Bacillus

J Am Chem Soc, 2004 Oct 6, 126(39), 12661 - 8
Water-assisted reaction mechanism of monozinc beta-lactamases; Dal Peraro M et al.; Hybrid Car-Parrinello QM/MM calculations are used to investigate the reaction mechanism of hydrolysis of a common beta-lactam substrate (cefotaxime) by the monozinc beta-lactamase from Bacillus cereus (BcII) . The calculations suggest a fundamental role for an active site water in the catalytic mechanism . This water molecule binds the zinc ion in the first step of the reaction, expanding the zinc coordination number and providing a proton donor adequately oriented for the second step . The free energy barriers of the two reaction steps are similar and consistent with the available experimental data . The conserved hydrogen bond network in the active site, defined by Asp120, Cys221, and His263, not only contributes to orient the nucleophile (as already proposed), but it also guides the second catalytic water molecule to the zinc ion after the substrate is bound . The hydrolysis reaction in water has a relatively high free energy barrier, which is consistent with the stability of cefotaxime in water solution . The modeled Michaelis complexes for other substrates are also characterized by the presence of an ordered water molecule in the same position, suggesting that this mechanism might be general for the hydrolysis of different beta-lactam substrates.

J Mol Biol, 2004 Oct 15, 343(2), 477 - 91
Crystal structures and electron micrographs of fungal volvatoxin A2; Lin SC et al.; Membrane adhesion and insertion of protein are essential to all organisms, but the underlying mechanisms remain largely unknown . Membrane pore-forming toxins (PFTs) are potential model systems for studying these mechanisms . We have determined the crystal structures of volvatoxin A2 (VVA2), a fungal PFT from Volvariella volvacea, using Br-multiple-wavelength anomalous diffraction (MAD) . The VVA2 structures obtained at pH 4.6, pH 5.5 and pH 6.5 were refined to resolutions of 1.42 A, 2.6 A and 3.2 A, respectively . The structures reveal that the VVA2 monomer contains a single alpha/beta domain . Most of the VVA2 surface is occupied by its oligomerization motif and two putative heparin-binding motifs . Residues Ala91 to Ala101 display several conformations at different pH values, which might be under the control of His87 . We also found that the shape of one putative heparin-binding motif in VVA2 appears similar to those found in fibroblast growth factors, and the other one displays a linear polypeptide . Our results suggest several possible intermediates of protein assembly in solution and protein adhering to cell membranes before conformational changes . The electron micrographs of VVA2 molecules in solution, at a protein concentration of 1 microg ml(-1), show that they can assemble into filament-like or braid-like oligomers in a pH-dependent way . In addition, the arc-shaped VVA2 structure obtained at pH 6.5 suggests that VVA2 could form a two-layered helical oligomer with 18 subunits per turn . The structures presented here could be used to elucidate the pore-formation mechanisms of VVA2 and its structural neighbors, Cyt toxins from Bacillus thuringiensis.

Toxicon, 2004 Oct, 44(5), 515 - 20
Immunoenzymatic visualization of tetrodotoxin (TTX) in Cephalothrix species (Nemertea: Anopla: Palaeonemertea: Cephalotrichidae) and Planocera reticulata (Platyhelminthes: Turbellaria: Polycladida: Planoceridae); Tanu MB et al.; Tetrodotoxin (TTX) was localized as brown color in different tissues of an undescribed species of the nemertean genus Cephalothrix (phylum Nemertea) and a turbellarian Planocera reticulata (phylum Platyhelminthes) on light microscopy by means of a monoclonal anti-TTX antibody . In the Cephalothrix sp., TTX was recognized in the vesicles apically arranged in the bacillary cells in the epidermis, basal lamina, the granular cells in the proboscis epithelium, rhynchocoel epithelium, and the vesicles in the basal portion of the intestinal wall near the blood vessels and rhynchocoel . The excretory system and the ovum also showed positive reaction of TTX antigen-antibody . On the other hand, the hermaphrodite flatworm P . reticulata exhibited TTX antigen-antibody complex only in their ovum . To our knowledge, this is the first experimental effort on micro-distribution of TTX in invertebrates.

Anal Biochem, 2004 Oct 15, 333(2), 289 - 95
A subnanogram assay for phospholipase activity based on a long-chain radioiodinatable phosphatidylcholine; Caramelo JJ et al.; Here, we introduce a radioiodinatable long-chain phosphatidylcholine (BHC12PC) which serves as the base for a very sensitive phospholipase assay . This compound has a 4-hydroxyphenyl group attached at the end of the fatty acyl chain located in position sn-2 . This feature enables this phospholipid to be radioiodinated . BHC12PC was tested as a substrate of Naja naja naja PLA(2) and Bacillus cereus PLC in a mixed micellar system with Triton X-100 . The detection limit for the assays was 0.25ng of PLA(2) and 0.05ng of PLC, thus becoming one of the most sensitive methods described so far . A low specific radioactivity (500microCi/mmol) suffices to achieve this level of sensitivity . In both cases, the behavior of BHC12PC was indistinguishable from that shown by phospholipids with n-acyl chains of similar length . The choice of spacer prevents any unfavorable interaction of the bulky 4-hydroxyphenyl group at the active site of the enzymes . The progress of the reaction as monitored by thin-layer chromatography is compared side by side with an alternative method based on the selective adsorption of BHC12PC to silica gel, which renders identical results in a simpler fashion . An additional advantage of BHC12PC is that the cost per Ci of the radioiodinated derivative is significantly lower than that of other labeled phospholipids ((3)H, (14)C, or (32)P).

Biochemistry, 2004 Oct 5, 43(39), 12523 - 31
Engineering of the pH optimum of Bacillus cereus beta-amylase: conversion of the pH optimum from a bacterial type to a higher-plant type; Hirata A et al.; The optimum pH of Bacillus cereus beta-amylase (BCB, pH 6.7) differs from that of soybean beta-amylase (SBA, pH 5.4) due to the substitution of a few amino acid residues near the catalytic base residue (Glu 380 in SBA and Glu 367 in BCB) . To explore the mechanism for controlling the optimum pH of beta-amylase, five mutants of BCB (Y164E, Y164F, Y164H, Y164Q, and Y164Q/T47M/Y164E/T328N) were constructed and characterized with respect to enzymatic properties and X-ray structural crystal analysis . The optimum pH of the four single mutants shifted to 4.2-4.8, approximately 2 pH units and approximately 1 pH unit lower than those of BCB and SBA, respectively, and their k(cat) values decreased to 41-3% of that of the wild-type enzyme . The X-ray crystal analysis of the enzyme-maltose complexes showed that Glu 367 of the wild type is surrounded by two water molecules (W1 and W2) that are not found in SBA . W1 is hydrogen-bonded to both side chains of Glu 367 and Tyr 164 . The mutation of Tyr 164 to Glu and Phe resulted in the disruption of the hydrogen bond between Tyr 164 Oeta and W1 and the introduction of two additional water molecules near position 164 . In contrast, the triple mutant of BCB with a slightly decreased pH optimum at pH 6.0 has no water molecules (W1 and W2) around Glu 367 . These results suggested that a water-mediated hydrogen bond network (Glu 367...W1...Tyr 164...Thr 328) is the primary requisite for the increased pH optimum of wild-type BCB . This strategy is completely different from that of SBA, in which a hydrogen bond network (Glu 380...Thr 340...Glu 178) reduces the optimum pH in a hydrophobic environment.

J Ind Microbiol Biotechnol, 2004 Nov, 31(10), 462 - 8 Epub 2004 Nov.
Mechanisms for solubilization of cobalt, copper and nickel from Indian Ocean nodules at near neutral pH by a marine isolate; Mukherjee A et al.; Polymetallic ocean nodules offer an alternative source for extracting valuable strategic metals like Cu, Co and Ni . A novel biodissolution process was carried out, employing the cell-free spent growth medium from a marine organism ( Bacillus M1) isolated from nodules; and Cu, Co and Ni solubilization from the nodules was observed to be beyond the theoretical solubility limits at near neutral pH . Different characterization techniques revealed the presence of phenolic substances in the spent growth medium, which might have formed soluble complexes with the transition metals . The low prevailing E(h) redox value in the medium suggested a strong reducing environment, favoring the reductive dissolution of the oxides . A correlation study of dissolution of Cu, Co and Ni with that of Mn and Fe in the nodules was made to investigate the mechanisms of metal solubilization by the marine isolate . Under the influence of a strong reducing environment coupled with complexation by a phenolic substance present in the spent growth medium, Mn and Fe oxides were solubilized from the nodules, resulting in concomitant dissolution of Cu, Co and Ni associated with them in the nodules.

Radiat Res, 2004 Oct, 162(4), 464 - 8
Photoabsorption study of Bacillus megaterium, DNA and related biological materials in the phosphorus K-shell edge region; Frigo SP et al.; We measured the X-ray transmission spectra of several biologically related samples in the phosphorus K-shell edge absorption region . These include red phosphorus, hydrated sodium phosphate (Na(3)PO(4).12 H(2)O), deoxyribonucleic acid (DNA), adenosine triphosphate (ATP), diolylphosphatidyl choline (DOPC), and Bacillus megaterium spores . Red phosphorus essentially displays an edge-jump . All other spectra are similar in form and energy position: Each is dominated by a narrower, more intense first peak and a broader but less intense second peak . The corresponding K-shell edge absorption thresholds are shifted toward higher energy relative to that for red phosphorus, as expected for increasing degrees of phosphorus oxidation . The B . megaterium spectrum has aspects common to both the phosphate and DNA spectra and is therefore interpreted as a composite of spectra arising from DNA, ribonucleic acid (RNA) and phosphates within the spore . The B . megaterium spore spectrum provides information for resonant radiation damage studies in the phosphorus K-shell edge absorption region by identifying candidate photoexcitations . In addition, the absorption spectra will be useful in X-ray microscopy and macromolecular crystallography studies at the phosphorus K-shell edge.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1611 - 5
Oribacterium sinus gen . nov., sp . nov., within the family 'Lachnospiraceae' (phylum Firmicutes); Carlier JP et al.; A hitherto unknown anaerobic bacillus isolated from sinus pus in a young child (strain AIP 354.02T) was characterized by using phenotypic and genotypic methods . 16S rRNA gene sequence analysis indicated that this strain was phylogenetically affiliated with several sequences of cloned 16S rRNA gene inserts previously deposited in the public databases . According to their 16S rRNA gene sequence similarities, these uncultivated bacteria, together with strain AIP 354.02T, formed a separate subgroup belonging to the family 'Lachnospiraceae' within the phylum Firmicutes . Oribacterium gen . nov . is proposed for this group of organisms and Oribacterium sinus gen . nov . sp . nov . for strain AIP 354.02T (= CIP 107991T = CCUG 48084T).

FEBS Lett, 2004 Sep 24, 575(1-3), 131 - 5
pKa of the essential Glu54 and backbone conformation for subunit c from the H+-coupled F1F0 ATP synthase from an alkaliphilic Bacillus; Rivera-Torres IO et al.; The conformation of the ATP synthase c-subunit and the pKa of its essential E54 residue were characterized in alkaliphilic Bacillus pseudofirmus OF4 . The c-subunit folds as a helix-loop-helix, with inter-helical contacts demonstrated by paramagnetic relaxation effects . The E54 pKa of 7.7 is significantly higher than in non-alkaliphiles, which likely prevents proton loss from the c-rotor at high pH . The E54 pKa was unchanged in a mutant, cP51A, that has a severe ATP synthesis defect at high pH only . cP51 must have some structural role that accounts for the mutant defect, such as different subunit-subunit interactions at high pH.

Arch Pathol Lab Med, 2004 Oct, 128(10), 1193 - 5
William H . Welch, MD, and the discovery of Bacillus welchii; Lucey BP et al.; William H . Welch, MD, and his colleagues performed an autopsy at The Johns Hopkins Hospital in October 1891 on a 38-year-old man and discovered a new bacterium, Bacillus aerogenes capsulatus . During the postmortem examination, gas bubbles were noted within many of the patient's blood vessels . Welch's laboratory personnel determined that a previously unknown bacterium was the source of the gas . Through a series of experiments, the organism's characteristics were described and its pathophysiology was detailed, findings that proved accurate in explaining gas gangrene during World War I . Welch never followed up these initial investigations with more experimentation . His subsequent writings regarding the bacterium that came to be known, appropriately, as Bacillus welchii consisted mostly of case reports from other medical institutions and summaries of previous data.

Curr Microbiol, 2004 Sep, 49(3), 165 - 9
Gene cloning and expression of an alkaline serine protease with dehairing function from Bacillus pumilus; Pan J et al.; A new gene (named AP gene) encoding an alkaline serine protease with dehairing function was cloned from Bacillus pumilus UN-31-C-42 and its nucleotide sequence was determined . The expression of AP gene was induced with IPTG in Escherichia coli after the mature protease region was cloned into pET15b and SDS-PAGE showed expressed product clearly, but no alkaline protease activity was detected . In order to express the AP gene in B . subtilis, a recombinant expression plasmid was constructed which contained a promoter Bp53 (also from B . pumilus), the AP gene and an E . coli-B . subtilis shuttle vector pSUGV4 . This plasmid was introduced into B . subtilis WB600 and the transformant displayed the hydrolyzed zone on a milk plate . The expressed product can be easily detected with SDS-PAGE and the fermentation fluid of the transformant showed low alkaline protease activity and dehairing activity . This is the first report of a gene cloned from B . pumilus, encoding an alkaline serine protease, which can alone accomplish the whole dehairing process.

J Econ Entomol, 2004 Aug, 97(4), 1425 - 31
Field and storage testing Bt potatoes for resistance to potato tuberworm (Lepidoptera: Gelichiidae); Douches DS et al.; Potato tuberworm, Phthorimaea operculella (Zeller), is the most serious insect pest of potatoes worldwide . The introduction of the Bacillus thuringiensis (Bt) toxin gene through genetic engineering offers host plant resistance for the management of potato tuberworm . We report on the field and storage studies to evaluate Bt-cry5 potato lines for resistance to potato tuberworm in Egypt under natural infestations and their agronomic performance in both Egypt and Michigan . From 1997 to 2001, field experiments were conducted at the International Potato Center (CIP) Research Station, Kafr El-Zyat, Egypt, and/or Agricultural Genetic Engineering Institute (AGERI), Giza, Egypt, to evaluate resistance to tuberworm . A total of 27 Bt-transgenic potato lines from six different Bt constructs were evaluated over a 5-yr period . After harvest and evaluation of the agronomic trials, storage evaluation of potato tuberworm damage was done at the CIP Research Station . The 1997 field trial was the first field test of genetically engineered crops in Egypt . Field tests to assess potato tuberworm resistance in Egypt were able to differentiate between the Bt-transgenic lines and the nontransgenic lines/cultivars in 1999, 2000, and 2001 . The Bt-cry5-Spunta lines (Spunta-G2, Spunta-G3, and Spunta-6a3) were the most resistant lines in field with 99-100% of tubers free of damage . In the 2001 storage study, these lines were also over 90% free of tuberworm damage after 3 mo . NYL235-4.13, which combines glandular trichomes with the Bt-cry5/gus fusion construct, also had a high percentage of clean tubers in the field studies . In agronomic field trials in Michigan from 1997 to 2001, the Bt-transgenic lines in most instances performed similar to the nontransgenic line in the agronomic trials; however, in Egypt (1998-1999), the yields were less than one-half of those in Michigan . Expression of the Bt-cry5 gene in the potato tuber and foliage will provide the seed producer and grower a tool in which to reduce potato tuberworm damage to the tuber crop in the field and storage.

J Econ Entomol, 2004 Aug, 97(4), 1413 - 24
Effects of insect population size on evolution of resistance to transgenic crops; Sisterson MS et al.; Models of the evolution of insect resistance to transgenic crops have often assumed that population size is infinite or that carrying capacity is fixed . To evaluate potential effects of population size on resistance evolution, we conducted sensitivity analyses by using a stochastic, spatially explicit model based partly on the interaction between pink bollworm and Bacillus thuringiensis (Bt) cotton . We examined interactions of carrying capacity, region size, dispersal, and percentage of fields planted with Bt cotton . The median and variance in the time to resistance decreased as region size increased, regardless of carrying capacity . This occurred because larger regions were more likely to have at least one field in which resistance evolved rapidly and served as a source from which resistance spread throughout the region . Carrying capacity significantly affected the median time to resistance with 75% of fields planted with Bt cotton, but not with 50% Bt cotton . In contrast, carrying capacity significantly influenced the variance in the time to resistance with 50% Bt cotton, but not with 75% Bt cotton . We also found resistance evolution was affected by interactions between carrying capacity, dispersal, and the percentage of fields planted with Bt cotton . The high variability observed in our simulations indicates that factors affecting stochastic events can play an important role in the evolution of resistance . Because population size determines the extent to which stochastic events are important, reasonable estimates of population size are essential for devising robust models of resistance evolution.

J Econ Entomol, 2004 Aug, 97(4), 1198 - 201
Growth, development, and survival of Nosema pyrausta-infected European corn borers (Lepidoptera: Crambidae) reared on meridic diet and Cry1Ab; Reardon BJ et al.; Transgenic corn, Zea mays L., hybrids expressing crystal protein endotoxin genes from Bacillus thuringiensis Berliner are an increasingly popular tactic for managing the European corn borer, Ostrinia nubilalis (Hubner), in North America . O . nubilalis populations also are often vulnerable to the ubiquitous entomopathogenic microsporidium Nosema pyrausta (Paillot) . We examined the effect of feeding meridic diet incorporated with purified Cry1Ab on growth, development, and survival of Nosema-infected and uninfected neonate O . nubilalis . Infected larvae developed more slowly than uninfected larvae . Increasing the concentration of Cry1Ab in diet reduced larval development, and this effect was amplified by microsporidiosis . Infected larvae weighed significantly less than uninfected larvae . The relationship among Nosema infection, Cry1Ab concentration, and larval weight was fitted to an exponential function . The LC50 of infected larvae was one-third that of uninfected larvae, indicating that infected larvae are more vulnerable to toxin . This work has implications for resistance management of O . nubilalis and demonstrates that it is important to determine whether N . pyrausta is present when testing susceptibility of larvae to transgenic corn hybrids.

Eur J Immunol, 2004 Nov, 34(11), 3276 - 84
Bacillus Calmette Guerin triggers the IL-12/IFN-gamma axis by an IRAK-4- and NEMO-dependent, non-cognate interaction between monocytes, NK, and T lymphocytes; Feinberg J et al.; The IL-12/IFN-gamma axis is crucial for protective immunity to Mycobacterium in humans and mice . Our goal was to analyze the relative contribution of various human blood cell subsets and molecules to the production of, or response to IL-12 and IFN-gamma . We designed an assay for the stimulation of whole blood by live M . bovis Bacillus Calmette-Guerin (BCG) alone, or BCG plus IL-12 or IFN-gamma, measuring IFN-gamma and IL-12 levels . We studied patients with a variety of specific inherited immunodeficiencies resulting in a lack of leukocytes, or T, B, and/or NK lymphocytes, or polymorphonuclear cells, or a lack of expression of key molecules such as HLA class II, CD40L, NF-kappaB essential modulator (NEMO), and IL-1 receptor-associated kinase-4 (IRAK-4) . Patients with deficiencies in IL-12p40, IL-12 receptor beta1 chain (IL-12Rbeta1), IFN-gammaR1, IFN-gammaR2, and STAT-1 were used as internal controls . We showed that monocytes were probably the main producers of IL-12, and that NK and T cells produced similar amounts of IFN-gamma . NEMO and IRAK-4 were found to be important for IL-12 production and subsequent IFN-gamma production, while a lack of CD40L or HLA class II had no major impact on the IL-12/IFN-gamma axis . The stimulation of whole blood by live BCG thus triggers the IL-12/IFN-gamma axis by an IRAK-4- and NEMO-dependent, non-cognate interaction between monocytes, NK, and T lymphocytes.

Biotechnol Bioeng, 2004 Oct 5, 88(1), 15 - 25
Development characterization and use of a high-performance enzymatic time-temperature integrator for the control of sterilization process' impacts; Guiavarc'h Y et al.; A small sized single-component enzymatic time temperature integrator (TTI) was developed . It consisted of glass beads coated with Bacillus licheniformis alpha-amylase (BLA) and stabilizing additives in a dehydrated form . Post heating residual enzymatic activity was used as a response property of the TTI . Under isothermal conditions, different batches of the system were characterized by z(TTI)-values around 13.5 degrees C in the temperature range 100-130 degrees C as well as by their ability to provide a response within 5 min after thermal processing . When used under non-isothermal conditions in a model food (silicone spheres), the system allowed to measure process-values (zTTI)F(121.1 degrees C) up to 60 min with an average error of 10.9% . The capabilities of the system were validated in a real solid/liquid food matrix sterilized by retorting . The combination of F(TTI)-values with heat transfer simulations based on finite difference calculations allowed for the determination of process values, which evaluated actual process-values (10 degrees C)F(121.1 degrees C) up to 90 min with an average error of 11.4% . The good performances of the system as well as its easiness of preparation and use, make the latter a valuable biological device for thermal process assessment.

J Immunol, 2004 Oct 1, 173(7), 4590 - 7
Activation of CD8 T cells by mycobacterial vaccination protects against pulmonary tuberculosis in the absence of CD4 T cells; Wang J et al.; We have investigated whether both primary CD8 T cell activation and CD8 T cell-mediated protection from Mycobacterium tuberculosis challenge could occur in mycobacterial-vaccinated CD4 T cell-deficient (CD4KO) mice . Different from wild-type C57BL/6 mice, s.c . vaccination with bacillus Calmette-Guerin (BCG) in CD4KO mice failed to provide protection from secondary M . tuberculosis challenge at 3 wk postvaccination . However, similar to C57BL/6 mice, CD4KO mice were well protected from M . tuberculosis at weeks 6 and 12 postvaccination . This protection was mediated by CD8 T cells . The maintenance of protective effector/memory CD8 T cells in CD4KO mice did not require the continuous presence of live BCG vaccine . As in C57BL/6 mice, similar levels of primary activation of CD8 T cells in CD4KO mice occurred in the draining lymph nodes at 3 wk after BCG vaccination, but different from C57BL/6 mice, the distribution of these cells to the spleen and lungs of CD4KO mice was delayed, which coincided with delayed acquisition of protection in CD4KO mice . Our results suggest that both the primary and secondary activation of CD8 T cells is CD4 T cell independent and that the maintenance of these CD8 T cells is also independent of CD4 T cells and no longer requires the presence of live mycobacteria . However, the lack of CD4 T cells may result in delayed distribution of activated CD8 T cells from draining lymph nodes to distant organs and consequently a delayed acquisition of immune protection . Our findings hold implications in rational design of tuberculosis vaccination strategies for humans with impaired CD4 T cell function.

Ann Surg Oncol, 2004 Oct, 11(10), 892 - 9 Epub 2004 Sep 20.
Peripheral blood CD4+ T-cell response before postoperative active immunotherapy correlates with clinical outcome in metastatic melanoma; Hsueh EC et al.; BACKGROUND: Canvaxin polyvalent specific active immunotherapeutic (CancerVax Corp., Carlsbad, CA) is a minimally toxic adjuvant after resection of regional metastatic melanoma . Because Canvaxin immunotherapeutic requires induction of an immune response, we hypothesized that survival would be directly correlated with cellular immune responses to Canvaxin cells prior to immunization . METHODS: We randomly selected 54 patients from a study of Canvaxin therapy after complete resection of American Joint Committee on Cancer (AJCC) stage III melanoma . Peripheral blood lymphocytes (PBLs) collected before immunotherapy were co-cultured with Canvaxin cells; cellular response was determined by flow cytometric measurement of the production of intracellular interleukin 4 (IL4) or interferon gamma (IFNgamma) by CD4(+) T-cells . Results were calculated as percent positive for double staining of CD4(+) plus IL4(+) or CD4(+) plus IFNgamma(+) . RESULTS: The mean (+/- SD) increase in cytokine-producing CD4(+) T-cells after Canvaxin stimulation was 4.8 +/- 2.3% for an IFN response and 5.1 +/- 2.0% for an IL4 response . Both increases were significantly correlated with overall survival by univariate analysis (P = .0471 for IFNgamma and 0.002 for IL4) . There was no significant correlation between unstimulated IFNgamma/IL4 responses and overall survival . Multivariate analysis showed that a CD4(+) T-cell IL4 response before Canvaxin therapy was a significant independent prognostic variable . CONCLUSIONS: In vitro cellular immune response to Canvaxin cells directly correlates with survival after subsequent initiation of immunotherapy for AJCC stage III melanoma . This finding will be evaluated in a multicenter phase III trial of Canvaxin plus bacille Calmette-Guerin (BCG) versus placebo plus BCG after resection of stage III melanoma.

Pest Manag Sci, 2004 Sep, 60(9), 887 - 93
Selection and heritability of resistance to Bacillus thuringiensis subsp kurstaki and transgenic cotton in Helicoverpa armigera (Lepidoptera: Noctuidae); Lu MG et al.; Compared with an unselected susceptible population, a cotton bollworm, Helicoverpa armigera (Hubner), population selected for 22 generations with transgenic cotton leaves (modified Cry1A) in the laboratory developed 11.0-fold resistance to Cry1Ac (one single-protein product MVPII) . Resistance to Bacillus thuringiensis Berliner subsp kurstaki (Btk) was selected for 22 generations with a 5.2-fold increase in LC50 . The estimated realized heritabilities (h2) of resistance for transgenic-cotton- and Btk-selected populations were 0.1008 and 0.2341, respectively . This reflects the higher phenotypic variation in response to Cry1Ac in the transgenic-cotton-selected population . This variation may have been caused by differences in protein toxin levels expressed in different growth stages of the transgenic cotton . Because of the different slopes of the probit regression lines between Cry1Ac and Btk, the estimated realized h2 cannot be used visually to compare resistance development to Cry1Ac and Btk in H armigera . Thus, the response quotient (Q) of resistance was also estimated . The Q values of resistance for transgenic-cotton- and Btk-selected populations were 0.0763 and 0.0836, respectively . This showed that the rate of resistance development would be similar in both selection populations . This result indicates that the selection of resistance using transgenic cotton is different from that selected using the single toxin . Resistance risk to transgenic cotton and Btk in field populations was assessed assuming different pressures of selection by using the estimated h2 . Assuming the h2 of resistance in a field population was half of the estimated h2, and the population received prolonged and uniform exposure to transgenic cotton or Btk causing >70% mortality in each generation, we predicted that resistance would increase 10-fold after <23 generations for Cry1Ac in transgenic cotton-selected-populations and after <21 generations for Btk in Btk-selected populations . Cross-resistance would be expected after <48 generations for Btk in transgenic-cotton-selected populations and after <21 generations for Cry1Ac in Btk-selected population . The results show that the potential to evolve resistance is similar in both transgenic-cotton- and Btk-selected populations, but that cross-resistance development to Btk is slower in transgenic-cotton-selected populations than cross-resistance development to Cry1Ac in Btk-selected populations.

Pest Manag Sci, 2004 Sep, 60(9), 839 - 41
Rapid report acetamiprid resistance and cross-resistance in the diamondback moth, Plutella xylostella; Ninsin KD; A 110-fold acetamiprid-resistant Plutella xylostella (L) strain was established after four selection experiments (in five generations) on a 9.5-fold resistant colony in the laboratory . The resistant strain did not show cross-resistance to chlorfluazuron or Bacillus thuringiensis subsp kurstaki Berliner, but displayed low resistance to cartap and phenthoate.

Arch Esp Urol, 2004 Jul-Aug, 57(6), 606 - 18
{Bacillus Calmette Guerin as adjuvant treatment for superficial bladder tumors using two different therapeutic schemes}; Iturralde Codina A et al.; OBJECTIVES: To demonstrate the importance of BCG as adjuvant treatment for superficial bladder tumors . To suggest the best dosage and treatment scheme for patients with this severe disease . To emphasize treatment-associated toxicity in our study with endovesical BCG . To evaluate relapse and progression rates of superficial bladder tumors . To describe histological grade and tumor location in our sample . To offer an economic and social benefit to both patient and our Country, improving the quality of life of our patients . METHODS: We performed a descriptive research in a series of cases, both retrospectively and prospectively, and a review of published advances in BCG adjuvant treatment for superficial bladder tumors . Between 1996 and 2000, 90 ambulatory patients were selected at the "Hospital Docente Clinico Quirurgico 10 de Octubre" to receive endovesical treatment with BCG in two different programs of treatment, 6 and 12 months long respectively, using statistical methods such as the homogeneicity test, chi-square test and the test for independence in selected tables and graphics . 45 controls were randomly selected from a group of patients to whom BCG was not administered between 1992 and 1996; their clinical records were retrospectively analyzed selecting the same variables of the 90 cases sample, being their results presented in tables and graphics . RESULTS: We evaluate and review a technology applied to patients with superficial bladder tumors . They were treated with Bacillus Calmette Guerin (BCG) after endoscopic resection (TUR) of bladder tumors and we observe that long-term treatment over 12 months is better than short-term over 6 months, improving quality of live with longer survivals . Using low doses of 50 mg/ml of a nationally produced BCG, economic savings were obtained for both patient and Country with fast patient's return to work, achieving the introduction and maintenance of the treatment in Cuba with relevant results during the "special period" . Comparisons between both treatment groups and control group showed greater benefits with the first two . CONCLUSIONS: In patients with superficial bladder tumors stage I, the use of low dose (50 mg/ml) Bacillus Calmette Guerin is an effective treatment with minimal side effects (cystitis) . Most tumors were located in the bladder trigone; histological grade 1 was predominant . Long-term treatment for 12 months was more effective than the short-term one, offering a social and economic benefit to patient and to our Country, by delaying or preventing tumor recurrences and progression, so that we recommend this modality of treatment should be part of the therapeutic armamentarium of the urologist in his fight against this severe health problem.

J Biosci, 2004 Sep, 29(3), 245 - 59
Enhanced functional and structural domain assignments using remote similarity detection procedures for proteins encoded in the genome of Mycobacterium tuberculosis H37Rv; Namboori S et al.; The sequencing of the Mycobacterium tuberculosis (MTB) H37Rv genome has facilitated deeper insights into the biology of MTB, yet the functions of many MTB proteins are unknown . We have used sensitive profile-based search procedures to assign functional and structural domains to infer functions of gene products encoded in MTB . These domain assignments have been made using a compendium of sequence and structural domain families . Functions are predicted for 78 % of the encoded gene products . For 69 % of these, functions can be inferred by domain assignments . The functions for the rest are deduced from their homology to proteins of known function . Superfamily relationships between families of unknown and known structures have increased structural information by approximately 11% . Remote similarity detection methods have enabled domain assignments for 1325 'hypothetical proteins' . The most populated families in MTB are involved in lipid metabolism, entry and survival of the bacillus in host . Interestingly, for 353 proteins, which we refer to as MTB-specific, no homologues have been identified . Numerous, previously unannotated, hypothetical proteins have been assigned domains and some of these could perhaps be the possible chemotherapeutic targets . MTB-specific proteins might include factors responsible for virulence . Importantly, these assignments could be valuable for experimental endeavors . The detailed results are publicly available at http://hodgkin.mbu.iisc.ernet.in/~dots.

Scand J Immunol, 2004 Oct, 60(4), 382 - 91
Mammalian cell-entry proteins encoded by the mce3 operon of Mycobacterium tuberculosis are expressed during natural infection in humans; Ahmad S et al.; The mammalian cell-entry (mce)3 operon is one of four homologous mce operons on Mycobacterium tuberculosis genome that encodes six putative invasin/ adhesin-like proteins (Mce3A-F) possibly involved in the entry and survival of this bacterium inside macrophages . To study the in vivo expression of the mce3 operon-encoded proteins during natural human infection, the genes encoding Mce3A-F were cloned and expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST) at the N-terminal and a x6 histidine (His) tag at the C-terminal end . The recombinant proteins appeared as major cellular proteins in SDS-PAGE gels and reacted with anti-GST and antipenta-His antibodies at the expected molecular mass of 72, 61, 78, 80, 66 and 78 kDa for GST-Mce3A, GST-Mce3B, GST-Mce3C, GST-Mce3D, GST-Mce3E and GST-Mce3F, respectively . In Western immunoblots, all the six fusion proteins, particularly GST-Mce3A, GST-Mce3C, GST-Mce3D and GST-Mce3E, reacted with antibodies in combined human serum from 11 tuberculosis (TB) patients . Pure Mce3A, Mce3D and Mce3E could be isolated by specific proteolytic cleavage by thrombin protease of the respective purified fusion protein followed by preparative SDS-PAGE . The pure Mce3A, Mce3D and Mce3E reacted to various extents with antibodies in serum samples from TB patients . The Mce3E reacted with 51 of 55 (93%) and all the three proteins reacted with 34 of 55 (62%) serum samples . The Mce3A, Mce3D and Mce3E proteins also reacted, albeit at lower frequency, with one of 23 (4%) serum sample obtained from M . bovis bacillus Calmette-Guerin-vaccinated healthy subjects and four of 18 (22%) serum samples from long-term contacts of TB patients showing reactivity with all the three Mce3 proteins . The data show that Mce3A, Mce3D and Mce3E encoded by mce3 operon of M . tuberculosis are expressed and elicit antibody responses in humans during natural infection with this pathogen.

Biochemistry, 2004 Sep 28, 43(38), 12349 - 57
A novel Bacillus thuringiensis (PS149B1) containing a Cry34Ab1/Cry35Ab1 binary toxin specific for the western corn rootworm Diabrotica virgifera virgifera LeConte forms ion channels in lipid membranes; Masson L et al.; The binary Bacillus thuringiensis PS149B1 insecticidal crystal (Cry) protein is comprised of two components, Cry34Ab1, a 14-kDa protein, and Cry35Ab1, a 44-kDa protein, the combination of which forms a novel binary toxin active on western corn rootworm larvae . The permeabilizing behavior of the native binary toxin and its two individual components expressed as recombinant proteins was studied using calcein efflux determination in liposomes and by ion channel activity measurements in planar lipid bilayers (PLBs) . Data obtained with solubilized native PS149B1 binary protein revealed it to be a pore-forming toxin that can permeabilize liposomes and form ion channels ( approximately 300-900 pS) in PLBs at pH 5.5 but not pH 9.0 . The 14-kDa component of the toxin also formed ion channels ( approximately 15-300 pS) at pH 5.5 but did not insert easily in PLBs . While the 44-kDa moiety did seldomly form resolvable ion channels ( approximately 15-750 pS) in PLBs, it did destabilize the membranes . It showed pH-dependent truncation to a stable 40-kDa protein . The purified 40-kDa truncated product formed channels ( approximately 10-450 pS) in PLBs at pH 5.5 . At that same pH, while a 3:1 molar mixture (14:44 kDa) of the individual components of the toxin induced channel activity that resembled that of the 14-kDa component alone, the 3:1 molar mixture of the 14-kDa component and 40-kDa truncated product induced channel activity ( approximately 20-800 pS) similar to that of PS149B1 in planar lipid bilayers . We conclude that the overall membrane permeabilization process of Cry34Ab1/Cry35Ab1 is a result of ion channel formation.

Biochemistry, 2004 Sep 28, 43(38), 12009 - 19
Two-site autoinhibition of the ADP-ribosylating mosquitocidal toxin (MTX) from Bacillus sphaericus by its 70-kDa ricin-like binding domain; Carpusca I et al.; The mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1 is an approximately 97-kDa arginine-specific ADP-ribosyltransferase that is activated by proteolytic cleavage, thereby releasing the active 27-kDa enzyme (MTX(30-264)) and a 70-kDa C-terminal fragment (MTX(265-870)) . In solution, the cleaved 70-kDa fragment is still a potent inhibitor of the ADP-ribosyltransferase activity of MTX . Here we studied the interaction of the 70-kDa fragment with the enzyme domain of MTX . Several C-terminal deletions of the 70-kDa fragment inhibited the enzymatic activity of MTX(30-264) . However, the IC(50) values were about 2 orders of magnitude higher for the deletions than for the 70-kDa fragment . A peptide covering amino acid residues 265-285 of the holotoxin exhibited the same inhibitory potency as the C-terminal deletions of the 70-kDa fragment . MTX(265-285) contains several acidic residues, of which D273 and D275 were found to be essential for the inhibitory effect . Exchange of these residues in the 70-kDa fragment (MTX(265-870)) reduced its inhibitory potency . Kinetic analysis showed that the peptide MTX(265-285) had no effect on the V(max) of MTX(30-264) but increased the K(m) for NAD . By contrast, the 70-kDa fragment deleted of residues Ile265 through Asn285 inhibited the enzyme activity of MTX(30-264) mainly by decreasing the V(max) of the enzyme . A second binding site for interaction of MTX(265-870) with MTX(30-264) was localized to the C-terminus within the region of residues 750-870 . The data support a two-site binding model for inhibition of the ADP-ribosyltransferase activity of MTX(30-264) by the 70-kDa fragment MTX(265-870) with an interaction of amino acid residues 265-285 at the active site and an allosteric inhibition by the C-terminal part of the 70-kDa fragment.

Proteomics, 2004 Oct, 4(10), 2942 - 53
Continued proteomic analysis of Mycobacterium leprae subcellular fractions; Marques MA et al.; Recently the sequence of the Mycobacterium leprae chromosome, the only known obligate intracellular mycobacterium, was completed . It has a dramatic reduction in functional genes, with a coding capacity of only 49.5%, the lowest one so far observed among bacterial genomes . The leprosy bacillus seems to preserve a minimal set of genes that allows its survival in the host . The identification of genes that are actually expressed by the bacterium is of high significance in the context of mycobacterial pathogenesis . In this current study, a proteomic approach was undertaken to identify the proteins present in the soluble/cytosol and membrane subcellular fractions obtained from armadillo derived M . leprae . Proteins from each fraction were separated by two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry . A total of 147 protein spots were identified from 2-DE patterns and shown to comprise products of 44 different genes, twenty eight of them corresponding to new proteins . Additionally, two highly basic proteins (with pI >10.0) were isolated by heparin affinity chromatography and identified by N-terminal sequencing . This study constitutes the first application of proteomics to a host-derived Mycobacterium.

J Infect Dis, 2004 Oct 15, 190(8), 1438 - 47 Epub 2004 Sep 10.
Development of Vgamma2Vdelta2+ T cell responses during active mycobacterial coinfection of simian immunodeficiency virus-infected macaques requires control of viral infection and immune competence of CD4+ T cells; Shen L et al.; Vgamma2Vdelta2+ T cells play a role in antimicrobial responses . It is unknown whether adaptive Vgamma2Vdelta2+ T cell responses during active mycobacterial coinfection of human immunodeficiency virus-infected humans can be generated during effective antiretroviral treatment . Here, simian immunodeficiency virus (SIV)mac-infected macaques previously exposed to bacille Calmette-Guerin (BCG) were reinfected with BCG, were treated either with tenofovir or tenofovir plus indinavir, and were assessed for the development of Vgamma2Vdelta2+ T cell responses during active BCG coinfection . A restored capacity of Vgamma2Vdelta2+ T cells to undergo major expansions and pulmonary migration during active BCG coinfection was detected after simultaneous BCG reinfection and treatment with tenofovir of the SIVmac-infected macaques . Interestingly, a restored expansion of Vgamma2Vdelta2+ T cells in the SIVmac/BCG-coinfected macaques was detectable, even though antiretroviral treatment was initiated 1 month after BCG reinfection . Importantly, the restored expansion of Vgamma2Vdelta2+ T cells coincided with increases in numbers of purified protein derivative-specific interferon- gamma -producing CD4+ T cells and increases in the magnitude of their proliferative responses . In contrast, the SIVmac-infected control macaques exhibited diminished responses of Vgamma2Vdelta2+ T cells and mycobacterium-specific CD4+ T cells during active BCG coinfection . Our results suggest that the development of adaptive immune responses of phosphoantigen-specific Vgamma2Vdelta2+ T cells during active mycobacterium/HIV coinfection requires control of viral infection and immune competence of peptide-specific CD4+ T cells .

