also fulfils the stereochemical constraints of the oxocarbenium ion-like transition state . Here, the 1.9 A structure of a nucleophile, E94A, mutant of the Xyn11 from Bacillus agaradhaerens in complex with xylotriose is presented . Intriguingly, this complex also adopts the (2,5)B conformation in the -1 subsite, with the vacant space provided by the Glu-->Ala mutation allowing the sugar to adopt the alpha-configuration at C1 . The structure of the covalent 2-deoxy-2-fluoroxylobiosyl-enzyme intermediate has been extended to atomic (1.1 A) resolution. Acta Crystallogr D Biol Crystallogr, 2001 Sep, 57(Pt 9), 1303 - 6 Epub 2001 Aug 23. Crystallization and preliminary X-ray diffraction analysis of the 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes; Tsai LC et al.; The truncated 1,3-1,4-beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase; E.C . 3.2.1.73) from Fibrobacter succinogenes was crystallized in four different forms by the vapour-diffusion method . Form A crystals have the largest trigonal P321 unit cell, diffracting to 3.0 A resolution with four to six molecules per asymmetric unit . Form B and C crystals belong to the same monoclinic space group P2(1), but the form B unit cell is twice as large as the unit cell of form C . Form B crystals diffract to 2.5 A resolution and contain four molecules per asymmetric unit . Form C crystals diffract to 2.1 A resolution and contain two molecules per asymmetric unit . Form D crystals have the smallest orthorhombic P2(1)2(1)2(1) unit cell, containing only one molecule per asymmetric unit, and diffract beyond 2.1 A resolution . The crystallization conditions for form B and C crystals are almost identical, except that form C crystals were grown in the presence of 2 mM Ca(2+) ions . It is likely that Ca(2+) directly binds to the glucanase, leading to unit-cell shrinkage as observed in other Bacillus glucanase crystals . A self-rotation search identified non-crystallographic twofold axes that combine with the crystallographic twofold dyads to give 222 symmetry for both form A and form B crystals, indicating that the glucanase has a tendency to pack in 222 symmetry. Acta Crystallogr D Biol Crystallogr, 2001 Sep, 57(Pt 9), 1300 - 2 Epub 2001 Aug 23. Crystallization and preliminary X-ray analysis of a thermoalkalophilic lipase from Bacillus stearothermophilus L1; Jeong ST et al.; A thermoalkalophilic lipase from Bacillus stearothermophilus L1 (L1 lipase) was crystallized in two different crystal forms using a low concentration of the enzyme and a calcium-exchange process . The first, needle-like, crystal form, which diffracts to about 3.5 A, belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.84, b = 72.96, c = 104.41 A . The second, monoclinic, crystal form, which behaves better than the first form for crystallographic analyses, belongs to the monoclinic space group C2 and has unit-cell parameters a = 119.62, b = 85.05, c = 98.36 A, beta = 99.73 degrees . From the monoclinic crystals, a native data set and a samarium-derivative data set were collected to 2.0 and 2.3 A resolution, respectively . The difference Patterson map between the two data sets shows strong heavy-atom peaks, indicating that the crystals are suitable for a high-resolution structure determination. Appl Environ Microbiol, 2001 Sep, 67(9), 4372 - 3 Susceptibility of a field-derived, Bacillus thuringiensis-resistant strain of diamondback moth to in vitro-activated Cry1Ac toxin; Sayyed AH et al.; Resistant and susceptible populations of the diamondback moth (Plutella xylostella) were tested with crystalline, solubilized, and partially and fully activated forms of the Bacillus thuringiensis Cry1Ac delta-endotoxin . Fully activated toxin greatly reduced the resistance ratio (ratio of the 50% lethal concentration for the resistant population to that for the susceptible population) of the resistant population, suggesting that a defect in toxin activation is a major resistance mechanism. Appl Environ Microbiol, 2001 Sep, 67(9), 3923 - 7 Effect of Bacillus thuringiensis Cry1 toxins in insect hemolymph and their neurotoxicity in brain cells of Lymantria dispar; Cerstiaens A et al.; Little information is available on the systemic effects of Bacillus thuringiensis toxins in the hemocoel of insects . In order to test whether B . thuringiensis-activated toxins elicit a toxic response in the hemocoel, we measured the effect of intrahemocoelic injections of several Cry1 toxins on the food intake, growth, and survival of Lymantria dispar (Lepidoptera) and Neobellieria bullata (Diptera) larvae . Injection of Cry1C was highly toxic to the Lymantria larvae and resulted in the complete inhibition of food intake, growth arrest, and death in a dose-dependent manner . Cry1Aa and Cry1Ab (5 microg/0.2 g {fresh weight} {g fresh wt}) also affected growth and food intake but were less toxic than Cry1C (0.5 microg/0.2 g fresh wt) . Cry1E and Cry1Ac (5 microg/0.2 g fresh wt) had no toxic effect upon injection . Cry1C was also highly toxic to N . bullata larvae upon injection . Injection of 5 microg/0.2 g fresh wt resulted in rapid paralysis, followed by hemocytic melanization and death . Lower concentrations delayed pupariation or gave rise to malformation of the puparium . Finally, Cry1C was toxic to brain cells of Lymantria in vitro . The addition of Cry1C (20 microg/ml) to primary cultures of Lymantria brain cells resulted in rapid lysis of the cultured neurons. Appl Environ Microbiol, 2001 Sep, 67(9), 3873 - 81 Putative virulence factor expression by clinical and food isolates of Bacillus spp . after growth in reconstituted infant milk formulae; Rowan NJ et al.; Forty-seven strains representing 14 different Bacillus species isolated from clinical and food samples were grown in reconstituted infant milk formulae (IMF) and subsequently assessed for adherence to, invasion of, and cytotoxicity toward HEp-2 and Caco-2 cells . Cell-free supernatant fluids from 38 strains (81%) were shown to be cytotoxic, 43 strains (91%) adhered to the test cell lines, and 23 strains (49%) demonstrated various levels of invasion . Of the 21 Bacillus cereus strains examined, 5 (24%) were invasive . A larger percentage of clinically derived Bacillus species (20%) than of similar species tested from the food environment were invasive . Increased invasion occurred after growth of selected Bacillus species in reconstituted IMF containing glucose . While PCR primer studies revealed that many different Bacillus species contained DNA sequences encoding the hemolysin BL (HBL) enterotoxin complex and B . cereus enterotoxin T, not all of these isolates expressed these diarrheagenic genes after growth in reconstituted IMF . Of the 