J Antibiot (Tokyo), 2004 Jul, 57(7), 436 - 45
Gene cluster in Micromonospora echinospora ATCC15835 for the biosynthesis of the gentamicin C complex; Unwin J et al.; Gentamicin is a 4,6-disubstituted aminocyclitol antibiotic complex synthesised by some members of the actinomycete genus Micromonospora . In a search for the gentamicin biosynthetic gene cluster we identified, using a cosmid library approach, a region of the M . echinospora ATCC15835 chromosome that encodes homologues of aminoglycoside biosynthesis genes including gntB-a close homologue of the 2-deoxy-scyllo-inosose synthase gene (btrC) from butirosin-producing Bacillus circulans . Insertional inactivation was achieved by homologous recombination with an internal gntB fragment-containing suicide plasmid, delivered by conjugal transfer from Escherichia coli . gntB disruptants were gentamicin nonproducing mutants as assayed by an ELISA antibiotic detection system, proving the association of gntB (or a downstream region) with gentamicin biosynthesis . The function of some open reading frames within the cluster, predicted by nucleotide database homology searching, is discussed with regards to their potential roles in gentamicin biosynthesis . The discovery of this genetic region represents the first report of a gene cluster involved in the biosynthesis of a 4,6-disubstituted aminocyclitol antibiotic.

Am J Ind Med, 2004 Oct, 46(4), 404 - 7
IgE sensitization to bacterial and fungal biopesticides in a cohort of Danish greenhouse workers: the BIOGART study; Doekes G et al.; BACKGROUND: The use of biopesticides in agriculture may implicate new risks of work-related allergic reactions . METHODS: Sera were tested from the BIOGART project, a longitudinal respiratory health study among >300 Danish greenhouse workers . IgE was measured by enzyme immunoassay (EIA) with extracts of biopesticide products containing Bacillus thuringiensis (BT) or Verticillium lecanii (Vert) . RESULTS: Many sera had detectable IgE to BT (23-29%) or Vert (9-21%) . IgE titers from the 2- and 3-year follow-up (n=230) were highly correlated, with discordant results in <15% . IgE titers to different BT, or to different Verticillium products were also significantly correlated (both r >0.70), whereas IgE anti-BT and anti-Verticillium showed no correlation at all . CONCLUSIONS: Exposure to these microbial biopesticides may confer a risk of IgE-mediated sensitization . In future research there is a need to identify allergenic components in the preparations, perform studies on non-exposed controls and analyze the relation between sensitization and health parameters.

Clin Exp Immunol, 2004 Oct, 138(1), 139 - 44
In vitro cellular immune responses to complex and newly defined recombinant antigens of Mycobacterium tuberculosis; Al-Attiyah R et al.; The immunological diagnosis and development of new antituberculosis vaccines require the characterization of Mycobacterium tuberculosis antigens inducing cell-mediated immune responses . In this study, we have tested peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients (n = 43) and Bacille Calmette-Guerin (BCG)-vaccinated healthy subjects (n = 24) for in vitro cellular immune responses, as indicated by antigen-induced proliferation and interferon (IFN)-gamma secretion, in response to a panel of complex (culture filtrate and cell wall preparations) and single recombinant antigens (Mtb8.4, Mtb9.8, Mtb9.9, Mtb32A, Mtb39A, Mtb40, Mtb41 and Ag85B) of M . tuberculosis . The results of cellular responses showed that the majority (ranging from 70 to 98%) of TB patients and healthy donors responded to the complex antigens in antigen-induced proliferation and IFN-gamma secretion assays . However, when PBMC from the same groups of patients and healthy donors were tested with the recombinant antigens, TB patients showed strong recognition (>50% responders) of Mtb9.8 and Mtb39A in proliferation assays (median SI = 6.2 and 6.4, respectively) and of Mtb9.8, Mtb39A, Mtb40 and Ag85B in IFN-gamma assays (median delta IFN-gamma= 15.5, 10.8, 7.8 and 8.1 U/ml, respectively) . BCG-vaccinated healthy donors showed weak (<30% responders) to moderate (31-50% responders) responses to all of the recombinant antigens in both assays . When PBMC of a subset of TB patients (n = 11) were tested for secretion of protective Th1 cytokines {IFN-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-12} and the immunosuppressive cytokine IL-10, the complex CF and CW antigens as well as the recombinant Mtb9.8, Mtb9.9, Mtb40 and Ag85B induced the secretion of both types of cytokines . On the other hand, Mtb41 induced only IL-10, while Mtb8.4, Mtb32Aand Mtb39A induced the secretion of one or more of Th1 cytokines, but not IL-10 . In conclusion, the recombinant antigens inducing the secretion of Th1 cytokines could be useful as subunit vaccine candidates against TB.

Anal Chem, 2004 Sep 1, 76(17), 5208 - 17
Microfabricated differential mobility spectrometry with pyrolysis gas chromatography for chemical characterization of bacteria; Schmidt H et al.; A microfabricated drift tube for differential mobility spectrometry (DMS) was used with pyrolysis-gas chromatography (py-GC) to chemically characterize bacteria through three-dimensional plots of ion intensity, compensation voltage from differential mobility spectra, and chromatographic retention time . The DMS analyzer provided chemical information for positive and negative ions simultaneously from chemical reactions between pyrolysis products in the GC effluent and reactant ions of H+(H2O)n and O2-(H2O)n in air at ambient pressure . Authentic standards for chemicals formed in the pyrolysis of bacteria showed favorable matches with plots from py-GC/DMS analysis and were supported by py-GC/MS results . These and other yet-unidentified constituents provided a means to distinguish Escherichia coli from Micrococcus luteus . A Gram-positive spore former (Bacillus megaterium) was distinguished by an abundant peak for crotonic acid evident in positive and negative ions and not observed with M . luteus . In contrast, plots from py-GC/DMS of lipid A and lipoteichoic acid showed poor matches to plots for a Gram-negative (E . coli) bacterium and a Gram-positive (M . luteus) bacterium and the differences were attributed to differences in genus sources of the biopolymers . A significant percentage of the chemical information available in py-GC/DMS is unidentified, and the analytical utility must be established . Precision in the chemical measurement was determined as +/- 0.2 V, 10% relative standard deviation (RSD), and +/- 0.05 min for compensation voltage, peak intensity, and retention time, respectively . The minimum number of total bacteria (cell forming units) detected was 6000 though detection limits and resolution could be varied by the magnitude of the separation voltage in the differential mobility spectrometer .

Eur J Gastroenterol Hepatol, 2004 Oct, 16(10), 1063 - 6
Mesenteric adenitis and portal vein thrombosis due to Fusobacterium nucleatum; El Braks R et al.; We report the first description of portal and mesenteric vein thrombosis associated with suppurative mesenteric adenitis in a 71-year-old woman . The bacterium detected in mesenteric lymph nodes was Fusobacterium nucleatum, an anaerobic Gram-negative bacillus . Our patient had a clinical syndrome of pharyngitis and fever preceding portal vein thrombosis . Abdominal symptoms improved with antibiotics and anticoagulant therapy . This location of F . nucleatum in mesenteric lymph nodes provides an interesting insight into the occurrence of septic thrombosis in the portal vein following pharyngo-tonsillar infection.

J Urol, 2004 Oct, 172(4 Pt 1), 1496 - 500
Autocrine over expression of fibronectin by human transitional carcinoma cells impairs bacillus Calmette-Guerin adherence and signaling; Zhang G et al.; PURPOSE: Bacillus Calmette-Guerin (BCG) binds to the tumor cell as a result of mycobacterial receptors for fibronectin (FN) . Cell surface bound FN serves as a bridge through which BCG attaches to the tumor cell . Despite the importance of FN studies have demonstrated an idiosyncratic decrease in BCG adherence in response to exogenous FN . We evaluated the effect of exogenous and autocrine FN on the ability of BCG to adhere to the tumor cell surface and initiate cellular signaling . MATERIALS AND METHODS: BCG adherence to parental 253J and FN over expressing 253JTGFbeta1-8 cells as well as to the intrinsic FN expressing cell line 647V was quantified using green fluorescent protein-BCG . Experiments were performed to assess the effect of FN on BCG initiated signal transduction through nuclear factor kappaB and AP1 . Finally, the integrity of the BCG activated signaling pathway in transforming growth factor-beta1/FN over expressors was assessed using antibody mediated cross-linking of the FN receptor . RESULTS: BCG adherence was decreased in cell lines with high autocrine expression of FN . Exogenous FN prevented BCG induced transactivation of nuclear factor kappaB and AP1 reporter constructs . No BCG stimulated signaling to these reporters could be detected in FN over expressing 253J cells . NonFN dependent alpha5beta1 cross-linking initiated signal transduction in FN over expressing cells . CONCLUSIONS: We propose that by saturating cellular and BCG receptors excess FN expression decreases the ability of cellular or mycobacterial bound FN to bind vacant receptors on BCG or on the cell . Excess FN inhibits BCG adherence and BCG initiated signal transduction.

J Urol, 2004 Oct, 172(4 Pt 1), 1490 - 5
Mechanisms of bacillus Calmette-Guerin mediated natural killer cell activation; Suttmann H et al.; PURPOSE: Natural killer (NK) cells are of crucial importance for bacillus Calmette-Guerin (BCG) mediated antitumor effects . We defined the mechanisms of BCG mediated NK cell activation in vitro . MATERIALS AND METHODS: A standard Cr release assay was used to measure the cytotoxicity of BCG activated NK cells . Using the MACS system (Miltenyi Biotec, Bergisch-Gladbach, Germany) we depleted various immune cell subpopulations from BCG stimulated peripheral blood mononuclear cells to phenotype activated NK cells . During the stimulation process anticytokine antibodies and recombinant cytokines were added to define their role in NK cell activation . For costimulation studies peripheral blood mononuclear cells were separated into lymphocytes and monocytes by counterflow-centrifugation (elutriation) . Inhibitory NK cell receptor expression on activated NK cells was measured by flow cytometry by antiCD3, antiCD56 and anti-inhibitory NK cell receptor triple staining . RESULTS: The accessory function of monocytes was indispensable for BCG mediated NK cell activation . However, the stimulatory potential of monocytes did not require direct cell-cell contact to NK cells or major histocompatibility complex dependent antigen presentation to T cells . Monocyte derived interleukin (IL)-12 and to a lesser extent interferon (IFN)-alpha were key mediators for stimulating BCG induced NK cell cytotoxicity and IFN-gamma production . In contrast, IL-10 inhibited NK cell cytotoxicity and IL-18 did not show any effect . Exogenous recombinant IFN-alpha and IL-12 enhanced BCG mediated secretion of IFN-gamma and yet BCG induced NK cell cytotoxicity remained unchanged . While the CD158a and CD158b subsets did not have a significant role, NKG2A cells represented the predominant cytolytic subset in BCG activated NK cells . CONCLUSIONS: Following BCG stimulation the monocyte derived TH1 cytokines IL-12 and IFN-alpha activate tumor cytotoxic CD3/CD56/NKG2A NK cells . Our results elucidate NK activating mechanisms that are operative during BCG immunotherapy for bladder cancer and are relevant for an early, innate antimycobacterial immune response.

J Urol, 2004 Oct, 172(4 Pt 1), 1286 - 90
Prospectively packaged lymph node dissections with radical cystectomy: evaluation of node count variability and node mapping; Bochner BH et al.; PURPOSE: Accumulating evidence supports the relationship between an increased number of lymph nodes (LNs) reported following radical cystectomy (RC) and overall outcome . We prospectively evaluated RC cases with transitional cell carcinoma of the bladder to determine which factors may contribute to the variability in the number of reported LNs . MATERIALS AND METHODS: We conducted a prospective evaluation in which 144 patients undergoing RC and pelvic lymph node dissection (PLND) between June 2001 and April 2003 were included . Lymph nodes were processed as individual packets . A standard method of evaluating nodal submissions was used . A mixed statistical model was used with neoadjuvant chemotherapy, node status, pathological stage, bacillus Calmette-Guerin exposure, age and number of days from transurethral resection as the fixed effects . Surgeon and pathologist were treated as random effects . RESULTS: The extended PLND group had a significantly greater lymph node yield (median 22.5 nodes) compared to standard PLND (median 8), however, no staging advantage was observed in the extended dissection group . Only the type of PLND performed was associated with node yield (p <0.001) . Subset analysis of patients with unexpected microscopic nodal involvement revealed that 33% had involvement of the common iliac nodes . CONCLUSIONS: In our series only the extent of the lymph node dissection was found to influence node yield significantly after radical cystectomy . Additionally, the observed risk of involvement of the common iliac chain in microscopically node positive cases suggests a need to include this region as part of the PLND for bladder cancer for cases without grossly involved LNs.

Cell, 2004 Sep 17, 118(6), 731 - 41
Structural basis for allosteric control of the transcription regulator CcpA by the phosphoprotein HPr-Ser46-P; Schumacher MA et al.; Carbon catabolite repression (CCR) is one of the most fundamental environmental-sensing mechanisms in bacteria and imparts competitive advantage by establishing priorities in carbon metabolism . In gram-positive bacteria, the master transcription regulator of CCR is CcpA . CcpA is a LacI-GalR family member that employs, as an allosteric corepressor, the phosphoprotein HPr-Ser46-P, which is formed in glucose-replete conditions . Here we report structures of the Bacillus megaterium apoCcpA and a CcpA-(HPr-Ser46-P)-DNA complex . These structures reveal that HPr-Ser46-P mediates a novel two-component allosteric DNA binding activation mechanism that involves both rotation of the CcpA subdomains and relocation of pivot-point residue Thr61, which leads to juxtaposition of the DNA binding regions permitting "hinge" helix formation in the presence of cognate DNA . The structure of the CcpA-(HPr-Ser46-P)-cre complex also reveals the elegant mechanism by which CcpA family-specific interactions with HPr-Ser46-P residues Ser46-P and His15 partition the high-energy CCR and low-energy PTS pathways, the latter requiring HPr-His15-P.

Gesnerus, 2004, 61(1-2), 37 - 56
{Diphtheria in Geneva at the end of the 19th century: the emergence of bacteriology and the use of serotherapy}; Kaba M; At the end of the 19th century, diphtheria was one of the infectious diseases striking children the most . With the development of laboratory medicine, appearing at this time throughout Europe, came the identification of the diphtherial bacillus, which not only causes a local infection, but also produces a poison attacking the whole organism . The discovery of this microbe will be an important vector for bacteriological research, leading to the elaboration of the antidiphtherial serum, considered the first specific and effective therapeutic product in the new experimental medicine . Through the study of diphtheria in the canton of Geneva, we examine the causes and consequences of the application of laboratory techniques and concepts in the sphere of clinical medicine.

Appl Microbiol Biotechnol, 2005 Jan, 66(4), 457 - 63 Epub 2004 Sep 11.
Quantifying bacterial population dynamics in compost using 16S rRNA gene probes; Schloss PD et al.; Composting provides a dynamic setting for studying ecological topics such as succession, competition, and community stability in a relatively short period of time . This study used hierarchical small sub-unit-based rRNA gene probes to quantify the change in the relative abundance of phylogenetic groups common to compost in laboratory scale reactors . Bacterial 16S rRNA gene targets accounted for only 37% of all small subunit (SSU) rRNA genes initially, but increased to a maximum of 83% of the total at 84 h . The sum of rRNA genes detected using probes specific to Pseudomonas and low-G+C Gram-positive rRNA genes represented between 16% and 87% of the total . The lack of hybridization to the taxon-specific probes was most pronounced between 36 h and 60 h, when the pH was between 4.6 and 4.8 . During this period the relative abundance of taxon-specific gene targets accounted for only 17-33% of the total bacterial rRNA gene targets . Pseudomonas-type 16S rRNA genes were the most abundant of the groups measured until 72 h . Those genes had their highest relative abundance at 12 h (78% of bacterial rRNA genes; 30% of all rRNA genes), after which time their relative abundance began to decline as the temperature increased . Prior to 72 h, 16S rRNA genes from low-G+C Gram-positive bacteria (LGC-GPB) represented less than 7% of the bacterial rRNA genes . However, by 84 h the relative abundance of LGC-GPB and Bacillus rRNA genes had increased to 60% and 18% of the bacterial rRNA gene targets, respectively (50% and 15% of all rRNA genes, respectively).

J Agric Food Chem, 2004 Sep 22, 52(19), 5939 - 42
Microbial transformation of aliphatic aldehydes by Bacillus megaterium to 2,3-dialkylacroleins; Abanda-Nkpwatt D et al.; The biotransformation of a series of aliphatic aldehydes (C(8)-C(12)) by Bacillus megaterium isolated from strawberry leaf surfaces was investigated . Products were isolated by liquid/liquid extraction and analyzed by gas chromatography (GC) combined with mass spectrometry (MS) . In addition to aliphatic alcohols and the remaining aldehydes, major transformation products included the corresponding acids as well as 2,3-dialkylacroleins, dehydrated aldol addition products, which were detected for the first time as biotransformation products . To verify the structures, 2,3-dialkylacroleins were chemically synthesized from the appropriate aldehydes by base-catalyzed aldol condensation reactions and characterized by (1)H and (13)C NMR spectroscopy . Time-course studies showed that the maximum yield of the acrolein derivatives was obtained after 6 days of incubation.

Korean J Intern Med, 2004 Jun, 19(2), 104 - 8
Stenotrophomonas maltophilia infection in patients receiving continuous ambulatory peritoneal dialysis; Baek JE et al.; BACKGROUND: Stenotrophomonas maltophilia is a gram-negative bacillus that has become increasingly recognized as an important nosocomial pathogen, particularly in individuals with severe debilitation or immunosuppression . S . maltophilia is also characterized by its resistance to multiple antibiotics . S maltophilia peritonitis in CAPD (continuous ambulatory peritoneal dialysis) patients is associated with a poor prognosis and loss of CAPD catheter . No report concerning this entity has been presented in Korea . Therefore, we describe and discuss five cases of the S . maltophilia infection associated with CAPD in three patients with peritonitis and two with exit-site infections . METHODS: We performed a retrospective search for episodes of S . maltophilia infections related to CAPD in our renal unit . The baseline levels of hemoglobin, albumin, cholesterol, BUN and creatinine were compared with age, sex and, if possible, the underlying disease-matched controls . RESULTS: All the patients with S . maltophilia peritonitis had diabetes mellitus as the underlying disease . The individual patients also had other significant combined morbidities, such as panhypopituitarism, COPD chronic obstructive pulmonary disease, cerebrovascular accident and myocardial infarction . The level of hemoglobin in these patients was significantly lower than in the controls, and the mean values of serum albumin, creatinine and BUN were also low . CONCLUSION: Immune dysfunction due to uremia, anemia, malnutrition, other comorbidities (e.g . diabetes mellitus), and also, an indwelling peritoneal catheter may be predisposing factors for the S . maltophilia infection in CAPD patients . Once the S . maltophilia infection is diagnosed in CAPD patient, the patient should be treated based on the understanding of this particular organism.

Monaldi Arch Chest Dis, 2004 Jan-Mar, 61(1), 71 - 4
Tuberculosis: a long fight against it and its current resurgence; Conti AA et al.; Unlike the discovery of penicillin in 1928 by A . Fleming, largely due to fortuitous circumstances, the isolation of streptomycin by S.A . Waksman was the result of a systematic research project carried out by a number of workers . In 1952, Waksman received the Nobel Prize in Medicine for having produced the first useful drug against tuberculosis . Before the tubercle bacillus was recognised as the causative agent of the disease, various sanatoria had been set up, as the only remedy for sufferers of tuberculosis . Between 1880 and 1930 sanatoria spread across Europe and North America, and they were partially effective against the ever worsening diffusion of tuberculosis: therefore in the United Kingdom a government-funded agency, the Medical Research Council (MRC), was created in 1913 . In 1947 streptomycin was put on the market, opening a new era in the history of modern medicine . Indeed, the first published report of the results of an (individually) randomised clinical trial was the 1948 paper by Bradford Hill and co-workers of the MRC's trial on the use of streptomycin . Streptomycin still represents a first-line agent in the recommended therapy of tuberculosis, whose burden is far higher in low-income countries . The current aim of any global intervention against tuberculosis should be the elimination of the pathology itself, an effort that will need both financial investments in scientific research and the targeted use of the fruits of that research to develop new, effective, preventive and therapeutic tools, such a tool as streptomycin proved to be more than fifty years ago.

Arch Microbiol, 2004 Dec, 182(6), 467 - 474 Epub 2004 Sep 10.
Global transcription profiles and intracellular pH regulation measured in Bacillus licheniformis upon external pH upshifts; Hornbaek T et al.; For optimization of propagation conditions for an industrially used Bacillus licheniformis, this study examines the effect of transferring cells at the early-stationary growth phase (pH 5.3) to fresh growth medium at pH 5.0-8.0 . Intracellular pH (pH(i)) was measured on a single-cell level, using fluorescence ratio imaging microscopy after staining with 5(6)-carboxyfluorescein diacetate succinimidyl ester . Transcription profiles were determined using a genome DNA microarray . The optimum extracellular pH (pH(ex)) value for growth of B . licheniformis was found to be pH 7.0, resulting in the shortest lag phase, highest maximum specific growth rate and maximum biomass formation . An average pH gradient (DeltapH = pH(i) - pH(ex)) of approx . 1.0 was found in B . licheniformis 15 min after transfer to pH(ex) 5.0-8.0 . Up-regulation of genes involved in sucrose uptake at pH 7.0 could be related to the optimum growth observed . Transcription profiles indicated that the organism was experiencing phosphate starvation upon transfer to pH 7.0 and pH 8.0 . Mechanisms involved in pH(i) regulation appeared to include changes in fatty acid synthesis to yield a more rigid cell membrane structure at low pH(ex) values and conversion of pyruvate to acetoin instead of acetate for neutralization of low pH(ex) values.

J Gen Physiol, 2004 Oct, 124(4), 349 - 56 Epub 2004 Sep 13.
Gating of the bacterial sodium channel, NaChBac: voltage-dependent charge movement and gating currents; Kuzmenkin A et al.; The bacterial sodium channel, NaChBac, from Bacillus halodurans provides an excellent model to study structure-function relationships of voltage-gated ion channels . It can be expressed in mammalian cells for functional studies as well as in bacterial cultures as starting material for protein purification for fine biochemical and biophysical studies . Macroscopic functional properties of NaChBac have been described previously (Ren, D., B . Navarro, H . Xu, L . Yue, Q . Shi, and D.E . Clapham . 2001 . Science . 294:2372-2375) . In this study, we report gating current properties of NaChBac expressed in COS-1 cells . Upon depolarization of the membrane, gating currents appeared as upward inflections preceding the ionic currents . Gating currents were detectable at -90 mV while holding at -150 mV . Charge-voltage (Q-V) curves showed sigmoidal dependence on voltage with gating charge saturating at -10 mV . Charge movement was shifted by -22 mV relative to the conductance-voltage curve, indicating the presence of more than one closed state . Consistent with this was the Cole-Moore shift of 533 micros observed for a change in preconditioning voltage from -160 to -80 mV . The total gating charge was estimated to be 16 elementary charges per channel . Charge immobilization caused by prolonged depolarization was also observed; Q-V curves were shifted by approximately -60 mV to hyperpolarized potentials when cells were held at 0 mV . The kinetic properties of NaChBac were simulated by simultaneous fit of sodium currents at various voltages to a sequential kinetic model . Gating current kinetics predicted from ionic current experiments resembled the experimental data, indicating that gating currents are coupled to activation of NaChBac and confirming the assertion that this channel undergoes several transitions between closed states before channel opening . The results indicate that NaChBac has several closed states with voltage-dependent transitions between them realized by translocation of gating charge that causes activation of the channel.

Vaccine, 2004 Sep 28, 22(29-30), 3848 - 57
Bacillus Calmette-Guérin shares with virulent Mycobacterium tuberculosis the capacity to subvert monocyte differentiation into dendritic cell: implication for its efficacy as a vaccine preventing tuberculosis; Gagliardi MC et al.; The only available vaccine against tuberculosis (TB) is Bacillus Calmette-Guerin (BCG) whose efficacy in preventing pulmonary tuberculosis is however controversial . Here, we show that BCG infection of monocytes causes their differentiation into mature dendritic cells (DCs) lacking CD1 molecules expression, coupled with suboptimal up-regulation of HLA class II, CD80 and CD40 molecules and a marked unresponsiveness to lipopolysaccharide stimulation . In addition, alloreactive naive T lymphocytes primed by these subverted DCs did not undergo defined functional polarization, as witnessed by their inability to produce IFN-gamma . Since efficient antigen presentation and IFN-gamma production by mycobacterial-specific T lymphocytes are required for protection against Mycobacterium tuberculosis, our data might provide additional explanation for the low efficacy of BCG vaccination.

FEMS Immunol Med Microbiol, 2004 Oct 1, 42(2), 197 - 204
Bacillus alcalophilus peptidoglycan induces IFN-alpha-mediated inhibition of vaccinia virus replication; Liu G et al.; Bacterial products such as cell walls (CW) and peptidoglycan (PGN) are known to activate macrophages and NK cells during microbial infections . In this report, we demonstrated that whole CW and PGN of four Gram-positive bacteria are capable of enhancing the anti-poxviral activity of murine macrophage RAW 264.7 cells . Among the major Bacillus alcalophilus CW components, PGN contributes the most to antiviral activity and induces remarkably higher levels of IFN-alpha . Anti-IFN-alpha/beta antibody, but not anti-IFN-gamma, anti-IFN-gamma receptor, or anti-IL-12, reversed the PGN-induced inhibition of vaccinia virus replication and reduced nitric oxide (NO) production . Our data thus suggest that PGN induce antiviral activity through IFN-alpha and to a lesser extent, through NO production.

Can J Public Health, 2004 Jul-Aug, 95(4), 249 - 55
Pediatric tuberculosis in Alberta First Nations (1991-2000): outbreaks and the protective effect of bacille Calmette-Guérin (BCG) vaccine; Long R et al.; BACKGROUND: The tuberculosis control strategy of vaccinating First Nations newborns with BCG (bacille Calmette-Guerin) is currently undergoing re-evaluation in Canada . Review of recent pediatric tuberculosis morbidity could inform this re-evaluation . METHODS: Potential source cases and pediatric cases of tuberculosis from Alberta First Nations were identified over the 10 years 1991-2000 . The distribution of pediatric disease was described . The effect of BCG on tuberculosis morbidity in two large outbreaks was determined . RESULTS: A total of 57 potential source cases and 41 pediatric cases of tuberculosis were reported from 17 (41.5%) and 8 (19.5%) of the 41 on-reserve First Nation Community Health Centres, respectively . Three outbreaks traceable to three source cases accounted for 34 (18, 3, and 13, respectively) of the 41 (82.9%) pediatric cases . Each outbreak was spatially and temporally separate from the other . Each outbreak strain of Mycobacterium tuberculosis had a unique DNA fingerprint . In the largest outbreaks, disease-to-infection ratios (secondary case rates) were higher in newly infected unvaccinated versus vaccinated close pediatric contacts (12/13 {92.3%} versus 7/15 {46.7%}, p=0.02), but the infection rate was almost certainly falsely high in the BCG vaccinated . One unvaccinated child had a brain tuberculoma in addition to primary pulmonary tuberculosis . CONCLUSION: For most Alberta First Nations communities, the spatial and temporal distribution of disease, and the meager impact on morbidity, challenge the rationale for continued use of BCG.

Acta Microbiol Immunol Hung, 2004, 51(1-2), 57 - 73
Properties of an immobilized lipase of Bacillus coagulans BTS-1; Kanwari SS et al.; Lipase (EC 3.1.1.3) is a tri-acylglycerol ester hydrolase, catalysing the hydrolysis of tri-, di-, and mono-acylglycerols to glycerol and fatty acids . To study the effect of adsorption of a lipase obtained from Bacillus coagulans BTS-1, its lipase was immobilized on native and activated (alkylated) matrices, i.e . silica and celite . The effect of pH, temperature, detergents, substrates, alcohols, organic solvent etc . on the stability of the immobilized enzyme was evaluated . The gluteraldahyde or formaldehyde (at 1% and 2% concentration, v/v) activated matrix was exposed to the Tris buffered lipase . The enzyme was adsorbed/entrapped more rapidly on to the activated silica than on the activated celite . The immobilized lipase showed optimal activity at 50 degrees C following one-hour incubation . The lipase was specifically more hydrolytic to the medium C-length ester (p-nitro phenyl caprylate than p-nitro phenyl laurate) . The immobilization/entrapment enhanced the stability of the lipase at a relatively higher temperature (50 degrees C) and also promoted enzyme activity at an acidic pH (pH 5.5) . Moreover, the immobilized lipase was quite resistant to the denaturing effect of SDS.

J Comput Chem, 2004 Nov 15, 25(14), 1677 - 92
PM3-compatible zinc parameters optimized for metalloenzyme active sites; Brothers EN et al.; Recent studies have shown that semiempirical methods (e.g., PM3 and AM1) for zinc-containing compounds are unreliable for modeling structures containing zinc ions with ligand environments similar to those observed in zinc metalloenzymes . To correct these deficiencies a reparameterization of zinc at the PM3 level was undertaken . In this effort we included frequency corrected B3LYP/6-311G* zinc metalloenzyme ligand environments along with previously utilized experimental data . Average errors for the heats of formation have been reduced from 46.9 kcal/mol (PM3) to 14.2 kcal/mol for this new parameter set, termed ZnB for "Zinc, Biological." In addition, the new parameter sets predict geometries for the Bacillus fragilis active site model and other zinc metalloenzyme mimics that are qualitatively in agreement with high-level ab initio results, something existing parameter sets failed to do.

Methods Mol Biol, 2005, 290, 91 - 103
Isolation and culture of murine macrophages; Davies JQ et al.; The two most convenient sources of primary murine macrophages are the bone marrow and the peritoneal cavity . Resident peritoneal macrophages can readily be harvested from mice and purified by adherence to tissue culture plastic . The injection of Bio-Gel polyacrylamide beads or thioglycollate broth into the peritoneal cavity produces an inflammatory response allowing the purification of large numbers of elicited macrophages . The production of an activated macrophage population can be achieved by using Bacillus-Calmette-Guerin as the inflammatory stimulus . Resident bone marrow macrophages can be isolated following enzymatic separation of cells from bone marrow plugs and enrichment on 30% fetal calf serum containing medium or Ficoll-Hypaque gradients . Bone marrow-derived macrophages can be produced by differentiating nonadherent macrophage precursors with medium containing macrophage colony-stimulating factor.

Int J Food Microbiol, 2004 Oct 1, 96(1), 75 - 83
Atmospheric oxygen and other conditions affecting the production of cereulide by Bacillus cereus in food; Jaaskelainen EL et al.; Factors influencing the production of cereulide, the emetic toxin of Bacillus cereus in food and laboratory media were investigated, using liquid chromatography-ion trap mass spectrometry and sperm motility inhibition bioassay for detection and quantitation . Oxygen was essential for production of the emetic toxin by B . cereus . When beans, rice or tryptic soy broth were inoculated with cereulide producing strains B203, B116 (recent food isolates) or the strain F-4810/72, high amounts (2 to 7 microg ml(-1) or g(-1) wet wt) of cereulide accumulated during 4-day storage at room temperature . In parallel cultures and foods, stored under nitrogen atmosphere (> 99.5% N2), less than 0.05 microg of cereulide ml(-1) or g(-1) wet wt accumulated . The outcome of the bioassay matched that of the chemical assay, with no indication of interference by substances in the rice or beans . Boiling for 20 to 30 min did not inactivate cereulide or cereulide producing strains in rice or the beans . Adding l-leucine and l-valine (0.3 g l(-1)) stimulated cereulide production 10- to 20-fold in R2A and in rice water agar . When the B . cereus strains were grown on agar media under permissive conditions (air, room temperature), cereulide was produced overnight with little or no increase when the incubation was extended to 4 days . In broth culture, the production of cereulide started later than 16-24 h . Anoxic storage prevented cereulide production also when the amino acids had been supplied . Packaging with modified atmosphere low in oxygen may thus be used to reduce the risk of cereulide formation during storage of food .

J Appl Microbiol, 2004, 97(4), 802 - 9
Morphological and phenotypical characterization of Bacillus sporothermodurans; Montanari G et al.; AIMS: Enumeration of resistant bacteria in ultra-high temperature (UHT) treated milk; morphological characterization and phenotyping of resistant strains by traditional and nontraditional methods and their identification by molecular biology . METHODS AND RESULTS: Modified standard plate count agar (PCA) and modified brain-heart infusion (BHI) agar were used for colony counts . Physiological culture traits were determined as suggested by Bergey's Manual of Systematic Bacteriology or in modified J-broth or in modified BHI agar . Scanning electron microscope (SEM) was used for microscopic examination . Strain identification was carried out by polymerase chain reaction (PCR) . A total of 125 (62.81% of 199) samples were positive and the bacterial load was higher than 10(5) CFU ml(-1) in 46 samples (28.80% of 125) . The 16S rRNA sequence of bacterial cultures obtained from UHT-treated milk was similar to that of Bacillus sporothermodurans M215 type strain((T)) and different biotypes were found by analysis of colony appearance, cell morphology and physiological traits . CONCLUSIONS: Bacillus sporothermodurans was the predominant sporigenous micro-organisms in UHT milk . SIGNIFICANCE AND IMPACT OF THE STUDY: BHI agar is more suitable than PCA for quality control of milk after UHT treatment . Modified J-broth medium is useful to determine selected physiological traits of B . sporothermodurans . The strains characterized and identified as B . sporothermodurans were significantly different compared with the type strain.

J Appl Microbiol, 2004, 97(4), 757 - 65
Expression of vip1/vip2 genes in Escherichia coli and Bacillus thuringiensis and the analysis of their signal peptides; Shi Y et al.; AIMS: To determine the expression time courses and high expression level of Vip2A(c) and Vip1A(c) in Bacillus thuringiensis, and survey their insecticidal toxicity and insecticidal spectrum . METHODS AND RESULTS: A kind of new vegetative insecticidal toxin genes encoded by a single operon from B . thuringiensis had been cloned and sequenced . The individual genes, 5-terminus truncated genes and the operon were respectively expressed in Escherichia coli . Only N-terminus deleted Vip2A(c) and Vip1A(c) proteins could be purified by Ni-NTA agarose, while others were processed and their N-terminal signal peptides were cleaved . The individual genes and the operon were also expressed in B . thuringiensis . Both proteins were mostly secreted into the cell supernatants . The expression level of Vip1A(c) was influenced because of the interruption of vip2A(c) gene on the operon . Bioassays showed that neither separate protein nor both performed any toxicity against tested lepidopteran and coleopteran insects . CONCLUSIONS: Vip2A(c) and Vip1A(c) have similar secretion mechanism in E . coli and B . thuringiensis . Vip1A(c) remained its high expression level only when being expressed with vip2A(c) gene as an operon in B . thuringiensis . SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of vip2A(c) and vip1A(c) genes in E . coli and B . thuringiensis were investigated . This would help to make clear the secretion mechanism of VIP proteins and study the function of ADP-ribosyltransferase Vip2.

Clin Infect Dis, 2004 Sep 1, 39(5), 667 - 72 Epub 2004 Aug 11.
Health care workers and the initiation of treatment for latent tuberculosis infection; Gershon AS et al.; BACKGROUND: Despite strong evidence and recommendations supporting the treatment of latent tuberculosis infection, many affected health care workers at risk of acquiring tuberculosis from and potentially transmitting tuberculosis to their patients do not receive treatment . The objective of this study was to determine whether health care workers were less likely than non-health care workers to initiate treatment for latent tuberculosis infection . METHODS: In this retrospective cohort study that used the disease management database from a specialized downtown Toronto tuberculosis clinic, patients with latent tuberculosis infection were included if they had risk factors for progression of disease and were excluded if they had contraindications to treatment . RESULTS: Our final cohort consisted of 308 patients with latent tuberculosis infection . The overall treatment initiation rate was 58% . We found that, when a number of confounding variables, including age, foreign birth, contact with persons with active tuberculosis, tuberculosis skin test conversion, bacille Calmette-Guerin vaccination, abnormal chest radiograph findings, comorbidities, and income, were considered and/or controlled for, the odds of a health care worker initiating treatment were approximately one-half of those of a non-health care worker (adjusted odds ratio, 0.55; 95% confidence interval, 0.32-0.93) . CONCLUSION: We conclude that, in our clinic, health care workers are less likely than non-health care workers to initiate treatment for latent tuberculosis infection.

Allergy, 2004 Oct, 59(10), 1068 - 73
Atopic disorders among Estonian schoolchildren in relation to tuberculin reactivity and the age at BCG vaccination; Annus T et al.; BACKGROUND: Published data about a relationship of atopic diseases to Bacillus Calmette-Guerin (BCG) vaccination and tuberculin responses are inconsistent . Our aim was to determine this association in a country with a low prevalence of allergies . METHODS: A random sample of 10-11-year-old schoolchildren in Tallinn was studied by a parental questionnaire (n = 979) and skin-prick tests (n = 643), according to the International Study of Asthma and Allergies in Childhood . Data about BCG vaccinations and tuberculin tests were obtained from school records (n = 723) . RESULTS: The prevalence of allergic symptoms and atopy was similar in children vaccinated during the first month of life and later . Positive tuberculin responses (> or =5 mm) were inversely related to symptoms of asthma {odds ratio (OR) 0.10 (95% confidence interval 0.00-0.68) for exercise-induced wheezing; OR 0.37 (0.12-0.99) for night cough}, and eczema {OR 0.53 (0.28-0.98)} but not to atopy . However, among BCG-revaccinated children, atopy tended to be more common in tuberculin responders, and the atopic children were significantly more likely to have a positive tuberculin response after the revaccination than would be predicted by their first test . CONCLUSIONS: We found no protective effect of early BCG vaccination against atopy in school age, although tuberculin responses and allergic symptoms were inversely related.

Clin Microbiol Infect, 2004 Sep, 10(9), 854 - 7
Acute and chronic otitis media and Turicella otitidis: a controversial association; Gomez-Garces JL et al.; Turicella otitidis is a non-fermenting Gram-positive bacillus isolated almost exclusively from ear exudates . Its significance in acute or chronic otitis media is controversial . Over a 12-month period, T . otitidis was isolated from nine ear exudates from seven patients . Most of these were cases of spontaneous drainage following recurrence of otitis media after antimicrobial therapy that was ineffective against T . otitidis . The MICs of penicillin, levofloxacin, linezolid and vancomycin were very low for all the isolates studied, but most isolates displayed high resistance to macrolides and lincosamides.

Annu Rev Entomol . 2004 Aug 18; {Epub ahead of print}
Effects of Plants Genetically Modified for Insect Resistance on Nontarget Organisms; O'Callaghan M et al.; Insect resistance, based on Bacillus thuringiensis (Bt) endotoxins, is the second most widely used trait (after herbicide resistance) in commercial genetically modified (GM) crops . Other modifications for insect resistance, such as proteinase inhibitors and lectins, are also being used in many experimental crops . The extensive testing on nontarget plant-feeding insects and beneficial species that has accompanied the long-term and wide-scale use of Bt plants has not detected significant adverse effects . GMplants expressing other insect-resistant proteins that have a broader spectrum of activity have been tested on only a limited number of nontarget species . Little is known about the persistence of transgene-derived proteins in soil, with the exception of Bt endotoxins, which can persist in soil for several months . Bt plants appear to have little impact on soil biota such as earthworms, collembolans, and general soil microflora . Further research is required on the effects of GM plants on soil processes such as decomposition . Assessment of nontarget impacts is an essential part of the risk assessment process for insect-resistant GM plants . Expected online publication date for the Annual Review of Entomology Volume 50 is December 3, 2004 . Please see for revised estimates.

Annu Rev Entomol . 2004 Aug 17; {Epub ahead of print}
The Evolution of Cotton Pest Management Practices in China; Wu KM et al.; The development of cotton pest management practices in China has followed a pattern seen for many crops that rely heavily on insecticides . Helicoverpa armigera resistance to chemical pesticides resulted in unprecedented pest densities of the early 1990s . Transgenic cotton that expresses a gene derived from the bacterium Bacillus thuringiensis (Bt) has been deployed for combating H . armigera since 1997 . The pest management tactics associated with Bt cotton have resulted in a drastic reduction in insecticide use, which usually results in a significant increase in populations of beneficial insects and thus contributes to the improvement of the natural control of some pests . Risk assessment analyses show that the natural refuges derived from the mixed-planting system of cotton, corn, soybean, and peanut on small-scale, single-family-owned farms play an important function in delaying evolution of cotton bollworm resistance, and that no trend toward Bt cotton resistance has been apparent despite intensive planting of Bt cotton over the past several years . Expected online publication date for the Annual Review of Entomology Volume 50 is December 3, 2004 . Please see for revised estimates.

Biochem Biophys Res Commun, 2004 Oct 8, 323(1), 52 - 7
Binding analyses of Cry1Ab and Cry1Ac with membrane vesicles from Bacillus thuringiensis-resistant and -susceptible Ostrinia nubilalis; Li H et al.; The binding properties of Bacillus thuringiensis toxins to brush border membrane vesicles of Dipel-resistant and -susceptible Ostrinia nubilalis larvae were compared using ligand-toxin immunoblot analysis, surface plasmon resonance (SPR), and radiolabeled toxin binding assays . In ligand-toxin immunoblot analysis, the number of Cry1Ab or Cry1Ac toxin binding proteins and the relative toxin binding intensity were similar in vesicles from resistant and susceptible larvae . Surface plasmon resonance with immobilized activated Cry1Ab toxin indicated that there were no significant differences in binding with fluid-phase vesicles from resistant and susceptible larvae . Homologous competition assays with radiolabeled Cry1Ab and Cry1Ac toxin and vesicles from resistant and susceptible larvae resulted in similar toxin dissociation constants and binding site concentrations . Heterologous competition binding assays indicated that Cry1Ab and Cry1Ac completely competed for binding, thus they share binding sites in the epithelium of the larval midguts of O . nubilalis . Overall, the binding analyses indicate that resistance to Cry1Ab and Cry1Ac in this Bt-resistant strain of O . nubilalis is not associated with a loss of toxin binding .

Acta Trop, 2004 Oct, 92(2), 109 - 18
Laboratory and field evaluation of Teknar HP-D, a biolarvicidal formulation of Bacillus thuringiensis ssp . israelensis, against mosquito vectors; Gunasekaran K et al.; Larvicidal efficacy of Teknar HP-D, an improved biolarvicidal formulation of Bacillus thuringiensis ssp . israelensis (Bti), against Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti was determined in the laboratory, and in field the efficacy of the formulation was tested against Cx . quinquefasciatus breeding in cesspits, unused wells and drains . The toxicity of the formulation to Gambusia affinis (larvivorous fish), Notonecta sp . and Diplonychus indicus (water bugs) was also evaluated in the laboratory . Teknar HP-D was field tested at three recommended dosages, 1, 1.5 and 2l/ha, selecting five habitats for each dosage . Another five habitats were kept untreated as controls . Ae . aegypti showed greatest susceptibility to the Bti toxin in the laboratory . In cesspits, all the three dosages caused >80% reduction of pupal recruitment up to day 6 post-treatment, indicating that a weekly application at the lowest would be necessary for sustained control . The residual activity of the formulation was longer in unused wells, causing >80% reduction of pupal recruitment for 17 days from the day of treatment . In controlling pupal recruitment the three dosages produced equal effect . Application of Teknar HP-D at 1 l/ha once in three weeks is therefore recommended to control Cx . quinquefasciatus in unused wells . However, in drains, >80% reduction of pupal recruitment was observed for only 3 days and hence, application of Teknar HP-D at 2 l/ha that caused significantly higher level of reduction twice in a week at 3-day interval is necessary . At dosages from 0.032 to 3.2 mg/l (ppm), Teknar HP-D was non-toxic to Gambusia fish . The two predatory water bugs, Notonecta sp . and Diplonychus indicus that fed on the surviving larvae of Cx . quinquefasciatus exposed to the sub-lethal doses (LC(50) and LC(80)) of Teknar HP-D were safe with out having any mortality.

J Insect Physiol, 2004 Sep, 50(9), 791 - 803
Patch-clamp study of the apical membrane of the midgut of Manduca sexta larvae: direct demonstration of endogenous channels and effect of a Bacillus thuringiensis toxin; Peyronnet O et al.; The patch-clamp technique was applied to the apical membrane of epithelial midgut cells of a lepidoptera, Manduca sexta L . Access to the apical membrane, the main target site of Bacillus thuringiensis (Bt) toxins, was achieved by using freshly isolated larval midgut preparations mounted onto holding glass pipettes . The epithelial cells retained their functional integrity, as evidenced by the magnitude of intracellular potentials recorded with microelectrodes . With standard 32 mM K(+) solution in the bath and the patch-clamp pipette, endogenous channel activity was detected in about 50% of experiments, mainly in moulting larvae and larvae that had been kept at reduced temperature for at least two days prior to the experiments . In both cell-attached and inside-out patch-clamp configurations, different types of channel were observed, with conductances varying between about 5 and 50 pS and different conducting properties . Addition of trypsin-activated Cry1Ac Bt toxin in the patch-clamp pipette triggered, after a delay, large conductances of a few nanosiemens . This is the first study allowing exploration, in the intact midgut, of the properties of apical membrane channels and the direct interaction between the apical membrane of epithelial cells and pathogenic agents such as Bt toxins.

Treat Respir Med, 2004, 3(4), 235 - 46
Current and emerging nonsteroidal anti-inflammatory therapies targeting specific mechanisms in asthma and allergy; Bjermer L et al.; Today inhaled corticosteroids (ICS) are regarded as the first-line controller anti-inflammatory treatment in the management of asthma . However, there is an increasing awareness of the risk of long-term adverse effects of ICS and that asthma is not only an organ-specific disease but also a systemic and small airway disease . This thinking has called for systemic treatment alternatives to treat asthma targeting more disease-specific mechanisms without influencing normal physiologic functions.Blocking of disease-specific mediators is a mechanism utilized by anti-leukotrienes and anti-immunoglobulin E treatment, each proven to be effective in both asthma and allergic rhinitis.Different cytokine-modifying strategies have been tested in clinical trials with variable results, some disappointing and some encouraging . Anti-interleukin (IL)-5 monoclonal antibody treatment effectively reduces the number of eosinophils locally in the airways and in peripheral blood in asthmatic patients . Unfortunately, this marked effect on eosinophils was not associated with an improvement in bronchial hyperresponsiveness and/or symptoms . Clinical trials with a recombinant soluble IL-4 receptor have been somewhat more successful at improving asthma control and allowing reduction of ICS therapy in asthma . Treatment with recombinant IL-12 had an effect on bronchial hyperresponsiveness and eosinophilic response, but was associated with unacceptable adverse effects . Other interesting cytokine-modulating treatments include those targeting IL-9, IL-10, IL-12 and IL-13.Immune-modulating treatment with bacterial antigens represents another strategy, originating from the hypothesis that some bacterial infections guide the immune system towards a T helper (Th) type 1 immune response . Mycobacterium vaccae, Bacille Calmette-Guerin (BCG) and immunostimulatory DNA sequences have all been tested in clinical trials, with encouraging results.Future asthma and allergy treatment will probably include not only one but also two or more disease-modifying agents administered to the same patient.

ScientificWorldJournal, 2004 Aug 12, 4, 622 - 7
Detection and characterization of beta-lactam resistance in Bacillus cereus PTCC 1015; Behravan J et al.; In the present study, detection, isolation, and characterization of beta-lactamases from Bacillus cereus PTCC 1015 were investigated . B . cereus was inoculated in nutrient broth containing ampicillin (50 mg x ml(-1)) for 24 h (35 degrees C, 200 rpm) . Activity measurements were carried out against ampicillin (0.1 mg x ml(-1)) and cephalexin (0.08 mg x ml(-1)) by a spectrophotometric method at different conditions (pH 6-10, temperatures 25-45 degrees C) . Maximum penicillinase and cephalosporinase activity was observed at pH 7 . The optimized temperatures for penicillinase and cephalosporinase activity were 30 and 40 degrees C, respectively . At the above conditions, maximum enzymatic activity was calculated as 0.89 +/- 0.014 and 0.037 +/- 0.001 units against ampicillin and cephalexin.

J Trauma, 2004 Aug, 57(2), 231 - 5
Treatment of field water with sodium hypochlorite for surgical irrigation; Cyr SJ et al.; BACKGROUND: Early irrigation and surgical debridement of high-energy wounds and open fractures effectively prevents infection . Rapid wound care has been maximized by the United States military's "forward surgical teams." However, the volume of sterile irrigant required to treat multiple patients with multiple wounds presents a significant logistical burden . Using ground-derived field water could eliminate this burden . METHODS: We collected 100 water samples from five sources . An initial bacterial count (CFU/mL) was determined before treatment . 5% sodium hypochlorite was then added to each sample to derive a concentration of 0.025% . After treatment, a final bacterial colony count was performed . RESULTS: We found no bacterial growth in 99/100 samples . One post-treatment sample grew a single colony of a Bacillus species not present in the pretreatment culture and was determined to be an air contaminant . CONCLUSIONS: Our field-expedient modification of Dakin's solution could substitute for sterile irrigation fluid when it is neither available nor logistically feasible.

Appl Environ Microbiol, 2004 Sep, 70(9), 5074 - 80
Display of bacterial lipase on the Escherichia coli cell surface by using FadL as an anchoring motif and use of the enzyme in enantioselective biocatalysis; Lee SH et al.; We have developed a novel cell surface display system by employing FadL as an anchoring motif, which is an outer membrane protein involved in long-chain fatty acid transport in Escherichia coli . A thermostable Bacillus sp . strain TG43 lipase (44.5 kDa) could be successfully displayed on the cell surface of E . coli in an active form by C-terminal deletion-fusion of lipase at the ninth external loop of FadL . The localization of the truncated FadL-lipase fusion protein on the cell surface was confirmed by confocal microscopy and Western blot analysis . Lipase activity was mainly detected with whole cells, but not with the culture supernatant, suggesting that cell lysis was not a problem . The activity of cell surface-displayed lipase was examined at different temperatures and pHs and was found to be the highest at 50 degrees C and pH 9 to 10 . Cell surface-displayed lipase was quite stable, even at 60 and 70 degrees C, and retained over 90% of the full activity after incubation at 50 degrees C for a week . As a potential application, cell surface-displayed lipase was used as a whole-cell catalyst for kinetic resolution of racemic methyl mandelate . In 36 h of reaction, (S)-mandelic acid could be produced with the enantiomeric excess of 99% and the enantiomeric ratio of 292, which are remarkably higher than values obtained with crude lipase or cross-linked lipase crystal . These results suggest that FadL may be a useful anchoring motif for displaying enzymes on the cell surface of E . coli for whole-cell biocatalysis.

J Immunol Methods, 2004 Aug, 291(1-2), 185 - 95
Novel application of a whole blood intracellular cytokine detection assay to quantitate specific T-cell frequency in field studies; Hanekom WA et al.; We optimized a whole blood intracellular cytokine assay to quantitate the frequency of specific CD4+ and CD8+ T cells in small volumes of whole blood from infants from developing countries . The assay is performed in two steps . First, whole blood is stimulated in the presence of specific antigens for 6-18 h, ending with cryopreservation of fixed white cells . These stimulation steps were specifically adapted to be practical and reliable in a rural, developing country field setting . Later, in a more resourceful setting, interferon-gamma producing CD4+ or CD8+ T cells are detected by flow cytometry . The assay proved sensitive and specific for detecting mycobacteria-specific immunity 10 weeks after Bacillus Calmette-Guerin (BCG) vaccination of newborns from a rural field site.

J Biol Chem, 2004 Nov 5, 279(45), 47344 - 51 Epub 2004 Sep 01.
The crystal structure of an oxidatively stable subtilisin-like alkaline serine protease, KP-43, with a C-terminal beta-barrel domain; Nonaka T et al.; The crystal structure of an oxidatively stable subtilisin-like alkaline serine protease, KP-43 from Bacillus sp . KSM-KP43, with a C-terminal extension domain, was determined by the multiple isomorphous replacements method with anomalous scattering . The native form was refined to a crystallographic R factor of 0.134 (Rfree of 0.169) at 1.30-A resolution . KP-43 consists of two domains, a subtilisin-like alpha/beta domain and a C-terminal jelly roll beta-barrel domain . The topological architecture of the molecule is similar to that of kexin and furin, which belong to the subtilisin-like proprotein convertases, whereas the amino acid sequence and the binding orientation of the C-terminal beta-barrel domain both differ in each case . Since the C-terminal domains of subtilisin-like proprotein convertases are essential for folding themselves, the domain of KP-43 is also thought to play such a role . KP-43 is known to be an oxidation-resistant protease among the general subtilisin-like proteases . To investigate how KP-43 resists oxidizing reagents, the structure of oxidized KP-43 was also determined and refined to a crystallographic R factor of 0.142 (Rfree of 0.212) at 1.73-A resolution . The structure analysis revealed that Met-256, adjacent to catalytic Ser-255, was oxidized similarly to an equivalent residue in subtilisin BPN' . Although KP-43, as well as proteinase K and subtilisin Carlsberg, lose their hydrolyzing activity against synthetic peptides after oxidation treatment, all of them retain 70-80% activity against proteinaceous substrates . These results, as well as the beta-casein digestion pattern analysis, have indicated that the oxidation of the methionine adjacent to the catalytic serine is not a dominant modification but might alter the substrate specificities.

Theor Appl Genet, 2004 Aug, 109(4), 806 - 14 Epub 2004 May 05.
Impact of ecological factors on the initial invasion of Bt transgenes into wild populations of birdseed rape (Brassica rapa); Vacher C et al.; The inevitable escape of transgenic pollen from cultivated fields will lead to the emergence of transgenic crop-wild plant hybrids in natural patches of wild plants . The fate of these hybrids and that of the transgene depend on their ability to compete with their wild relatives . Here we study ecological factors that may enhance the fitness of genetically modified hybrids relative to wild plants for a Bacillus thuringiensis ( Bt) transgene conferring resistance to insects . Mixed stands of wild plants and first-generation hybrids were grown under different conditions of herbivore pressure and density, with Bt oilseed rape ( Brassica napus) as the crop and B . rapa as the wild recipient . Biomass and fitness components were measured from plant germination to the germination of their offspring . The frequency of transgenic seedlings in the offspring generation was estimated using the green fluorescent protein marker . The biomass of F(1) Bt-transgenic hybrids relative to that of wild-type plants was found to be sensitive to both plant density and herbivore pressure, but herbivore pressure appeared as the major factor enhancing their relative fitnesses . In the absence of herbivore pressure, Bt hybrids produced 6.2-fold fewer seeds than their wild neighbors, and Bt plant frequency fell from 50% to 16% within a single generation . Under high herbivore pressure, Bt hybrids produced 1.4-fold more seeds, and Bt plant frequency was 42% in the offspring generation . We conclude that high-density patches of highly damaged wild plants are the most vulnerable to Bt-transgene invasion . They should be monitored early to detect potential transgene spread.

Naturwissenschaften, 2004 Sep, 91(9), 451 - 4 Epub 2004 Aug 24.
Transgenic insect-resistant corn affects the fourth trophic level: effects of Bacillus thuringiensis-corn on the facultative hyperparasitoid Tetrastichus howardi; Prutz G et al.; As hyperparasitoids may have a considerable influence on the control of herbivorous arthropods, analyzing the host-mediated impact of Bacillus thuringiensis-plants ("Bt-plants") on hyperparasitoids is of interest . Laboratory-scale experiments were conducted in order to assess the potential effect of Bt-corn leaf material on the facultative hyperparasitoid Tetrastichus howardi (Olliff) (Hymenoptera: Eulophidae), mediated through the herbivore Chilo partellus Swinhoe (Lepidoptera: Crambidae) and its primary parasitoid Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) . In the Bt-group, significantly less C . flavipes larvae per host spun cocoons and pupated than in the control, and the mean fresh weight of a single C . flavipes cocoon was significantly reduced compared to the control . All C . flavipes cocoons of one host formed cocoon clusters . T . howardi females of the Bt-group parasitized significantly less cocoon clusters than in the control . Moreover, significantly fewer C . flavipes cocoons per cocoon cluster were successfully parasitized as compared to the control . As a consequence, T . howardi females of the control had more offspring than in the Bt-group . Adult female T . howardi offspring of the Bt-group weighed significantly less than in the control, but there was no significant weight difference between males of both groups . Our results suggest that transgenic insect-resistant plants could affect hyperparasitoids indirectly . However, it remains to be determined whether facultative hyperparasitoids prefer to develop as primary or secondary parasitoids under field conditions.

FEMS Microbiol Lett, 2004 Sep 1, 238(1), 107 - 13
Colony immunoblot assay for the detection of hemolysin BL enterotoxin producing Bacillus cereus; Moravek M et al.; Bacillus cereus strains involved in food poisoning cases of the diarrheal type may produce two different enterotoxin complexes . To facilitate the identification of hemolysin BL-enterotoxin complex (HBL) and/or the nonhemolytic enterotoxin (NHE) producing colonies a colony immunoblot procedure was developed, which allows a fast and easy identification of the respective colonies from blood agar plates . The enterotoxins were transferred from the blood agar medium to a nitrocellulose membrane and the immobilized toxins were probed with monoclonal antibodies . The antibodies 2A3 and 1A8 allowed the specific detection of the B component of HBL and the nheA component of NHE . The assay enabled the reliable identification of HBL expressing colonies and differentiation from NHE producing but HBL negative colonies.

Expert Opin Biol Ther, 2004 Sep, 4(9), 1493 - 504
Tuberculosis vaccine development: research, regulatory and clinical strategies; Brennan MJ et al.; In the past decade, while the global tuberculosis (TB) epidemic has continued to devastate mankind, considerable progress has nevertheless been made in the development of new and improved vaccines for this ancient disease . Recombinant bacillus Calmette-Guerin strains, DNA-based vaccines, live attenuated Mycobacterium tuberculosis vaccines and subunit vaccines formulated with novel adjuvants have shown promise in preclinical animal challenge models . Three of these vaccines are being evaluated at present in human clinical studies, and several other vaccine preparations are being targeted for clinical trials in the near future . Although the preclinical characterisation and testing of new TB vaccines has clearly led to exciting new findings, complex regulatory and clinical trial design issues remain as a challenge to TB vaccine development . This report reviews some of the exciting advances in TB research that have led to the development of new TB vaccines, and addresses the unique regulatory and clinical issues associated with the testing of novel anti-TB preparations in human populations.

Hinyokika Kiyo, 2004 Jul, 50(7), 469 - 73
{Intravesical Bacillus Calmette-Guerin (BCG: Tokyo 172 strain) instillation for carcinoma in situ of the bladder: results with 6 successive instillations of 40 mg BCG}; Mugiya S et al.; We performed a study to evaluate the usefulness of intravesical Bacillus Calmette-Guerin (BCG: Tokyo 172 strain) instillation on carcinoma in situ (CIS) of the bladder . Between 1998 and 2003, 43 patients were treated for CIS of the bladder with a median follow-up period of 45 months (range: 12 to 69 months) . The patients (35 males and 8 females) ranged in age from 45 to 89 years (average: 67.5 years) . They underwent intravesical instillation of 40 mg of BCG once a week for 6 weeks . A complete response (CR) was achieved in 83.7% of the patients . Among these patients, 97.2% and 70.7% remained recurrence-free during follow up for one year and three years, respectively . The median duration of CR was 31.5 months . Although total cystectomy was performed on 1 patient, none of the patients died of bladder cancer . Adverse effects included bladder irritability in 48.8%, pyuria in 46.5%, gross hematuria in 18.6%, and fever (temperature over 37.5 degrees C) in 9.3% . No clinically significant side effects were observed . These results indicate that intravesical instillation of BCG at a dose of 40 mg given 6 times was as effective as the routine dose of 80 mg, and could decrease systemic adverse effects such as high fever.

J Surg Oncol, 2004 Sep 1, 87(3), 146 - 51
Kaposi's sarcoma: an update; Schwartz RA; While there have been many important advances in the study of Kaposi's sarcoma (KS), it remains both a challenge and an enigma in many ways . Kaposi's original description of "multiple idiopathic hemorrhagic sarcoma{s}" in patients who died within 2-3 years resembles KS in AIDS more than classic KS in elderly men of Italian, Jewish, or Mediterranean lineage, in whom the disease is usually benign . KS had been evident in about one-third of those with early AIDS, often as its presenting sign, a pattern markedly reduced in recent times since the introduction of highly active anti-retroviral therapy (HAART) . The most important advance has been the convincing etiologic linkage of KS with human herpesvirus 8 (HHV-8), which is necessary but not sufficient . It has a low prevalence in the general population of the USA and UK, with an intermediate rate in Italy and Greece, and a high one in Uganda . KS risk may be significantly lower in AIDS patients with a history of anti-herpes therapy . Many aspects of HHV-8, including its transmission pattern and different genospecies, are being scrutinized . The diagnosis of KS may be difficult . One should be aware of KS clinical variants, including telangiectatic, eccymotic, and keloidal KS . One must consider a number of other disorders, including bacillary angiomatosis . HHV-8 DNA sequences in dermatofibromas and other tumors should probably not be viewed as representing a marker for KS . Therapeutic options vary for KS . Intralesional and low-dose outpatient intravenous vinblastine may be valuable, as immunosuppression with KS is not a good idea if it can be avoided . Anti-herpes virus therapy may have potential for wide use, especially in preventing the development of KS in at risk populations, such as HHV-8 seropositive individuals undergoing transplantation surgery .

J Food Prot, 2004 Aug, 67(8), 1770 - 3
Detection of Bacillus cereus on selected retail chicken products; Smith DP et al.; Samples from five chicken meat products, obtained at retail stores, were evaluated for the presence of Bacillus cereus . The products tested were as follows: breaded, fully cooked, frozen nuggets (NUGGETS); breaded, fully cooked, frozen tenders (TENDERS); fully cooked, frozen, white-meat fajita-style strips (STRIPS); raw, refrigerated, boneless, skinless, marinated breast fillets (FILLETS); and raw, refrigerated, cut-up, tray-pack bone-in parts (PARTS), either split breasts or thighs . Four packages of each item were obtained on three different days (n = 60) . Frozen and refrigerated products were held overnight in their respective environments as appropriate; then packages were opened aseptically, and a total of 25 g of tissue was excised from multiple pieces within a package . The 25-g samples were enriched in 225 ml of Trypticase soy-polymixin broth for 18 to 24 h at 30 degrees C and then plated on mannitol-egg yolk-polymixin agar and incubated for 18 to 24 h at 30 degrees C . Colonies characteristic of B . cereus were chosen and replated for isolation on mannitol-egg yolk-polymixin agar . Suspect colonies were confirmed as Bacillus spp . by Gram stain, hemolysis on blood agar, and a biochemical test strip . Isolates were further confirmed as B . cereus using Bacteriological Analytical Manual procedures, including tests for motility, rhizoid growth, hemolysis, and protein toxin crystal production . B . cereus was detected in 27 of 60 total samples . By product, the prevalence levels were as follows: NUGGETS, 11 of 12 positive; TENDERS, 8 of 12 positive; STRIPS, 6 of 12 positive; FILLETS, 0 of 12 positive; and PARTS, 2 of 12 positive . Isolates were tested by PCR for presence of the toxin-encoding genes bceT, nheABC, hblACD, and cytK . Results indicate that B . cereus organisms were present on four of the five retail poultry products tested in this study, with the highest rates reported for the three fully cooked items, especially the two breaded products . All strains isolated contained the gene(s) for at least one of the toxins, although none of the strains contained the cytK gene.

J Food Prot, 2004 Aug, 67(8), 1694 - 701
A PCR assay based on a sequence-characterized amplified region marker for detection of emetic Bacillus cereus; Nakano S et al.; A PCR assay for the detection of Bacillus cereus strains able to produce an emetic toxin (cereulide) was developed in this study based on a sequence-characterized amplified region (SCAR) derived from a random amplified polymorphic DNA (RAPD) fragment . One of the RAPD fragments generated was selected, cloned, and sequenced . A set of PCR primers was newly designed from the SCAR obtained (the sequence of the cloned RAPD fragment) and used in this assay . To determine the specificity of the assay, 30 different B . cereus strains, 8 other Bacillus strains (of six species), and 16 other non-Bacillus strains (from 16 genera) were tested . Results were positive for every emetic B . cereus strain and for only one nonemetic B . cereus strain . For all other bacterial strains, results were negative . Bacterial DNA for PCR was prepared by a simple procedure using Chelex 100 resin from the bacterial colony on the agar plate or from culture after growth in brain heart infusion medium . This PCR assay enabled us to detect the bacteria of emetic B . cereus grown on agar plates but not the bacteria of nonemetic B . cereus . To test this PCR assay for the monitoring of the emetic bacteria, 10 to 70 CFU of B . cereus DSM 4312 (emetic) per g of food was inoculated into several foods as an indicator, followed by a 7-h enrichment culture step . Because this PCR assay based on the SCAR derived from the RAPD fragment was able to detect bacterial cells, this assay should be useful for rapid and specific detection of emetic B . cereus.

J Nippon Med Sch, 2004 Aug, 71(4), 263 - 9
Effect of adjuvant transarterial infusion of anticancer agents after transurethral resection in patients with pT2a muscle invasive transitional cell carcinoma of the bladder: five and ten-year outcome; Shimizu H et al.; Five- and ten-year survival rates in patients who had transurethral resection for pT2aN0M0 muscle invasive transitional cell carcinoma of the bladder was studied with emphasis on the effect of adjuvant transarterial infusions of anticancer agents (TAI) . A total of 17 patients out of 290 patients with bladder cancer who had initial treatment in our department between January 1991 and May 2001 were found to be pT2aN0M0 and were included in the present study . Fifteen patients received intravesical instillations of BCG after transurethral resection of the bladder tumor and 8 patients received TAI which was given two weeks after bacillus Calmette-Guerin therapy, with the exception of 1 patient . Five- and ten-year overall survival rates were 76.0% and 52.1%, respectively, and both five- and ten-year cancer-specific survival rates were 76.0% with an average follow up of 71 months . Significant prognostic factor associated with survival rates was only the infiltration pattern of the tumor cells between the alpha and beta group and gamma group (P = 0.0420) in cancer-specific survival rates . The data support conservative management of transurethral resection followed by intravesical instillation of BCG and TAI in patients with pT2a bladder cancer.

Zh Obshch Biol, 2004 May-Jun, 65(3), 195 - 210
{Highest level of division in classification of organisms . 3 . Monodermata and Didermata}; Shatalkin AI; The deepening our knowledge and embrassing the larger array of the investigated organisms leads to replacement of typological classifications with phylogenetic ones . This process seems to be the main stream of modern systematics . But typological classifications have not lost the value, remaining the important tool of the description of phylogeny . It is especially obvious today when molecular reconstructions are using so widely . However resulted phylogenetic classifications are difficult for understandable interpretation . Therefore phylogeneticist is interested in elaboration of typological classifications that can help to explain the results . As an example the phylogenetic classifications of organisms proposed recently by Cavalier-Smith (1998, 2002) and Gupta (1998, 2000) are considered . The modified system of Gupta is the most adequate description of organism phylogeny . Basal clostridia and togobacteria have to the greatest degree kept features of a common ancestor of organisms . From this common ancestor evolution spread by two phyletic lines . One of them included Gram-negative bacteria . The main groups of them have branched of from a common ancestor in the following order: (Deinococci, Chloroflexi) --> (Cyanobacteria) --> (Chlamydia, CFB, Fibrobacteria, Chlorobia) --> (Aquificae) --> --> (Epsilonproteobacteria, Deltaproteobacteria) --> (Alfaproteobacteria) --> (Betaproteobacteria) --> --> (Gammaproteobacteria) . In other phyletic line the main groups were separated in the following order: (Thermotogae) --> (Clostridia, Fusobacteria) --> (Bacillae) --> (Actinobacteria) . Exact position of archaebacteria and eukaryotes related to this line remains unclear . Typological division of organisms into Didermata and Monodermata (Gupta, 1998) corresponds to these two branches of a cladogram . The cell of the diderm organisms is covered with two membranes, plasmatic and outer . The cell of the monoderm organisms has only one plasmatic membrane . Development of the cellular organization at the earliest stages of evolution of a life went through use of non-lamellar (non-bilayer) lipids which could give a cell with one membrane (not two membranes as in the scenario of Cavalier-Smith (2001)) . Membranes appeared at the earliest stages of the evolution of life . Therefore their distinction is quite logical to take as a principle the first typological division of organisms . At the same time the typological classifications considered beyond the framework of phylogenetics, have no independent value . Typological classifications do not give monothetic division into groups . Always there are exceptions . So, among Monodermata there are Gram-negative forms (Acidaminococcaceae, Syntrophomonadaceae, some Thermoanaerobacteriaceae), which are didermic.

Arch Med Res, 2004 Jul-Aug, 35(4), 348 - 58
Severe combined immunodeficiency syndrome associated with colonic stenosis; Lopez-Herrera G et al.; BACKGROUND: This is the first report in Mexico of a case of severe combined immunodeficiency syndrome (SCID) associated with colonic stenosis . The patient was an 8-month-old Mexican female who died at this age . She suffered infections due to microorganisms such as Mycobacterium tuberculosis, bacille Calmette-Guerin (BCG), Candida sp., and Pneumocystis carinii; and had frequent diarrhea . She was HIV-negative without familial history of immunodeficiency . The aim of the work was to analyze the immunologic status of this patient . METHODS: Peripheral blood from the patient and from a healthy matched control were analyzed by flow cytometry to determine peripheral leukocytes and production of cytokines and their receptors in T-lymphocytes and monocytes . Immunohistochemical analysis was performed in spleen and lymph node sections from the patient and control samples to assess alterations in architectural and cellular distribution within these lymphoid tissues . RESULTS: Peripheral blood analysis demonstrated reduced numbers of both T and B cells and defective expression of cytokines by activated T cells . Postmortem analysis revealed very small T and B cell zones in spleen and lymph nodes, absence of germinal centers and follicular dendritic cell networks, and two zones of stenosis at level of colon sigmoides . CONCLUSIONS: As a whole, these data are consistent with severe combined immunodeficiency (SCID) syndrome; thus, we conclude that this patient may have had a variant of SCID syndrome associated with intestinal stenosis.

Shi Yan Sheng Wu Xue Bao, 2004 Jun, 37(3), 205 - 11
{Characterization of resistant cell line of Trichoplusia ni to Bacillus thuringiensis toxin Cry1Ac}; Liu KY et al.; The cell line (BTI-TN-5B1-4) of Trichoplusia ni had been selected by Bacilllus thuringiensis (Bt) toxin Cry1Ac for 86 generations continuously . A high level of resistance was achieved and the partial characterizations of the resistance-selected BTI-TN-5B1-4 cells were studied in order to explore the mechanism of the resistance to Bt toxin . The cells selected by Cry1Ac had low level of cross-resistance (19.7 fold) to Cry1C, their ability of tolerance in the hypotonic solution increased evidently (2.5 fold), and there were differential membrane proteins between resistance-selected and unselected susceptible cells on two dimensional electrophoresis maps . We supposed the changes of membrane proteins in resistant cells would be linked to the cross-resistance to Cry1C and the increase of tolerance in hypotonic solution.

Biosci Biotechnol Biochem, 2004 Aug, 68(8), 1794 - 7
Identification of amino acid residues essential for the catalytic reaction of Bacillus kaustophilus leucine aminopeptidase; Chi MC et al.; The functional significance of amino acid residues Lys-265, Asp-270, Lys-277, Asp-288, Asp-347, Glu-349, and Arg-351 of Bacillus kaustophilus leucine aminopeptidase was explored by site-directed mutagenesis . Variants with an apparent molecular mass of approximately 54 kDa were overexpressed in Escherichia coli and purified to homogeneity by nickel-chelate chromatography . The purified mutant enzymes had no LAP activity, implying that these residues are important for the catalytic reaction of the enzyme.

J Immunol, 2004 Sep 1, 173(5), 3287 - 96
CCR5-deficient mice control Mycobacterium tuberculosis infection despite increased pulmonary lymphocytic infiltration; Algood HM et al.; The control of Mycobacterium tuberculosis infection is dependent on the development of an adaptive immune response, which is mediated by granulomas . The granuloma is a dynamic structure that forms in the lung and consists primarily of macrophages and lymphocytes . For this structure to be effective in containment of the bacillus, it must develop in an organized and timely manner . The formation of the granuloma is dependent on recruitment of activated cells through adhesion molecules and chemokines . M . tuberculosis infection causes an increase in the expression of beta-chemokines CCL3, CCL4, and CCL5, and their receptor CCR5, in the lungs . In this study, we demonstrate that CCR5-transgenic knockout mice were capable of recruiting immune cells to the lung to form granulomas . CCR5(-/-) mice successfully induced a Th1 response and controlled infection . Unexpectedly, M . tuberculosis infection in these mice resulted in greater numbers of lymphocytes migrating to the lung and higher levels of many inflammatory cytokines, compared with wild-type mice, without apparent long-term detrimental effects . In the absence of CCR5, there were more dendritic cells in the lung-draining lymph nodes and more primed T lymphocytes in these mice . Bacterial numbers in the lymph nodes were also higher in CCR5(-/-) mice . Therefore, CCR5 may play a role in the migration of dendritic cells to and from the lymph nodes during M . tuberculosis infection.

Infect Immun, 2004 Sep, 72(9), 5235 - 46
Activation of dendritic cells by liposomes prepared from phosphatidylinositol mannosides from Mycobacterium bovis bacillus Calmette-Guerin and adjuvant activity in vivo; Sprott GD et al.; Liposome vesicles could be formed at 65 degrees C from the chloroform-soluble, total polar lipids (TPL) extracted from Mycobacterium bovis bacillus Calmette-Guerin (BCG) . Mice immunized with ovalbumin (OVA) entrapped in TPL liposomes produced both anti-OVA antibody and cytotoxic T lymphocyte responses . Murine bone marrow-derived dendritic cells were activated to secrete interleukin-6 (IL-6), IL-12, and tumor necrosis factor upon exposure to antigen-free TPL liposomes . Three phosphoglycolipids and three phospholipids comprising 96% of TPL were identified as phosphatidylinositol dimannoside, palmitoyl-phosphatidylinositol dimannoside, dipalmitoyl-phosphatidylinositol dimannoside, phosphatidylinositol, phosphatidylethanolamine, and cardiolipin . The activation of dendritic cells by liposomes prepared from each purified lipid component of TPL was evaluated in vitro . A basal activity of phosphatidylinositol liposomes to activate proinflammatory cytokine production appeared to be attributable to the tuberculosteric fatty acyl 19:0 chain characteristic of mycobacterial glycerolipids, as similar lipids lacking tuberculosteric chains showed little activity . Phosphatidylinositol dimannoside was identified as the primary lipid that activated dendritic cells to produce amounts of proinflammatory cytokines several times higher than the basal level, indicating the importance of mannose residues . Although the activity of phosphatidylinositol dimannoside was little influenced by palmitoylation of mannose at C-6, a further palmitoylation at inositol C-3 diminished the induction levels of IL-6 and IL-12 . Further, OVA entrapped in palmitoyl-phosphatidylinositol dimannoside liposomes was delivered to dendritic cells for major histocompatibility complex class I presentation more effectively than TPL OVA-liposomes . BCG liposomes containing mannose lipids caused up-regulation of costimulatory molecules and CD40 . Thus, the inclusion of pure phosphatidylinositol mannosides of BCG in lipid vesicle vaccines represents a simple and efficient option for targeting antigen delivery and providing immune stimulation.

FEMS Microbiol Lett, 2004 Aug 15, 237(2), 289 - 95
The Bacillus thuringiensis phage GIL01 encodes two enzymes with peptidoglycan hydrolase activity; Verheust C et al.; Bacteriophage GIL01, infecting Bacillus thuringiensis serovar israelensis, possesses a linear dsDNA genome of 14,931 bp with proteins attached to its 5' extremities and terminal inverted repeats at both ends . Viral particles are sensitive to organic solvents, suggesting that a lipid membrane is present in the capsid . All these characteristics are reminiscent of those found in members of the Tectiviridae family . Sequence analysis of GIL01 revealed the presence of two open reading frames (ORF25 and ORF30) encoding potential lytic enzymes, which were cloned and overexpressed in Escherichia coli . The muralytic activity of these two proteins, designated Mur1 and Mur2 respectively, was confirmed in situ using renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gels containing bacterial cell wall preparations . While Mur2 degrading activity is limited to B . thuringiensis israelensis, Mur1 has a broader cleavage spectrum.

Scand J Immunol, 2004 Sep, 60(3), 273 - 7
The influence of remaining live BCG organisms in vaccinated mice on the maintenance of immunity to tuberculosis; Olsen AW et al.; The only available vaccine against Mycobacterium tuberculosis, the bacille Calmette-Guerin (BCG) vaccine, is at present being used as a reference for the efficacy of novel vaccines . Herein, we demonstrate that viable BCG can be detected at late time points after vaccination in C57BL/6J mice . If BCG is cleared by antibiotic treatment, the number of mycobacteria-reactive effector cells in the spleen gradually reverts to low levels and consequently immunity in this organ wanes, while resistance in the lung remains stable . The implications for comparing BCG vaccination with experimental vaccines including non-replicating vaccines are discussed.

Biochem J, 2004 Dec 15, 384(Pt 3), 507 - 13
Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains II and III in specificity towards Spodoptera exigua larvae; Herrero S et al.; Several mutants of the Bacillus thuringiensis Cry1Ca toxin affected with regard to specific activity towards Spodoptera exigua were studied . Alanine was used to replace single residues in loops 2 and 3 of domain II (mutant pPB19) and to replace residues 541-544 in domain III (mutant pPB20) . Additionally, a Cry1Ca mutant combining all mutations was constructed (mutant pPB21) . Toxicity assays showed a marked decrease in toxicity against S . exigua for all mutants, while they retained their activity against Manduca sexta, confirming the importance of these residues in determining insect specificity . Parameters for binding to the specific receptors in BBMV (brush border membrane vesicles) of S . exigua were determined for all toxins . Compared with Cry1Ca, the affinity of mutant pPB19 was slightly affected (2-fold lower), whereas the affinity of the mutants with an altered domain III (pPB20 and pPB21) was approx . 8-fold lower . Activation of Cry1Ca protoxin by incubation with S . exigua or M . sexta BBMV revealed the transient formation of an oligomeric form of Cry1Ca . The presence of this oligomeric form was tested in the activation of the different Cry1Ca mutants, and we found that those mutated in domain II (pPB19 and pPB21) could not generate the oligomeric form when activated by S . exigua BBMV . In contrast, when oligomerization was tested using BBMV prepared from M . sexta, all of the Cry1Ca mutants showed the formation of a similar oligomeric form as did the wild-type toxin . Our results show how modification of insect specificity can be achieved by manipulation of different parts of the toxin structure involved in different steps of the mode of action of B . thuringiensis toxins.

Ying Yong Sheng Tai Xue Bao, 2004 May, 15(5), 871 - 4
{Revulsive response of protein and enzyme activity of degradative Bacilluss strains by phenanthrene}; Zhong M et al.; Three strains of Bacillus sp . BAll, BA19 and BA27, which can grow when phenanthrene is taken as the sole source of carbon and energy, were isolated from an oil-contaminated soil . Their crude extract was used to test the activities of polyphenol oxidase and catalase . The results demonstrated that all the three strains did not show a significant change in the activity of both enzymes when phenanthrene was lower than 200 mg x L(-1) . Under different phenanthrene concentration, the enzyme activities of strains BA19, BA27 showed a higher stabilization than BA11 . Using the SDS-PAGE contrasted the difference of proteins between the strains in normal medium and the strains growing on phenanthrene, the results showed that BA27 growing on phenanthrene synthesized a new protein with 27000 dal of molecular weight . At the same time, the synthesis of some proteins was cancelled . We conjecture that the new protein induced by phenanthrene may be related to the ability of phenanthrene degradation and the stabilization of enzyme activity by the strain.