47 Bacillus isolates examined, 3 isolates of B . cereus and 1 isolate of B . subtilis produced the HBL enterotoxin after 18 h of growth in brain heart infusion broth . However, eight isolates belonging to the species B . cereus, B . licheniformis, B . circulans, and B . megaterium were found to produce this enterotoxin after growth in reconstituted IMF when assessed with the B . cereus enterotoxin (diarrheal type) reversed passive latex agglutination (RPLA) kit . It is concluded that several Bacillus species occurring occasionally in clinical specimens and food samples are of potential medical significance due to the expression of putative virulence factors. Gan To Kagaku Ryoho, 2001 Aug, 28(8), 1094 - 8 {Minimally invasive therapy for bladder and prostate cancer}; Uchida T et al.; Recently, minimally invasive therapy has been a key word in the medical field . Many new therapies have been developed in the field of urology . In this area, bacillus Calmette-Guerin (BCG) instillation therapy, transurethral resection of the bladder tumor and intra-arterial infusion with irradiation therapy are noted as minimally invasive therapies for bladder cancer . Laparoscopic prostatectomy, brachytherapy, three-dimensional conformal radiotherapy (3D-CRT) and high-intensity focused ultrasound (HIFU) have also been developed as minimally invasive therapies for prostate cancer . Though the establishment of the validity of each treatment will still take time, the best treatment for each patient should be chosen case by case, including considerations of postoperative quality of life and economic efficiency. Clin Infect Dis, 2001 Sep 15, 33 Suppl 3, S233 - 7 Does the dose matter? Craig WA. Pharmacokinetic/pharmacodynamic (PK/PD) parameters, such as the ratio of peak to minimum inhibitory concentration (peak/MIC ratio), ratio of 24-hour area under the curve to MIC (24-h AUC/MIC ratio), and the time above MIC, are good indicators of the drug dose-organism interaction . Time above the MIC is the important determinant of the activity of beta-lactams, macrolides, clindamycin, and linezolid . Free drug serum levels of these drugs should be above the MIC for at least 40%-50% of the dosing interval to produce adequate clinical and microbiological efficacy . Peak/MIC and 24-h AUC/MIC ratios are major determinants of the activity of aminoglycosides and fluoroquinolones . In general, peak/MIC ratios should exceed 8 and 24-h AUC/MIC values should be >100 to successfully treat gram-negative bacillary infections and to prevent the emergence of resistant organisms during therapy . The successful treatment of pneumococcal infections with fluoroquinolones and azithromycin appear to require 24-h AUC/MIC ratios of only 25-35 . Mutation prevention concentrations are being reported for various fluoroquinolones with different pathogens, but their clinical significance has not yet been established . More information is needed on the role of PK/PD parameters and their magnitude for preventing mutations and the emergence of resistant organisms for most classes of antibiotics. Biochemistry, 2001 Sep 4, 40(35), 10411 - 6 A novel engineered subtilisin BPN' lacking a low-barrier hydrogen bond in the catalytic triad; Stratton JR et al.; The low-barrier hydrogen bond (LBHB) between the Asp and His residues of the catalytic triad in a serine protease was perturbed via the D32C mutation in subtilisin BPN' (Bacillus protease N') . This mutant enzyme catalyzes the hydrolysis of N-Suc-Ala-Ala-Pro-Phe-SBzl with a k(cat)/K(m) value that is only 8-fold reduced from that of the wild-type (WT) enzyme . The value of k(cat)/K(m) for the corresponding p-nitroanilide (pNA) substrate is only 50-fold lower than that of the WT enzyme (DeltaDeltaG++ = 2.2 kcal/mol) . The pK(a) controlling the ascending limb of the pH versus k(cat)/K(m) profile is lowered from 7.01 (WT) to 6.53 (D32C), implying that any hydrogen bond replacing that between Asp32 and His64 of the WT enzyme most likely involves the neutral thiol rather than the thiolate form of Cys32 . It is shown by viscosity variation that the reaction of WT subtilisin with N-Suc-Ala-Ala-Pro-Phe-SBzl is 50% (sucrose) to 100% (glycerol) diffusion-controlled, while that of the D32C construct is 29% (sucrose) to 76% (glycerol) diffusion-controlled . The low-field NMR resonance of 18 ppm that has been assigned to a proton shared by Asp32 and His64, and is considered diagnostic of a LBHB in the WT enzyme, is not present in D32C subtilisin . Thus, the LBHB is not an inherent requirement for substantial rate enhancement for subtilisin. J Mass Spectrom, 2001 Aug, 36(8), 929 - 36 Factors contributing to peak broadening and mass accuracy in the characterization of intact spores using matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry; Ramirez J et al.; Factors contributing to peak broadening, accuracy and precision in mass assignment in the matrix-assisted laser desorption/ionization characterization of a lipopeptide desorbed from intact Bacillus spores were investigated . These spores were studied as an example of a thick, topologically irregular sample, which present a more difficult target than a pure peptide or protein . The type of matrix, matrix:sample ratio, laser fluence, and localized repetitive laser irradiation were all found to affect the full-width at half maximum of the biomarker . Both in-source and post-source phenomena were shown to contribute . Sample thickness had less effect . Precision and accuracy of mass assignment were also affected by matrix:sample ratio and laser fluence . In general, this sample was responsive to the same experimental variables as pure peptides, and the use of an internal standard produced significant improvements in precision and accuracy . Protein Eng, 2001 Jul, 14(7), 505 - 12 The determinants of alpha-amylase pH-activity profiles; Nielsen JE et al.; The glycosyl hydrolases present a large family of enzymes that are of great significance for industry . Consequently, there is considerable interest in engineering the enzymes in this family for optimal performance under a range of very diverse conditions . Until recently, tailoring glycosyl hydrolases for specific industrial processes mainly involved stability engineering, but lately there has also been considerable interest in engineering their pH-activity profiles . We mutated four neutral residues (N190, F290, N326 and Q360) in the chimeric Bacillus Ba2 alpha-amylase to both charged and neutral amino acids . The results show that the pH-activity profile of the Ba2 alpha-amylase can be changed by inserting charged residues close to the active site . The changes in the pH-activity profile for these neutral --> charged