Ying Yong Sheng Tai Xue Bao, 2004 May, 15(5), 845 - 8
{Response of parasitic wasps of cotton bollworm to different cotton varieties with transgenic Bacillus thringienthsis genes}; Yu Y et al.; The relationship between the parasitic wasp population number of cotton bollworm (Helicoverpa armigera) and the varieties of cotton was studied by releasing and collecting the eggs and larvae of cotton bollworm on transgenic and non-transgenic cottons . The research results from 2000 to 2003 showed that the parasitic rates of the eggs and larvae of cotton bollworm were notably lower in transgenic cotton field than in non-transgenic cotton field . At the same time, the effect of transgenic and non-transgenic cottons on the selection behavior of the parasitic wasp Microplitis mediatar of the larvae of cotton bollworm was studied by using "Y" olfactometer . The results showed that the selection response about the parasitic wasp Microplitis mediatar of cotton bollworm on non-transgenic cotton was prior to the transgenic cotton.

J Infect Dis, 2004 Sep 15, 190(6), 1167 - 76 Epub 2004 Aug 11.
Induction of apoptosis and release of interleukin-1 beta by cell wall-associated 19-kDa lipoprotein during the course of mycobacterial infection; Ciaramella A et al.; Mycobacterium tuberculosis induces apoptosis in human monocyte-derived macrophages (MDMs) during the early stages of infection . We investigated the proapoptotic role of cell wall-associated mycobacterial 19-kDa lipoprotein and the possible association between 19-kDa lipoprotein signaling and production of proinflammatory cytokines . Purified mycobacterial 19-kDa lipoprotein, 19-kDa lipoprotein-expressing M . smegmatis (M . smegmatis 19+), 19-kDa lipoprotein knockout (KO) M . tuberculosis, and 19-kDa lipoprotein KO M . bovis bacille Calmette-Guerin (BCG) strains were analyzed for their ability to induce apoptosis in MDMs . The 19-kDa lipoprotein and infection with M . smegmatis 19+ induced apoptosis in MDMs . M . tuberculosis and BCG KO strains had significantly decreased abilities to induce apoptosis . The 19-kDa lipoprotein proapoptotic signal was mediated by Toll-like receptor 2 but not by tumor necrosis factor-alpha . Only the release of interleukin (IL)-1 beta was decreased after infection with 19-kDa lipoprotein KO strains . These findings indicate that the 19-kDa lipoprotein is the main signal required to trigger both apoptosis and the release of IL-1 beta during the early stages of mycobacterial infection.

Extremophiles, 2004 Dec, 8(6), 447 - 54 Epub 2004 Aug 14.
Characterization and gene cloning of a novel beta-mannanase from alkaliphilic Bacillus sp . N16-5; Ma Y et al.; An alkaline beta-mannanase was purified to homogeneity from a culture broth of alkaliphilic Bacillus sp . N16-5 . The enzyme had optimum activity at pH 9.5 and 70 degrees C . It was composed of a single polypeptide chain with a molecular weight of 55 kDa deduced from SDS-PAGE, and its isoelectric point was around pH 4.3 . The enzyme efficiently hydrolyzed galactomannan and glucomannan, producing a series of oligosaccharides and monosaccharides . The beta-mannanase gene (manA) contained an open reading frame (ORF) of 1,479 bp, encoding a 32-amino acids signal peptide, and a mature protein of 461 amino acids, with a calculated molecular mass of 50,743 Da . Strain N16-5 ManA, deduced from the manA ORF, exhibited relatively high amino acid similarity to the members of the glycosyl hydrolase family 5 . The eight conserved active-site amino acids in family 5 glycosyl hydrolase were found in the deduced amino acid sequence of strain N16-5 ManA.

Proc Natl Acad Sci U S A, 2004 Aug 24, 101(34), 12598 - 603 Epub 2004 Aug 16.
The RD1 virulence locus of Mycobacterium tuberculosis regulates DNA transfer in Mycobacterium smegmatis; Flint JL et al.; Conjugal DNA transfer occurs by an atypical mechanism in Mycobacterium smegmatis . The transfer system is chromosomally encoded and requires recipient recombination functions for both chromosome and plasmid transfer . Cis-acting sequences have been identified that confer mobility on nontransferable plasmids, but these are larger and have different properties to canonical oriT sites found in bacterial plasmids . To identify trans-acting factors required for mediating DNA transfer, a library of transposon insertion mutants was generated in the donor strain, and individual mutants were screened for their effect on transfer . From this screen, a collection of insertion mutants was isolated that increased conjugation frequencies relative to wild type . Remarkably, the mutations map to a 25-kb region of the M . smegmatis chromosome that is syntenous with the RD1 region of Mycobacterium tuberculosis, which is considered to be the primary attenuating deletion in the related vaccine strain Mycobacterium bovis bacillus Calmette-Guerin . The genes of the RD1 region encode a secretory apparatus responsible for exporting Cfp10- and Esat-6, both potent antigens and virulence factors . In crosses using two M . smegmatis donors, we show that wild-type cells can suppress the elevated transfer phenotype of mutant donors, which is consistent with the secretion of a factor that suppresses conjugation . Most importantly, the RD1 region of M . tuberculosis complements the conjugation phenotype of the RD1 mutants in M . smegmatis . Our results indicate that the M . tuberculosis and M . smegmatis RD1 regions are functionally equivalent and provide a unique perspective on the role of this critical secretion apparatus.

Proc Natl Acad Sci U S A, 2004 Aug 24, 101(34), 12652 - 7 Epub 2004 Aug 16.
Mycobacterium tuberculosis (Mtb) isocitrate dehydrogenases show strong B cell response and distinguish vaccinated controls from TB patients; Banerjee S et al.; Proteins released from Mycobacterium tuberculosis (Mtb) during late logarithmic growth phase are often considered candidate components of immunogenic or autolysis markers . One such protein is isocitrate dehydrogenase (ICD), a key regulatory enzyme in the citric acid cycle . We have evaluated the immunogenic properties of two isoforms of Mtb ICD and compared them with the control antigens heat-shock protein 60 and purified protein derivative (PPD) . PPD lacks the sensitivity to distinguish between bacillus Calmette-Guerin (BCG)-vaccinated and tuberculosis (TB)-infected populations, and, therefore, epidemiological relevance of PPD in BCG-vaccinated regions is debatable . We show that Mtb ICDs elicit a strong B cell response in TB-infected populations and can differentiate between healthy BCG-vaccinated populations and those with TB . The study population (n = 215) was categorized into different groups, namely, patients with fresh infection (n = 42), relapsed TB cases (n = 32), patients with extrapulmonary TB (n = 35), clinically healthy donors (n = 44), nontuberculous mycobacteria patients (n = 30), and non-TB patients (culture negative for acid-fast bacteria but carrying other infections, n = 32) . The Mtb ICDs showed statistically significant antigenic distinction between healthy BCG-vaccinated controls and TB patients (P < 0.0001) and those with other infections . Although extrapulmonary infections could not be discriminated from healthy controls by heat-shock protein 60 (P = 0.2177), interestingly, the Mtb ICDs could significantly (P < 0.0001) do so . Our results highlight the immunodominant, immunosensitive, and immunospecific nature of Mtb ICDs and point to an unusual property of this tricarboxylic acid energy cycle enzyme.

J Mol Biol, 2004 Jul 30, 341(1), 107 - 17
The structure of endo-beta-1,4-galactanase from Bacillus licheniformis in complex with two oligosaccharide products; Ryttersgaard C et al.; The beta-1,4-galactanase from Bacillus licheniformis (BLGAL) is a plant cell-wall-degrading enzyme involved in the hydrolysis of beta-1,4-galactan in the hairy regions of pectin . The crystal structure of BLGAL was determined by molecular replacement both alone and in complex with the products galactobiose and galactotriose, catching a first crystallographic glimpse of fragments of beta-1,4-galactan . As expected for an enzyme belonging to GH-53, the BLGAL structure reveals a (betaalpha)(8)-barrel architecture . However, BLGAL betaalpha-loops 2, 7 and 8 are long in contrast to the corresponding loops in structures of fungal galactanases determined previously . The structure of BLGAL additionally shows a calcium ion linking the long betaalpha-loops 7 and 8, which replaces a disulphide bridge in the fungal galactanases . Compared to the substrate-binding subsites predicted for Aspergillus aculeatus galactanase (AAGAL), two additional subsites for substrate binding are found in BLGAL, -3 and -4 . A comparison of the pattern of galactan and galactooligosaccharides degradation by AAGAL and BLGAL shows that, although both are most active on substrates with a high degree of polymerization, AAGAL can degrade galactotriose and galactotetraose efficiently, whereas BLGAL prefers longer oligosaccharides and cannot hydrolyze galactotriose to any appreciable extent . This difference in substrate preference can be explained structurally by the presence of the extra subsites -3 and -4 in BLGAL.

Biotechnol Appl Biochem . 2004 Aug 16; {Epub ahead of print}
The combined effects of two anti-aggregatory agents: alpha-cyclodextrin and Ca 2+ ion, on the refolding process of denatured alpha-amylase; Yazdanparast R et al.; It is now well known that alpha-cyclodextrin (alpha-CD) and Ca 2+ ion are two important folding agents in reactivation and the refolding processes of many denatured enzyme solutions . Bacillus sp . alpha-amylase was extensively denatured in 6 M guanidinium chloride (GdmCl) solution overnight . Under suitable renaturation conditions, 20-30% more activity was recovered in the presence of various concentrations of alpha-CD (0-100 mM) . Similarly, 25-30% more activity was recovered under the influence of different concentrations of Ca 2+ ion (0-100 mM) . Regardless of these data, both alpha-CD and Ca 2+ at 100 mM concentrations were separately capable of preventing the extent of aggregate formation by 86% and 77%, respectively . These observations clearly indicate that the effect of alpha-CD and/or Ca 2+ ion on the process of aggregation and refolding (activity recovery) are not synchronized: both agents mostly act as anti-aggregatory agents than as refolding aids.

J Urol, 2004 Sep, 172(3), 937 - 42
Treatment of urethral recurrence following radical cystectomy and ileal bladder substitution; Varol C et al.; PURPOSE: With the introduction of orthotopic bladder substitution after radical cystectomy in patients with invasive bladder cancer urethral recurrences have become a therapeutic challenge . MATERIALS AND METHODS: We retrospectively evaluated our patients with urethral recurrences treated with a urethra sparing approach after orthotopic bladder substitution . Depending on the extension of recurrence and eventual concomitant metastases patients were treated with urethrectomy, no treatment, systemic chemotherapy or intraurethral bacillus Calmette-Guerin (BCG) . Three times the common dose of BCG (ImmuCyst, Aventis, Paris, France or OncoTICE, Organon, West Orange, New Jersey) in 150 ml NaCl 0.9% was used for intraurethral BCG perfusion therapy according to an institutional protocol using a modified Foley catheter . This regimen was repeated weekly for 6 weeks and patients were followed prospectively . RESULTS: Between 1985 and 2001, 15 of 371 patients (4%) who received an orthotopic bladder substitute had urethral recurrence . Two patients were treated with systemic chemotherapy (methotrexate, vinblastine, doxorubicin and cisplatin) alone due to metastatic disease and 10 received intraurethral BCG therapy . Five of 6 patients (83%) with carcinoma in situ remained free of recurrence following treatment with BCG, while in 4 with papillary or invasive disease treatment failed . Three patients underwent urethrectomy, including 2 following failed BCG therapy for papillary disease . CONCLUSIONS: Carcinoma in situ urethral recurrence following orthotopic bladder substitution can be treated successfully with intraurethral BCG perfusion therapy in approximately 80% of patients . However, papillary and invasive transitional cell urethral recurrence should be treated with urethrectomy.

J Urol, 2004 Sep, 172(3), 888 - 93
Interim results from a national multicenter phase II trial of combination bacillus Calmette-Guerin plus interferon alfa-2b for superficial bladder cancer; O'Donnell MA et al.; PURPOSE: Interim results are provided from a large multicenter trial of combination bacillus Calmette-Guerin (BCG) plus interferon (IFN) alfa-2b for BCG naive (BCG-N) and previous BCG failure (BCG-F) cases of superficial bladder cancer . MATERIALS AND METHODS: A total of 490 patients enrolled from May 1999 to May 2000 with a median of 24 months of followup were analyzed . The BCG-N group (259) was treated with a 6-week induction course of standard dose BCG plus 50 million units of IFN followed by 3, 3-week maintenance cycles of reduced dose BCG (1/3 to 1/10) plus 50 million units IFN at 3, 9 and 15 months after induction . The BCG-F group (231) was treated similarly except induction therapy began at a decreased (1/3 to 1/10) BCG dose . RESULTS: The simple tumor recurrence rates for BCG-N and BCG-F groups were 40% and 52%, and the Kaplan-Meier estimates for disease freedom at 24 months were 57% and 42%, respectively . Progression to muscle invasion occurred in 5% and 4.3% while metastasis occurred in 2.3% and 2.6%, respectively . In each group 3.9% of patients underwent cystectomy and 2 patients in each group died of bladder cancer . Serious adverse events occurred in 5.5% with infection related serious adverse events being less prevalent in the BCG-F group (2.6% vs 5.4%) . Toxicity related dropout, treatment delay and/or further BCG dose reduction, and need for symptomatic drugs were similar . Moderate to severe local side effects during induction were higher in the BCG-F group (6.2% vs 16.9%) but equilibrated during maintenance therapy . Systemic reactions were rare . CONCLUSIONS: This multicenter trial provides a benchmark for the efficacy and safety of combination BCG and IFN as up front and salvage therapy . The incremental value of IFN cannot be determined from this study.

Methods Mol Biol, 2005, 286, 215 - 26
Green fluorescent protein quantification in whole plants; Halfhill MD et al.; As future biotechnology applications utilize recombinant proteins as commercial products, nondestructive assays will be necessary to determine protein concentrations accurately within plant tissues . Green fluorescent protein (GFP) has been proposed as a potential marker for the monitoring of transgenic plants and quantifying recombinant protein levels under field conditions . This chapter discusses the utility of using GFP fluorescence as an indicator of protein concentrations and the methods used to quantify GFP fluorescence in whole plant tissues . Furthermore, we discuss the accuracy and effectiveness of the portable General Fluorescence Plant Meter (GFP Meter, Opti-Sciences, Inc.) compared to a laboratory-based spectrofluorometer (Fluoro-Max2, Jobin Yvon & Glen Spectra) . In whole plants, GFP fluorescence was shown to be variable at each leaf position over time and among different leaves on the same plant . A leaf had its highest GFP fluorescence after emergence, and subsequently, its fluorescence intensity decreased over time . Younger leaves were significantly more fluorescent than older leaves on the same plant . GFP fluorescence intensity was directly correlated with the concentration of soluble protein per unit wet mass and with another genetically linked recombinant protein (Bacillus thuringiensis {Bt} cry1Ac endotoxin protein).

World J Gastroenterol, 2004 Sep 15, 10(18), 2690 - 6
Protective effect of melatonin against liver injury in mice induced by Bacillus Calmette-Guerin plus lipopolysaccharide; Wang H et al.; AIM: To investigate the effects and mechanisms of melatonin on immunological liver injury in mice . METHODS: A model of liver injury was induced by tail vein injection of Bacillus Calmette Guerin (BCG) and lipopolysaccharide (LPS) in mice . Kupffer cells and hepatocytes were isolated and cultured according to a modified two-step collagenase perfusion technique . Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and nitric oxide (NO), content of malondiadehyde (MDA), activity of superoxide dismutase (SOD), were measured by biochemical methods . Tumor necrosis factor-alpha (TNF-alpha) activity was determined by RIA . Interleukin (IL)-1 activity was measured by thymocyte proliferation bioassay . Hepatic tissue sections were stained with hematoxylin and eosin and examined under a light microscope . RESULTS: Immunological liver injury induced by BCG+LPS was successfully duplicated . Serum transaminase (ALT, AST) activities were significantly decreased by melatonin (0.25, 1.0, 4.0 mg/kg bm) . Meanwhile, MDA content was decreased and SOD in liver homogenates was upregulated . Furthermore, pro-inflammatory mediators (TNF-alpha, IL-1, NO) in serum and liver homogenates were significantly reduced by melatonin . Histological examination demonstrated that melatonin could attenuate the area and extent of necrosis, reduce the immigration of inflammatory cells . In in vitro experiment, TNF-alpha was inhibited at the concentrations of 10(-8)-10(-6) mol/L of melatonin, while IL-1 production of Kupffer cells induced by LPS (5 microg/mL) was decreased only at the concentration of 10(-6) mol/L of melatonin, but no effect on NO production was observed . Immunological liver injury model in vitro was established by incubating hepatocytes with BCG- and LPS-induced Kupffer cells . Activities of ALT, TNF-alpha, IL-1, and MDA in supernatant were significantly increased . Melatonin had little effect on the level of ALT, but reduced the content of TNF-alpha and MDA at concentrations of 10(-7)-10(-5) mol/L and decreased the content of IL-1 at concentrations of 10(-6)-10(-5) mol/L . CONCLUSION: Melatonin could significantly protect liver injury in mice, which was related to free radical scavenging, increased SOD activity and pro-inflammatory mediators.

Plant Cell Rep, 2004 Nov, 23(5), 319 - 26 Epub 2004 Aug 11.
A variable region of the sugarcane bacilliform virus (SCBV) genome can be used to generate promoters for transgene expression in sugarcane; Braithwaite KS et al.; Four promoters derived from sugarcane bacilliform virus (SCBV) were compared and characterised . Three were obtained by PCR amplification of purified virion DNA extracted from three sugarcane cultivars . The fourth promoter was obtained by subcloning from an almost genome-length clone of SCBV . All promoters were able to drive stable expression of beta-glucuronidase in sugarcane . The PCR-derived promoter sequences shared more DNA homology with banana streak virus than to the subcloned SCBV . The subcloned promoter was the strongest expressing and was able to drive reporter gene expression in vitro and in the leaves, meristems and roots of glasshouse-grown sugarcane . Expression levels were at least equal to or higher than those measured for the maize polyubiquitin promoter.

Appl Microbiol Biotechnol . 2004 Aug 7; {Epub ahead of print}
Enrichment and molecular characterization of chloropicrin- and metam-sodium-degrading microbial communities; Ibekwe AM et al.; Chloropicrin (CP) and metam sodium are commonly used as fumigants in agricultural soils in order to provide effective control of nematodes, soil-borne pathogens, and weeds in preparation for planting of high-value cash crops . Repeated application of these compounds to agricultural soils for many years may result in the enrichment of microorganisms capable of degrading them . In this study, a microcosm-enrichment approach was used to investigate bacterial populations that may be components of metam-sodium- and CP-degrading microorganisms in compost-amended soils . After 6 months incubation, with repeated application of metam sodium and CP, degradation was >/=70% faster in compost-manure-amended (CM) soil compared to </=50% in the unamended soils . The accelerated fumigant degradation may have been due to the addition of compost or to the development of new microbial populations with enhanced degradation capacity . Denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified regions of 16S rRNA genes were used to identify dominant bacterial populations responsible for the accelerated fumigant degradation . The DGGE results indicated that specific bacterial types had been enriched and these were similar to strains isolated from basal minimal media . Fragments from DGGE bands and colonies were cloned, sequenced, and compared with published 16S rRNA sequences . Cloned sequences were dominated by Pseudomonas, Bacillus, Arthrobacter, Mycobacterium and uncultured bacterial species . The addition of organic amendment to soil during fumigation practices has the potential to increase the diversity of different microbial species, thereby accelerating fumigant degradation and reducing atmospheric emissions.

J Biol Chem, 2004 Oct 22, 279(43), 45245 - 56 Epub 2004 Aug 11.
Analyzing the handoff of DNA from UvrA to UvrB utilizing DNA-protein photoaffinity labeling; DellaVecchia MJ et al.; To better define the molecular architecture of nucleotide excision repair intermediates it is necessary to identify the specific domains of UvrA, UvrB, and UvrC that are in close proximity to DNA damage during the repair process . One key step of nucleotide excision repair that is poorly understood is the transfer of damaged DNA from UvrA to UvrB, prior to incision by UvrC . To study this transfer, we have utilized two types of arylazido-modified photoaffinity reagents that probe residues in the Uvr proteins that are closest to either the damaged or non-damaged strands . The damaged strand probes consisted of dNTP analogs linked to a terminal arylazido moiety . These analogs were incorporated into double-stranded DNA using DNA polymerase beta and functioned as both the damage site and the cross-linking reagent . The non-damaged strand probe contained an arylazido moiety coupled to a phosphorothioate-modified backbone of an oligonucleotide opposite the damaged strand, which contained an internal fluorescein adduct . Six site-directed mutants of Bacillus caldotenax UvrB located in different domains within the protein (Y96A, E99A, R123A, R183E, F249A, and D510A), and two domain deletions (Delta2 and Deltabeta-hairpin), were assayed . Data gleaned from these mutants suggest that the handoff of damaged DNA from UvrA to UvrB proceeds in a three-step process: 1) UvrA and UvrB bind to the damaged site, with UvrA in direct contact; 2) a transfer reaction with UvrB contacting mostly the non-damaged DNA strand; 3) lesion engagement by the damage recognition pocket of UvrB with concomitant release of UvrA.

Endocrinology, 2004 Nov, 145(11), 5075 - 9 Epub 2004 Aug 12.
Graves' hyperthyroidism and the hygiene hypothesis in a mouse model; Nagayama Y et al.; Graves' hyperthyroidism is an organ-specific autoimmune disease mediated by stimulatory autoantibodies against the TSH receptor (TSHR; thyroid-stimulating antibodies), causing thyroid hyperplasia and hyperthyroidism . Development of this ailment is well known to be under polygenic and environmental control . For example, we recently demonstrated that parasite helminth Schistosoma mansoni infection suppressed a T helper cell type 1 (Th1)-type anti-TSHR immune response and prevented disease development in our mouse model of Graves' disease using adenovirus coding for the TSHR . In the present study we examined the outcome of infection with Mycobacterium bovis bacillus Calmette-Guerin (BCG), a Th1-promoting infectious pathogen, on Graves' disease . Our results show that prior infection with M . bovis BCG differentiates the TSHR-specific immune response toward a Th1 phenotype, as demonstrated by enhanced secretion of a Th1 cytokine interferon-gamma and impaired production of a Th2 cytokine IL-10 from splenocytes stimulated in vitro with TSHR antigen . M . bovis BCG also significantly suppressed disease induction . These data together with our recent report that coinjection of adenovirus expressing the Th1 cytokine IL-12 induced a Th1-polarized, TSHR-specific immune response without affecting disease development support the hygiene hypothesis, rather than Th1-mediated disease suppression . Thus, some infectious pathogens may influence the development of Graves' disease regardless of their ability to modify the Th1/Th2 balance.

Vaccine, 2004 Sep 3, 22(25-26), 3375 - 85
No epidemiological evidence for infant vaccinations to cause allergic disease; Koppen S et al.; CONTEXT: The prevalence of allergic diseases has increased considerably over the last decades . The hygiene hypothesis has emerged, linking reduced microbial exposure and infections early in life with the development of allergic diseases . Especially some of currently available non-replicating infant vaccines are unlikely to mimic a natural infection-mediated immune response that protects against the development of allergic diseases . Moreover, several studies suggested infant vaccinations to increase the risk of allergic diseases . OBJECTIVE: To determine whether infant vaccinations increase the risk of developing allergic disease . DATA SOURCES: We searched MEDLINE from 1966 to March 2003 and bibliography lists from retrieved articles, and consulted experts in the field to identify all articles relating vaccination to allergy . STUDY SELECTION AND DATA EXTRACTION: We selected epidemiological studies with original data on the correlation between vaccination with diphtheria, pertussis, tetanus (DPT), measles, mumps, rubella (MMR) and Bacillus Calmette-Guerin (BCG) vaccine in infancy and the development of allergic diseases, and assessed their quality and validity . DATA SYNTHESIS: Methodological design and quality varied considerably between the studies we reviewed . Many studies did not address possible confounders, such as the presence of lifestyle factors, leaving them prone to bias . The studies that offer the stronger evidence, including the only randomized controlled trial at issue published to date, indicate that the infant vaccinations we investigated do not increase the risk of developing allergic disease . Furthermore, BCG does not seem to reduce the risk of allergies . CONCLUSIONS: The reviewed epidemiological evidence indicates that, although possibly not contributing to optimal stimulation of the immune system in infancy, current infant vaccines do not cause allergic diseases.

Pest Manag Sci, 2004 Aug, 60(8), 827 - 32
Genetics of spinosad resistance in a multi-resistant field-selected population of Plutella xylostella; Sayyed AH et al.; Resistance to the bacteria-derived insecticides spinosad (Conserve), abamectin (Vertimec), Bacillus thuringiensis var kurstaki (Btk) (Dipel), B thuringiensis var aizawai (Bta) (Xentari), B thuringiensis crystal endotoxins Cry1Ac and Cry1Ca, and to the synthetic insecticide fipronil was estimated in a freshly-collected field population (CH1 strain) of Plutella xylostella (L) from the Cameron Highlands, Malaysia . Laboratory bioassays at G1 indicated significant levels of resistance to spinosad, abamectin, Cry1Ac, Btk, Cry1Ca, fipronil and Bta when compared with a laboratory insecticide-susceptible population . Logit regression analysis of F1 reciprocal crosses indicated that resistance to spinosad in the CH1 population was inherited as a co-dominant trait . At the highest dose of spinosad tested, resistance was close to completely recessive, while at the lowest dose it was incompletely dominant . A direct test of monogenic inheritance based on a back-cross of F1 progeny with CH1 suggested that resistance to spinosad was controlled by a single locus.

Pest Manag Sci, 2004 Aug, 60(8), 735 - 8
Activity of Bacillus thuringiensis toxins against cocoa pod borer larvae; Santoso D et al.; Twelve Cry proteins from Bacillus thuringiensis Berliner were tested in bioassays on cacao plantations in Indonesia for activity against the larvae of cocoa pod borer (Conopomorpha cramerella (Snellen)), an insect pest of the cacao tree . Through the damage caused by their feeding, the larvae of cocoa pod borer cause the pods of the cocoa tree to ripen prematurely . They are difficult to control with conventional measures . Preliminary assays identified five toxins that were more active than others . In two subsequent bioassays the activity of selected toxins was determined more accurately . Three Cryl proteins with relatively little homology were all found to be toxic, opening perspectives for controlling cocoa pod borer by expression of Cry proteins in transgenic plants.

Philos Transact A Math Phys Eng Sci, 2004 Feb 15, 362(1815), 337 - 47; discussion 347-9
Potential for detection of explosive and biological hazards with electronic terahertz systems; Choi MK et al.; The terahertz (THz) regime (0.1-10 THz) is rich with emerging possibilities in sensing, imaging and communications, with unique applications to screening for weapons, explosives and biohazards, imaging of concealed objects, water content and skin . Here we present initial surveys to evaluate the possibility of sensing plastic explosives and bacterial spores using field-deployable electronic THz techniques based on short-pulse generation and coherent detection using nonlinear transmission lines and diode sampling bridges . We also review the barriers and approaches to achieving greater sensing-at-a-distance (stand-off) capabilities for THz sensing systems . We have made several reflection measurements of metallic and non-metallic targets in our laboratory, and have observed high contrast relative to reflection from skin . In particular, we have taken small quantities of energetic materials such as plastic explosives and a variety of Bacillus spores, and measured them in transmission and in reflection using a broadband pulsed electronic THz reflectometer . The pattern of reflection versus frequency gives rise to signatures that are remarkably specific to the composition of the target, even though the target's morphology and position is varied . Although more work needs to be done to reduce the effects of standing waves through time-gating or attenuators, the possibility of mapping out this contrast for imaging and detection is very attractive.

Chem Pharm Bull (Tokyo), 2004 Aug, 52(8), 1029 - 30
Three new cytotoxic cyclic acylpeptides from marine Bacillus sp; Zhang HL et al.; Three new cytotoxic cyclopeptides belonging to iturin class have been isolated from marine bacterium Bacillus sp . These three compounds have the cytotoxic activities . Their structures were determined based on chemical analysis and all kinds of spectroscopic techniques.

Appl Biochem Biotechnol, 1999 May, 80(2), 121 - 40
Molecular Cloning and Expression of an Endo-beta-1,4-D-glucanase I (Avicelase I) Gene from Bacillus cellulyticus K-12 and Characterization of the Recombinant Enzyme; Lee TK et al.; Bacillus cellulyticus K-12 Avicelase (Avicelase I; EC 3.2.1.4) gene (ace A) has been cloned in Escherichia coli by using the vector pT7T3U19 and HindIII- HindIII libraries of the chromosomal inserts . The libraries were screened for the expression of avicelase by monitoring the immunoreaction of the antiavicelase (immunoscreening) . Positive clones (Ac-3, Ac-5, and Ac-7) contained the identical 3.5-kb HindIII fragment as determined by restriction mapping and Southern hybridization, and expressed avicelase efficiently and constitutively using its own promoter in the heterologous host . From the immunoblotting analysis, a polypeptide that showed a carboxymethylcellulase (CMCase) activity with an Mr of 64,000 was detected . The recombinant endo 1,4-beta-D-glucanase I was purified to homogeneity from an intracellular fraction of E . coli by DEAE-Toyopearl M650, Phenyl Toyoperal M650, and TSK gel HW50S chromatography . The enzyme had a monomeric structure, its relative molecular mass being 65 kDa by gel filtration and 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The pI was 5.3 and the optimal pH was 4.6, and the enzyme was stable at pH 4.0-10.5 . The enzyme had a temperature optimum of 50 degrees C and was stable at 55 degrees C for 48 h, and retained approx 20% of its activity after 30 min at 70 degrees C . It showed high activity toward carboxymethylcellulose (CMC) as well as p-nitrophenyl-beta-D-cellobioside, 4-methylumbelliferyl cellobioside, Avicel, filter paper, and some cellooligosaccharides . Km values for CMC and Avicel were 7.6 and 85.2 mg/mL, respectively, whereas Vmax values were 201 and 9.2 micromol . min-1 . mg-1, respectively . Cellotetraose (G4) was preferentially cleaved into cellobiose (G2) and cellopentaose (G5) was cleaved into G2 + cellotriose (G3), whereas cellohexaose (G6) was cleaved into G4 + G2 and, to a lesser extent, into G3 + G3 . G3 was not cleaved at all . G2 was the main product of Avicel hydrolysis . G2 inhibited whereas Mg++ stimulated the activity of CMCase and Avicelase . Hydrolysis of CMC took place with a rapid decrease in viscosity but a slow liberation of reducing sugars . Based on these results, it appeared that the cellulase should be regarded as endo type, although it hydrolyzed Avicel.

Appl Biochem Biotechnol, 1999 Jan, 76(1), 57 - 64
Studies on Alkaline Protease Produced by Bacillus sp . NG312; S J et al.; An alkalophilic hyperproducer of alkaline protease, Bacillus sp . NG312, was isolated, and the enzyme showed maximum activity at pH 11.0 and 60 degrees C . The temperature optimum was increased by 10 degrees C in presence of Ca2+ . The crude enzyme was found to have half-life of 11 d at 37 degrees C and maximum stability at pH 9.0-10.0 . It also exhibited very good stability in presence of detergent components and some locally available commercial detergent powders.

J Environ Biol, 2004 Jan, 25(1), 113 - 6
Assessment of phytoplankton density in relation to environmental variables in Gopalaswamy pond at Chitradurga, Karnataka; Harsha TS et al.; Surface water samples of the Gopalaswamy pond were studied at two different sites during September 1997 to January 1998 and phytoplanktons were studied in relation to ten environmental variables during this period . A total of 30 species of phytoplanktons were identified under four classes, Chlorophyceae, Cyanophyceae, Bacillariophyceae and Euglenophyceae . Among these, maximum density was recorded under Cyanophyceae, Chlorophyceae, Cyanophyceae and Bacillariophyceae showed considerable fluctuations with environmental variables.

J Biol Chem, 2004 Oct 29, 279(44), 46153 - 61 Epub 2004 Aug 09.
Structural evidence that alanine racemase from a D-cycloserine-producing microorganism exhibits resistance to its own product; Noda M et al.; Alanine racemase (ALR), an enzyme that catalyzes the interconversion of Ala enantiomers, is essential for the synthesis of the bacterial cell wall . We have shown that it is harder to inhibit the catalytic activity of ALR from D-cycloserine (DCS)-producing Streptomyces lavendulae than that from Escherichia coli by DCS . To obtain structural evidence for the fact that Streptomyces ALR displays resistance to DCS, we determined the precise nature of the x-ray crystal structures of the cycloserine-free and cycloserine enantiomer-bound forms of Streptomyces ALR at high resolutions . Streptomyces ALR takes a dimer structure, which is formed by interactions between the N-terminal domain of one monomer with the C-terminal domain of its partner . Each of the two active sites of ALR, which is generated as a result of the formation of the dimer structure, is composed of pyridoxal 5'-phosphate (PLP), the PLP-binding residue Lys(38), and the amino acids in the immediate environment of the pyridoxal cofactor . The current model suggests that each active site of Streptomyces ALR maintains a larger space and takes a more rigid conformation than that of Bacillus stearothermophilus ALR determined previously . Furthermore, we show that Streptomyces ALR results in a slow conversion to a final form of a pyridoxal derivative arising from either isomer of cycloserine, which inhibits the catalytic activity noncompetitively . In fact, the slow conversion is confirmed by the fact that each enzyme bound cycloserine derivative, which is bound to PLP, takes an asymmetric structure.

Clin Exp Rheumatol, 2004 Jul-Aug, 22(4), 481 - 2
Cryoglobulinemia vasculitis following intravesical instillations of bacillus Calmette-Guerin; Granel B et al.; Infections and/or immune-mediated reactions may occur after intravesical instillation of bacillus Calmette-Guerin for the treatment of bladder carcinoma . We report herein a cryoglobulinemia vasculitis occurring after intravesical BCG instillation for a superficial papillary transitional cell bladder carcinoma . The patient, an 80-year-old man, presented peripheral ischemia 10 days after the second course of intravesical BCG instillation . Biological evaluation revealed autoimmune thrombocytopenia, hypergammaglobulinemia, low C3 and C4 complement fraction levels related to mixed cryoglobulinemia and lupus anticoagulant . The patient was treated with heparin and prostacyclins with a good outcome . All of the immune anomalies spontaneously regressed within 3 months . To our knowledge, cryoglobulinemia has only been reported once in the literature and lupus anti-coagulant has never been reported as a complication of intravesical BCG instillation.

Eur J Clin Microbiol Infect Dis, 2004 Sep, 23(9), 725 - 8 Epub 2004 Aug 06.
Bacillus cereus pneumonia in a patient with acute lymphoblastic leukemia; Frankard J et al.; Reported here is a case of Bacillus cereus pneumonia that occurred in a patient with acute lymphoblastic leukemia . The presentation was severe, essentially marked by respiratory distress and pleuritic chest pain . Classic empirical treatment initiated for febrile neutropenia did not cover this rare pathogen and appropriate therapy was therefore delayed . B . cereus is most often a culture contaminant, but it can also be responsible for self-limited gastrointestinal intoxication and, more rarely, severe systemic diseases . Virulence in the case of systemic disease is attributed to tissue necrosis mediated by toxin release . B . cereus pneumonia, as described in the English-language literature, mainly affects immunocompromised patients and most often has a fatal outcome . Thus, the identification of B . cereus in clinical specimens of severely ill immunocompromised patients should lead physicians to question its clinical significance .

Nat Med, 2004 Sep, 10(9), 935 - 41 Epub 2004 Aug 08.
Methylation-dependent T cell immunity to Mycobacterium tuberculosis heparin-binding hemagglutinin; Temmerman S et al.; Although post-translational modifications of protein antigens may be important componenets of some B cell epitopes, the determinants of T cell immunity are generally nonmodified peptides . Here we show that methylation of the Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA) by the bacterium is essential for effective T cell immunity to this antigen in infected healthy humans and in mice . Methylated HBHA provides high levels of protection against M . tuberculosis challenge in mice, whereas nonmethylated HBHA does not . Protective immunity induced by methylated HBHA is comparable to that afforded by vaccination with bacille Calmette et Guerin, the only available anti-tuberculosis vaccine . Thus, post-translational modifications of proteins may be crucial for their ability to induce protective T cell-mediated immunity against infectious diseases such as tuberculosis.

Curr Opin Urol, 2004 Sep, 14(5), 271 - 5
Second-line intravesical therapy versus cystectomy for bacille Calmette-Guérin (BCG) failures; Joudi FN et al.; PURPOSE OF REVIEW: To give an update on the new modalities in treating patients with superficial bladder cancer who have failed bacille Calmette-Guerin . RECENT FINDINGS: The addition of interferon to bacille Calmette-Guerin has proven to be an effective combination therapy for bacille Calmette-Guerin failures . Electromotive intravesical mitomycin C as well as local microwave hyperthermia have been shown to improve drug delivery and increase response rates . Intravesical gemcitabine has shown some promising results in phase I studies and is being investigated in phase II trials . Photodynamic therapy is proposed as a second-line therapy for bacille Calmette-Guerin failures . SUMMARY: New treatment modalities are being introduced and existing ones improved to treat bacille Calmette-Guerin-refractory superficial bladder cancer . These agents need to be studied in large randomized trials . Until these agents prove to decrease recurrence rates and delay progression of high-risk superficial bladder cancer, cystectomy remains the standard of care for the patient who is a good surgical candidate and willing to undergo such major surgery.

Acta Crystallogr D Biol Crystallogr, 1997 Jul, 53(Pt 4), 477 - 9
Purification, crystallization and preliminary X-ray analysis of the M.BseCI DNA methyltransferase from Bacillus stearothermophilus; Athanasiadis A; The DNA methyltransferase M.BseC1 from B . stearothermophilus methylates the N6 atom of the 3' adenine in the sequence 5'-ATCGAT-3' . The 579-residue protein has been isolated and crystallized using seeding and microdialysis techniques . The crystals are monoclinic, space group P2(1) with cell dimensions a = 53.7, b = 85.7, c = 151.8 A and beta = 95.1 degrees, two molecules in the asymmetric unit and diffract to at least 2.5 A resolution.

Acta Crystallogr D Biol Crystallogr, 1995 Jul, 51(Pt 4), 575 - 89
Refined 3.2 A structure of glycosomal holo glyceraldehyde phosphate dehydrogenase from Trypanosoma brucei brucei; Vellieux FM; The three-dimensional crystal structure of the enzyme glyceraldehyde phosphate dehydrogenase from the kinetoplastid Trypanosoma brucei brucei has been determined at 3.2 A resolution from a 37% complete data set collected using the Laue method . The crystals used in the structure determination contain one and a half tetrameric enzyme molecules in the asymmetric unit, i.e . six identical subunits . Initial phasing was carried out by the method of molecular replacement using the refined coordinates of holo glyceraldehyde phosphate dehydrogenase from Bacillus stearothermophilus as a search model . The initial electron-density distribution, obtained from the molecular-replacement solution, was greatly improved by a procedure consisting of 36 cycles of iterative non-crystallographic density averaging . During the averaging procedure, the missing reflections (63% of the data) were gradually introduced as map-inversion structure factors . At completion of the procedure, the R-factor between averaged map-inversion amplitudes and observed structure-factor amplitudes was 19.0% for all data between 7.0 and 3.2 A resolution, and that between the map-inversion amplitudes and later recorded structure-factor amplitudes was 41.9% . After model building into the resulting averaged electron-density map, refinement by molecular-dynamics procedures with X-PLOR provided the current model, which has an R-factor of 17.6% for 34 835 reflections between 7.0 and 3.2 A resolution . The refined model, comprising 2735 protein atoms plus one NAD(+) molecule and two sulfate ions per subunit, has r.m.s . deviations from ideality of 0.02 A for bond lengths and 3.6 degrees for bond angles . All subunits, located either within the tetrameric molecule or within the half tetramer present in the asymmetric unit, are related to each other by almost exact twofold symmetry . The overall structure of the glycosomal glyceraldehyde phosphate dehydrogenase subunit and its quaternary arrangement in the tetrameric molecule are similar to that of the enzyme of lobster and Bacillus stearothermophilus (with r.m.s . differences between equivalent Calpha positions of 0.71 and 0.64 A, respectively) . The main differences between the structures is the presence of three insertions, plus the substitution of a beta-strand by a short alpha-helix, both occurring at the surface of the glycosomal enzyme subunit.

Acta Crystallogr D Biol Crystallogr, 1995 Sep, 51(Pt 5), 856 - 7
Crystallization and preliminary X-ray crystallographic analysis of chitosanase from Bacillus circulans MH-K1; Saito JI; Chitosanase, an enzyme which hydrolyzes chitosan, isolated from Bacillus circulans MH-K1, was crystallized by a vapor-diffusion procedure at 293 K using ammonium sulfate as a precipitant . Rod-shaped colorless crystals, which grew to 0.05 x 0.15 x 1.2 mm within a week, belong to the orthorhombic system and the space group P222(1) or P2(1)2(1)2 with unit-cell dimensions of a = 43.3, b = 57.7, and c = 128.0 A . The asymmetric unit is thought to contain one chitosanase molecule (29 024 Da) . The crystals diffract X-rays to at least 2.3 A resolution and are suitable for high-resolution X-ray structure analysis.

Acta Crystallogr D Biol Crystallogr, 1995 Sep, 51(Pt 5), 835 - 6
Crystallization and preliminary X-ray crystallographic studies of glutamic acid-specific proteinase from Bacillus licheniformis complex with Z-Leu-Glu-CH(2)Cl; Kitadokoro K; A glutamic acid specific proteinase from Bacillus licheniformis has been crystallized as a complex with the inhibitor Z-Leu-Glu-CH(2)Cl . Crystals were grown by the vapor-diffusion method using sodium formate as a precipitant . The crystals diffracted to about 2.0 A resolution and belonged to the trigonal space group P3(1)21 (P3(2)21) with unit-cell parameters a = b = 134.3, c = 109.7 A . A total of 26 964 independent reflections were obtained up to 2.2 A resolution, the merging R-factor being 0.05 for 42 614 measurements.

Acta Crystallogr D Biol Crystallogr, 1995 Sep, 51(Pt 5), 830 - 2
Crystallization of glycerol dehydrogenase from Bacillus stearothermophilus; Wilkinson KW; The NAD(+)-linked glycerol dehydrogenase from Bacillus stearothermophilus is a member of the so-called 'iron- containing' class of polyol dehydrogenases . This enzyme has been crystallized in three different forms in the presence of a range of divalent cations and glycerol or NAD+ using the hanging-drop method of vapour diffusion with ammonium sulfate as the precipitant . X-ray photographs have established that the crystals grown in the presence of zinc and glycerol (form A) most likely belong to space group I4(1)22 with cell parameters a = b = 102 and c = 728 A . The crystals grown with zlnc and NAD(+) (form B) belong to the tetragonal system and probably belong to the space group P42(1)2 with cell parameters a = b = 150 and c = 220 A . The crystals grown with lead and glycerol (form C) belong to a primitive orthorhombic system with cell parameters a = 127, b = 178 and c = 173 A . Experiments using the synchrotron radiation source at the DRAL Daresbury laboratory have shown all three crystal types diffract to at least 3 A resolution . Elucidation of the three-dimensional structure of this enzyme will provide a structural framework for this class of polyol dehydrogenases, which are not represented in the database at present, and enable comparisons to be made with enzymes belonging to the other classes.

Acta Crystallogr D Biol Crystallogr, 1996 May, 52(Pt 3), 543 - 50
E144S Active-Site Mutant of the Bacillus cereus Thermolysin-Like Neutral Protease at 2.8 A Resolution; Lister SA; The X-ray crystal structure of the Bacillus cereus neutral protease (CNP) active-site mutant E144S, in which the putative general base proposed for the thermolysin-like zinc neutral proteases, Glu144, has been replaced by serine, has been determined to a resolution of 2.8 A . This represents the first crystal structure of an active-site mutant of a zinc neutral protease . The E 144S mutant was crystallized in the hexagonal space group, P6(5)22, with unit-cell dimensions a = b = 76.57, c = 201.91 A . Although the ligands involved in zinc coordination in the active site are identical to those found in the wild-type protein, the mutation results in a modified environment around the zinc ion; particularly with respect to the water molecules . While the structure of the mutant is similar to that of wild type, its protease activity is reduced to 0.16% that of the wild-type CNP and the protein is virtually resistant to autolysis in the presence of calcium . The lowered protease activity of the mutant is consistent with the role proposed for Glu144 as the general base in the catalysis of thermolysin-like neutral proteases {Matthews (1988) . Acc . Chem . Res . 21, 333-340} . We suggest that the residual activity of the E144S mutant arises from a water molecule, which is found within hydrogen-bonding distance of Ser144, acting as a general base in the catalytic function of the mutant.

Acta Crystallogr D Biol Crystallogr, 1996 Jul, 52(Pt 4), 866 - 8
Crystallization and preliminary X-ray study of the 8-amino-7-oxopelargonate synthase from Bacillus sphaericus; Spinelli S; The 8-amino-7-oxopelargonate synthase (AOPS) cloned from Bacillus sphaericus, overproduced in Escherichia coli, has been crystallized in the pyridoxal 5'-phosphate (PLP)-bound form at pH 7.5, using polyethylene glycol as the precipitant . One crystal form corresponds to a tetragonal space group, with unit-cell dimensions a = b = 66, c = 181 A . These crystals do not diffract beyond 5 A, with conventional X-ray sources and cannot be used in the structure elucidation . A second crystal form is obtained when crystallization conditions are varied slightly by the addition of 0.2 M ammonium sulfate . The space group is P2(1)2(1)2(1), with unit-cell dimensions a = 68.9, b = 85.5, c = 125.9 A, indicating the presence of two molecules in the asymmetric unit (V(m) = 2.26 A(3) Da(-1); 46% water) . These crystals diffract X-rays up to 3.2 A using in-house facilities and a preliminary data set has been collected . A second data set using the synchrotron radiation source W32 at LURE (Paris) has shown the crystals to diffract to at least 3 A, resolution, with good statistics . The structure determination of AOPS will provide a structural framework for the other alpha-amino ketone synthases for which no three-dimensional structure is yet available.

Acta Crystallogr D Biol Crystallogr, 1996 Nov, 52(Pt 6), 1136 - 45
X-ray Structure of Cyclodextrin Glucano-transferase from Alkalophilic Bacillus Sp . 1011 . Comparison of Two Independent Molecules at 1.8 A Resolution; Harata K; Cyclodextrin glucanotransferase (CGTase) is an enzyme which produces cyclodextrins by the degradation of starch . The enzyme from alkalophilic Bacillus sp . 1011, consisting of 686 amino acid residues, was crystallized from the solution containing 20% PEG 3000 and 20% 2-propanol at pH 5.6 adjusted with citrate buffer . The space group was P1 and the unit cell contained two molecules (V(m) = 2.41 A(3) Da(-1)) . The structure was solved by the molecular replacement method and refined to a conventional R value of 0.161 (R(free) = 0.211) for the reflections in the resolution range 1.8-10 A by energy minimization combined with simulated annealing . The molecule consists of five domains, designated A-E, and its backbone structure is similar to the structure of other bacterial CGTases . The molecule has two calcium binding sites where calcium ions are coordinated by seven ligands, forming a distorted pentagonal bipyramid . The two independent molecules are related by a pseudotwofold symmetry and are superimposed with an r.m.s . deviation value of 0.32 A for equivalent C(alpha) atoms . Comparison of these molecules indicated the relatively large mobility of domains C and E with respect to domain A . The active site is filled with water molecules forming a hydrogen-bond network with polar side-chain groups . Two water molecules commonly found in the active center of both molecules link to several catalytically important residues by hydrogen bonds and participate in maintaining a similar orientation of side chains in the two independent molecules.

Acta Crystallogr D Biol Crystallogr, 1994 Mar, 50(Pt 2), 231 - 3
Crystallization and preliminary investigation of xylose isomerase from Bacillus coagulans; Rasmussen H; Xylose isomerase from Bacillus coagulans has been crystallized in two different crystal forms . One crystal form is in space group P2(1)2(1)2, cell dimensions a = 462, b = 165, c = 82 A . The other is in space group I422, cell dimensions a = b = 113, c = 153 A.

Acta Crystallogr D Biol Crystallogr, 1994 Mar, 50(Pt 2), 202 - 9
Structure of the ADP complex of the 3-phosphoglycerate kinase from Bacillus stearothermophilus at 1.65 A; Davies GJ; The structure of the ADP complex of the enzyme 3-phosphoglycerate kinase (PGK, E.C . 2.7.2.3) from Bacillus stearothermophilus NCA-1503 has been determined by the method of molecular replacement . The structure has been refined to an R factor of 0.16 for all data between 10.0 and 1.65 A resolution, using data collected on the Hendrix-Lentfer imaging plate at the EMBL outstation in Hamburg . The r.m.s . deviations from stereochemical ideality are 0.010 and 0.011 A for bonds and planes, respectively . Although crystallized in the presence of the nucleotide product MgATP, the high-resolution structure reveals the bound nucleotide to be MgADP reflecting the low intrinsic ATPase activity of PGK . Although the two domains of this enzyme are found to be some 4.5 degrees closer together than is found in the yeast and horse-muscle apo-enzyme structures, this structure represents the 'open' rather than the 'closed', catalytically competent form, of the enzyme.

Acta Crystallogr D Biol Crystallogr, 1994 Jul, 50(Pt 4), 650 - 6
Packing selection of Bacillus lentus subtilisin and a site-specific variant; Dauberman JL; The crystallization of a variant of Bacillus lentus subtilisin and the native enzyme was achieved using identical conditions . The variant B . lentus was found to grow in two crystal forms, form 1 and form 2, whereas the native B . lentus subtilisin enzyme crystallized in only one, form 1 . Form 2 crystals, once obtained, were found to grow much more rapidly than form 1 crystals . The lattice contacts and structural changes giving both crystal forms have been examined . The results show that crystal form 2 has a more complex network of interactions . There is also a small surface conformational change in the form 2 structure relative to the native and variant form 1 crystals and at least two solvent molecules bound to the enzyme in crystal form 1 are displaced in crystal form 2 . In addition, a site specific substitution in the variant at position 27 induces a 'short' lattice contact which does not exist in the native B . lentus or the form 2 variant B . lentus . These results suggest that in some circumstances engineered variants could be designed to crystallize more rapidly than the native enzyme.

Acta Crystallogr D Biol Crystallogr, 1994 Jul, 50(Pt 4), 572 - 90
Quantitative analysis in the characterization and optimization of protein crystal growth; Carter CW CW; Protein crystal growth often depends on the combination of many different factors . Some affect protein solubility directly; others may act indirectly by causing conformational changes . Systematic characterization of these factors can be important for generating good crystals . It can also provide useful insight into the biochemical behavior of the protein being crystallized . Here we focus on statistical methods to achieve these two objectives . (1) Characterization of a protein system by analyzing patterns of crystal polymorphism under different levels of biochemical parameters, such as ligands and pH . Tests of the reproducibility of crystal growth experiments indicate that quantitative scales of crystal quality can be statistically significant . Analysis of variance for a replicated, full-factorial design in which four factors were tested at two levels has been used to demonstrate highly significant, biochemically relevant, two-factor interactions strongly implicating pH and ligand-dependent conformational changes . (2) Optimization of crystal growth via response-surface methods . 'Minimum predicted variance' designs provide for efficient response-surface experiments aimed at constructing quadratic models in several dimensions . We have used such models to improve crystal size and quality significantly for three forms of Bacillus stearothermophilus tryptophanyl-tRNA synthetase . In one case we can now avoid having to increase the size by repeated seeding, a difficult procedure that also produces unwanted growth of satellite crystals . Graphs of two-dimensional level surfaces reveal a number of ridges, where the same result is obtained for many combinations of the factors usually varied when trying to improve crystals . An important inference is that it may be better to sample simultaneously for the effects of protein concentration and supersaturation . For a system involving only one crystallizing agent, supersaturation can be approximated as the product of protein and precipitant concentrations . Use of this search direction significantly improves the performance of response-surface experiments . Advantages of growing crystals at stationary points of their response surfaces include better crystals and higher reproducibility, since crystal growth at stationary points is insulated from the deleterious effects of experimental fluctuations . This arises because the derivatives of the response are by definition zero with respect to the experimental variables . Quantitative analysis of appropriately designed crystal growth experiments can thus be a powerful way to characterize complex and interacting biochemical dependencies in macromolecular systems and optimize parameters important to the crystallography.

Acta Crystallogr D Biol Crystallogr, 1994 Sep, 50(Pt 5), 790 - 2
Crystallization and preliminary X-ray diffraction studies of bacterial ribosomal protein L14; Davies C; Based on amino-acid sequence homology, it is predicted that ribosomal protein L14 is a member of a recently identified family of structurally related RNA-binding proteins . To verify this, the gene for Bacillus stearothermophilus L14 has been cloned, and the protein has been purified and crystallized . The crystals are in space group C2 with cell dimensions a = 67.0, b = 32.7, c = 49.4 A, and beta = 101.8 degrees, and there is one molecule in the asymmetric unit (V(m) = 2.0 A(3) Da(-1)) . They are of high quality, and a native data set has been collected to a resolution of 1.6 A, with an R(merge) of 5.3%.

Acta Crystallogr D Biol Crystallogr, 1995 Mar, 51(Pt 2), 199 - 206
Structure of a new alkaline serine protease (M-protease) from Bacillus sp . KSM-K16; Yamane T; An alkaline serine protease, M-protease, from Bacillus sp . KSM-K16 has been crystallized . Two morphologically different crystal forms were obtained . Crystal data of form 1: space group P2(1)2(1)2(1), a = 47.3, b = 62.5, c = 75.6 A, V = 2.23 x 10(5) A(3), Z = 4 and V(m) = 2.09 A(3) Da(-1) . Crystal data of form 2: space group P2(1)2(1)2(1), a = 75.82 (2), b = 57.79 (2), c = 54.19 (1) A, V = 2.29 (2) x 10(5) A(3), Z = 4 and V(m) = 2.15 A(3) Da(-1) . The crystal structure of M-protease in form 2 has been solved by molecular replacement using the atomic model of subtilisin Carlsberg (SBC) which is 60% homologous with M-protease, and refined to the crystallographic R-factor of 0.189 for 7004 reflections with F(o)/sigma(F) > 3 between 7 and 2.4 A resolution . The final model of M-protease contains 1882 protein atoms, two calcium ions and 44 water molecules . The three-dimensional structure of M-protease is essentially similar to other subtilisins of known structure . The 269 C(alpha) positions of M-protease have an r.m.s . difference of 1.06 A with the corresponding positions of SBC . The crystal data of form 2 are close to those of SBC, though the structure determination of form 2 made it clear that it is not isomorphous to the crystal structure of SBC . The deletions of amino acids occur at the residues 36' and 160'-163' compared with SBC (numerals with primes show the numbering for SBC) . The deletion of the four residues (160'-163') may significantly affect the lack of isomorphism between M-protease and SBC.

Cancer Epidemiol Biomarkers Prev, 2004 Aug, 13(8), 1385 - 91
Serologic assessment of type 1 and type 2 immunity in healthy Japanese adults; Birmann BM et al.; We assessed the informativeness of several serologic biomarkers of immune function using serum specimens collected in the Miyazaki Cohort Study from subjects who were seronegative for anti-human T-cell lymphotrophic virus I and anti-hepatitis C virus . To broadly characterize type 1 immune status, we measured EBV antibody titers, because titer profiles associated with cellular immune suppression are well described . We also tested for three type 2 biomarkers: total serum IgE, soluble CD23, and soluble CD30 . Nonreactivity to a tuberculin purified protein derivative (PPD) skin test is indicative of diminished delayed-type hypersensitivity (type 1) responsiveness in the study population due to a history of tuberculosis exposure or Bacillus Calmette-Guerin vaccination . We therefore evaluated the serologic markers as predictors of PPD nonreactivity using logistic regression . Subjects whose EBV antibody profiles were consistent with deficient type 1 immunity were more than thrice as likely to be PPD nonreactive as persons with "normal" antibody titers . Elevated total IgE was also strongly associated with PPD nonreactivity (odds ratio 3.4, 95% confidence interval 1.2-9.9); elevated soluble CD23 had a weaker, but positive, odds ratio, whereas soluble CD30 levels were not predictive of PPD status . Therefore, PPD nonreactivity is associated, in this population, with a pattern of serum biomarkers that is indicative of diminished type 1 and elevated type 2 immunity . We conclude that, with the exception of soluble CD30, the serologic markers are informative for the characterization of type 1/type 2 immune status using archived sera from study populations of healthy adults.

Bull World Health Organ, 2004 May, 82(5), 373 - 80
The future incidence of leprosy: a scenario analysis; Meima A et al.; OBJECTIVE: To investigate the impact of the current strategy for the elimination of leprosy on its incidence and to assess the consequences of failure to sustain this strategy . METHODS: Scenarios for assessing the impact of the elimination strategy were implemented in a computer simulation program . The scenarios reflected the assumptions made regarding contagiousness, transmission and bacille Calmette-Guerin (BCG) vaccination . The trend in case detection rate for the main countries in which leprosy was endemic during 1985-98 was fitted, and incidence up to 2020 was projected . FINDINGS: Owing to the gradual shortening of delays in detection up to 1998, and because of the low relapse rate that occurs with multidrug treatment MDT, incidence is predicted to decrease beyond 2000 in all scenarios . The annual decline was a few per cent higher when favourable assumptions were made about protection and coverage of BCG vaccination . Overall, the predicted annual decline in incidences ranged from 2% to 12% . CONCLUSION: The elimination strategy reduces transmission, but the decline may be slow . Relaxation of control after 2005 is unjustified given the uncertainty about the rate of decline and the adverse effects of longer delays in detection . A long-term strategy for leprosy control should be adopted.

Curr Microbiol, 2004 Jul, 49(1), 22 - 7
Competition of Bacillus thuringiensis Cry1 toxins for midgut binding sites: a basis for the development and management of transgenic tropical maize resistant to several stemborers; Rang C et al.; Binding and competition of five Bacillus thuringiensis toxins--Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ca, and Cry1Ea--for midgut binding sites from three pests, Spodoptera frugiperda, Diatraea saccharalis, and Diatraea grandiosella, were investigated as part of a strategy to develop tropical transgenic maize resistant to several stemborers . On S . frugiperda, Cry1Ab and Cry1Ac compete for the same binding site; Cry1Ba and Cry1Ca compete for a second binding site . Cry1Ea recognizes a third specific binding site in S . frugiperda and does not compete with any of the other toxins . On D . grandiosella and D . saccharalis, Cry1Ac competes with Cry1Ab and not with Cry1Ba and Cry1Ca . Cry1Ba and Cry1Ca recognize each a specific binding site and do not compete with any of the other four toxins . Cry1Ea does not recognize any binding site on Diatraea species . Combinations of toxins are proposed to develop transgenic maize resistant to the three stemborers while allowing resistance management.

J Clin Microbiol, 2004 Aug, 42(8), 3675 - 80
Population genetic analysis of Bartonella bacilliformis isolates from areas of peru where Carrion's disease is endemic and epidemic; Hambuch TM et al.; Carrion's disease is caused by infection with the alpha-proteobacterium Bartonella bacilliformis . Distribution of the disease is considered coincident with the distribution of its known vector, the sand fly Lutzomyia verrucarum . Recent epidemics of B . bacilliformis infections associated with atypical symptomatology in nonendemic regions have raised questions regarding the historic and present distribution of this bacterium and the scope of disease that infection causes . Phylogenetic relationships and genomic diversity of 18 B . bacilliformis isolates (10 isolates from a region where Carrion's disease is epidemic, Cuzco, Peru, and 8 isolates from a region where Carrion's disease is endemic, Caraz, Peru) were assessed using genomic data generated by infrequent restriction site PCR and gene sequence analysis of the flagellin gltA and ialB genes . A population genetic analysis of the genomic diversity suggests that what was once considered an epidemic region of Peru did not result from the recent introduction of B . bacilliformis.

Vaccine, 2004 Aug 13, 22(23-24), 3061 - 8
Recombinant cholera toxin B subunit (rCTB) as a mucosal adjuvant enhances induction of diphtheria and tetanus antitoxin antibodies in mice by intranasal administration with diphtheria-pertussis-tetanus (DPT) combination vaccine; Isaka M et al.; Recombinant cholera toxin B subunit (rCTB) which is produced by Bacillus brevis carrying pNU212-CTB acts as a mucosal adjuvant capable of enhancing host immune responses specific to unrelated, mucosally co-administered vaccine antigens . When mice were administered intranasally with diphtheria-pertussis-tetanus (DPT) combination vaccine consisting of diphtheria toxoid (DTd), tetanus toxoid (TTd), pertussis toxoid (PTd), and formalin-treated filamentous hemagglutinin (fFHA), the presence of rCTB elevated constantly high values of DTd- and TTd-specific serum ELISA IgG antibody titres, and protective levels of diphtheria and tetanus toxin-neutralizing antibodies but the absence of rCTB did not . Moreover, the addition of rCTB protected all mice against tetanic symptoms and deaths . DPT combination vaccine raised high levels of serum anti-PT IgG antibody titres regardless of rCTB and protected mice from Bordetella pertussis challenge . These results suggest that co-administration of rCTB as an adjuvant is necessary for induction of diphtheria and tetanus antitoxin antibodies on the occasion of intranasal administration of DPT combination vaccine.

Biotechnol Prog, 2004 Jul-Aug, 20(4), 1267 - 9
Enzymatic hydrolysis of gelatin layers on used lith film using thermostable alkaline protease for recovery of silver and PET film; Masui A et al.; To develop a new efficient and potential industrial enzymatic process for the recovery of silver and poly(ethylene terephthalate) (PET) from used lith film for printing, which has not been recycled at all, enzymatic hydrolysis of gelatin layers on lith film was investigated using the thermostabilized mutant enzyme of the alkaline protease from alkaliphilic Bacillus sp . B21-2 . The rate of gelatin hydrolysis of lith film in a stirred-tank reactor increased with the temperature and enzyme concentration . The time required to complete the hydrolysis of gelatin on lith film was longer than that on X-ray film because of the tightly cross-linked structure of the gelatin layers of lith film . The time required to complete the hydrolysis by using the mutant enzyme was less than that using the wild-type enzyme . The gelatin hydrolysis of lith film was well explained by a model that took into consideration a number of physical processes in addition to the chemical process.

Pediatr Infect Dis J, 2004 Aug, 23(8), 784 - 6
Calmette-Guérin bacillus sternal osteomyelitis diagnosed by DNA sequencing analysis of PNC A; Lin WJ et al.; Sternal osteomyelitis is an uncommon complication of Calmette-Guerin bacillus vaccination . Herein we describe a 4-month-old Taiwanese infant with a growing parasternal mass resulting from sternal osteomyelitis . By using DNA sequencing analysis, we identified the etiology as Calmette-Guerin bacillus vaccination.

Appl Environ Microbiol, 2004 Aug, 70(8), 4889 - 98
Binary toxins from Bacillus thuringiensis active against the western corn rootworm, Diabrotica virgifera virgifera LeConte; Baum JA et al.; The western corn rootworm, Diabrotica virgifera virgifera LeConte, is a significant pest of corn in the United States . The development of transgenic corn hybrids resistant to rootworm feeding damage depends on the identification of genes encoding insecticidal proteins toxic to rootworm larvae . In this study, a bioassay screen was used to identify several isolates of the bacterium Bacillus thuringiensis active against rootworm . These bacterial isolates each produce distinct crystal proteins with approximate molecular masses of 13 to 15 kDa and 44 kDa . Insect bioassays demonstrated that both protein classes are required for insecticidal activity against this rootworm species . The genes encoding these proteins are organized in apparent operons and are associated with other genes encoding crystal proteins of unknown function . The antirootworm proteins produced by B . thuringiensis strains EG5899 and EG9444 closely resemble previously described crystal proteins of the Cry34A and Cry35A classes . The antirootworm proteins produced by strain EG4851, designated Cry34Ba1 and Cry35Ba1, represent a new binary toxin . Genes encoding these proteins could become an important component of a sustainable resistance management strategy against this insect pest.

Appl Environ Microbiol, 2004 Aug, 70(8), 4784 - 91
Characterization of two Bacillus thuringiensis genes identified by in vivo screening of virulence factors; Fedhila S et al.; Bacillus thuringiensis vegetative cells are known to be highly pathogenic when injected into the hemocoel of susceptible insect larvae . This pathogenicity is due to the capacity of B . thuringiensis to cause septicemia in the host . We screened a B . thuringiensis mini-Tn10 insertion library for loss of virulence against Bombyx mori larvae on injection into the hemocoel . Three clones with attenuated virulence were isolated, corresponding to two different mini-Tn10 insertions mapping to the yqgB/yqfZ locus . Single disruptions of the yqgB and yqfZ genes did not affect virulence against B . mori . In contrast, the inactivation of both genes simultaneously reproduced the effect of the mini-Tn10 insertion and resulted in a significant delay to infection . The double DeltayqgB DeltayqfZ mutant was also nonmotile, and its growth was affected at 25 degrees C . We analyzed lacZ transcriptional fusions and detected promoter activity upstream from yqgB at 25 and 37 degrees C . Overall, our findings suggest that the yqgB and yqfZ genes encode adaptive factors that may act in synergy, enabling the bacteria to cope with the physical environment in vivo, facilitating colonization of the host.

Appl Environ Microbiol, 2004 Aug, 70(8), 4522 - 31
Purification and characterization of chitosanase from Bacillus sp . strain KCTC 0377BP and its application for the production of chitosan oligosaccharides; Choi YJ et al.; For the enzymatic production of chitosan oligosaccharides from chitosan, a chitosanase-producing bacterium, Bacillus sp . strain KCTC 0377BP, was isolated from soil . The bacterium constitutively produced chitosanase in a culture medium without chitosan as an inducer . The production of chitosanase was increased from 1.2 U/ml in a minimal chitosan medium to 100 U/ml by optimizing the culture conditions . The chitosanase was purified from a culture supernatant by using CM-Toyopearl column chromatography and a Superose 12HR column for fast-performance liquid chromatography and was characterized according to its enzyme properties . The molecular mass of the enzyme was estimated to be 45 kDa by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The enzyme demonstrated bifunctional chitosanase-glucanase activities, although it showed very low glucanase activity, with less than 3% of the chitosanase activity . Activity of the enzyme increased with an increase of the degrees of deacetylation (DDA) of the chitosan substrate . However, the enzyme still retained 72% of its relative activity toward the 39% DDA of chitosan, compared with the activity of the 94% DDA of chitosan . The enzyme produced chitosan oligosaccharides from chitosan, ranging mainly from chitotriose to chitooctaose . By controlling the reaction time and by monitoring the reaction products with gel filtration high-performance liquid chromatography, chitosan oligosaccharides with a desired oligosaccharide content and composition were obtained . In addition, the enzyme was efficiently used for the production of low-molecular-weight chitosan and highly acetylated chitosan oligosaccharides . A gene (csn45) encoding chitosanase was cloned, sequenced, and compared with other functionally related genes . The deduced amino acid sequence of csn45 was dissimilar to those of the classical chitosanase belonging to glycoside hydrolase family 46 but was similar to glucanases classified with glycoside hydrolase family 8.

Protein Expr Purif, 2004 Sep, 37(1), 32 - 8
Purification of ArcR, an oxidation-sensitive regulatory protein from Bacillus licheniformis; Wohlkonig A et al.; In Bacillus licheniformis, ArcR, a transcriptional activator of the Crp/Fnr family, is required for expression of the anaerobic pathway of arginine catabolism, the arginine deiminase pathway . The method described here allows the purification of milligram quantities of functional ArcR from a recombinant Escherichia coli strain . The solubility properties of ArcR were much exploited during the purification process . The protein appeared highly sensitive to oxidation . Oxidation-induced precipitation of the protein was attributed to the formation of intermolecular disulfide bridges . Alkylation of mutant proteins with single substitutions showed that both cysteine residues of the protein, C178 and C205, are involved in formation of the disulfide bridges . Substitution of both cysteines yielded a functional protein insensitive to oxidation and able to form a complex with its cognate target on the DNA.

Protein Expr Purif, 2004 Sep, 37(1), 27 - 31
Two-step purification of Bacillus circulans chitinase A1 expressed in Escherichia coli periplasm; Chen CT et al.; A protein purification procedure was developed to efficiently and effectively purify the target enzyme, chitinase A1 of Bacillus circulans WL-12, from Escherichia coli DH5alpha carrying the chiA gene with its natural promoter in the plasmid pNTU110 . Chitinase A1 was purified to apparent homogeneity from E . coli periplasm with a final recovery of 90.6% . Two main steps were included in this protein purification procedure, ammonium sulfate precipitation (40% saturation) and anion-exchange chromatography at pH 6.0 using Q Ceramic HyperD column . The yield of chitinase A1 was estimated at 95 microg/L . A polyclonal antibody against chitinase A1 was raised by immunizing BALB/c mice with chitinase A1 purified from E . coli DH5alpha(pNTU110) . As indicated by Western blot analysis, a 3000-fold diluted antibody detected purified chitinase A1 from E . coli DH5alpha(pNTU110) in an amount of at least 1 ng and specifically detected chitinase A1 produced by B . circulans WL-12.

Spectrochim Acta A Mol Biomol Spectrosc, 2004 Sep, 60(11), 2517 - 21
Detection of Bacillus endospores using total luminescence spectroscopy; Smith CB et al.; Detection and analysis of bacteria from environmental samples (e.g . water, air, and food) are usually accomplished by standard culture techniques or by analyses that target specific DNA sequences, antigens or chemicals . For large cell numbers in aqueous suspensions, an alternative technique that has proven useful is total luminescence spectroscopy (TLS) . TLS is the acquisition of fluorescence data that records the unique excitation-emission matrix (EEM) of compound fluorophores . Past work has shown that one type of bacterial endospore, Bacillus megaterium, possessed a distinct EEM pattern useful for differentiating it in complex biological fluids and suspensions . The work described here extends those observations to establish some limits on the sensitivity and specificity of TLS for the detection and analysis of bacterial endospores versus (bacterial) vegetative cells in aqueous culture . Our findings show Bacillus endospores exhibit a dramatic blue shift of 130 nm in excitation and a smaller shift of 50 nm in emission when compared to ancillary endospore and non-endospore forming bacterial cells.

East Afr Med J, 2004 Mar, 81(3), 124 - 9
Missed opportunities and inappropriately given vaccines reduce immunisation coverage in facilities that serve slum areas of Nairobi; Borus PK; OBJECTIVES: To quantify missed opportunities for immunisation, document reasons for their occurrence and evaluate the extent of inappropriately given vaccine doses . DESIGN: A cross sectional study of children under two years of age attending health facilities . SETTING: Six health facilities predominantly serving the slums of Nairobi . Methodology: Information on vaccination was extracted from child immunisation cards as well as from mothers or guardians of children . RESULTS: Effective immunisation coverage for Bacille-Callmette Guerin (BCG) was 91% . Coverage for the birth dose, first, second, and third doses of oral polio vaccine (OPV0, OPVI, OPV2, and OPV3) was 44%, 83%, 79% and 75% respectively . Effective coverage for first, second and third doses of diphtheria-pertusis-tetanus (DPTI, DPT2 and DPT3) vaccine was 88%, 87% and 85% respectively . Measles coverage was 80% . Immunisation coverage for all antigens except OPV0 and OPV3 would have been increased to over 90% had missed immunisation opportunities and inappropriately administered vaccination been avoided . There would have been an 11% increase in OPV3 coverage to 86% . Increases in coverage for OPVI and OPV2 would have been 16% and 18% respectively . Coverage would have increased by 10% for diphtheria pertusistetanus (DPT) doses DPTI and DPT2, and 7% for DPT3 . Measles immunisation coverage would have increased by 19% had missed immunisation opportunities and inappropriately administered vaccinations been avoided . The overall missed opportunities rate was 3% . The proportions of missed opportunities were higher for the OPV series than DPT series . CONCLUSION: Missed immunisation opportunities among clinic attendees in Nairobi occur and routine supervision should be strengthened in these health facilities in order to minimise such missed opportunities and inappropriately administered vaccines.

Microbiol Res, 2004, 159(2), 135 - 40
Laundry detergent compatibility of the alkaline protease from Bacillus cereus; Banik RM et al.; The endogenous protease activity in various commercially available laundry detergents of international companies was studied . The maximum protease activity was found at 50 degrees C in pH range 10.5-11.0 in all the tested laundry detergents . The endogenous protease activity in the tested detergents retained up to 70% on incubation at 40 degrees C for 1 h, whereas less than 30% activity was only found on incubation at 50 degrees C for 1 h . The alkaline protease from an alkalophilic strain of Bacillus cereus was studied for its compatibility in commercial detergents . The cell free fermented broth from shake flask culture of the organism showed maximum activity at pH 10.5 and 50 degrees C . The protease from B . cereus showed much higher residual activity (more than 80%) on incubation with laundry detergents at 50 degrees C for 1 h or longer . The protease enzyme from B . cereus was found to be superior over the endogenous proteases present in the tested commercial laundry detergents in comparison to the enzyme stability during the washing at higher temperature, e.g., 40-50 degrees C.

Microbiol Res, 2004, 159(2), 129 - 33
Bme585 I {5'-CCCGC(4/6)-3'}, a new isoschizomer of restriction endonuclease Fau I, isolated from a strain of Bacillus mesentericus; Davalieva K et al.; Bme585 I is a new member of the restriction endonuclease type IIS family . It was partially purified from the heterothrophic, mesophilic bacterial strain Bacillus mesentericus 585 by ammonium sulphate precipitation and phosphocellulose column chromatography . Bme585 I is a monomeric protein with a molecular mass of 62 kD . The enzyme is active over a broad pH range from 7.0 to 8.8, has a temperature optimum of 37 degrees C and tolerance of NaCl in reaction buffer from 0 to 400 mM . Bme585 I recognizes the asymmetric sequence 5'-CCCGC(4/6)-3' and is therefore an isoschizomer of restriction endonuclease Fau I.

Appl Microbiol Biotechnol, 2004 Aug, 65(2), 177 - 82 Epub 2004 Feb 24.
A cloned Bacillus halodurans multicopper oxidase exhibiting alkaline laccase activity; Ruijssenaars HJ et al.; The gene product of open reading frame bh2082 from Bacillus halodurans C-125 was identified as a multicopper oxidase with potential laccase activity . A homologue of this gene, lbh1, was obtained from a B . halodurans isolate from our culture collection . The encoded gene product was expressed in Escherichia coli and showed laccase-like activity, oxidising 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid), 2,6-dimethoxyphenol and syringaldazine (SGZ) . The pH optimum of Lbh1 with SGZ is 7.5-8 (at 45 degrees C) and the laccase activity is stimulated rather than inhibited by chloride . These unusual properties make Lbh1 an interesting biocatalyst in applications for which classical laccases are unsuited, such as biobleaching of kraft pulp for paper production.

J Natl Cancer Inst, 2004 Aug 4, 96(15), 1128 - 32
Randomized trial of adjuvant therapy in colon carcinoma: 10-year results of NSABP protocol C-01; Smith RE et al.; BACKGROUND: The National Surgical Adjuvant Breast and Bowel Project C-01 trial reported in 1988 that, for patients with adenocarcinoma of the colon, compared with surgery alone, 1) postoperative chemotherapy with 1-(2-chloroethyl)-3-(4-trans-methylcyclohexyl)-1-nitrosourea (i.e., MeCCNU or semustine), vincristine, and 5-fluorouracil was associated with better 5-year disease-free and overall survival and 2) postoperative immunotherapy with bacillus Calmette-Guerin was associated with better 5-year overall, but not disease-free, survival . We now provide a 10-year update of this trial . METHODS: Between November 11, 1977, and February 28, 1983, 1166 patients with resected Dukes' stage B and C adenocarcinoma of the colon were stratified by Dukes' stage, sex, and age (<65 years or > or =65 years) and then randomly assigned to receive no further treatment (surgery alone; 394 patients), adjuvant chemotherapy (379 patients), or adjuvant immunotherapy (393 patients) . Those eligible for follow-up included 375 (95.2%) patients in the surgery-alone group, 349 (92.1%) patients in the adjuvant-chemotherapy group, and 372 (94.7%) patients in the adjuvant-immunotherapy group . All statistical tests were two-sided . RESULTS: No difference was observed between patients in the chemotherapy group and those in the surgery-alone group in 10-year disease-free survival (hazard ratio {HR} = 1.14, 95% confidence interval {CI} = 0.94 to 1.39;P =.17) or overall survival (HR = 1.12, 95% CI = 0.91 to 1.38; P=.27) . Immunotherapy did not appear to prevent tumor relapse after 10 years (for surgery alone versus immunotherapy, relative risk {RR} = 0.99, 95% CI = 0.78 to 1.25; P =.93) but had a beneficial effect on 10-year overall survival (for surgery alone versus immunotherapy, RR = 1.27, 95% CI = 1.03 to 1.56; P =.02) that apparently results from a reduction in deaths associated with comorbidities in the immunotherapy group . CONCLUSION: The disease-free and overall survival benefit associated with chemotherapy in this patient population is of limited duration, disappearing after 10 years.

BJU Int, 2004 Aug, 94(3), 311 - 6
Superficial papillary urothelial carcinomas in young and elderly patients: a comparative study; Migaldi M et al.; OBJECTIVE: To compare the clinicopathological and immunohistochemical findings of superficial papillary transitional cell carcinomas in "young" and "elderly" patients, as the natural history and prognosis of bladder tumours in young patients remains a matter of debate . PATIENTS AND METHODS: Tumours from 50 patients with superficial urothelial tumours of the bladder diagnosed before 45 years old ("young" group, follow-up 25-119 months) were compared with 90 similar tumours developed in patients aged >55 years ("elderly", follow-up 24-102 months) . All the patients had a transurethral resection with curative intent, and none had received any therapy before surgery . After surgery only patients diagnosed with pT1 tumours were treated by intravesical bacille Calmette-Guerin (BCG) instillations; all received intravesical BCG if there was a recurrence . The clinicopathological variables, recurrence and disease-free interval to recurrence were assessed . Proliferative activity (MIB-1) and expression of cell-cycle regulation proteins cyclin D1, p53 and p27(kip1) were detected by immunohistochemistry in the tumours of both groups . RESULTS There were statistically significant differences in tumour grade, stage and occurrence between the "young" and "elderly" groups . The 'young' group had a longer disease-free interval to recurrence . Among the immunohistochemical markers analysed, only MIB-1 and cyclin D1 were associated with an increased risk of recurrence in the "young" group (P < 0.04 and <0.01, respectively) in a univariate analysis . CONCLUSIONS Superficial papillary urothelial tumours of the bladder in "young" patients had a better prognosis than those in the "elderly" group, showing a lower grade and stage at diagnosis, and a lower recurrence rate . Proliferative activity and cyclin D1 expression levels were of prognostic significance for the risk of recurrence in these patients.