mutations do not, however, correlate with the predictions from calculations of the p K(a) values of the active site residues . More surprisingly, the neutral --> neutral mutations change the pH-activity profile as much as the neutral --> charged mutations . From these results, it is concluded that factors other than electrostatics, presumably the dynamic aspects of the active site, are important for the shape of the pH-activity profiles of the alpha-amylases. Protein Eng, 2001 Jul, 14(7), 501 - 4 Thermostabilization by replacement of specific residues with lysine in a Bacillus alkaline cellulase: building a structural model and implications of newly formed double intrahelical salt bridges; Ozawa T et al.; An alkaline, mesophilic endo-1,4-beta-glucanase from alkaliphilic Bacillus sp . strain KSM-64 was significantly thermostabilized by replacement of both Asn179 and Asp194 with lysine by site-directed mutagenesis . Structural remodeling of the mutant enzyme newly generated by the double mutation suggested that Glu175-->Lys179 and Glu190-->Lys194 were the most plausible ion pairs, both of which involved side chains at the i and i + 4 positions on the alpha(4)-helix from Glu175 to Ser195 . By molecular dynamics simulations, the N(zeta) hydrogens of Lys179 and Lys194 were found to coordinate with the carbonyl O(varepsilon1) and O(varepsilon2) of Glu175 and the carbonyl O(varepsilon1) of Glu190, respectively, with distances of around 2 A for all . These results confirm that the formation of these double intrahelical ion pairs (salt bridges) is responsible for the thermostabilization by the double mutation. Drug Metabol Drug Interact, 2001, 18(1), 69 - 77 Determination of beta-lactamase activities and antibiotic susceptibility of some Bacillus strains causing food poisoning; Uraz G et al.; Some Bacillus species are important food pathogens . For example, B . cereus is an opportunistic pathogen found in raw milk that is a common cause of food poisoning . It is of interest to investigate the virulant profiles of Bacillus strains isolated from foods and samples associated with food-poisoning outbreaks . Nineteen Bacillus strains were isolated from various milk samples . Beta-lactamase enzyme activities of these Bacillus strains were evaluated with iodometric and chromogenic cephalosporin (nitrocefin) test methods . Five of 19 Bacillus strains isolated were positive for beta-lactamase activity . Clavulanate-amoxycillin and cephazolin were chosen to test the antibiotic susceptibilities of the beta-lactamase positive and negative Bacillus strains . Of the five beta-lactamase positive Bacillus strains, three were susceptible, and two strains intermediate to clavulanate-amoxycillin; one was susceptible, and four strains were intermediate to cephazolin . None of the beta-lactamase positive Bacillus strains was resistant to both antibiotics . Of the 14 beta-lactamase negative strains, five were susceptible to clavulanate-amoxycillin, four strains were intermediate, and five strains were resistant; three were susceptible, one intermediate, and ten beta-lactamase negative strains were resistant to cephazolin. FEMS Microbiol Lett, 2001 Aug 21, 202(2), 189 - 93 BceS1, a new addition to the type III restriction and modification family; Hegna IK et al.; The nucleotide sequence of an 11-kb chromosomal BglII fragment from Bacillus cereus American Type Culture Collection (ATCC) 10987 strain revealed two closely adjacent open reading frames organized in an operon, of which the deduced amino acids showed identity to the type III restriction and modification (R/M) subunits described in Gram-negative bacteria . An enhanced transcription level was revealed when the culture was grown in the presence of foreign DNA . A cell-free extract from this culture restricted pUC19, whereas from a plain medium the restriction was very weak . The in vitro methylation protected pUC 19 from restriction . The R/M system was designated BceS1 as this endonuclease required ATP and Mg2+ as cofactors like other type III endonucleases . BceS1 is the first chromosomal type III R/M system characterized in a Gram-positive bacterium. J Chromatogr A, 2001 Aug 3, 925(1-2), 123 - 32 Automated mass analysis of low-molecular-mass bacterial proteome by liquid chromatography-electrospray ionization mass spectrometry; Dunlop KY et al.; Due to the presence of a large number of proteins in cell extracts, ion chromatograms of cell extracts obtained by electrospray ionization mass spectrometry (ESI-MS) can be quite complicated . It is found that the elevated baseline in an ion chromatogram contains many protein signals . One deficiency of current commercially available LC-ESI-MS data interpretation software is found to be the lack of functional operation that allows automated mass spectral integration and interpretation over signals hidden in the baseline . This current limitation can be overcome by a technique that involves the introduction of artificial pulses to an ion chromatogram by removing the solvent mixer in the HPLC pump . These artificial pulses are treated as chromatographic peaks by the software, thereby allowing automated spectral integration over the duration of a pulse . The reliability of mass analysis from the integrated spectra is shown to be dependent on spectral interpretation parameters such as mass spectral baseline threshold . The application of this method is demonstrated for rapid detection and mass analysis of low-molecular-mass proteins from cell extracts of Escherichia coli or Bacillus globigii. Syst Appl Microbiol, 2001 Jul, 24(2), 311 - 20 Molecular microbial diversity of a spacecraft assembly facility; Venkateswaran K et al.; In ongoing investigations to map and archive the microbial footprints in various components of the spacecraft and its accessories, we have examined the microbial populations of the Jet Propulsion Laboratory's Spacecraft Assembly Facility (JPL-SAF) . Witness plates made up of spacecraft materials, some painted with spacecraft qualified paints, were exposed for approximately 7 to 9 months at JPL-SAF and examined the particulate materials collected for the incidence of total cultivable aerobic heterotrophs and heat-tolerant (80 degrees C for 15-min.) spore-formers . The results showed that the witness plates coated with spacecraft qualified paints attracted more dust particles than the non-coated stainless steel witness plates . Among the four paints tested, witness plates coated with NS43G accumulated the highest number of particles, and hence attracted more cultivable microbes . The conventional microbiological examination revealed that the JPL-SAF harbors mainly Gram-positive microbes and mostly spore-forming