Int J Biometeorol, 2005 Jan, 49(3), 167 - 78 Epub 2004 Jul 29.
Climate factors influencing bacterial count in background air samples; Harrison RM et al.; Total (as opposed to culturable) bacterial number counts are reported for four sites in the United Kingdom measured during campaigns over four separate seasons . These are interpreted in relation to simple climatic factors, i.e . temperature, wind speed and wind direction . Temperature has a marked effect at all four sites with data for a rural coastal site conforming best to a simple exponential model . Data for the other rural and urban locations show a baseline similar to that determined at the coastal rural location, but with some very significant positive excursions . The temperature dependence of bacterial number is found to conform to that typical of bacterial growth rates . At the coastal rural location, bacterial numbers normalised for temperature show no dependence on wind speed whilst at the inland sites there is a decrease with increasing wind speed of the form expected for a large area source . Only one site appeared to show a systematic relationship of bacterial concentrations to wind direction that being a site in the suburbs of Birmingham with highest number concentrations observed on a wind sector approaching from the city centre . PCR techniques have been used to identify predominant types of bacteria and results are presented which show that Bacillus was the dominant genus observed at the three inland sites during the winter and summer seasons . Pseudomonas appeared with comparable frequency at certain sites and seasons . There was in general a greater diversity of bacteria at the coastal site than at the inland sites.

Appl Microbiol Biotechnol, 2005 Jan, 66(4), 434 - 42 Epub 2004 Jul 29.
Enrichment of chitinolytic microorganisms: isolation and characterization of a chitinase exhibiting antifungal activity against phytopathogenic fungi from a novel Streptomyces strain; Hoster F et al.; Thirteen different chitin-degrading bacteria were isolated from soil and sediment samples . Five of these strains (SGE2, SGE4, SSL3, MG1, and MG3) exhibited antifungal activity against phytopathogenic fungi . Analyses of the 16S rRNA genes and the substrate spectra revealed that the isolates belong to the genera Bacillus or Streptomyces . The closest relatives were Bacillus chitinolyticus (SGE2, SGE4, and SSL3), B . ehimensis (MG1), and Streptomyces griseus (MG3) . The chitinases present in the culture supernatants of the five isolates revealed optimal activity between 45 degrees C and 50 degrees C and at pH values of 4 (SSL3), 5 (SGE2 and MG1), 6 (SGE4), and 5-7 (MG3) . The crude chitinase preparations of all five strains possessed antifungal activity . The chitinase of MG3 (ChiIS) was studied further, since the crude enzyme conferred strong growth suppression of all fungi tested and was very active over the entire pH range tested . The chiIS gene was cloned and the gene product was purified . The deduced protein consisted of 303 amino acids with a predicted molecular mass of 31,836 Da . Sequence analysis revealed that ChiIS of MG3 is similar to chitinases of Streptomyces species, which belong to family 19 of glycosyl hydrolases . Purified ChiIS showed remarkable antifungal activity and stability.

Microbiology, 2004 Aug, 150(Pt 8), 2699 - 705
Comparison of cytotoxin cytK promoters from Bacillus cereus strain ATCC 14579 and from a B . cereus food-poisoning strain; Brillard J et al.; The cytotoxin CytK produced by Bacillus cereus is believed to be involved in food-borne diseases . The transcriptional activity of the cytK promoter region in a food-poisoning strain was studied using a reporter gene and compared with that in the reference B . cereus strain ATCC 14579 . In the food-poisoning strain, cytK is more strongly transcribed, possibly explaining the pathogenicity . The global regulator PlcR in B . cereus controls several putative virulence factors . It was found that PlcR regulates cytK in this clinical strain despite a mismatch in the PlcR recognition site, as currently defined . This suggests that the PlcR box consensus should be reconsidered and that the PlcR regulon might be larger than suspected . It is also shown that the high level of cytK transcription is not caused by a modification in the PlcR recognition site.

Microbiology, 2004 Aug, 150(Pt 8), 2689 - 97
Genetic and functional analysis of the cytK family of genes in Bacillus cereus; Fagerlund A et al.; CytK is a pore-forming toxin of Bacillus cereus that has been linked to a case of necrotic enteritis . PCR products of the expected size were generated with cytK primers in 13 of 29 strains . Six strains were PCR-positive for the related gene hly-II, which encodes haemolysin II, a protein that is 37 % identical to the original CytK . Five of the strains were positive for both genes . The DNA sequences of putative cytK genes from three positive strains were determined, and the deduced amino acid sequences were 89 % identical to that of the original CytK . The authors have designated this new cytK variant cytK-2, and refer to the original cytK as cytK-1 . The CytK-2 proteins from these three strains were isolated, and their identity was verified by N-terminal sequencing . blast analysis using the cytK-2 gene sequences revealed very high homology with two cytK-2 sequences in the genomes of B . cereus strains ATCC 14579 and ATCC 10987 . The differences between CytK-1 and the CytK-2 proteins were clustered to certain regions of the proteins . The isolated CytK-2 proteins were haemolytic and toxic towards human intestinal Caco-2 cells and Vero cells, although their toxicity was about 20 % of that of CytK-1 . Both native and recombinant CytK-2 proteins from B . cereus 1230-88 were able to form pores in planar lipid bilayers, but the majority of the channels observed were of lower conductance than those created by CytK-1 . It is likely that CytK-2 toxins contribute to the enterotoxicity of several strains of B . cereus, although not all of the CytK-2 toxins may be as harmful as the CytK-1 originally isolated.

Br J Haematol, 2004 Aug, 126(4), 517 - 26
Tolerance associated with cord blood transplantation may depend on the state of host dendritic cells; Liu E et al.; Allogeneic cord blood (CB) transplantation is associated with less severe graft-versus-host disease (GvHD), thought to be due to the immaturity of CB T cells, but how T cells interact with host and donor-derived dendritic cells (DCs) to initiate GvHD has not been elucidated . We therefore investigated the responses of CB and adult blood CD4(+) T cells co-cultured with adult host DCs of different maturities . Primed by adult host DCs, CB and adult blood CD4(+) T cells underwent similar changes in the expression of CD45RA/45RO, CD25, CD40L and CTLA-4 . However, CB CD4(+) T cells, when primed by either immature or Bacillus Calmette-Guerin mycobacteria-treated adult host DCs, produced lower interferon-gamma (IFN-gamma) and higher interleukin-10 (IL-10), which is a regulatory T cell-like cytokine profile, as compared with adult blood CD4(+) T cells . In contrast, lipopolysaccharide (LPS)-treated adult host DCs significantly up-regulated IFN-gamma and down-regulated IL-10 production levels from CB CD4(+) T cells to that from adult blood CD4(+) T cells . The sustained low IFN-gamma and high IL-10 production from CB CD4(+) T cells co-cultured with adult blood DCs might account for the less severe GvHD occurrence after CB transplantation, which could be reversed by LPS-treated adult blood DCs.

Lett Appl Microbiol, 2004, 39(3), 246 - 51
Use of a fluorescent viability stain to assess lethal and sublethal injury in food-borne bacteria exposed to high-intensity pulsed electric fields; Yaqub S et al.; AIMS: To apply scanning electron microscopy, image analysis and a fluorescent viability stain to assess lethal and sublethal in food-borne bacteria exposed to high-intensity pulsed electric fields (PEF) . METHODS AND RESULTS: A rapid cellular staining method using the fluorescent redox probes 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 4',6-diamidino-2-phylindole was used for enumerating actively respiring cells of Listeria mononcytogenes, Bacillus cereus and Escherichia coli . This respiratory staining (RS) approach provided good agreement with the conventional plate count agar method for enumerating untreated and high-intensity PEF-treated bacteria suspended in 0.1% (w/v) peptone water . However, test organisms subjected to similar levels of lethality by heating at 56 degrees C resulted in ca 3-log-unit difference in surviving cell numbers ml(-1) when enumerated by these different viability indicators . PEF-treated bacteria were markedly altered at the cellular level when examined by scanning electron microscopy . CONCLUSIONS: While PEF-treatment did not produce sublethally injured cells (P < 0.05), substantial subpopulations of test bacteria rendered incapable of forming colonies by heating may remain metabolically active . SIGNIFICANCE AND IMPACT OF THE STUDY: The fluorescent staining method offers interesting perspectives on assessing established and novel microbial inactivation methods . Use of this approach may also provide a better understanding of the mechanisms involved in microbial inactivation induced by PEF.

Rev Inst Med Trop Sao Paulo, 2004 May-Jun, 46(3), 171 - 4 Epub 2004 Jul 20.
Carrion's disease (Bartonellosis bacilliformis) confirmed by histopathology in the High Forest of Peru; Maco V et al.; Bartonellosis or Carrion's disease is endemic in some regions of Peru, classically found in the inter-Andean valleys located between 500 and 3200 meters above sea level . We report the case of a 43 year-old male patient, farmer, who was born in the Pichanaki district (Chanchamayo, Junin), located in the High Forest of Peru . He presented with disseminated, raised, erythematous cutaneous lesions, some of which bled . The distribution of these lesions included the nasal mucosa and penile region . Additionally subcutaneous nodules were distributed over the trunk and extremities . Hematologic exams showed a moderate anemia . Serologic studies for HIV and Treponema pallidum were negative . The histopathologic results of two biopsies were compatible with Peruvian wart . Oral treatment with ciprofloxacin (500 mg BID) was begun . Over 10 days, the patient showed clinical improvement . This is the first report of a confirmed case of bartonellosis in the eruptive phase originating from the Peruvian High Forest, showing the geographical expansion of the Carrion's disease.

Indian J Exp Biol, 2004 Feb, 42(2), 164 - 73
Development and mechanisms of resistance to Bacillus thuringiensis endotoxin Cry1Ac in the American bollworm, Helicoverpa armigera (Hübner); Chandrashekar K et al.; The American bollworm, H . armigera, evolved 31-fold resistance to selection pressure of B . thuringiensis endotoxin Cry1Ac within six generations . The Cry1Ac selected larvae of H . armigera showed cross-resistance to Cry1Aa and Cry1Ab both in terms of mortality and growth reduction . Studies on mechanisms of resistance to Cry1Ac showed that proteases of resistant insects degraded Cry1Ac faster than those of susceptible insects, which led to the relative unavailability of toxin of about 58 kDa for binding and perforation of midgut epithelial membrane of the target insect . Besides, resistant and susceptible populations of H . armigera differed in the binding of their receptors with Cry1Ac toxin . These results suggest the possibility of both mechanisms existing in imparting resistance . These findings mandate the necessity of B . thuringiensis resistance management for usage of B . thuringiensis either as a conventional insecticide or through transgenic crops.

Indian J Exp Biol, 2004 Feb, 42(2), 157 - 63
Potentiation of insecticidal activity of Bacillus thuringiensis subsp . kurstaki HD-1 by proteinase inhibitors in the American bollworm, Helicoverpa armigera (Hübner); Gujar T et al.; The effect of crude proteinase inhibitor extracts from seeds of different crop plants (black gram, chickpea, chickling vetch, finger millet, French bean, green gram, horse gram, lentil, pea and soybean) on the insecticidal activity of B . thuringiensis var . kurstaki HD-1 was investigated against neonate larvae of H . armigera by diet incorporation method . The larval mortality due to crude proteinase inhibitors alone (5% seed weight equivalent) ranged from 4.1 to 19.1%; the maximum mortality with finger millet and the minimum with pea var . DDR-23 . A mixture of B . thuringiensis var . kurstaki HD-1 (10 ppm) and proteinase inhibitor (5% seed weight equivalent) was synergistic in larval mortality with respect to proteinase inhibitors of pea var . DMR-16, chickling vetch var . RLK-1098 and B101-212, lentil var . ILL-8095 and L-4076, soybean var . PK-1042, PK-416 and Pusa-22, chickpea var . Pusa-413, French bean (Chitra) and black gram; and antagonistic with respect to those of finger millet, horse gram and kidney bean . The larval growth reduction with crude proteinase inhibitors alone ranged from 17.9 to 53.1%; the maximum growth reduction with soybean var . PK-1042 and minimum with lentil var . L-4076 . A mixture of B . thuringiensis var . kurstaki and proteinase inhibitor was synergistic in growth reduction with respect to proteinase inhibitors of lentil var . ILL-8095, and L-4626 and antagonistic with respect to that of finger millet . The midgut proteinase inhibition with crude seed extracts (3.3% seed weight equivalent) ranged from 9.3 to 60.9% and was negatively correlated with larval mortality . These results showed that interactive effect of B . thuringiensis var . kurstaki HD-1 and proteinase inhibitors in the larvae of H . armigera depended upon the quality and quantity of proteinase inhibitors, which vary widely in different plants.

Int J Food Microbiol, 2004 Sep 1, 95(2), 205 - 18
Estimation of the non-isothermal inactivation patterns of Bacillus sporothermodurans IC4 spores in soups from their isothermal survival data; Periago PM et al.; The isothermal survival curves of the heat resistant spores of Bacillus sporothermodurans IC4, in the range of 117-125 degrees C, were determined in chicken, mushroom and pea soups by the capillary method . They were all non-linear with a noticeable upper concavity and could be described by the equation log(10) {N(t)/N(0)}=-b(T)t(n) with a fixed power, n, of the order of 0.7-0.8 . The temperature dependence of b(T) could be described by the equation b(T)=log(e){1+exp{k(T-T(c))}}, where T(c) is the temperature where intensive inactivation starts and k is the slope of b(T) at temperatures well above T(c) . They were 121-123 degrees C and 0.2-0.4 degrees C(-1), respectively, depending on the soup . These parameters were used to estimate the survival curves of the spores in two non-isothermal heat treatments using the procedure originally proposed by Peleg and Penchina {Crit . Rev . Food Sci . Nutr . 40 (2000) 159} . The results were compared with experimental survival curves, determined by the direct injection method, in another laboratory . There was a general agreement, although not perfect, between the predicted and experimentally observed survival ratios . Also, the isothermal survival parameters, estimated directly from the non-isothermal inactivation data using the model, were in general agreement with those calculated from the isothermal data . This suggests that the heat inactivation patterns of B . sporothermodurans IC4 spores in soups can be at least roughly estimated using the same general survival model, which has until now only been experimentally validated for vegetative bacterial cells.

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1369 - 75
Bacillus indicus sp . nov., an arsenic-resistant bacterium isolated from an aquifer in West Bengal, India; Suresh K et al.; Strain Sd/3T (=MTCC 4374T=DSM 15820T), an arsenic-resistant bacterium, was isolated from a sand sample obtained from an arsenic-contaminated aquifer in Chakdah district in West Bengal, India (23 degrees 3' N 88 degrees 35' E) . The bacterium was Gram-positive, rod-shaped, non-motile, endospore-forming and yellowish-orange pigmented . It possessed all the characteristics that conform to the genus Bacillus, such as it had A4beta murein type (L-orn-D-Asp) peptidoglycan variant, MK-7 as the major menaquinone and iso-C15 : 0 and anteiso-C15 : 0 as the major fatty acids . Based on its chemotaxonomic and phylogenetic characteristics, strain Sd/3T was identified as a species of the genus Bacillus . It exhibited maximum similarity (95%) at the 16S rRNA gene level with Bacillus cohnii; however, DNA-DNA similarity with B . cohnii was 60.7% . Strain Sd/3T also exhibited a number of phenotypic differences from B . cohnii (DSM 6307T) . These data suggest that Sd/3T represents a novel species of the genus Bacillus . The name Bacillus indicus sp . nov . is proposed.

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1355 - 64
Bacillus farraginis sp . nov., Bacillus fortis sp . nov . and Bacillus fordii sp . nov., isolated at dairy farms; Scheldeman P et al.; Forty-eight bacterial strains were isolated at dairy farms from raw milk, the milking apparatus, green fodder or feed concentrate after a heat treatment of 30 min at 100 degrees C . In this way, spore-forming bacteria with a very high intrinsic heat resistance were selected for . The aerobic spore-forming isolates were subjected to a polyphasic taxonomical study, including repetitive element sequence-based PCR typing, whole-cell protein profiling, 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base composition, fatty acid analysis, and morphological and biochemical characteristics . A comparison of the REP- and (GTG)5-PCR and whole-cell protein SDS-PAGE profiles resulted in three clusters of similar strains . Analysis of the 16S rDNA sequences and DNA-DNA relatedness data showed that these clusters represented three novel species . The highest 16S rDNA similarity to a recognized species found for the three groups was around 94% with Bacillus lentus and Bacillus sporothermodurans . Further phenotypic characterization supported the proposal of three novel species in the genus Bacillus, Bacillus farraginis, Bacillus fortis and Bacillus fordii . The respective type strains are R-6540T (=LMG 22081T=DSM 16013T), R-6514T (=LMG 22079T=DSM 16012T) and R-7190T (=LMG 22080T=DSM 16014T); their G+C DNA base contents are 43.7, 44.3 and 41.9 mol%, respectively . Although in variable amounts, a predominance of the branched fatty acids iso-C(15 : 0) and anteiso-C(15 : 0) was observed in all three novel species.

J Econ Entomol, 2004 Jun, 97(3), 1058 - 64
Genetics of resistance to transgenic Bacillus thuringiensis poplars in Chrysomela tremulae (Coleoptera: Chrysomelidae); Augustin S et al.; The area under genetically engineered plants producing Bacillus thuringiensis (Bt) toxins is steadily increasing . This increase has magnified the risk of alleles conferring resistance to these toxins being selected in natural populations of target insect pests . The speed at which this selection is likely to occur depends on the genetic characteristics of Bt resistance . We selected a strain of the beetle Chrysomela tremulae Fabricius on a transgenic Bt poplar clone Populus tremula L . x Populus tremuloides Michx producing high levels of B . thuringiensis Cry3Aa toxin . This strain was derived from an isofemale line that generated some F2 offspring that actively fed on this Bt poplar clone . The resistance ratio of the strain was >6400 . Susceptibility had decreased to such an extent that the mortality of beetles of the strain fed Bt poplar leaves was similar to that of beetles fed nontransgenic poplar leaves . Genetic crosses between susceptible, resistant, and F1 hybrids showed that resistance to the Cry3Aa toxin was almost completely recessive (D(LC) = 0.07) and conferred by a single autosomal gene . The concentration of Cry3Aa produced in the transgenic Bt poplar used in this study was 6.34 times higher than the LC99 of the F1 hybrids, accounting for the complete recessivity (D(ML) = 0) of survival on Bt poplar leaves . Overall, the genetic characteristics of the resistance of C . tremulae to the Cry3Aa toxin are consistent with the assumptions underlying the high-dose refuge strategy, which aims to decrease the selection of Bt resistance alleles in natural target pest populations.

J Econ Entomol, 2004 Jun, 97(3), 1049 - 57
Cross-resistance of Cry1Ab-selected Ostrinia nubilalis (Lepidoptera: Crambidae) to Bacillus thuringiensis delta-endotoxins; Siqueira HA et al.; Corn plants expressing the toxin from Bacillus thuringiensis (Berliner) have proven to be effective in controlling lepidopteran pests such as the European corn borer, Ostrinia nubilalis (Hubner) (Lepidoptera: Crambidae) . Several Bt toxins are being tested and incorporated into crop genomes, although tests for cross-resistance among different toxins have been limited by a lack of resistant colonies . Four different colonies of O . nubilalis selected with full-length Cry1Ab incorporated into artificial diet developed significant levels of resistance (2.0- to 10-fold) within 10 generations . Additionally, selection with Cry1Ab resulted in decreased susceptibility to a number of other toxins to which the selected colonies were not previously exposed . Significantly, levels of resistance were highest to Cry1Ac with resistance ratios up to 51.0-fold . Low levels (less than five-fold) of cross-resistance were detected with Cry1F . In contrast, Cry9C susceptibility was unaffected by selection with Cry1Ab . These results indicate that the availability of multiple toxins could improve resistance management strategies, provided cross-resistance among toxins is not a factor.

J Econ Entomol, 2004 Jun, 97(3), 774 - 80
Two new bacterial pathogens of Colorado potato beetle Coleoptera: Chrysomelidae); Martin PA et al.; Other than Bacillus thuringiensis Berliner, few bacteria are lethal to the Colorado potato beetle (Leptinotarsa decemlineata {Say}), a major pest of potatoes and eggplant . Expanded use of biologicals for the control of Colorado potato beetle will improve resistance management, reduce pesticide use, and produce novel compounds for potential use in transgenic plants . Using freeze-dried, rehydrated artificial diet in pellet form to screen bacteria lethal to other insects, we determined that strains of Photorhabdus luminescens killed Colorado potato beetle larvae . The LC50 for second instar larvae of strain HM5-1 was 6.4 +/- 1.87 x 10(7) cells per diet pellet . In an attempt to find additional naturally occurring P . luminescens strains toxic to Colorado potato beetle larvae, we recovered, from soil, bacteria that produced a purple pigment . This bacterial strain, identified as Chromobacterium sp . by 16S ribosomal DNA sequencing, was also toxic to Colorado potato beetle larvae within 3 d . The LC50 for second instar larvae for these bacteria was 2.0 +/- 0.79 x 10(8) cells per diet pellet, while the LC50 was approximately 1 log lower for third instar larvae . P . luminescens appeared to kill by means of a protein toxin that may be similar to the described lepidopteran protein toxins . Based on the heat and acid stability, the toxin or toxins that Chromobacterium sp . produces, while not fully characterized, do not appear to be typical proteins . In both bacteria, the toxins are made after exponential growth ceases.

J Econ Entomol, 2004 Jun, 97(3), 721 - 6
Shared genetic basis of resistance to Bt toxin Cry1ac in independent strains of pink bollworm; Tabashnik BE et al.; Classical and molecular genetic analyses show that two independently derived resistant strains of pink bollworm, Pectinophora gossypiella (Saunders), share a genetic locus at which three mutant alleles confer resistance to Bacillus thuringiensis (Bt) toxin Cry1Ac . One laboratory-selected resistant strain (AZP-R) was derived from individuals collected in 1997 from 10 Arizona cotton fields, whereas the other (APHIS-98R) was derived from a long-term susceptible laboratory strain . Both strains were previously reported to show traits of "mode 1" resistance, the most common type of lepidopteran resistance to Cry1A toxins . Inheritance of resistance to a diagnostic concentration of Cry1Ac (10 microg per gram of diet) was recessive in both strains . In interstrain complementation tests for allelism, F1 progeny from crosses between the two strains were resistant to the diagnostic concentration of Cry1Ac . These results indicate that a major resistance locus is shared by the two strains . Analysis of DNA from the pink bollworm cadherin gene (BtR) using allele-specific polymerase chain reaction (PCR) tests showed that the previously identified resistance alleles (r1, r2, and r3) occurred in both strains, but their frequencies differed between strains . In conjunction with previous findings, the results reported here suggest that PCR-based detection of the three known cadherin resistance alleles might be useful for monitoring resistance to Cry1Ac-producing Bt cotton in field populations of pink bollworm.

Med Sci Monit, 2004 Aug, 10(8), CS37 - 40 Epub 2004 Jul 23.
A case of tuberculous granuloma at the supra-sternal notch that was difficult to differentiate from a thyroid tumor; Asayama I et al.; BACKGROUND: Despite a decline after World War II, the rate of tuberculosis remains higher in Japan than in other countries . We report a case of tuberculous granuloma at the supra-sternal notch that was difficult to differentiate from a thyroid tumor . CASE REPORT: The patient was a 75-year-old Japanese woman who was referred to our hospital for further investigations and treatment of an anterior neck tumor, that was diagnosed as a suspected of thyroid malignancy by another institute . The thyroid function and biological data were normal except for an elevated erythrocyte sedimentation rate . Imaging studies showed a mass at the supra-sternal notch, and the border between the tumor and the thyroid gland was indistinct . The tuberculosis bacillus group was identified by fine needle aspiration cytology . The patient was treated surgically for tuberculous granuloma, and histopathological findings revealed that the lymph node tuberuculosis had invaded the thyroid gland . We started anti-tuberculous therapy after the operation . The post-operative course was uneventful with good wound healing . CONCLUSIONS: When a markedly elevated erythrocyte sedimentation rate and c-reactive protein value are associated with an anterior neck mass, tuberculosis should be considered in the differential diagnosis of thyroid swelling . Fine needle aspiration cytology is a rapid, simple and effective diagnostic method for extra-pulmonary tuberculous lesions involving the neck . When there is abscess formation or features of compression, or if the mass cannot be differentiated from a thyroid tumor, combined therapy involving anti-tuberculous agents and surgery must be considered.

Biosci Biotechnol Biochem, 2004 Jul, 68(7), 1533 - 40
Gene cloning and characterization of a Bacillus vietnamensis metalloprotease; Kim M et al.; A Bacillus vietnamensis metalloprotease (BVMP) with high affinity toward collagen was isolated and purified from the culture supernatant of Bacillus vietnamensis 11-4 occurring in Vietnamese fish sauces . The BVMP gene was cloned and its nucleotide and coded amino acid sequences determined . BVMP consists of 547 amino acid residues, with the zinc-binding sites conserved in common metalloproteases . It shares 57% amino acid identity with thermolysin originating from Bacillus thermoproteolyticus . The three-dimensional structure of BVMP was deduced by computer-aided modeling with the use of the known three-dimensional thermolysin structure as a template . Like thermolysin, BVMP cleaved the oxidized insulin B-chain at the peptide bonds involving the N-terminal sides of hydrophobic and aromatic amino acids . BVMP also showed high hydrolytic activity toward gelatin, collagen, casein, and elastin, especially toward the skeletal proteins at increased NaCl concentration . The high activity was found to be due to enhanced affinity to the substrates . Kinetical data on BVMP indicated that the Km values for the hydrolysis of Cbz-GPGGPA as a collagen model decreased as the concentration of added NaCl increased . Some contribution of this enzyme during the aging of fish sauces at high salt concentrations can thus be expected.

Biosci Biotechnol Biochem, 2004 Jul, 68(7), 1414 - 23
Cloning and characterization of the catabolite control protein A gene from Bacillus stearothermophilus No . 236; Choi ID et al.; The gene encoding the catabolite control protein A (CcpA) of Bacillus stearothermophilus No . 236, a strong xylanolytic bacterium, was cloned, sequenced, and expressed in Escherichia coli . The nucleotide sequence of the ccpA gene corresponded to an open reading frame of 1,005 bp that encodes a polypeptide of 334 amino acid residues with a calculated molecular mass of 36,902 kDa . The CcpA protein belonging to the LacI/GalR family of transcriptional regulators was produced by a recombinant E . coli strain expressing the B . stearothermophilus No . 236 ccpA gene and purified to apparent homogeneity . The transcription start site was mapped at a position 63 nucleotides upstream of the translation initiation codon, and a presumed promoter sequence was also identified . The deduced amino acid sequence of the ccpA gene product contained the helix-turn-helix motif found in many DNA-binding proteins, and showed the highest identity (62%) with CcpA from B . subtilis . The B . stearothermophilus No . 236 ccpA gene was demonstrated to be able to complement a B . subtilis ccpA mutant that exhibited two distinct mutant phenotypes: a growth defect and a release of carbon catabolite repression (CCR) . These results indicate that the ccpA gene product of B . stearothermophilus No . 236 is functionally active also in B . subtilis . Electrophoretic mobility shift assay with the purified CcpA revealed that the CcpA of B . stearothermophilus No . 236 bound specifically to the xynA creB (catabolite responsive element B) sequence . Taken together, these results strongly suggest that the CcpA protein participates in CCR of B . stearothermophilus No . 236 xynA gene.

Paediatr Respir Rev, 2004 Sep, 5(3), 225 - 30
Tuberculosis in HIV-infected children; Swaminathan S; Tuberculosis (TB) is the most common opportunistic infection in human immunodeficiency virus (HIV)-infected people worldwide . HIV-positive children are at risk of diagnostic error as well as delayed diagnosis of TB because of overlapping clinical and radiographic features with other lung diseases . Acute pneumonias and chronic lung diseases such as bronchiectasis and lymphocytic interstitial pneumonitis are difficult to distinguish from TB . TB manifestations are more severe in HIV-positive children and progression to death is more rapid than in HIV-negative children . The response to standard short-course therapy in HIV-positive children is not as good as in HIV-negative children due to lower cure rates and higher mortality . TB hastens the progression of HIV disease by increasing viral replication and reducing CD4 counts further . Although Bacille Calmette-Guerin vaccination could lead to disseminated Mycobacterium bovis disease in the presence of immunosuppression, this has been rarely reported . More studies are required to assess the role of newer diagnostic tests, TB preventive therapy and co-administration of anti-retroviral therapy in the control of TB among HIV-infected children.

Parasitol Today, 1997 Dec, 13(12), 485 - 7
Microbial control of mosquitoes: Management of the upper rhine mosquito population as a model programme; Becker N; For more than a decade, Bacillus thuringiensis israelensis (Bti) and B . sphaericus have been used successfully as biological control agents against mosquitoes in Germany . Over 1000 km(2) of breeding areas have been treated with Bti, resulting in a yearly reduction of the mosquito population by more than 90%, without evidence of any harmful impact on the environment . Here, Norbert Becker describes the control strategy and operational use of Bti and B . sphaericus in Germany.

Biochemistry, 2004 Aug 3, 43(30), 9589 - 99
Structural stability and unfolding properties of thermostable bacterial alpha-amylases: a comparative study of homologous enzymes; Fitter J et al.; In a comparative investigation on two thermostable alpha-amylases {Bacillus amyloliquefaciens (BAA), T(m) = 86 degrees C and Bacillus licheniformis (BLA), T(m) = 101 degrees C}, we studied thermal and guanidine hydrochloride (GndHCl)-induced unfolding using fluorescence and CD spectroscopy, as well as dynamic light scattering . Depletion of calcium from specific ion-binding sites in the protein structures reduces the melting temperature tremendously for both alpha-amylases . The reduction is nearly the same for both enzymes, namely, in the order of 50 degrees C . Thus, the difference in thermostability between BLA and BAA (DeltaT(m) approximately 15 degrees C) is related to intrinsic properties of the respective protein structures themselves and is not related to the strength of ion binding . The thermal unfolding of both proteins is characterized by a full disappearance of secondary structure elements and by a concurrent expansion of the 3D structure . GndHCl-induced unfolding also yields a fully vanishing secondary structure but with more expanded 3D structures . Both alpha-amylases remain much more compact upon thermal unfolding as compared to the fully unfolded state induced by chemical denaturants . Such rather compact thermal unfolded structures lower the conformational entropy change during the unfolding transition, which principally can contribute to an increased thermal stability . Structural flexibilities of both enzymes, as measured with tryptophan fluorescence quenching, are almost identical for both enzymes in the native states, as well as in the unfolded states . Furthermore, we do not observe any difference in the temperature dependence of the structural flexibilities between BLA and BAA . These results indicate that conformational dynamics on the time scale of our studies seem not to be related to thermal stability or to thermal adaptation.

Langmuir, 2004 Aug 3, 20(16), 6841 - 6
Influence of calcium on the self-assembly of partially hydrolyzed alpha-lactalbumin; Graveland-Bikker JF et al.; Self-assembly of alpha-lactalbumin after partial hydrolysis by a protease from Bacillus licheniformis can result in nanotubular structures, which show many similarities to microtubules . Calcium plays a crucial role in this process . The objective of this investigation was to study the role of calcium in more detail . The kinetics of the hydrolysis step and the self-assembly step were monitored by respectively liquid chromatography-mass spectrometry and dynamic light scattering . The microstructure of the gels finally formed was investigated by transmission electron microscopy . This investigation demonstrates that calcium accelerated the kinetics of the self-assembly, but it had no effect on the hydrolysis kinetics . As a result of the accelerated self-assembly kinetics at a high calcium concentration, the time of gelation decreased as well . A minimum concentration of calcium needed to obtain the tubular alpha-lactalbumin structures was determined . Below R = 1.5 (mole calcium/mole alpha-lactalbumin), turbid gels with randomlike structure were obtained . Between R = 1.5 and R = 6, translucent gels with a fine stranded network of tubules were formed, while higher calcium concentrations had a negative effect on the tubule formation, resulting in amorphous structures . The optimum calcium concentration for alpha-lactalbumin nanotube formation seemed to be around R = 3.

Mem Inst Oswaldo Cruz, 2004 May, 99(3), 283 - 7 Epub 2004 Jul 19.
Pulmonary tuberculosis: evaluation of interferon-gamma levels as an immunological healing marker based on the response to the Bacillus Calmette-Guerin; Moura EP et al.; Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis whose interaction with the host may lead to a cell-mediated protective immune response . The presence of interferon gamma (IFN-gamma) is related to this response . With the purpose of understanding the immunological mechanisms involved in this protection, the lymphoproliferative response, IFN-gamma and other cytokines like interleukin (IL-5, IL-10), and tumor necrosis factor alpha (TNF-alpha) were evaluated before and after the use of anti-TB drugs on 30 patients with active TB disease, 24 healthy household contacts of active TB patients, with positive purified protein derivative (PPD) skin tests (induration > 10 mm), and 34 asymptomatic individuals with negative PPD skin test results (induration < 5 mm) . The positive lymphoproliferative response among peripheral blood mononuclear cells of patients showed high levels of IFN-gamma, TNF-alpha, and IL-10 . No significant levels of IL-5 were detected . After treatment with rifampicina, isoniazida, and pirazinamida, only the levels of IFN-gamma increased significantly (p < 0.01) . These results highlight the need for further evaluation of IFN-gamma production as a healing prognostic of patients treated.

Southeast Asian J Trop Med Public Health, 2004 Mar, 35(1), 68 - 78
Space spraying of bacterial and chemical insecticides against Anopheles balabacensis Baisas for the control of malaria in Sabah, East Malaysia; Seleena P et al.; A pilot study was undertaken to determine the effectiveness of space application of insecticides for the control of malaria in Ranau, a district in Sabah . A village each was treated monthly: with chemical adulticide--alpha cypermethrin (Fendona SC(R)/10SC(R)) at 2 g a.i./10,000 m2 in Pahu; with biological larvicides--Bacillus thuringiensis israelensis (Vectobac 12AS(R)) at 500 ml/10,000 m2 or B . sphaericus (Vectolex WG(R)) at 500 g/10,000 m2 in Pinawantai; and with a mixture of chemical adulticide and biological larvicide in Togop Laut . All sprayings were conducted using a portable mist blower . During the study period all villages, including Tarawas the untreated village, received the conventional malaria control measures . Entomological and epidemiological surveillance was used to measure the effectiveness of the space application . The entomological surveillance indicated that the An . balabacensis population was significantly reduced by alpha cypermethrin in Pahu and Togop Laut and B . sphaericus in Pinawantai; but was not reduced by B.t.i . in Pinawantai . There was a significant reduction in the number of malaria cases and in the slide positivity rate in the treated villages during the study period . The pilot study does indicate that space application of larvicides/adulticides or a mixture of both is able to reduce the malaria vector population and the malaria transmission . A larger scale study needs to be undertaken in a malarious village/province to determine whether space application of insecticides together with other malaria control measures will be able to eradicate malaria.

Acta Crystallogr D Biol Crystallogr, 2004 Aug, 60(Pt 8), 1490 - 2 Epub 2004 Jul 21.
Crystallization and preliminary X-ray study of alkaline mannanase from an alkaliphilic Bacillus isolate; Akita M et al.; An alkaline mannanase (EC 3.2.1.78) from the alkaliphilic Bacillus sp . strain JAMB-602 was cloned and sequenced . The deduced amino-acid sequence of the enzyme suggested that the enzyme consists of a catalytic and unknown additional domains . The recombinant enzyme expressed by B . subtilis was crystallized using the hanging-drop vapour-diffusion method at 277 K . X-ray diffraction data were collected to 1.65 A . The crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 70.7, b = 79.5, c = 80.4 A . The asymmetric unit contains one protein molecule, with a corresponding VM of 2.26 A3 Da(-1) and a solvent content of 45.6% . Molecular replacement for initial phasing was carried out using the three-dimensional structure of a mannanase from Thermomonospora fusca as a search model, which corresponds to the catalytic domain of the alkaline mannanase . It gave sufficient phases to build the unknown domain.

Acta Crystallogr D Biol Crystallogr, 2004 Aug, 60(Pt 8), 1404 - 12 Epub 2004 Jul 21.
Two orthorhombic crystal structures of a galactose-specific lectin from Artocarpus hirsuta in complex with methyl-alpha-D-galactose; Rao KN et al.; Based on their carbohydrate specificity, the jacalin family of lectins can be divided into two groups: galactose-specific and mannose-specific . The former are cytoplasmic proteins, whereas the latter are localized in the storage vacuoles of cells . It has been proposed that the post-translational modification in some of the lectins that splits their polypeptide chains into two may be crucial for galactose specificity . The mannose-specific members of the family are single-chain proteins that lack the above modification . Although the galactose-specific and the mannose-specific jacalin-type lectins differ in their sequences, they share a common fold: the beta-prism I fold, which is characteristic of Moraceae plant lectins . Here, two crystal structures of a jacalin-related lectin from Artocarpus hirsuta, which is specific for galactose, in complex with methyl-alpha-D-galactose are reported . The lectin crystallized in two orthorhombic forms and one hexagonal form under similar conditions . The crystals had an unusually high solvent content . The structure was solved using the molecular-replacement method using the jacalin structure as a search model . The two orthorhombic forms were refined using data to 2.5 and 3.0 A resolution, respectively . The structures of the A . hirsuta lectin and jacalin are identical . In orthorhombic form I the crystal packing provides three different micro-environments for sugar binding in the same crystal . The observed difference in the specificity for oligosaccharides between the A . hirsuta lectin and jacalin could only be explained based on differences in the molecular associations in the packing and variation of the C-terminal length of the beta-chain . The observed insecticidal activity of A . hirsuta lectin may arise from its similar fold to domain II of the unrelated delta-endotoxin from Bacillus thuringiensis.

J Med Microbiol, 2004 Aug, 53(Pt 8), 807 - 11
Chemotactic response of Helicobacter pylori to human plasma and bile; Worku ML et al.; To clarify further the role of chemotaxis in Helicobacter pylori colonization, the in vitro bacterium response to human plasma and bile (secretions containing chemoeffector compounds that are present in the gastric mucus layer) was examined . Human plasma, after dilution to 1 % (v/v) with buffer, was found to be a chemoattractant for the motile bacillus . Human gall-bladder bile, after dilution to 2 % (v/v) with buffer, was found to be a chemorepellent, but did not cause the motility of the bacillus to be diminished after prolonged exposure . The basis of the chemoattractant effect of plasma was explored by examining how urea and 12 amino acids found in plasma affected the taxis of H . pylori . Urea and the amino acids histidine, glutamine, glycine and arginine were the strongest chemoattractants . Other amino acids were chemoattractants, with the exceptions of aspartic and glutamic acids, which were chemorepellents . The basis of the chemorepellent effect of bile was explored by examining how the six most abundant conjugated bile acids in human bile affected the taxis of H . pylori . All the bile acids were chemorepellents, with the greatest effects being demonstrated by taurocholic and taurodeoxycholic acids . The implications of these findings for H . pylori colonization of gastric epithelium are discussed .

J Evol Biol, 2004 Jul, 17(4), 904 - 12; discussion 913-8
Delaying evolution of insect resistance to transgenic crops by decreasing dominance and heritability; Tabashnik BE et al.; The refuge strategy is used widely for delaying evolution of insect resistance to transgenic crops that produce Bacillus thuringiensis (Bt) toxins . Farmers grow refuges of host plants that do not produce Bt toxins to promote survival of susceptible pests . Many modelling studies predict that refuges will delay resistance longest if alleles conferring resistance are rare, most resistant adults mate with susceptible adults, and Bt plants have sufficiently high toxin concentration to kill heterozygous progeny from such matings . In contrast, based on their model of the cotton pest Heliothis virescens, Vacher et al . (Journal of Evolutionary Biology, 16, 2003, 378) concluded that low rather than high toxin doses would delay resistance most effectively . We demonstrate here that their conclusion arises from invalid assumptions about larval concentration-mortality responses and dominance of resistance . Incorporation of bioassay data from H . virescens and another key cotton pest (Pectinophora gossypiella) into a population genetic model shows that toxin concentrations high enough to kill all or nearly all heterozygotes should delay resistance longer than lower concentrations .

Biochem Soc Trans, 2004 Aug, 32(Pt 4), 626 - 8
BCG (Bacille Calmette-Guérin) HspCs (heat-shock protein-peptide complexes) induce T-helper 1 responses and protect against live challenge in a murine aerosol challenge model of pulmonary tuberculosis; Colaco CA et al.; The need for an effective TB (tuberculosis) vaccine remains acute, with tuberculosis still one of the major killers worldwide and 3 million new infections annually . We report here on the immune responses elicited by HspCs (heat-shock protein-peptide complexes) isolated from BCG (Bacille Calmette-Guerin) vaccine . These HspCs elicit both the appropriate cellular and protective immune responses required to merit their further development as TB vaccine candidates.