Bacillus species . Most of the isolated microbes were heat resistant to 80 degrees C and proliferate at 60 degrees C . The phylogenetic relationships among 23 cultivable heat-tolerant microbes were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations . By 16S rDNA sequence analysis, the isolates fell into seven clades: Bacillus licheniformis, B . pumilus, B . cereus, B . circulans, Staphylococcus capitis, Planococcus sp . and Micrococcus lylae . In contrast to the cultivable approach, direct DNA isolation, cloning and 16S rDNA sequencing analysis revealed equal representation of both Gram-positive and Gram-negative microorganisms. Syst Appl Microbiol, 2001 Jul, 24(2), 267 - 76 Toxic Bacillus pumilus from indoor air, recycled paper pulp, Norway spruce, food poisoning outbreaks and clinical samples; Suominen I et al.; Forty-four B . pumilus isolates of food poisoning, clinical, environmental and industrial origins were investigated for toxin production using the boar spermatozoan motility assay, previously shown to be a sensitive method for detecting non-protein toxins from B . cereus and B . licheniformis . The three toxic isolates originated from live tree, indoor air and recycled paper pulp and were more toxic than the previously described food poisoning isolates of B . licheniformis, whereas the B . pumilus food poisoning and clinical isolates were lower in toxicity . The type strain also produced inhibitory substances . The toxic substances were insensitive to heat (100 degrees C, 20 min), to pH 2 or pH 10 and to digestion with pronase . The substances were readily soluble in methanol and chloroform, but less soluble in toluene . Exposure of boar spermatozoa to 1-10 microg ml(-1) (EC50) of methanol soluble substance from the four strains disrupted the plasma membrane permeability barrier, induced abnormalities in the postacrosomal sheath, collapsed the mitochondrial and suppressed cytoplasmic NAD reduction . No change was observed in human peripheral blood lymphocytes exposed to concentrations of B . pumilus extract that affected spermatozoa . The toxin producing isolates were 99.4 to 99.6% similar in 16SrDNA (500 bp) to the type strain and could not be distinguished from the 41 non-toxic isolates by biochemical properties or whole cell fatty acid composition. In Vitro Cell Dev Biol Anim, 2001 Jun, 37(6), 348 - 52 Control of life, death, and differentiation in cultured midgut cells of the lepidopteran, Heliothis virescens; Loeb MJ et al.; Differentiated cells in the insect midgut depend on stem cells for renewal . We have immunologically identified Integrin beta1, a promotor of cell-cell adhesion that also induces signals mediating proliferation, differentiation, and apoptosis on the surfaces of cultured Heliothis virescens midgut cells; clusters of immunostained integrin beta1-like material, indicative of activated integrin, were detected on aggregating midgut columnar cells . Growth factor-like peptides (midgut differentiation factors 1 and 2 {MDF1 and MDF2}), isolated from conditioned medium containing Manduca sexta midgut cells, may be representative of endogenous midgut signaling molecules . Exposing the cultured midgut cells to Bacillus thuringiensis (Bt) toxin caused large numbers of mature differentiated cells to die, but the massive cell death simultaneously induced a 150-200% increase in the numbers of midgut stem and differentiating cells . However, after the toxin was washed out, the proportions of cell types returned to near-control levels within 2 d, indicating endogenous control of cell-population dynamics . MDF1 was detected immunologically in larger numbers of Bt-treated columnar cells than controls, confirming its role in inducing the differentiation of rapidly produced stem cells . However, other insect midgut factors regulating increased proliferation, differentiation, as well as inhibition of proliferation and adjustment of the ratio of cell types, remain to be discovered. Int Immunopharmacol, 2001 Aug, 1(8), 1559 - 69 Interferon gamma-producing ability in blood lymphocytes of patients with lung cancer through activation of the innate immune system by BCG cell wall skeleton; Matsumoto M et al.; An in vitro assay system was developed to assess the potency of the human innate immune system by measurement of IL-12, IL-18, IL-10 and IFNgamma in the supernatants of bacillus Calmette-Guerin cell wall skeleton (BCG-CWS)-stimulated blood samples . BCG-CWS is a ligand for Toll-like receptor (TLR) 2 and 4, and activates monocytes to macrophages (Mphi), and immature dendritic cells to mature antigen-presenting cells (APC) . This system was found to allow the discrimination of immune suppressive states in patients with lung cancer from normal immune states in light of the cytokine profile . The following results were deduced from analyses of BCG-CWS-stimulated blood samples of lung cancer patients with reference to normal subjects . (1) The levels of production of IFNgamma and IL-10 by lymphocytes were decreased . (2) IL-12 p40 production by monocytes/Mphi was upregulated, while that of IL-10 was downregulated . (3) IL-18 was detected in all patients in a range similar to normal subjects . (4) Responses of lymphocytes to IL-2 and IL- 18 in terms of IFNgamma production were diminished . (5) The upregulated IL-12 levels were recovered to within the normal range in most patients after tumor resection . (6) Male patients showed more severe suppression of IL-12/IL-18-mediated IFNgamma production than female patients . Thus, the lesser IFNgamma production observed in patients' blood with high IL-12 p40 levels in response to BCG-CWS may reflect the production of p40 dimers or IL-23 instead of p70, or the presence of some unknown pathways to prohibit the interface between the innate and acquired immune systems . BCG-CWS-mediated Toll signaling may participate in IFNgamma induction for lymphocytes through Mphi/APC IL-12/I-18 modulation. Phytomedicine, 2001 Jul, 8(4), 302 - 5 Antimicrobial evaluation of coumarins and flavonoids from the stems of Daphne gnidium L; Cottiglia F et al.; The antimicrobial activity of stems methanol extract from Daphne gnidium L . collected from Sardinia (Italy) was evaluated against 6 strains of standard and clinical isolated gram (+/-) bacteria . The antimicrobial effect on two strains of fungi was also tested . The extract in toto exhibited antibacterial activity against Bacillus lentus and Escherichia coli, but was inactive against fungi . Four coumarins (daphnetin, daphnin, acetylumbelliferone, daphnoretin) and seven flavonoids (luteolin, orientin, isoorientin, apigenin-7-O-glucoside, genkwanin, 5-O-beta-D-primeverosyl genkwanine, 2,5,7,4'-tetrahydroxyisoflavanol) present in the plant extract were also investigated against the same strains of bacteria and fungi assayed for the crude extract . The most active compounds were daphnetin, genkwanin, and 2,5,7,4'-tetrahydroxyisoflavanol. Protein Sci, 2001 Sep, 10(9), 1835 - 46 A cofactor-dependent phosphoglycerate mutase homolog from Bacillus stearothermophilus is actually a broad specificity phosphatase; Rigden DJ et al.; The distribution of phosphoglycerate mutase (PGM) activity in bacteria is complex, with some organisms possessing both a cofactor-dependent and a cofactor-independent PGM and others having only one of these enzymes . Although Bacillus species contain only a cofactor-independent PGM, genes homologous to those encoding cofactor-dependent PGMs have been detected in this group of bacteria, but in at least one case the encoded protein lacks significant PGM activity . Here we apply sequence analysis, molecular modeling, and enzymatic assays to the cofactor-dependent PGM homologs from B . stearothermophilus and B . subtilis, and show that these enzymes are phosphatases with broad substrate specificity . Homologs from other gram-positive bacteria are also likely to possess phosphatase activity . These studies clearly show that the exploration of genomic sequences through three-dimensional modeling is capable of producing useful predictions regarding function . However, significant methodological improvements will be needed before such analysis can be carried out automatically. Biochemistry, 2001 Aug 28, 40(34), 10115 - 39 Dissecting the electrostatic interactions and pH-dependent activity of a family 11 glycosidase; Joshi MD et al.; Previous studies of the low molecular mass family 11 xylanase from Bacillus circulans show that the ionization state of the nucleophile (Glu78, pK(a) 4.6) and the acid/base catalyst (Glu172, pK(a) 6.7) gives rise to its pH-dependent activity profile . Inspection of the crystal structure of BCX reveals that Glu78 and Glu172 are in very similar environments and are surrounded by several chemically equivalent and highly conserved active site residues . Hence, there are no obvious reasons why their apparent pK(a) values are different . To address this question, a mutagenic approach was implemented to determine what features establish the pK(a) values (measured directly by (13)C NMR and indirectly by pH-dependent activity profiles) of these two catalytic carboxylic acids . Analysis of several BCX variants indicates that the ionized form of Glu78 is preferentially stabilized over that of Glu172 in part by stronger hydrogen bonds contributed by two well-ordered residues, namely, Tyr69 and Gln127 . In addition, theoretical pK(a) calculations show that Glu78 has a lower pK(a) value than Glu172 due to a smaller desolvation energy and more favorable background interactions with permanent partial charges and ionizable groups within the protein . The pK(a) value of Glu172 is in turn elevated due to electrostatic repulsion from the negatively charged glutamate at position 78 . The results also indicate that all of the conserved active site residues act concertedly in establishing the pK(a) values of Glu78 and Glu172, with no particular residue being singly more important than any of the others . In general, residues that contribute positive charges and hydrogen bonds serve to lower the pK(a) values of Glu78 and Glu172 . The degree to which a hydrogen bond lowers a pK(a) value is largely dependent on the length of the hydrogen bond (shorter bonds lower pK(a) values more) and the chemical nature of the donor (COOH > OH > CONH(2)) . In contrast, neighboring carboxyl groups can either lower or raise the pK(a) values of the catalytic glutamic acids depending upon the electrostatic linkage of the ionization constants of the residues involved in the interaction . While the pH optimum of BCX can be shifted from -1.1 to +0.6 pH units by mutating neighboring residues within the active site, activity is usually compromised due to the loss of important ground and/or transition state interactions . These results suggest that the pH optima of an enzyme might be best engineered by making strategic amino acid substitutions, at positions outside of the "core" active site, that electrostatically influence catalytic residues without perturbing their immediate structural environment. J Synchrotron Radiat, 2001 Mar 1, 8(Pt 2), 199 - 203 High-resolution X-ray spectroscopy of rare events: a different look at local structure and chemistry; Bergmann U et al.; The combination of large-acceptance high-resolution X-ray optics with bright synchrotron sources permits quantitative analysis of rare events such as X-ray fluorescence from very dilute systems, weak fluorescence transitions or X-ray Raman scattering . Transition-metal Kbeta fluorescence contains information about spin and oxidation state; examples of the characterization of the Mn oxidation states in the oxygen-evolving complex of photosystem II and Mn-consuming spores from the marine bacillus SG- are presented . Weaker features of the Kbeta spectrum resulting from valence-level and 'interatomic' ligand to metal transitions contain detailed information on the ligand- atom type, distance and orientation . Applications of this spectral region to characterize the local structure of model compounds are presented . X-ray Raman scattering (XRS) is an extremely rare event, but also represents a unique technique to obtain bulk-sensitive low-energy (<600 eV) X-ray absorption fine structure (XAFS) spectra using hard (approximately 10 keV) X-rays . A photon is inelastically scattered, losing part of its energy to promote an electron into an unoccupied level . In many cases, the cross section is proportional to that of the corresponding absorption process yielding the same X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) features . XRS finds application for systems that defy XAFS analysis at low energies, e.g . liquids or highly concentrated complex systems, reactive compounds and samples under extreme conditions (pressure, temperature) . Recent results are discussed. Adv Perit Dial, 2001, 17, 172 - 5 Treatment of mycobacterial exit-site infections in patients on continuous ambulatory peritoneal dialysis; Kleinpeter MA et al.; Exit-site infections (ESIs) are frequently due to gram-positive organisms and occasionally to gram-negative organisms . Initial empiric antibiotic therapy is therefore directed against these organisms until culture reports are available . Two cases of ESI associated with Mycobacterium are here reported . The first patient, a 63-year-old man with type 2 diabetes, recently treated for Staphylococcus epidermidis