Biotechnol Lett, 2004 May, 26(10), 823 - 8
Starch hydrolysing Bacillus halodurans isolates from a Kenyan soda lake; Hashim SO et al.; Fourteen obligate alkaliphilic and halotolerant bacterial isolates, exhibiting extracellular amylase activity at 55 degrees C and pH 10, were isolated from hot springs around Lake Bogoria, Kenya . From 16S rDNA sequence analysis, nine isolates shared 100% identity with Bacillus halodurans strain DSM 497T, while the rest shared 99% identity with alkaliphilic Bacillus species A-59 . PCR of the intergenic spacer region between 16S and 23S rRNA genes (ISR-PCR) divided the isolates into two groups, while tDNA-PCR divided them into three groups . Bacillus halodurans DSM 497T had a different ISR pattern from the isolates, while it had a tDNA-PCR profile similar to the group that shared 99% identity with alkaliphilic Bacillus species A-59 . All isolates hydrolysed soluble starch as well as amylose, amylopectin and pullulan . The amylase activity (1.2-1.8 U ml(-1)) in the culture broths had an optimum temperature of 55-65 degrees C, was stimulated by 1 mm Ca2+, and was either partially (16-30%) or completely inhibited by 1 mM EDTA . Activity staining of the cell-free culture supernatant from the isolates revealed five alkaline active amylase bands.

Biotechnol Lett, 2004 Jun, 26(11), 929 - 32
BpaI and BpnI: novel type II restriction endonucleases from Bacillus pasteurii and Bacillus pantothenticus; Jutur PP et al.; Two novel type II restriction endonucleases, designated as BpaI and BpnI, were isolated from Bacillus pasteurii strain1761 and Bacillus pantothenticus strain1639, respectively . They were partially purified and SDS-PAGE indicated Mr values of 28 and 67 kDa for BpaI, 28 and 48 kDa for BpnI . The partially purified endonucleases hydrolyzed DNA into discrete fragments: pUC18 (2.6 kb for BpaI; 1.8 and 0.8 kb for BpnI), pBR322 (2.5 and 1.8 kb for BpaI; 2.6 and 1.7 kb for BpnI) and phix174 DNA (3.2 and 2.1 kb for BpaI; 4 and 1.3 kb for BpnI).

Biotechnol Lett, 2004 Jun, 26(11), 865 - 8
Fermentation of sugar cane bagasse hemicellulose hydrolysate to L(+)-lactic acid by a thermotolerant acidophilic Bacillus sp; Patel M et al.; Sugar cane bagasse hemicellulose, hydrolyzed by dilute H2SO4, supplemented with mineral salts and 0.5% corn steep liquor, was fermented to L(+)-lactic acid using a newly isolated strain of Bacillus sp . In batch fermentations at 50 degrees C and pH 5, over 5.5% (w/v) L(+)-lactic acid was produced (89% theoretical yield; 0.9 g lactate per g sugar) with an optical purity of 99.5%.

Clin Cancer Res, 2004 Jul 15, 10(14), 4717 - 23
Sequential immunization of melanoma patients with GD3 ganglioside vaccine and anti-idiotypic monoclonal antibody that mimics GD3 ganglioside; Chapman PB et al.; GD3 ganglioside is an attractive target for immunotherapy of melanoma because it is abundantly expressed on all melanomas but not expressed on most normal tissues . Although GD3 has proven to be one of the least immunogenic gangliosides, our recent studies showed that anti-GD3 antibodies can be induced in patients immunized either with GD3-lactone-KLH (GD3-L-KLH) plus QS-21 adjuvant or with BEC2 anti-idiotypic monoclonal antibody vaccine, which mimics GD3, plus Bacillus Calmette-Guerin . We compared the immunogenicity of these two vaccines and tested whether one vaccine could prime an antibody response to the other . This is the first clinical trial immunizing patients with both antigen and anti-idiotypic monoclonal antibody vaccine . Twenty-four melanoma patients were randomized to be immunized with either BEC2 followed by GD3-L-KLH or in the opposite order . Our prior study suggested that a 25-microg dose of BEC2 was more immunogenic than our standard dose of 2.5 mg and therefore was used in this trial . Overall, 10 of 24 patients (42%) developed anti-GD3 antibodies detectable by ELISA, five in each cohort . All antibody responses were in response to the GD3-L-KLH vaccine . We found no evidence of priming by either vaccine . Antibody responses did not correlate with survival outcomes . Cellular responses were detected by enzyme-linked immunospot against BEC2, Bacillus Calmette-Guerin, and KLH, but not against GD3 . We confirmed that GD3-L-KLH vaccine induces anti-GD3 antibodies, but we were unable to confirm our previous finding that a 25-microg dose of BEC2 is immunogenic . Future multivalent ganglioside vaccines should include the GD3-L-KLH vaccine.

Biotechnol Lett, 2004 Jul, 26(14), 1153 - 6
Cloning and nucleotide sequence of a novel cry gene from Bacillus thuringiensis; Stobdan T et al.; A cry1Ab-type gene was cloned from a new isolate of Bacillus thuringiensis by PCR . When restriction pattern was compared with that of known genes it was found to have additional restriction site for ClaI . Nucleotide sequencing and homology search revealed that the toxin shared 95% homology with the known Cry1Ab proteins as compared to more than 98% homology among the other reported Cry1Ab toxins . The gene encoded a sequence of 1,177 amino acids compared to 1,155 amino acids encoded by all the other 16 cry1Ab genes reported so far . An additional stretch of 22 amino acids after the amino acid G793 in the new toxin sequence showed 100% homology with several other cry genes within cry1 family . Homology search indicated that the new cry1Ab-type gene might have resulted by nucleotide rearrangement between cry1Ab and cry1Aa/cry1Ac genes.

Eur J Biochem, 2004 Aug, 271(15), 3127 - 35
Characterization of a Cry1Ac-receptor alkaline phosphatase in susceptible and resistant Heliothis virescens larvae; Jurat-Fuentes JL et al.; We reported previously a direct correlation between reduced soybean agglutinin binding to 63- and 68-kDa midgut glycoproteins and resistance to Cry1Ac toxin from Bacillus thuringiensis in the tobacco budworm (Heliothis virescens) . In the present work we describe the identification of the 68-kDa glycoprotein as a membrane-bound form of alkaline phosphatase we term HvALP . Lectin blot analysis of HvALP revealed the existence of N-linked oligosaccharides containing terminal N-acetylgalactosamine required for {125I}Cry1Ac binding in ligand blots . Based on immunoblotting and alkaline phosphatase activity detection, reduced soybean agglutinin binding to HvALP from Cry1Ac resistant larvae of the H . virescens YHD2 strain was attributable to reduced amounts of HvALP in resistant larvae . Quantification of specific alkaline phosphatase activity in brush border membrane proteins from susceptible (YDK and F1 generation from backcrosses) and YHD2 H . virescens larvae confirmed the observation of reduced HvALP levels . We propose HvALP as a Cry1Ac binding protein that is present at reduced levels in brush border membrane vesicles from YHD2 larvae.

J Agric Food Chem, 2004 Jul 28, 52(15), 4737 - 9
Evaluation of calcium-alginate gel as an artificial diet medium for bioassays on common cutworms; Morimoto M et al.; A calcium-alginate gel diet was developed for Spodoptera litura larvae, and its reliability as a carrier for incorporating antifeedants as well as insecticides was investigated . The alginate gel diet was prepared with a simple protocol, which does not involve any heating process . When tested using this diet, acephate, a Bacillus thuringiensis endotoxin formulation and rotenone reproducibly showed insecticidal activity against the larvae, while neem oil and scabequinone deterred the larval feeding effectively . However, not only the insecticidal activity of acephate but also the antifeedant activity of neem oil was reduced by replacing the alginate component by agar in the diet, suggesting the usefulness of the alginate gel diet as an assay tool for testing a broad range of samples against the larvae .

J Am Mosq Control Assoc, 2004 Jun, 20(2), 171 - 5
Effects of algae on the efficacy of Bacillus thuringiensis var . israelensis against larval black flies; Stephens MS et al.; Personnel from several black fly control programs have reported that the efficacy of Bacillus thuringiensis var . israelesis (Bti) is reduced during periods when algal concentrations are high in the waterways . Although the reduction in Bti-induced mortality in black fly larvae is presumed to be related to the presence of algae, no scientific data support this theory . In this study, 4 genera of algae (Microcytis, Scenedesmus, Dictrosphaerium, and Chlorella) commonly detected in Pennsylvania rivers where Bti-induced mortality in black fly larvae has been reduced were assessed to determine their respective effects on Bti-induced mortality by using an orbital shaker bioassay with laboratory-reared black fly larvae (Simulium vittatum cytospecies IS-7) . A significant reduction in Bti-induced mortality was observed when Scenedesmus was present in the flasks at concentrations > or = 16,000 cells/ml . The Bti-induced mortality of larvae was not significantly reduced when Chlorella, Dictyosphaerium, or Microcytis was present in the flasks, even at concentrations > or = 250,000 cells/ml . These results indicate that the presence of certain types of algae can reduce the mortality of black flies exposed to Bti . Although not clearly defined, the mechanisms involved may be related to algal morphology due to overall size and structures associated with certain types of algae, and possible interference with feeding.

J Am Mosq Control Assoc, 2004 Jun, 20(2), 165 - 70
Influence of temperature and concentration of VectoBac on control of the salt-marsh mosquito, Ochlerotatus squamiger, in Monterey County, California; Christiansen JA et al.; Laboratory susceptibility bioassays were conducted to determine the efficacy of VectoBac TP (Bacillus thuringiensis var . israelensis {Bti}) at different concentrations and temperatures against the salt-marsh mosquito Ochlerotatus squamiger . Bioassays on late 3rd- and early 4th-stage larvae, read at 72 h and 14 degrees C produced an LD90 of 0.223 mg/liter, whereas more than double this dose was required to produce similar mortality at 6 degrees C . A field trial in the winter of 2001-02 of an aerially applied VectoBac TP formulation in Salinas, CA, corroborated laboratory bioassay observations by producing 97-100% control of Oc . squamiger at 72 h postapplication . Inconsistencies in mortality with field applications of VectoBac TP previously observed by North Salinas Valley Mosquito Abatement personnel were most likely caused by uneven application rates and varying temperatures and water volumes.

J Struct Funct Genomics, 2004, 5(1-2), 95 - 102
Production of selenomethionine-labeled proteins in two-liter plastic bottles for structure determination; Stols L et al.; A simplified approach developed recently for the production of heterologous proteins in Escherichia coli uses 2-liter polyethylene terephthalate beverage bottles as disposable culture vessels {Sanville Millard, C . et al . 2003 . Protein Expr . Purif . 29, 311-320} . The method greatly reduces the time and effort needed to produce native proteins for structural or functional studies . We now demonstrate that the approach is also well suited for production of proteins in defined media with incorporation of selenomethionine to facilitate structure determination by multiwavelength anomalous diffraction . Induction of a random set of Bacillus stearothermophilus target genes under the new protocols generated soluble selenomethionyl proteins in good yield . Several selenomethionyl proteins were purified in good yields and three were subjected to amino acid analysis . Incorporation of selenomethionine was determined to be greater than 95% in one protein and greater than 98% in the other two . In the preceding paper {Zhao et al., this issue, pp . 87-93}, the approach is further extended to production of {U-15N}- or {U-13C, U-15N}-labeled proteins . The approach thus appears suitable for high-throughput production of proteins for structure determination by X-ray crystallography or nuclear magnetic resonance spectroscopy.

Insect Biochem Mol Biol, 2004 Aug, 34(8), 753 - 62
Comparative analysis of proteinase activities of Bacillus thuringiensis-resistant and -susceptible Ostrinia nubilalis (Lepidoptera: Crambidae); Li H et al.; Proteinase activities were compared in soluble and membrane fractions of guts obtained from larvae of Bacillus thuringiensis-resistant and -susceptible Ostrinia nubilalis . Overall, serine proteinases from soluble fractions of the susceptible strain were more active than those of the resistant strain . The soluble trypsin-like proteinase activity of the resistant strain was approximately half that of the susceptible strain . The number and relative molecular masses of soluble and membrane serine proteinases were different . However, there were no significant differences in the activities of serine proteinases and aminopeptidases extracted from midgut membranes of the two strains . Cry1Ab protoxin hydrolysis by soluble proteinase extracts of the resistant strain was reduced approximately 20-30% relative to that of the susceptible strain . Reduced protoxin processing due to decreased activities of Bt protoxin activation proteinases may be associated with resistance to Bt toxin in this resistant strain of O . nubilalis.

J Invertebr Pathol, 2004 Jul, 86(3), 104 - 10
Factors affecting the toxicity of Bacillus thuringiensis var . israelensis and Bacillus sphaericus to fourth instar larvae of Chironomus tepperi (Diptera: Chironomidae); Stevens MM et al.; Laboratory bioassays (48h duration, 25+/-1 degrees C) were used to determine the toxicity of Bacillus thuringiensis var . israelensis (B.t.i.) and Bacillus sphaericus to fourth instar larvae of Chironomus tepperi, a major pest of rice in southern Australia . Bioassays were conducted using different combinations of larval ages and densities to determine if these factors affected toxicity . The effects of temperature and substrate type on B.t.i . toxicity were also investigated . Tests were conducted using a commercial B.t.i . formulation (VectoBac WDG, 3000ITU/mg), a spore/crystal mixture derived from the VectoBac WDG strain, and VectoLex WDG, a commercial B . sphaericus formulation (650ITU/mg) . VectoBac WDG was highly toxic to fourth instar C . tepperi in bioassays using a sand substrate (LC(50) 0.46mg/L, older larvae); younger fourth instar larvae were more susceptible (LC(50) 0.20mg/L) . Increasing larval densities (from 10 to 30 per bioassay cup) increased LC(50) values for both age groups, significantly so in the case of older larvae (higher density LC(50) 0.80mg/L) . Use of a soil substrate increased the LC(50) value (older larvae, 10 per cup) to 0.99mg/L . Similar differences in toxicity relative to larval age and substrate type were found in bioassays using the B.t.i . spore/crystal mixture . VectoBac WDG and the spore/crystal mixture both showed similar (approximately 6-fold) declines in activity between 30 and 17.5 degrees C . At lower temperatures (between 17.5 and 15 degrees C), activity of the spore/crystal mixture declined much more rapidly than that of VectoBac WDG . VectoLex WDG showed very low toxicity to C . tepperi larvae, and the overall impact of larval age and density was relatively minor (LC(50) values 1062-1340mg/L) . Autoclaving VectoLex WDG did not substantially reduce its toxicity (LC(50) 1426mg/L), suggesting that formulation additives (i.e., surfactants and other adjuvants) are responsible for much of the toxicity occurring at the high product concentrations required to cause C . tepperi mortality . Whilst VectoLex WDG was ineffective against C . tepperi, VectoBac WDG has the potential to provide selective control of this rice pest at economically viable application rates.

Aquat Toxicol, 2004 Aug 10, 69(2), 189 - 98
Seasonal variations in the effect of zinc pyrithione and copper pyrithione on pelagic phytoplankton communities; Maraldo K et al.; The relationship between environmental factors, community composition and the sensitivity of pelagic phytoplankton to the antifouling agents zinc pyrithione (ZPT) and copper pyrithione (CPT) was studied using phytoplankton communities collected from March until August 2001 in Roskilde fjord, Denmark . Sensitivity to ZPT and CPT was measured as EC50 values obtained from dose-response curves of photosynthesis to ZPT and CPT . EC50 for ZPT and CPT varied between 2 and 60 nM and 4 and 25 nM, respectively . Changes in sensitivity throughout the season were related to changes in phytoplankton community composition and density, and to nutrient levels . It was found that the variation in sensitivity of ZPT and CPT was related to the abundance of the groups Cryptophyceae, Bacillariophycaea and Dinophyceae when they were dominating the community . Furthermore, the sensitivity to ZPT was increased at low concentrations of phosphate per cell (<0.2 nmol/cell) . For CPT there was a negative correlation between toxicity and phosphate concentration in the water . Consequently, in aquatic environments where phytoplankton is phosphate limited the effect of ZPT and CPT may be enhanced.

Int J Tuberc Lung Dis, 2004 Jul, 8(7), 842 - 7
The value of counting BCG scars for interpretation of tuberculin skin tests in a tuberculosis hyperendemic shantytown, Peru; Saito M et al.; SETTING: The tuberculin skin test (TST) is widely used as a diagnostic or screening test for Mycobacterium tuberculosis infection and disease . A peri-urban shantytown in the desert hills of south Lima, Peru, highly endemic for tuberculosis, and where bacille Calmette-Guerin (BCG) vaccine had been given in multiple doses until 1995 . OBJECTIVE: To analyze the effect of multiple BCG vaccines on TST in a community-based setting . DESIGN: Point-prevalence survey of TST reactions of 572 people aged 6-26 years from 255 households . TST reactions were compared to the observed number of BCG scars and other potential risk factors (age, living with a TST-positive person, and contact with active tuberculosis) . RESULT: People with two or more scars had significantly larger reactions, even after adjusting for potential risk factors . The adjusted population attributable fraction of being TST-positive and having two or more BCG scars was 26% . CONCLUSION: There is no demonstrated benefit of repeat BCG vaccination . We therefore recommend that physicians take into consideration the number of BCG scars when interpreting the TST and that programs give no more than one BCG vaccination.

Indian J Exp Biol, 2004 Jun, 42(6), 575 - 80
Caffeine in tea plants {Camellia sinensis (L) O . Kuntze}: in situ lowering by Bacillus licheniformis (Weigmann) Chester; Ramarethinam S et al.; Tea plants (Camellia sinensis) contain 5-6% caffeine that is responsible for the stimulating effect of the beverage . As the tolerance to caffeine varies among individuals, low caffeine tea would be an ideal alternative . While assessing the potential of a few selected bacteria-Bacillus licheniformis, B . subtilis and B . firmus, to multiply on nutrient medium supplemented with glucose (5%) and tea leaf extract (2%), it was observed that only B . licheniformis could proliferate on this medium . Hence, B . licheniformis was used for further studies . Tea plants were sprayed with a suspension of B . licheniformis at a dilution of 5 x 10(8) CFU/ml containing 0.1% Tween 80 as surfactant . In situ lowering of caffeine from tea leaves was evident without affecting the quality of the other tea components . Further, there was no change in the morphological and physiological characteristics as well . It is suggested that spraying of B . licheniformis may be useful in yielding decaffeinated tea with good flavour and aroma.

Water Sci Technol, 2004, 49(10), 147 - 54
Biotechnology of intensive aerobic conversion of sewage sludge and food waste into fertilizer; Wang JY et al.; Biotechnology for intensive aerobic bioconversion of sewage sludge and food waste into fertilizer was developed . The wastes were treated in a closed reactor under controlled aeration, stirring, pH, and temperature at 60 degrees C, after addition of starter bacterial culture Bacillus thermoamylovorans . The biodegradation of sewage sludge was studied by decrease of volatile solids (VS), content of organic carbon and autofluorescence of coenzyme F420 . The degradation of anaerobic biomass was faster than biodegradation of total organic matter . The best fertilizer was obtained when sewage sludge was thermally pre-treated, mixed with food waste, chalk, and artificial bulking agent . The content of volatile solid and the content of organic carbon decreased at 24.8% and 13.5% of total solids, respectively, during ten days of bioconversion . The fertilizer was a powder with moisture content of 5% . It was stable, and not toxic for the germination of plant seeds . Addition of 1.0 to 1.5% of this fertilizer to the subsoil increased the growth of different plants tested by 113 to 164% . The biotechnology can be applied in larger scale for the recycling of sewage sludge and food wastes in Singapore.

Trans R Soc Trop Med Hyg, 2003 Jul-Aug, 97(4), 469 - 72
Immunotherapy of american cutaneous leishmaniasis in Venezuela during the period 1990-99; Convit J et al.; Of a total of 11532 Venezuelan patients with American cutaneous leishmaniasis (ACL) receiving immunotherapy with a combined vaccine containing heat-killed Leishmania promastigotes and bacille Calmette-Guerin (BCG) during the period 1990-99, we evaluated 5341 from 4 widely separated geographical states . Clinical healing varied from 91.2 to 98.7%, with an average of 95.7% . Adverse reactions were mild and limited to those associated with BCG vaccination alone . Immunotherapy failures in 143 patients included 54.5% with typical localized ulcers and 45.5% with non-mucosal intermediate cutaneous leishmaniasis (ICL) . Less than 2% of the patients in this study had lesions suggestive of ICL . The disproportionately large number of immunotherapy failures in the ICL group suggests that it should not be used as monotherapy in this group . Weaker reactivity to purified protein derivative in immunotherapy failures, while not statistically significant in the small group reported here, suggests the possibility that these patients develop a relatively torpid immune response . The high percentage of clinical cures achieved with immunotherapy, associated with few secondary effects and low cost, support the use of immunotherapy in the routine treatment of localized ACL.

World J Gastroenterol, 2004 Aug 1, 10(15), 2267 - 71
FR167653 attenuates murine immunological liver injury; Yao HW et al.; AIM: To study the effect of FR167653 on immunological liver injury (ILI) in mice . METHODS: ILI was established by tail vein injection of 2.5 mg Bacillus Calmette-Guerin (BCG), and 10 d later with 10 mg lipopolysaccharide (LPS) in 0.2 mL saline (BCG plus LPS) . Alanine aminotransferase (ALT), aspartate aminotransferase (AST) in sera and malondialdehyde (MDA), glutathione peroxidase (GSHpx) contents in liver homogenates were assayed by spectrophotometry . The levels of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) levels in sera were determined using ELISA . Interleukin-1 (IL-1) produced by peritoneal macrophages was determined by the method of (3)H-infiltrated cell proliferation . The nuclear factor-kappa B (NF-kappaB) p65 in liver tissue was analyzed with reverse transcription polymerase chain reaction (RT-PCR) . Liver samples collected were stained with hematoxylin and eosin . RESULTS: FR167653 (50, 100, 150 mg/kg) could significantly decrease the serum transaminase (ALT, AST) activity and MDA content in liver homogenate, and improve reduced GSHpx level of liver homogenate . Liver histopathological examination showed FR167653 (100, 150 mg/kg) significantly reduced inflammatory cells infiltration and liver cells necrosis . FR167653 (50, 100, 150 mg/kg) significantly lowered TNF-alpha and NO levels in serum, and IL-1 produced by peritoneal macrophages . Moreover, expression of NF-kappaB mRNA in liver tissue of ILI induced by BCG plus LPS was significantly reduced by FR167653 . CONCLUSION: All results showed that FR167653 had significant inhibitory action on ILI in mice.

Eur J Immunol, 2004 Aug, 34(8), 2220 - 9
Characterization of HLA-DR- and TCR-binding residues of an immunodominant and genetically permissive peptide of the 16-kDa protein of Mycobacterium tuberculosis; Caccamo N et al.; The 16-kDa protein of Mycobacterium tuberculosis represents an important antigenic target during bacillary latency and, consequently, should be considered as candidate subunit vaccine component . In this study, we have used CD4 T cell clones that recognize the peptide p91-110, an immunodominant and genetically permissive epitope, in the context of five different HLA-DR molecules and truncated and substituted variants of this peptide, to identify the minimal binding sequence (HLA-DR-binding core) and the minimal stimulatory sequence (TCR-binding core), as well as the residues that contact HLA-DR molecules and the TCR . We have found a common 9-mer sequence, spanning amino acids 93-101, as the binding core for HLA-DR1, -DR11, -DR13 and -DR7, but a longer (13-mer) sequence spanning amino acids 92-104 was required for binding to the HLA-DR15 molecules . F(93) was required for binding to all the tested HLA-DR molecules, hence allowing us to identify it as the N-terminal primary anchor residue (P1) . Additionally, the binding requirements for other residues varied considerably between the tested alleles: A(94) for HLA-DR15, V(99) for HLA-DR1, -DR15, -DR11 and -DR7, R(100) for HLA-DR11 and -DR13, and L(104) for HLA-DR15 . Concerning the residues of p91-110 peptide required for binding to the TCR, the pepscan analysis results would support the contention that P(-1) E(92), P6 F(98) would be important TCR contact sites because their substitutions led to full loss of T cell activation . Moreover, P8 R(100) is found to be critical residue in binding to HLA-DR11- and -DR13-restricted T cell clones, without influencing binding to the relevant HLA-DR molecule . Our results could be useful to design peptides with altered HLA anchor residues or TCR interaction sites to achieve remarkable increase in activity and to study their vaccine potential.

Proc Natl Acad Sci U S A, 2004 Jul 27, 101(30), 10995 - 1000 Epub 2004 Jul 15.
Mitogen-activated protein kinase pathways defend against bacterial pore-forming toxins; Huffman DL et al.; Cytolytic pore-forming toxins are important for the virulence of many disease-causing bacteria . How target cells molecularly respond to these toxins and whether or not they can mount a defense are poorly understood . By using microarrays, we demonstrate that the nematode Caenorhabditis elegans responds robustly to Cry5B, a member of the pore-forming Crystal toxin family made by Bacillus thuringiensis . This genomic response is distinct from that seen with a different stressor, the heavy metal cadmium . A p38 mitogen-activated protein kinase (MAPK) kinase and a c-Jun N-terminal-like MAPK are both transcriptionally up-regulated by Cry5B . Moreover, both MAPK pathways are functionally important because elimination of either leads to animals that are (i) hypersensitive to a low, chronic dose of toxin and (ii) hypersensitive to a high, brief dose of toxin such that the animal might naturally encounter in the wild . These results extend to mammalian cells because inhibition of p38 results in the hypersensitivity of baby hamster kidney cells to aerolysin, a pore-forming toxin that targets humans . Furthermore, we identify two downstream transcriptional targets of the p38 MAPK pathway, ttm-1 and ttm-2, that are required for defense against Cry5B . Our data demonstrate that cells defend against pore-forming toxins by means of conserved MAPK pathways.

South Med J, 2004 Jun, 97(6), 590 - 2
Renal failure and hypercalcemia as initial manifestations of extrapulmonary sarcoidosis; Ponce C et al.; Sarcoidosis is a granulomatous, multisystem disease . Rarely, sarcoidosis may present with both renal failure and hypercalcemia . A 27-year-old black man presented with severe abdominal pain and renal failure . A kidney biopsy demonstrated features of both interstitial nephritis and membranous glomerulopathy thought to be secondary to nonsteroidal anti-inflammatory drugs . His renal function and symptoms improved with short-term prednisone therapy . Discontinuation of steroids led to a recurrence of renal failure and severe hypercalcemia . On the basis of an elevated angiotensin-converting enzyme level of 160 U/L and anemia, a bone marrow biopsy was performed . Acid-fast bacillus-negative, noncaseating granulomas suggested the diagnosis of sarcoidosis . The patient recovered after restarting prednisone . Sarcoidosis may cause both interstitial and membranous nephritis from direct infiltration . Hypercalcemia results from increased calcium absorption secondary to 1,25-dihydroxyvitamin D production by sarcoid granulomas . Sarcoidosis must be considered in the differential diagnosis of renal failure in black patients . Serum calcium and angiotensin-converting enzyme levels may aid the diagnosis.

FEMS Microbiol Lett, 2004 Jul 15, 236(2), 325 - 31
Cloning, expression, and fibrin (ogen)olytic properties of a subtilisin DJ-4 gene from Bacillus sp . DJ-4; Choi NS et al.; Previously, we purified a strong fibrinolytic enzyme (subtilisin DJ-4) from Bacillus sp . DJ-4 and characterized its enzymatic activity . Here, we cloned the gene subtilisin DJ-4, and determines its nucleotide sequence, which showed 97% identity with subtilisin BPN' from B . amyloliquefacens . Recombinant full-subtilisin DJ-4 (rf-subDJ-4) and mature-subtilisin DJ-4 (rm-subDJ-4) were expressed using a pET29 vector system, and their fibrin (ogen)olytic and plasminogen activator activities were studied . rf-subDJ-4 was found to have a higher stability to heat (60 degrees C) and to acidic conditions (pH 3.0-4.0) than the native subtilisin DJ-4 of Bacillus sp . DJ-4 . The plasminogen activator activity of rf-subDJ-4 was 2.75 times greater than that of plasmin on a molar basis . And its specific activity (F/C, the ratio of fibrinolytic activity to caseinolytic activity) was 2.67 and 3.97 times higher than those of subtilisin BPN' and subtilisin Carlsberg, respectively . rf-subDJ-4 rapidly hydrolyzed the Aalpha-, Bbeta-, and gamma-chains of fibrinogen within 5 min . But, unlike subtilisin BPN' at a very low concentration (50 ng), the gamma-chain was not cleaved . On the other hand, rm-subDJ-4 did not show enzyme activity.

FEMS Microbiol Lett, 2004 Jul 15, 236(2), 319 - 24
Evidence for non-ribosomal peptide synthetase production of cereulide (the emetic toxin) in Bacillus cereus; Horwood PF et al.; Little is known about the process whereby the emetic toxin (or cereulide) of Bacillus cereus is produced . Two cereulide-producing strains of B . cereus were cloned and sequenced following polymerase chain reaction (PCR) amplification with primers that were specific for conserved regions of non-ribosomal peptide synthetase (NRPS) genes . The cloned regions of the B . cereus strains were highly homologous to conserved regions of other peptide synthetase nucleotide sequences . Primers were designed for two variable regions of the NRPS gene sequence to ensure specificity for the emetic strains . A total of 86 B . cereus strains of known emetic or non-emetic activity were screened using these primers . All of the emetic strains (n=30) displayed a 188 bp band following amplification and gel electrophoresis . We have developed an improved method of identifying emetic strains of B . cereus and provided evidence that cereulide is produced by peptide synthetases.

Res Microbiol, 2004 Jul-Aug, 155(6), 483 - 90
Genomic and phenotypic comparison of Bacillus fumarioli isolates from geothermal Antarctic soil and gelatine; De Clerck E et al.; Bacillus fumarioli was originally isolated from geothermal soils in continental and maritime Antarctica, and recently, it has been shown to be a frequent contaminant of gelatine extracts obtained from European and American production plants . These habitats are geographically widely separated, share similar temperature and pH conditions, but have substantially different organic loads . Because of the prevalence in gelatine extracts and the dissimilarity of this habitat to geothermal soil, a comparative study was performed to assess the diversity among B . fumarioli strains and reveal possible intraspecies differences that might correspond to their niches of origin . Genomic (rep-PCR, 16S rDNA sequencing, DNA-DNA hybridisations) and phenotypic techniques (analysis of fatty acid content, total cellular proteins, metabolic and morphological traits) illustrate the very close relationship between isolates from the two niches . An abundant protein band was demonstrated for gelatine isolates only . This band was shown to result from a protein with high similarity to a stress response protein . Furthermore, subtractive hybridisation revealed genomic differences between Antarctic and gelatine isolates that may indicate adaptive evolution to a specific environment.

J Ind Microbiol Biotechnol, 2004 Jul, 31(6), 273 - 7 Epub 2004 Jul 10.
Fusion of Bacillus stearothermophilus leucine aminopeptidase II with the raw-starch-binding domain of Bacillus sp . strain TS-23 alpha-amylase generates a chimeric enzyme with enhanced thermostability and catalytic activity; Hua YW et al.; Bacillus stearothermophilus leucine aminopeptidase II (LAPII) was fused at its C-terminal end with the raw-starch-binding domain of Bacillus sp . strain TS-23 alpha-amylase . The chimeric enzyme (LAPsbd), with an apparent molecular mass of approximately 61 kDa, was overexpressed in IPTG-induced Escherichia coli cells and purified to homogeneity by nickel-chelate chromatography . The purified enzyme retained LAP activity and adsorbed raw starch . LAPsbd was stable at 70 degrees C for 10 min, while the activity of wild-type enzyme was completely abolished under the same environmental condition . Compared with the wild-type enzyme, the twofold increase in the catalytic efficiency for LAPsbd was due to a 218% increase in the k(cat) value.

Bioresour Technol, 2004 Nov, 95(2), 187 - 90
Evaluation of fly ash as a carrier for diazotrophs and phosphobacteria; Gaind S et al.; Fly ash and its different combinations with soil (w/w) were tested to explore its possible use as a potential carrier for diazotrophs and phosphobacteria . Azotobacter chroococcum, Azospirillum brasilense and Bacillus circulans showed their maximum viability in fly ash alone whereas Pseudomonas striata proliferated most in soil:fly ash (1:1) combination.

Int J Food Microbiol, 2004 Aug 1, 94(3), 301 - 12
Occurrence and significance of Bacillus thuringiensis on wine grapes; Bae S et al.; Wine grapes harvested at different stages during cultivation from several vineyards in New South Wales, Australia, harboured Bacillus thuringiensis at viable populations of 10(2)-10(6) cfu/g . Commercial preparations of B . thuringiensis had been sprayed onto the grapes as a biological insecticide . B . thuringiensis (10(1)-10(3) cfu/ml) was isolated from grape juice and fermenting grape juice in a commercial winery . Although B . thuringiensis remained viable when inoculated at 10(3)-10(4) cfu/ml into grape juice and wine (pH 3.0-6.0), it did not grow . Using in vitro agar culture assays, B . thuringiensis inhibited several grape-associated yeasts and bacteria as well as various species of fungi associated with grape spoilage and ochratoxin A production . B . thuringiensis did not inhibit Saccharomyces cerevisiae in agar culture or during alcoholic fermentation of grape juice . B . thuringiensis inhibited the malolactic bacterium, Oenococcus oeni, in agar culture but not during mixed cultures in a liquid medium.

Clin Exp Dermatol, 2004 Jul, 29(4), 377 - 9
Severe sporotrichoid infection with Mycobacterium szulgai; Kapur N et al.; Mycobacterium szulgai is a nontuberculous, acid-fast bacillus or atypical mycobacteria, which prior to 1972 was not thought of as a pathogen . Since then most cases reported in the literature have been of pulmonary disease with only a few case reports of cutaneous disease . Our patient, who had an underlying, uncategorized, immunosuppressive condition, presented with multiple severe ulcers spreading proximally up the arms in a sporotrichoid pattern with more scatttered lesions on his legs . He made a full recovery with appropriate antimicrobial treatment.

J Appl Microbiol, 1998 Jan, 84(1), 68 - 71
Effect of probiotic CenBiot on the control of diarrhoea and feed efficiency in pigs; Zani JL et al.; The production and testing of a probiotic formulated with a strain of Bacillus cereus (Probiotic CenBiot) is reported . The strain was grown in fed batch fermenters, desiccated, and mixed with cornflour . To test the effect of the probiotic in controlling piglet diarrhoea, two groups of sows and their respective litters received feed supplemented with probiotic CenBiot or Furazolidone; the control group received the same basic feed but not supplemented . Probiotic CenBiot was as effective as Furazolidone, reducing the prevalence of diarrhoea to half that in the control group . It also significantly improved feed conversion, daily weight gain and total weight gain in pigs in the nursery phase . Feed conversion ratios of weaned pigs were 1.904, 2.146 and 2.099 for the groups that received Probiotic CenBiot, a commercial probiotic, and basic feed, respectively . In terms of food consumption, the Probiotic CenBiot group consumed 12.6% less than the commercial probiotic group, and 10% less than the control group, to achieve the same weight.

J Infect Dis, 2004 Aug 1, 190(3), 588 - 97 Epub 2004 Jul 06.
Immune response to postprimary tuberculosis in mice: Mycobacterium tuberculosis and Miycobacterium bovis bacille Calmette-Guérin induce equal protection; Mollenkopf HJ et al.; We addressed the question of whether protective immunity induced by natural infection with Mycobacterium tuberculosis and that induced by vaccination with Mycobacterium bovis bacille Calmette-Guerin (BCG) differ in the murine model . We infected mice with M . tuberculosis Erdman, cured them by chemotherapy, and subsequently reinfected them with a low dose of M . tuberculosis H37Rv . The course of tuberculosis was compared with that in mice previously vaccinated with BCG Danish 1331 . Protection against postprimary M . tuberculosis infection did not differ significantly between the 2 groups . After challenge infection, numbers of interferon- gamma -positive splenocytes did not differ between mice with primary infection and vaccinated mice . Splenocytes from primary M . tuberculosis-infected mice conferred marginally higher protection than did those from BCG-vaccinated mice . Serum transfer did not protect against reinfection in either group . Our data emphasize that natural infection with M . tuberculosis and vaccination with BCG do not differ in their capacity to induce protective immunity against tuberculosis and support the notions that reinfection contributes to the development of active disease and that any novel vaccine against tuberculosis has to perform better than both vaccination with BCG and immunity evoked by natural infection.

Theor Appl Genet, 2004 Sep, 109(5), 1051 - 7 Epub 2004 Jul 09.
A QTL that enhances and broadens Bt insect resistance in soybean; Walker DR et al.; Effective strategies are needed to manage insect resistance to Bacillus thuringiensis (Bt) proteins expressed in transgenic crops . To evaluate a multiple resistance gene pyramiding strategy, eight soybean (Glycine max) lines possessing factorial combinations of two quantitative trait loci (QTLs) from plant introduction (PI) 229358 and a synthetic Bt cry1Ac gene were developed using marker-assisted selection with simple sequence repeat markers . Field studies were conducted in 2000 and 2001 to evaluate resistance to corn earworm (Helicoverpa zea) and soybean looper (Pseudoplusia includens), and detached leaf bioassays were used to test antibiosis resistance to Bt-resistant and Bt-susceptible strains of tobacco budworm (TBW; Heliothis virescens) . Based on defoliation in the field and larval weight gain on detached leaves, lines carrying a combination of cry1Ac and the PI 229358 allele at a QTL on linkage group M were significantly more resistant to the lepidopteran pests, including the Bt-resistant TBW strain, than were the other lines . This is the first report of a complementary additive effect between a Bt transgene and a plant insect resistance QTL with an uncharacterized mode of action that was introgressed using marker-assisted selection.

Indian J Exp Biol, 2003 Sep, 41(9), 986 - 90
Biosorption and elution of chromium from immobilized Bacillus coagulans biomass; Srinath T et al.; Bacillus coagulans, a tannery wastewater isolate, previously shown to bind dissolved Cr(VI), retained its ability to biosorb Cr(VI) in different matrices . Polymeric materials like agar, agarose, calcium alginate and polyacrylamide were screened . Agarose emerged as the suitable candidate for biomass immobilization mainly due to its higher stability and integrity in acidic pH . Aptness of agarose as the matrix for B . coagulans biomass was revealed during Cr(VI) biosorption from natural wastewater.

J Leukoc Biol, 2004 Oct, 76(4), 827 - 34 Epub 2004 Jul 07.
Dendritic cells derived from BCG-infected precursors induce Th2-like immune response; Martino A et al.; Human monocytes can differentiate into dendritic cells (DCs) according to the nature of environmental signals . We tested here whether the infection with the live tuberculosis vaccine bacillus Calmette-Guerin (BCG), which is known to be limited in preventing pulmonary tuberculosis, modulates monocyte and DC differentiation . We found that monocytes infected with BCG differentiate into CD1a- DCs (BCG-DCs) in the presence of granulocyte macrophage-colony stimulating factor and interleukin (IL)-4 and acquired a mature phenotype in the absence of maturation stimuli . In addition, BCG-DCs produced proinflammatory cytokines (tumor necrosis factor alpha, IL-1beta, IL-6) and IL-10 but not IL-12 . BCG-DCs were able to stimulate allogeneic T lymphocytes to a similar degree as DCs generated in the absence of infection . However, BCG-DCs induced IL-4 production when cocultured with human cord-blood mononuclear cells . The induction of IL-4 production by DCs generated by BCG-infected monocytes could explain the failure of the BCG vaccine to prevent pulmonary tuberculosis.