peritonitis, presented with acute purulent drainage at the catheter exit site, accompanied by pain and erythema . No tunnel abscess was identified by ultrasound . Empiric antibiotic therapy was initiated with ofloxacin and vancomycin . A rapid-growing acid-fast bacillus (AFB) noted four days after culture was eventually identified as Mycobacterium fortuitum . Ofloxacin was continued, vancomycin was discontinued, and clarithromycin was added . The ESI initially showed improvement; therapy was therefore continued for several months . However, cultures remained positive for M . fortuitum, and the catheter was removed 5 months after therapy was initiated . The second patient, a 28-year-old woman, presented with severe pain and tenderness at the exit site without erythema or drainage . Empiric therapy with cefazolin, gentamicin, and cephalexin was initiated . Gram-positive cocci and an AFB were identified from the exit-site culture, and antibiotics were initially changed to clarithromycin, trimethoprim/sulfamethoxazole, and ofloxacin . The organisms were subsequently identified as M . chelonae-M . abscessus complex and coagulase-negative Staphylococcus . The patient continued to improve after 3 weeks of antibiotic therapy . However, despite the initial improvement in the ESI, the M . chelonae-M . abscessus complex continued to grow, and amikacin was added intravenously . Despite continued treatment, the ESI did not resolve, and the catheter was removed after 4 months of therapy . Despite unusual exist-site infections with rapidly growing AFBs, both patients continued continuous ambulatory peritoneal dialysis (CAPD) while undergoing treatment for ESI . Catheters were left intact, as improvement was initially seen with no evidence of tunnel infection or peritonitis . Rapid-growing AFB should be considered another possible causative agent for ESI . Two cases of atypical mycobacterial exit-site infection are presented to illustrate the difficulties in managing this complication of peritoneal dialysis . Ofloxacin--or other quinolones--may provide a better spectrum of coverage when choosing empiric therapy in patients presenting with ESI. Zhonghua Gan Zang Bing Za Zhi, 2001 Jul, 9 Suppl, 50 - 2 {Modulation of angelica sinensis polysaccharides on the expression of nitric oxide synthase and Bax, Bcl-2 in liver of immunological liver-injured mice}; Ding H et al.; OBJECTIVE: To investigate the modulation of Angelica Sinensis Polysaccharides on the expression of nitric oxide synthase and Bax, Bcl-2 in the liver of immunological liver injured mice . METHODS: Immunological liver injury was induced by lipopolysaccharide (ip, 2mug/10g) in bacillus calmette-guerin (ip, 1mg/10g, qd/1 d) primed mice . Angelica Sinensis Polysaccharides was administrated to the mice with 30mg/kg, 60mg/kg, respectively . The effects of Angelica Sinensis Polysaccharides on the expression of nitric oxide synthase, Bcl-2, and Bax gene were assessed by immunohistochemistry, and the nitric oxide (NO) production, alanine transaminase (ALT) activity, glutathione S-transferase (GST) activity in serum were determined . RESULTS: The levels of NO, sALT, and sGST were significantly higher in immunological liver-injured mice than controls . The contents of iNOS and Bax were 17.8 times (P<0.001) and 1.31 times (P<0.05) of control values, respectively . cNOS expression was not obviously changed, and no Bcl-2 expression was found in immunological liver-injured mice . Angelica Sinensis Polysaccharides of 30mg/kg could reduce the levels of NO, sALT, and sGST by 24.6%, 40.8%, and 18.4%, respectively; the expression of iNOS and Bax decrease by 84.2% and 37.1%; and the expression of cNOS, Bcl-2 increase by 66.8% and 3.38 times, respectively . The influence of Angelica Sinensis Polysaccharides of 60mg/kg was not so obvious as that of 30mg/kg on sGST, NO, cNOS, Bax, and Bcl-2, but was more effective on sALT and iNOS . CONCLUSIONS: NO production may play a role in the LPS-induced hepatotoxicity . Angelica Sinensis Polysaccharides can alleviate the immune liver injury by modulating the expression of cNOS, iNOS, Bax, Bcl-2. Intern Med, 2001 Jul, 40(7), 654 - 7 Bacillus cereus brain abscesses occurring in a severely neutropenic patient: successful treatment with antimicrobial agents, granulocyte colony-stimulating factor and surgical drainage; Sakai C et al.; Multiple brain and liver abscesses developed immediately after Bacillus cereus bacteremia in a neutropenic patient with acute lymphoblastic leukemia . After even 8 weeks of antimicrobial chemotherapy together with administration of granulocyte colony-stimulating factor, every infectious process disappeared but the patient's headache has still persisted . Because the wall of one brain abscess became thin and was in danger of rupturing into the ventricle, surgical drainage was performed, resulting in disappearance of headache and resolution of brain abscess . The present case indicates that a combined medical and surgical approach is mandatory to treat patients with brain abscesses. Nucleic Acids Res . 2001 Aug 15;29(16):E76. Conditional cell ablation by stringent tetracycline-dependent regulation of barnase in mammalian cells; Leuchtenberger S et al.; Conditional expression of suicide genes in vivo has a wide range of applications in biological research and requires a minimal basal promoter activity in the uninduced state . To reduce basal activity of tetracycline (tc)-inducible target promoters we combined synthetic tet operators in varying numbers with a core promoter derived from the plant viral 35S promoter . An optimized promoter, P(TF), was found to exert a stringent regulation of luciferase in combination with tTA and rtTA in different mammalian cell lines . We linked P(TF) to the barnase gene, coding for a highly active RNase from Bacillus amyloliquefaciens . Stable cell clones expressing barnase under control of tTA exerted cell death only after tc withdrawal, correlating with a 10-fold induction of barnase mRNA expression . Directing tTA expression through a neuron-specific enolase promoter (P(NSE)) leads to barnase expression and cell death in neuronal cells after tc withdrawal . Taken together, our data demonstrate that a stringent control of barnase expression in the uninduced state improves cell ablation studies, as high frequencies of transgene propagation in both cell lines and in transgenic mice are observed. J Dairy Res, 2001 May, 68(2), 277 - 86 Molecular self-assembly of partially hydrolysed alpha-lactalbumin resulting in strong gels with a novel microstructure; Ipsen R et al.; Gelation of alpha-lactalbumin (alpha-la) incubated with a protease from Bacillus licheniformis (BLP) at 50 degrees C for 4 h was monitored using small oscillatory shear and the large deformation properties of final gels were characterized by uniaxial compression . Transmission electron microscopy was used to visualize the microstructure . Gels made from alpha-la (10 g/l) using BLP were almost transparent, although somewhat whitish, and they were more than 20 times stiffer (measured as complex modulus) than equivalent gels made from beta-lactoglobulin (beta-lg) at the same concentration . The microstructure of the gels consisted of non-branching, apparently hollow strands with a uniform diameter close to 20 nm, similar in overall structure to microtubules . Adding Ca2+ in amounts of 50 or 100 mM changed the spatial distribution of the strands and resulted in a reduction in the failure stress recorded in uniaxial compression . Apart from affecting the microstructure, Ca2+ was shown to be essential for the formation of the gels . It is proposed . that the mechanism behind the self-assembly of the partially hydrolysed alpha-la into long tubes is a spatially restricted creation of ionic bonds between Ca2+ and carboxyl acid groups on peptide fragments resulting from the action of BLP on alpha-la . Proteolysis of alpha-la with BLP in the presence of Ca2+ thus results in formation of a strong gel with a microstructure not previously observed in food protein systems. J Dairy Res, 2001 May, 68(2), 251 - 65 Correlation of base consumption with the degree of hydrolysis in enzymic protein hydrolysis; Camacho F et al.; It is fairly easy to control the enzymic hydrolysis of proteins in alkaline conditions by measuring the base consumption required to keep the pH constant in the reactor . Unfortunately, however, base consumption is not related in any simple way to the degree of hydrolysis reached at any given moment and to establish this relationship it is essential to find out the mean pK of the alpha-amino groups released during the hydrolytic process . We have shown here that the correct mean pK value varies according to the pH of the working conditions and that the relationship between these values may depend upon the kind of protein and protease used . We have put forward a method for determining this relationship experimentally by using a given protein-protease system, consisting of an alkaline titration of the raw protein and when partially hydrolysed . We have tested the results predicted by our theoretical model by applying it to the hydrolysis of whey proteins with a bacterial protease from Bacillus licheniformis at 50 degrees C, pH 8.0 . This model can easily be applied to any hydrolytic process involving the appearance of functional groups that are partially protonizable under the working conditions in question in order to follow the kinetics of the reaction via the consumption of the neutralizing agent required to keep pH constant. J Dairy Res, 2001 May, 68(2), 189 - 96 Contamination of pasteurized milk by Bacillus cereus in the filling machine; Eneroth A et al.; The contamination of pasteurized milk by Bacillus cereus during the filling process was studied in two dairy plants . Samples of pasteurized milk were taken at four different sites along the production line . The samples were stored at 7 degrees C for 7 d, or at 10 degrees C for 5 d, before plate counting and random selection of B . cereus isolates . Isolates of B . cereus were typed by the polymerase chain reaction (PCR)-based method randomly amplified polymorphic DNA (RAPD) . Samples taken at three different sites between the pasteurizer and the filling machine were all holding similar low concentrations of B . cereus, while an increase of the B . cereus count was seen in the consumer packages . More B . cereus of different RAPD types was growing in the consumer packages than in samples taken just before the filling machine . Several RAPD types found in the consumer packages were not detected in the samples taken just before the filling machine. Arch Environ Contam Toxicol, 2001 Oct, 41(3), 369 - 73 Secondary effects of the pesticide Bacillus thuringiensis kurstaki on chicks of spruce grouse (Dendragapus canadensis); Norton ML et al.; The objective of this study was to determine if application of the biological pesticide Bacillus thuringiensis kurstaki (B.t.k.) had secondary effects on spruce grouse (Dendragapus canadensis) chicks in the southern boreal forest of Ontario, Canada . B.t.k . kills larvae of Lepidoptera that might otherwise be food . This may affect types of food ingested and influence chick growth rates by reducing the availability of a high-protein food . Amount of ingested grit, which is diet-dependent, may also be influenced, which could alter exposure to anthropogenic toxic metals, such as cadmium (Cd) and lead (Pb), associated with the grit . Chick growth rate from 0 to 14 days of age was determined, and gizzard contents were analyzed for types of foods and levels of macronutrients and trace metals in chicks raised in a plot treated with B.t.k . compared to chicks raised on a control plot . Chick growth rates were significantly different (p < 0.05); chicks from the treatment plot increased body mass 30% less as compared to chicks raised on the control plot . Control chicks contained on average 2.5-fold the mass (in g) of grit and nearly twofold the mass of food, which included larvae, in their gizzards compared to those exposed to B.t.k . whose diets were dominated by spiders and ants . Concentrations of magnesium (Mg), calcium (Ca), phosphorous (P), iron (Fe), manganese (Mn), sulfur (S) and the trace metals zinc (Zn), copper (Cu), nickel (Ni), and chromium (Cr) did not differ between experimental and control birds (p > 0.05) . In contrast, control chicks had higher levels of aluminum (Al), Cd, cobalt (Co), and Pb in their gizzards than chicks from the sprayed area (p < 0.05), attributed to greater amounts of consumed grit . Although birds from the sprayed area had lower levels of the toxic metals Pb and Cd in their gizzards, a protein-deficient diet could have been a factor that contributed to decreased rates of growth in B.t.k.-exposed birds as compared to those birds held on nonsprayed sites . Reduced growth during the first 2 weeks of avian development has important consequences at the population level, and it is recommended that this secondary effect be considered for all species who rely on Lepidoptera larvae as a food resource prior to wide spread application of B.t.k . for insect control. Diagn Microbiol Infect Dis, 2001 Jul, 40(3), 125 - 7 Identification of Arcobacter cryaerophilus isolated from a traffic accident victim with bacteremia by 16S ribosomal RNA gene sequencing; Woo PC et al.; Traditional ways of identifying slow growing bacteria is slow and often difficult . In this study, a small, Gram-negative, facultative anaerobic, slow growing bacillus was isolated from the blood culture of a 7-year old traffic accident victim . The bacterium was non-hemolytic, catalase and oxidase positive . An attempt to use the Vitek system (GNI+) and the API system (20NE) to identify the strain was unsuccessful as the growth controls showed negative results . 16S ribosomal RNA gene sequencing showed that there was 1 base difference between the isolate and Arcobacter cryaerophilus (GenBank Accession no . U25805), 1 base difference between the isolate and A . cryaerophilus (GenBank Accession no . U34387), 10 base differences between the isolate and A . cryaerophilus (GenBank Accession no . L14624), 34 base differences between the isolate and A . butzleri (GenBank Accession no . U34386), 34 base differences between the isolate and A . butzleri (GenBank Accession no . U34387), and 38 base differences between the isolate and A . butzleri (GenBank Accession no . L14626), indicating that the isolate most closely resembled a strain of A . cryaerophilus . Identification of the isolate in our case by conventional methods was difficult, as the absence of a curved morphology has made it confused with other Gram-negative non-fermentative bacteria, and the slow growth rate has made it unidentifiable by both the Vitek and API systems . Although the exact source of infection and route of transmission in our case remains elusive, we speculate that the bacteria were transmitted through the respiratory tract while the boy was suffocated in the mud . The present report represents an example of showing the usefulness of 16S rRNA gene sequencing for identification of slow growing bacteria. Folia Microbiol (Praha), 2000, 45(6), 552 - 4 Antifungal activity of Bacillus sp . isolated from compost; Czaczyk K et al.; Four strains of Bacillus isolated from lupine compost exhibited an antifungal activity against six plant fungal pathogens (Rhizoctonia solani, Bipolaris sorokiniana, Sclerotinia sclerotiorum, Trichothecium roseum, Fusarium solani, Fusarium oxysporum) . It was significantly influenced by the composition of the cultivation media. J Environ Sci Health A Tox Hazard Subst Environ Eng, 2001, 36(6), 1135 - 44 Degradation of dimethylphthalate by cells of Bacillus sp . immobilized in calcium alginate and polyurethane foam; Niazi JH et al.; A Bacillus sp . which is capable of degrading dimethylphthalate (DMP) was immobilized in calcium alginate and polyurethane foam for efficient and long term degradation of DMP . Freely suspended cells (10(12) cfu ml-1) degraded a maximum of 20 mM DMP . Whereas, alginate-(10(12)cfu g-1 beads) and polyurethane foam-entrapped (0.34 x 10(6-9) cfu g-1 foam cubes) cells degraded a maximum of 40 mM DMP within 12-15 days of incubation . Polyurethane foam-entrapped cells degraded 30 mM of DMP at 4 days and alginate-entrapped cells degraded within 10 to 12 days of incubation irrespective of the cell population . When the initial concentration of DMP increased to 50 mM, the DMP degrading ability of the immobilized cells was not increased even after 20 days . Repeated batch cultures by alginate-entrapped cells with initial 35 mM DMP loading could be reused for a maximum of 20 cycles . However, the degradation rate was gradually decreased when the beads were reused for more than 15 cycles . On the other hand, the foam-entrapped cells, with the same initial DMP loading there was no decrease in DMP degrading ability and could be reused for more than 20 cycles . The packed bed reactor with alginate-entrapped cells (1 x 10(10-12) cfu g-1 bead) could be continuously operated for 7-8 days with an initial 25 mM DMP at a flow rate of 50 ml h-1 . Whereas, the polyurethane foam-entrapped cells (1 x 10(6-9) cfu g-1 foam cubes) could be operated continuously for more than 90 days with the same initial DMP loading at a flow rate of 100 ml h-1 . Thus the enhanced degradation of DMP could be achieved by immobilizing the cells of Bacillus sp . in calcium alginate and polyurethane foam as compared to that of freely suspended cells. J Biotechnol, 2001 Aug 23, 89(2-3), 147 - 53 Thermo-alkali-stable catalases from newly isolated Bacillus sp . for the treatment and recycling of textile bleaching effluents; Paar A et al.; Three thermoalkaliphilic bacteria, which were grown at pH 9.3-10 and 60-65 degrees C were isolated out of a textile wastewater drain . The unknown micro-organisms were identified as thermoalkaliphilic Bacillus sp . Growth conditions were studied and catalase activities and stabilities compared . Catalases from Bacillus SF showed high stabilities at 60 degrees C and pH 9 (t1/2=38 h) and thus this strain was chosen for further investigations, such as electron microscopy, immobilization of catalase and hydrogen peroxide degradation studies . Degradation of hydrogen peroxide with an immobilized catalase from Bacillus SF enabled the reuse of the water for the dyeing process . In contrast, application of the free enzyme for treatment of bleaching effluents, caused interaction between the denaturated protein and the dye, resulting in reduced dye uptake, and a higher color difference of 1.3DeltaE* of dyed fabrics compared to 0.9DeltaE* when using the immobilized enzyme. J Invertebr Pathol, 2001 Jul, 78(1), 45 - 51 Gene knockout demonstrates that vip3A contributes to the pathogenesis of Bacillus thuringiensis toward Agrotis ipsilon and Spodoptera exigua; Donovan WP et al.; Vip3A is an 89-kDa protein secreted by Bacillus thuringiensis during vegetative growth . To determine the importance of Vip3A for the insect pathogenicity of B . thuringiensis the vip3A gene was deleted from strain HD1, yielding strain HD1Deltavip3A . Compared with HD1, strain HD1Deltavip3A was one-fourth as toxic to Agrotis ipsilon larvae and less than one-tenth as toxic to Spodoptera exigua larvae . When streptomycin was included in the S . exigua diet the toxicity of HD1Deltavip3A was approximately half that of HD1 . Addition of HD1 spores increased the toxicity of purified Cry1 protein more than 600-fold against S . exigua, whereas addition of HD1Deltavip3A spores increased toxicity of Cry1 protein approximately 10-fold . These results demonstrate that an important component of B . thuringiensis insecticidal activity against S . exigua is the synthesis of Vip3A protein by B . thuringiensis cells after ingestion of spores and crystal proteins by insect larvae . J Invertebr Pathol, 2001 Jul, 78(1), 37 - 44 Construction and characterization of a recombinant Bacillus thuringiensis subsp . israelensis strain that produces Cry11B; Park HW et al.; The mosquitocidal bacterium