J Appl Microbiol, 2004, 97(2), 338 - 46
Improvement of bioinsecticides production through mutagenesis of Bacillus thuringiensis by u.v . and nitrous acid affecting metabolic pathways and/or delta-endotoxin synthesis; Ghribi D et al.; AIMS: The present work aimed to obtain bioinsecticide over-producing mutants through classical mutagenesis of vegetative cells of Bacillus thuringiensis (Bt) by using u.v . and nitrous acid, and to evidence the involvement of cell metabolism in delta-endotoxin synthesis . METHODS AND RESULTS: Vegetative cells of Bt were treated by nitrous acid (0.17 mg ml(-1)) or exposed to u.v . rays (emitted at a wave length of 240 nm) . The isolated survivors were screened on the basis of the production of delta-endotoxins and biomass in glucose and/or in gruel-based media at two aeration conditions . Bioinsecticide over-producing mutants were obtained with high frequencies because random mutations were shown to affect cell metabolism at different pathways related to the regulation of delta-endotoxin synthesis . CONCLUSIONS: Classical mutagenesis of Bt cells lead to the isolation of a large variety of delta-endotoxin over-producing mutants that could be classified into six groups based on the location of the mutations, particularly in metabolism pathways and delta-endotoxin synthesis . SIGNIFICANCE AND IMPACT OF THE STUDY: High frequencies of delta-endotoxin over-producing mutants of Bt could be obtained through classical mutagenesis of vegetative cells . This should contribute to a significant reduction of production and utilization costs of Bt bioinsecticides.

Am J Trop Med Hyg, 2004 Jul, 71(1), 53 - 55
REPORT OF AN UNUSUAL CASE OF PERSISTENT BACTEREMIA BY BARTONELLA BACILLIFORMIS IN A SPLENECTOMIZED PATIENT; Henriquez C et al.; We report a case of a 56-year-old man with a history of splenectomy for idiopathic thrombocytopenic purpura who developed persistent bacteremia in the acute phase of human bartonellosis . This patient did not develop hemolytic anemia . Only after several courses of antibiotic treatment was the infection eradicated . This is an unusual case of overwhelming post-splenectomy infection by Bartonella bacilliformis, which provides clinical evidence that the spleen is a critical effector organ of clearance of this infection as well as the effector organ of bartonellosis-associated hemolytic anemia.

J Occup Environ Hyg, 2004 Jul, 1(7), 430 - 5
UV-induced inactivation rates for airborne Mycobacterium bovis BCG; Peccia J et al.; Engineering ultraviolet irradiation systems as a control against infectious airborne diseases requires a knowledge of intrinsic ultraviolet (UV) inactivation rates of airborne bacteria . Ultraviolet inactivation rates for airborne Mycobacterium bovis bacillus Calmette-Guerin (BCG) were determined at 50% and 95% relative humidity (RH) in a 0.8 m3 bioaerosol reactor . Ultraviolet inactivation response of waterborne M . bovis BCG pure cultures was also determined . At 50% RH the airborne UV inactivation rates observed were two times greater than those observed in saturated air (RH = 95%), and rates at 95% RH were similar to those observed in otherwise identical cultures suspended in water . Intrinsic UV inactivation rates for M . bovis BCG were statistically similar to rates observed for Mycobacterium parafortuitum at 50% and 95% RH, indicating that M . parafortuitum is a valid surrogate for studying airborne UV responses of M . bovis BCG and Mycobacterium tuberculosis . Results also confirm that UV inactivation responses for bacteria suspended in water cannot be used to estimate UV dose response in unsaturated air .

J Mol Biol, 2004 Jul 23, 340(5), 941 - 56
Crystal structures of the ADP and ATP bound forms of the Bacillus anti-sigma factor SpoIIAB in complex with the anti-anti-sigma SpoIIAA; Masuda S et al.; Cell type-specific transcription during Bacillus sporulation is established by sigma(F), the activity of which is controlled by a regulatory circuit involving the anti-sigma factor and serine kinase SpoIIAB, and the anti-anti-sigma SpoIIAA . When ATP is present in the nucleotide-binding site of SpoIIAB, SpoIIAA is phosphorylated, followed by dissociation . The nucleotide-binding site of SpoIIAB is left bound to ADP . SpoIIAB(ADP) can bind an unphosphorylated molecule of SpoIIAA as a stable binding partner . Thus, in this circuit, SpoIIAA plays a dual role as a substrate of the SpoIIAB kinase activity, as well as a tight binding inhibitor . Crystal structures of both the pre-phosphorylation complex and the inhibitory complex, SpoIIAB(ATP) and SpoIIAB(ADP) bound to SpoIIAA, respectively, have been determined . The structural differences between the two forms are subtle and confined to interactions with the phosphoryl groups of the nucleotides . The structures reveal details of the SpoIIAA:SpoIIAB interactions and how phosphorylated SpoIIAA dissociates from SpoIIAB(ADP) . Finally, the results confirm and expand upon the docking model for SpoIIAA function as an anti-anti-sigma in releasing sigma(F) from SpoIIAB.

Int J Pediatr Otorhinolaryngol, 2004 Aug, 68(8), 1069 - 74
Prevalence of otitis media with effusion in first and second grade primary school students and its correlation with BCG vaccination; Keles E et al.; OBJECTIVE: The object of this study was to determine the prevalence of otitis media with effusion (OME) in first and second grade primary school students, to analyze the causes of the difference in the prevalence, to define the effect of OME on the academic performance of the children and to investigate a correlation between the prevalence of OME and Bacillus Calmette-Guerin (BCG) vaccination . STUDY PLAN AND METHODS: The study was conducted during the September-November, 2002 period in Elazig . A total of 3675 clinically healthy primary school children attending primary schools in the central district of Elazig, living in the same region and of a similar socioeconomic status and age group were included in the study . Of the 3675 children, 2042 were in their first and 1633 in their second grade of primary school . The routine ear-nose and throat examinations of the children were carried out at their schools by the same medical team . The tympanometric test was performed in children diagnosed with OME following otoscopic examination . A scale measuring the academic performance of the children was developed . This scale was filled in by the student's teachers prior to physical examination . RESULTS: OME was diagnosed in 64 out of 2042 (3.1%) first grade and in 25 out of 1633 (1.5%) second grade students . The difference between the percentages of OME in first and second grade students was statistically significant (P < 0.05) . The frequency of other ear-nose and throat pathologies accompanying OME was similar to those in children without OME . There was no statistically significant difference between the academic performances of children with and without OME (P > 0.05) . CONCLUSION: The analysis of risk factors for OME revealed no difference between first and second grade students . The reason for the difference in the prevalence of OME between first and second grade students may be the positive effect on the immune system of the BCG vaccine which had been administered to the second grade students 4 months previously.

Arq Neuropsiquiatr, 2004 Jun, 62(2A), 342 - 6 Epub 2004 Jun 23.
Whipple's disease with neurological manifestations: case report; Franca MC Jr et al.; Whipple's disease (WD) is an uncommon multisystem condition caused by the bacillus Tropheryma whipplei . Central nervous system involvement is a classical feature of the disease observed in 20 to 40% of the patients . We report the case of a 62 year old man with WD that developed neurological manifestations during its course, and discuss the most usual signs and symptoms focusing on recent diagnostic criteria and novel treatment regimens.

J Biol Chem, 2004 Sep 17, 279(38), 39798 - 806 Epub 2004 Jul 02.
Extracellular mycobacterial DNA-binding protein 1 participates in mycobacterium-lung epithelial cell interaction through hyaluronic acid; Aoki K et al.; Mycobacterium tuberculosis infects not only host macrophages but also nonprofessional phagocytes, such as alveolar epithelial cells . Glycosaminoglycans (GAGs) are considered as the component of mycobacterial adherence to epithelial cells . Here we show that extracellularly occurring mycobacterial DNA-binding protein 1 (MDP1) promotes mycobacterial infection to A549 human lung epithelial cells through hyaluronic acid (HA) . Both surface plasmon resonance analysis and enzyme-linked immunosorbent assay revealed that MDP1 bound to HA, heparin, and chondroitin sulfate . Utilizing synthetic peptides, we next defined heparin-binding site of 20 amino acids from 31 to 50 of MDP1, which is responsible for the specific DNA-binding site of MDP1 . MDP1 bound to A549 cells, and exogenous DNA and HA interfered with the interaction . The binding was also abolished by treatment of A549 cells with hyaluronidase, suggesting that HA participates in the MDP1-A549 cell interaction . Adherence of bacillus Calmette-Guerin (BCG) and M . tuberculosis to A549 cells was inhibited by addition of HA, DNA, and anti-MDP1 antibody, showing that MDP1 participates in the interaction between mycobacteria-alveolar epithelial cells . Simultaneous treatment of intratracheal BCG-infected mice with HA reduced the growth of BCG in vivo . Taken together, theses results suggest that HA participates in Mycobacterium-lung epithelium interaction and has potential for therapeutic and prophylactic interventions in mycobacterial infection.

Indian J Exp Biol, 2004 May, 42(5), 515 - 21
Purification and characterization of a small size protease from Bacillus sp . APR-4; Kumar D et al.; A thermostable extracellular protease of Bacillus sp . APR-4 was purified by size-exclusion and ion-exchange chromatographic methods and its properties were studied . The purified enzyme had a specific activity of 21,000 U/mg of protein and gave single band on SDS/PAGE with a molecular mass of 16.9 KDa . This protease had an optimal pH of 9 and exhibited its highest activity at 60 degrees C . The enzyme activity was inhibited by EDTA, suggesting the presence of metal residue at the active site . Ca2+ (5 mM) had stabilising effect on the activity of protease, but Cu2+ (5 mM) had inhibitory effect . The enzyme exhibited highest specificity towards casein (1%) and had a Km of 26.3 mg/ml and a Vmax of 47.6 U/mg with casein as a substrate . The stability of this enzyme was evaluated in the presence of some organic solvents and the enzyme was stable in methanol, petroleum ether and ethanol . Detergents (Wheel, Farishta) had stimulatory effect on the activity of this enzyme.

PDA J Pharm Sci Technol, 2004 May-Jun, 58(3), 147 - 58
Evaluation of Blow/Fill/Seal extrusion through processing polymer contaminated with bacterial spores and endotoxin; Leo F et al.; A study has been carried out to further the understanding of the extrusion process and its impact upon the quality of Blow/Fill/Seal product . Controlled challenges to the extrusion system, comprising low-density polyethylene granulate contaminated with characterized levels of Bacillus atrophaeus (ATCC 9372) endospores and Escherichia coli 055:B5 bacterial endotoxin, have been conducted . Batches of spore contaminated polymer, at challenge levels varying from 10(3) to 10(6) spores g(-1) polymer with derived D160 values ranging from 1.22 to 2.07 minutes, and endotoxin contaminated polymer, at challenge levels varying from 10(2) to 10(4) EU g(-1) polymer, were processed through a Blow/Fill/Seal machine employing Tryptone Soya Broth and Water for Injection as the fill mediums, respectively . Relationships have been established between the levels of challenge for each of spores and endotoxin and the extent of product contamination . The relationships allow for prediction of microbiological and pyrogenic quality based upon the microbiological and pyrogenic attributes of unprocessed polymeric granulate and for rationalized choices of polymeric granulate acceptance limits . It is stressed that the findings apply only to the particular Blow/Fill/Seal machine and to the specific conditions of machine operation.

Environ Toxicol Chem, 2004 Jul, 23(7), 1662 - 71
Susceptibility of the leaf-eating beetle, Galerucella calmariensis, a biological control agent for purple loosestrife (Lythrum salcaria), to three mosquito control larvicides; Lowe TP et al.; We evaluated the susceptibility of Galerucella calmariensis, a species used to control purple loosestrife (Lythrum salicaria), to three mosquito control larvicides . Larvae and adults were fed loosestrife cuttings dipped in Abate (< or = 375 g x L(-1)), Altosid (< or = 250 g x L(-1)), and Bacillus thuringiensis var israeliensis (Bti) (< or = 110 g x L(-1)) . Eggs on cuttings were dipped in the same concentrations . Pupae were immersed in Abate and Altosid solutions (< or = 474.4 microg x L(-1) and < or = 1,169.2 microg x L(-1), respectively) . Hatching success of eggs dipped in Abate (> or = 3.75 g x L(-1)) was reduced significantly and survival was significantly lower among larvae and adults eating cuttings dipped in Abate (> or = 0.17 g x L(-1) and > or = 2.27 g x L(-1), respectively) . Hatching success of eggs dipped in Altosid (> or = 2.52 g x L(-1)) was reduced significantly . With exposure to Altosid, larval survival to pupation and adult emergence was reduced significantly at concentrations of > or = 2.92 g x L(-1) and > or = 0.63 g x L(-1), respectively . Altosid (> or = 0.23 g x L(-1)) also delayed the onset of pupation and adult emergence among larvae that survived to pupate . Larvae that survived with exposure to Altosid (> or = 1.72 g x L(-1)) grew to 70% larger than those exposed to lower concentrations . Pupal survival was unaffected with exposure to Abate and Altosid and adult survival was unaffected with exposure to Altosid . Bacillus thuringiensis var israeliensis did not adversely affect any life stage of G . calmariensis . The mean Abate concentration on cuttings exposed to operational spraying was in the range that reduced egg hatchability and adult survival but was higher than concentrations that caused complete mortality of larvae . The mean Altosid concentration on cuttings exposed to operational spraying was in the range that reduced hatching success in eggs and delayed pupation and adult emergence of larvae.

Probl Tuberk Bolezn Legk, 2004, (5), 35 - 7
{Influence of environmental factors on the epidemiology of tuberculosis in the Astrakhan region}; Strel'tsova EN et al.; The epidemiology of tuberculosis in the Astrakhan Region depends on many causes among which environmental factors do not take last places; a significant positive correlation with which was observed in 41.5 and 52% cases in the rural areas of the Astrakhan Region and in Astrakhan, respectively . The proportion of environmental factors among the causes in noticeably higher in destructive pulmonary tuberculosis, in chronic destructive forms in particular, and in first detected bacillary patients.

Mol Microbiol, 2004 Jul, 53(2), 457 - 67
IS231-MIC231 elements from Bacillus cereus sensu lato are modular; De Palmenaer D et al.; Summary IS231A was originally discovered in Bacillus thuringiensis as a typical 1.6 kb insertion sequence (IS) displaying 20 bp inverted repeats (IR) flanking a transposase gene . A first major variation of this canonical organization was found in MIC231A1 . This mobile insertion cassette (MIC), delineated by IS231A-related extremities, contained an active d-stereospecific endopeptidase (adp) gene instead of a transposase . Interestingly, it was shown that MIC231A1 can be mobilized in trans by the IS231A transposase . In this paper, we show that this family of IS231-MIC231 elements can be extended to a broad range of related entities displaying higher levels of structural complexity . Several IS231A-like elements contained, upstream of their transposase gene, passenger genes coding for putative antibiotic resistances or regulatory factors . Furthermore, the diversity of the MIC231 elements ranged from empty cassettes to structures carrying up to three passenger genes . Among these, MIC231V carried, in addition to an adp gene, an active fosfomycin resistance determinant . In vivo transposition assays showed that MIC231V is also trans-activated by the IS231A transposase . These results lend further support to the potential contribution of these modular mobile elements to the genome plasticity of the Bacillus cereus/B . thuringiensis group.

Anal Chem, 2004 Jul 1, 76(13), 3707 - 15
Thermosiphon-based PCR reactor: experiment and modeling; Chen Z et al.; A self-actuated, flow-cycling polymerase chain reaction (PCR) reactor that takes advantage of buoyancy forces to continuously circulate reagents in a closed loop through various thermal zones has been constructed, tested, and modeled . The heating required for the PCR is advantageously used to induce fluid motion without the need for a pump . Flow velocities on the order of millimeters per second are readily attainable . In our preliminary prototype, we measured a cross-sectionally averaged velocity of 2.5 mm/s and a cycle time of 104 s . The flow velocity is nearly independent of the loop's length, making the device readily scalable . Successful amplifications of 700- and 305-bp fragments of Bacillus cereus genomic DNA have been demonstrated . Since the device does not require any moving parts, it is particularly suitable for miniature systems.

Trans R Soc Trop Med Hyg, 2003 May-Jun, 97(3), 365 - 8
Alum-precipitated autoclaved Leishmania major plus bacille Calmette-Guérrin, a candidate vaccine for visceral leishmaniasis: safety, skin-delayed type hypersensitivity response and dose finding in healthy volunteers; Kamil AA et al.; In a previous efficacy study, autoclaved Leishmania major (ALM) + bacille Calmette-Guerrin (BCG) vaccine was shown to be safe, but not superior to BCG alone, in protecting against visceral leishmaniasis . From June 1999 to June 2000, we studied the safety and immunogenicity of different doses of alum-precipitated ALM + BCG vaccine mixture administered intradermally to evaluate whether the addition of alum improved the immunogenicity of ALM . Twenty-four healthy adult volunteers were recruited and sequentially allocated to receive either 10 microg, 100 microg, 200 microg, or 400 microg of leishmanial protein in the alum-precipitated ALM + BCG vaccine mixture . Side effects were minimal for all doses and confined to the site of injection . All volunteers in the 10 microg, 100 microg, and 400 microg groups had a leishmanin skin test (LST) reaction of > or = 5 mm by day 42 and this response was maintained when tested after 90 d . Only 1 volunteer out of 5 in the 200 microg group had a LST reaction of > or = 5 mm by day 42 and the reasons for the different LST responses in this group are unclear . This is the first time that an alum adjuvant with ALM has been in used in humans and the vaccine mixture was safe and induced a strong delayed type hypersensitivity (DTH) reaction in the study volunteers . On the basis of this study we suggest that 100 1 microg of leishmanial protein in the vaccine mixture is a suitable dose for future efficacy studies, as it induced the strongest DTH reaction following vaccination.

Nihon Kokyuki Gakkai Zasshi, 2004 Jun, 42(6), 519 - 22
{A case of lung infection due to Mycobacterium abscessus (M . abscessus) complicated with primary macroamylasemia}; Matsuzawa K et al.; We report a case of lung infection due to Mycobacterium abscessus (M . abscessus), complicated with primary macroamylasemia . A 76-year-old man was admitted to our hospital in August 2002 because of bloody sputum and an abnormal shadow found on chest radiography . The patient had had pulmonary tuberculosis from 1998 to 2000 . He was found to be antacid bacillus-positive (Gaffky 5) on examination of the sputum in August 2002, but after hospitalization was negative for tuberculosis bacillus on sputum examination by the PCR method . We had suspected the presence of non-tuberculous mycobacterial disease since the patient's admission, and had started a regime of three drugs: clarithromycin, rifampicin, and ethambutol . The bacteria were identified as M . abscessus in a later sputum culture examination . It was noticed that the blood amylase level was high, and the disease was diagnosed as primary macroamylasemia . Such a case of lung infection due to M . abscessus complicated with macroamylasemia has rarely been reported in Japan.

Mol Microbiol, 2004 Jul, 53(1), 29 - 40
Differentiate to thrive: lessons from the Legionella pneumophila life cycle; Molofsky AB et al.; When confronted by disparate environments, microbes routinely alter their physiology to tolerate or exploit local conditions . But some circumstances require more drastic remodelling of the bacterial cell, as sporulation by the Bacillus and Streptomyces species of soil bacteria vividly illustrates . Cellular differentiation is also crucial for pathogens, the challenge for which is to colonize one host, then be transmitted to the next . Using the Gram-negative Legionella pneumophila as a model intracellular pathogen, we describe how biogenesis of the replication vacuole is determined by the developmental state of the bacterium . Subsequently, when replicating bacteria have exhausted the nutrient supply, the pathogens couple their conversion to stationary phase physiology with expression of traits that promote transmission to a new host . The cellular differentiation of L . pneumophila is co-ordinated by a regulatory circuit that integrates several elements that are broadly conserved in the microbial world . The alarmone (p)ppGpp promotes transcription directed by the alternative sigma factors RpoS, FliA and, probably, RpoN, and also post-transcriptional control mediated by a two-component regulatory system, LetA/S (GacA/S), and an mRNA-binding protein, CsrA (RsmA) . By applying knowledge of microbial differentiation in combination with tools to screen the complete genomes of pathogens, experiments can be designed to identify two distinct classes of virulence traits: factors that promote replication and those dedicated to transmission.

J Environ Qual, 2004 May-Jun, 33(3), 832 - 6
Soil microbial communities associated with Bt and non-Bt corn in three soils; Blackwood CB et al.; The effects of expression of Cry endotoxin by Bt corn (transgenic corn engineered to express Bacillus thuringiensis toxin) on soil microbial community structure were assessed in a growth chamber experiment . Two lines of transgenic corn expressing different Cry endotoxins were compared with their respective non-transgenic isolines in three soil types with differing textures . Phospholipid fatty acid (PLFA) profiles from bulk soil and community-level physiological profiles (CLPP) from the rhizosphere community were used to assess community structure . Differences in PLFA profiles due to soil type were significant, accounting for 73% of the total variability in the dataset . Differences in bacterial and fungal CLPP profiles due to soil type were statistically significant, but probably not biologically important, accounting for 6.3 and 3.8% of the total variability, respectively . Neither expression of Cry endotoxin nor corn line had a significant effect on microbial profiles, except in the high-clay soil where both factors significantly affected bacterial CLPP profiles (accounting for 6.6 and 6.1% of the variability in that soil, respectively) . Expression of Cry endotoxin also significantly reduced the presence of eukaryotic PLFA biomarker in bulk soils, although it is unclear which groups of eukaryotes were affected . We conclude that the effects of transgenic Bt corn in this short-term experiment are small, and longer-term investigations are necessary.

Appl Microbiol Biotechnol, 2004 Jul, 65(1), 56 - 60 Epub 2004 Feb 04.
Aerobic production of alanine by Escherichia coli aceF ldhA mutants expressing the Bacillus sphaericus alaD gene; Lee M et al.; Alanine was produced from glucose in an Escherichia coli aceF ldhA double mutant strain that contained the pTrc99A- alaD plasmid expressing Bacillus sphaericus alanine dehydrogenase . The aceF gene encodes one of the proteins of the pyruvate dehydrogenase complex, and therefore this strain required acetate as an additional carbon source . The ldhA gene encodes fermentative lactate dehydrogenase, a competitor of alanine dehydrogenase for the substrate pyruvate . Fermentations included an oxygenated growth phase followed by an oxygen-limited alanine production phase . The lowest value for the mass transfer coefficient ( k(L)a) studied during the production phase yielded the greatest alanine . With feeding of glucose and NH(4)Cl, 32 g/l alanine accumulated in 27 h with a yield of 0.63 g alanine generated per gram glucose consumed.

Appl Microbiol Biotechnol, 2004 Sep, 65(4), 407 - 13 Epub 2004 Jun 18.
Production of autoproteolytically subunit-assembled 7-beta-(4-carboxybutanamido)cephalosporanic acid (GL-7ACA) acylase from Pseudomonas sp . C427 using a chitin-binding domain; Nagao K et al.; 7-Beta-(4-Carboxybutanamido)cephalosporanic acid (GL-7ACA) acylase from Pseudomonas sp . C427 is known as a proteolytically processed bacterial enzyme . GL-7ACA acylase from Pseudomonas sp . C427 (C427) consists of alpha- and beta-subunits that are processed from a precursor peptide by removing the spacer peptide . A chitin-binding domain (CBD) of chitinase A1 derived from Bacillus circulans was genetically fused into four different positions of the C427-encoding gene . In the four enzymes thereby produced, Nalpha427, SP427, Calpha427, and Cbeta427, it was fused, respectively, to the N-terminal region of the alpha-subunit; the C-terminal region of the alpha-subunit; the three-amino-acid upper region of the C-terminal of the alpha-subunit; and to the C-terminal region of the beta-subunit . All of the fusion enzymes, expressed in Eschericha coli, were successfully processed into active forms and had GL-7ACA acylase activity . The affinity-binding activity to crystalline chitin was affected by the fusing position of CBD . Nalpha427, Calpha427, and Cbeta427 remained fused to the CBD after their processing steps and could bind to chitin, but in the case of SP427 the fused CBD was cleaved away during the processing steps and binding activity was no longer observed . These results indicate that CBD is functional in such autoproteolytically subunit-assembled acylases.

Int J Lepr Other Mycobact Dis, 2004 Mar, 72(1), 1 - 7
Relapses in multibacillary patients treated with multi-drug therapy until smear negativity: findings after twenty years; Norman G et al.; The Schieffelin Leprosy Research and Training Center at Karigiri, India participated in several of the World Health Organization (WHO) trials . The first trial on combined therapy in multi-bacillary leprosy was initiated in 1981 . The main objectives of this field trial were to evaluate the efficacy of WHO recommended regimens in preventing relapses, especially drug resistance relapses . This paper reports on the relapses twenty years after patients were inducted into the WHO field trial.Between 1981 and 1982, 1067 borderline lepromatous and lepromatous patients were inducted into the WHO field trial for combined therapy in multi-bacillary leprosy trial . Among them, 357 patients were skin smear positive . During the follow-up in 2002, only 173 of them could be traced and assessed . The mean duration of follow-up was 16.4 +/- 1.83 years . Two patients relapsed 14 and 15 years after being released from treatment, the relapse rate being 0.07 per 100 person years follow-up . Drug susceptibility tests done on one of the relapsed patients revealed drug sensitive organisms to all multi-drug therapy drugs.

Microbiol Immunol, 2004, 48(6), 457 - 63
Effects of recombinant cholera toxin b subunit (rCTB) on cellular immune responses: enhancement of delayed-type hypersensitivity following intranasal co-administration of Mycobacterium bovis-BCG with rCTB; Maeyama J et al.; Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant . To gain insight into the mechanism underlying the adjuvant effect of rCTB, the effects of rCTB on cell-mediated immune responses of mice and guinea pigs were examined after intranasal administration of Mycobacterium bovis -bacillus Calmette-Guerin (BCG) with and without rCTB . Delayed-type hypersensitivity, for skin reactions in guinea pigs and for footpad swelling reactions in mice, to purified protein derivative (PPD) were enhanced by intranasal co-administration of BCG and rCTB, as compared to giving BCG alone to these animals . Moreover, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma production of spleen cells and antigen specific spleen cell proliferation, stimulated with PPD, were enhanced in the presence of rCTB . These results strongly suggest that rCTB enhances cellular as well as humoral immune responses.

Biosci Biotechnol Biochem, 2004 Jun, 68(6), 1299 - 305
A chitinase indispensable for formation of protoplast of Schizophyllum commune in basidiomycete-lytic enzyme preparation produced by Bacillus circulans KA-304; Yano S et al.; KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S . commune mycelia . alpha-1,3-Glucanase, which was isolated from an ammonium sulfate fraction of 0-30% saturation of KA-prep, gave the protoplast-forming activity to an ammonium sulfate fraction of 30-50% saturation of KA-prep, which contained chitinase(s) and beta-glucanase(s) but was inactive in the protoplast formation . Chitinase(s) and beta-glucanase(s) in the ammonium sulfate fraction of 30-50% saturation were separated by DEAE-cellulofine A-500 column chromatography, and the protoplast-forming activity appeared when the chitinase preparation was mixed with the alpha-1,3-glucanase . The beta-glucanase preparation was not effective for the protoplast formation whereas its addition enhanced the protoplast-forming activity of the mixture of alpha-1,3-glucanase and the chitinase preparation . The chitinase preparation contained two chitinases (chitinase I and II) . Chitinase I showed the protoplast-forming activity with alpha-1,3-glucanase, but chitinase II did not . Chitinase I, a monomeric protein with a molecular weight of 41,000, was active toward colloidal chitin and ethylene glycol chitin . Chitinase I produced predominantly N,N'-diacetylchitobiose and N,N',N"-triacetylchitotriose from colloidal chitin, and the enzyme was inactive to p-NP-beta-D-N-acetylglucosaminide, suggesting that it was an endo-type enzyme . The N-terminal amino acid sequence of chitinase I (A L A T P T L N V S A S S G M) had no sequential identity to those of known chitinases.

Syst Appl Microbiol, 2004 May, 27(3), 301 - 7
Bacillus algicola sp . nov., a novel filamentous organism isolated from brown alga Fucus evanescens; Ivanova EP et al.; A slightly yellowish, Gram-positive, filamentous with 'cross-like' branching, aerobic, spore-forming bacterium was isolated from enrichment culture during degradation of the thallus of the brown alga Fucus evanescens . The bacterium studied was chemoorganotrophic, tolerant to 3% NaCl, alkalitolerant, and alginolytic . The predominant cellular fatty acid was ai15:0 which accounted more than 65% of total fatty acids, while i14:0, il5:0 i16:0, and ai17:0 made up 25% . DNA base composition was 37 mol% GC . Phylogenetic analysis of 16S rDNA gene revealed that this isolate was a member of the genus Bacillus, with no close relatives at the species level (16S rRNA gene sequence similarity less 97%) . On the basis of the significant differences demonstrated in the phenotypic and chemotaxonomic characteristics, it is suggested that the bacterium be classified as a novel species; the name Bacillus algicola sp . nov . is proposed . The type strain is KMM 3737T (= CIP 107850T).

Syst Appl Microbiol, 2004 May, 27(3), 286 - 9
Melanin pigment formation and increased UV resistance in Bacillus thuringiensis following high temperature induction; Ruan L et al.; The pigment melanin is well known to protect against the damaging effects of UV radiation . In this study, we show that thirty-five of thirty-seven tested Bacillus thuringiensis strains possess the potential to produce melanin in the presence of L-tyrosin at elevated temperature (42 degrees C) . These findings offer a method of protecting insecticidal toxins produced by B . thuringiensis from UV degredation and may therefore have important applications in the field of crop protection . Toxicity assays on Heliothis armigera suggested that the insecticidal activity of B . thuringiensis that produced melanin was significantly higher after UV irradiation than when melanin was not produced.

Protein J, 2004 May, 23(4), 295 - 302
Inactivation of Bacillus stearothermophilus leucine aminopeptidase II by hydrogen peroxide and site-directed mutagenesis of methionine residues on the enzyme; Kuo LY et al.; Leucine aminopeptidases (LAPs) are exopeptidases that remove the N-terminal L-leucine from peptide substrates . Oxidative stability assay showed that the recombinant Bacillus stearothermophilus LAP II (rLAPII) was sensitive to oxidative damage by hydrogen peroxide at the elevated temperature . The H2O2-treated enzyme experienced obvious changes in the secondary structure when the oxidant concentration increased to 300 mM . To investigate the role of methionine residues on the oxidative inactivation, each of the five methionine residues in the rLAPII was replaced with leucine by site-directed mutagenesis . The mutant enzymes with an apparent Mr of approximately 44.5 kDa were overexpressed in Escherichia coli and were purified to homogeneity by nickel-chelate chromatography . The specific activities for Met82Leu, Met88Leu, Met254Leu, and Met382Leu were similar to that of the wild-type enzyme, whereas a reduced activity was observed in Met136Leu . The 50% decrease in the catalytic efficiency (kcat/Km) for Met136Leu was caused by 47% decrease in kcat value . As compared with the wild-type enzyme, all mutant proteins were more sensitive to the oxidant, implying that the methionine residues of B . stearothermophilus LAP II are important for the protection of the enzyme from oxidative inactivation.

Can J Microbiol, 2004 May, 50(5), 361 - 8
A glycerol-inducible thermostable lipase from Bacillus sp.: medium optimization by a Plackett-Burman design and by response surface methodology; Gupta N et al.; The production of a neutral lipase from a Bacillus sp . was improved tremendously (193-fold) following media optimization involving both the "one-at-a-time" and the statistical designing approaches . The present lipase was poorly induced by oils, instead its production was induced in the presence of sugars and sugar alcohols, mainly galactose, lactose, glycerol, and mannitol . A high inoculum density of 15% v/v (A550 = 0.8) led to maximum lipase production . Interestingly, the enzyme induction was growth independent, a property very different from most of the lipases investigated to date . The optimal composition of the growth medium to achieve maximum lipase production was determined to be as follows: NH4Cl, 35 g x L(-1); glycerol, 10 mL x L(-1); K2HPO4, 3 g x L(-1); KH2PO4, 1 g x L(-1); MgSO4.7H2O, 0.1 g x L(-1); glucose, 2 g x L(-1); MgCl2, 0.6 mmol x L(-1), with 15% inoculum density and an incubation period of 24 h . About 62 U x mL(-1) of enzyme production was achieved in the optimized medium.

Acta Crystallogr D Biol Crystallogr, 2004 Jul, 60(Pt 7), 1315 - 8 Epub 2004 Jun 22.
Crystallization and preliminary crystallographic study of the functional form of the Bacillus thuringiensis mosquito-larvicidal Cry4Aa mutant toxin; Boonserm P et al.; The 65 kDa functional form of the mosquito-larvicidal Cry4Aa-R235Q mutant toxin has been crystallized . The crystals belong to space group C222(1), with unit-cell parameters a = 91.2, b = 202.1, c = 98.7 A, and contain one molecule per asymmetric unit . The crystals diffract to approximately 2.9 A using synchrotron radiation and a complete native data set has been collected . The structure has been solved using a molecular-replacement method with the Cry4Ba toxin protein as a search model.

Infect Immun, 2004 Jul, 72(7), 3855 - 62
Overexpression of Interleukin-15 increases susceptibility to lipopolysaccharide-induced liver injury in mice primed with Mycobacterium bovis bacillus Calmette-Guerin; Yajima T et al.; Mice primed with Mycobacterium bovis bacillus Calmette-Guerin (BCG) are highly sensitive to lipopolysaccharide (LPS)-induced liver injury and lethality . We found that interleukin-15 (IL-15) transgenic (Tg) mice primed with BCG were more susceptible to LPS-induced liver injury than non-Tg mice . The numbers of CD44+ CD8+ T cells expressing intracellular gamma interferon (IFN-gamma) significantly increased in the livers of BCG-primed IL-15 Tg mice after LPS injection, and the depletion of CD8+ T cells from BCG-primed IL-15 Tg mice completely abolished the susceptibility to LPS-induced lethality . Liver T cells from BCG-primed IL-15 Tg mice produced IFN-gamma in vitro in response to LPS, which was inhibited by the addition of anti-IL-12 monoclonal antibody (MAb) . In vivo treatment with anti-IL-12 MAb inhibited the appearance of CD44+ CD8+ T cells expressing intracellular IFN-gamma after LPS injection . These results suggest that the overexpression of IL-15 increases susceptibility to LPS-induced liver injury in BCG-primed mice via bystander activation of CD8+ T cells.

FEMS Microbiol Lett, 2004 Jul 1, 236(1), 145 - 51
The effect of inoculum age and solid versus liquid propagation on inoculum quality of an industrial Bacillus licheniformis strain; Hornbaek T et al.; Shorter lag phases were obtained in cultivations of Bacillus licheniformis using early-compared to late-stationary growth phase inocula and using liquid versus solid propagation medium . Flow cytometry and fluorescence ratio imaging microscopy (FRIM) after staining with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), confirmed that liquid early-stationary growth phase inoculum had a higher vitality and was more homogeneous than solid late-stationary growth phase inoculum . DNA-microarray analyses indicated that liquid early-stationary growth phase inoculum was in a more active state in terms of cell multiplication whereas solid late-stationary growth phase inoculum was induced to some spore formation potentially causing delayed growth initiation.

J Agric Food Chem, 2004 Jun 30, 52(13), 4149 - 58
Composition of grain and forage from corn rootworm-protected corn event MON 863 is equivalent to that of conventional corn (Zea mays l.); George C et al.; Insect-protected corn hybrids containing event MON 863 protect corn plants against feeding damage from corn rootworm (Diabrotica), a major North American insect pest . Corn event MON 863 contains a gene that expresses an amino acid sequence variant of the wild-type Cry3Bb1 insecticidal protein from Bacillus thuringiensis . The purpose of this study was to compare the composition of corn containing event MON 863 with that of conventional nontransgenic corn . Compositional analyses were conducted to measure proximates, fiber, amino acids, fatty acids, minerals, folic acid, thiamin, riboflavin, vitamin E, antinutrients, and certain secondary metabolites in grain and proximates and fiber content in forage collected from a total of eight field sites in the U.S . and Argentina . Compositional analyses demonstrated that the grain and forage of event MON 863 are comparable in their nutritional content to the control corn hybrid and conventional corn . These comparisons, together with the history of the safe use of corn as a common component of animal feed and human food, support the conclusion that corn event MON 863 is compositionally equivalent to, and as safe and nutritious as, conventional corn hybrids grown commercially today.

Biotechnol Bioeng, 2004 Jul 5, 87(1), 61 - 8
Extracellular enzyme loss during polyelectrolyte flocculation of cells from fermentation broth; Pearson CR et al.; Association of extracellular protein product with flocculated cells reduces product yield . Here, partitioning of the enzyme subtilisin between the liquid and polyelectrolyte-flocculated and sedimented Bacillus increased as the polymer dosage was increased beyond that necessary to obtain optimum floc character (brain floc) for cell removal by centrifugation . Partitioning to the cell floc is partly physical entrapment at all polymer dosages; however, at higher levels there is also direct interaction between the polyelectrolyte and enzyme . Enzyme loss was not likely due to pH denaturation during the flocculation process because conditions were within the stable pH range of the enzyme . The direct interaction between polyelectrolyte and enzyme was characterized through turbidimetric titrations and partitioning studies . Neither changes in the polymer feed concentration nor the method of polymer addition reduced the enzyme loss at dosages optimal for cell removal .

Biochim Biophys Acta, 2004 Jul 1, 1700(1), 35 - 41
Antibodies specific to modified glyceraldehyde-3-phosphate dehydrogenase induce inactivation of the native enzyme and change its conformation; Arutyunova EI et al.; The antibodies specific to an inactive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus prepared by the treatment of the tetrameric holoenzyme with glutaraldehyde were obtained . They were purified from the pool of polyclonal rabbit antibodies to GAPDH with the use of immobilized GAPDH cross-linked by glutaraldehyde as an affinity sorbent . Such antibodies were capable of interacting with the native enzyme, inducing its time-dependent inactivation; the effect was different with the apo- and holoenzyme forms . Differential scanning calorimetry of the purified {GAPDH}.{antibody} complex revealed a large shift of the temperature corresponding to the maximal heat capacity of the holoenzyme towards the lower temperature . Again, the effect appeared to be different with the apoenzyme . Together, the results are consistent with the hypothesis that a specific antibody is able to exercise a certain strain on the target protein, altering its conformation toward the structure of the species which served to select the antibody . The possibility of preparing selective enzyme inhibitors based on the antibodies specific to inactive enzyme conformations is considered.

Echocardiography, 2004 Jul, 21(5), 423 - 7
A rare case of Listeria monocytogenes presenting as prosthetic valve bacterial endocarditis and aortic root abscess; Makaryus AN et al.; Listeria monocytogenes is a gram-positive bacillus that is rarely associated with infections in the general population . Those susceptible to this pathogen include neonates, pregnant women, and the immunocompromised . The most common clinical manifestations of listeriosis are bacteremia and meningitis . Endocarditis caused by L . monocytogenes is rare with less than 60 cases reported in the world literature . We report the case of an 81-year-old man who suffered aortic prosthetic valve listeria endocarditis, and examine the literature regarding this rare manifestation of human listeriosis.

Mol Biotechnol, 2004 Jun, 27(2), 109 - 18
The injection of plasmid DNA in mouse muscle results in lifelong persistence of DNA, gene expression, and humoral response; Armengol G et al.; The duration of the immune response against any vaccine is critical . The present study was performed to determine the stability of injected plasmid deoxyribonucleic acid (DNA), the duration of gene expression in mouse muscle, as well as the duration of the immune response generated in mice after injection of plasmid pSO2C1 harboring the cry11Bb gene of Bacillus thuringiensis serovar . medellin . The localization and the persistence of the inoculated gene were determined by in situ hybridization and polymerase chain reaction (PCR) . The results demonstrated that plasmid DNA can persist in mouse muscle for up to 2 yr . Moreover, immunohistochemical analysis showed that Cry11Bb protein was expressed for the lifetime of the mice at a low but significant level . Finally, production of Cry11Bb-specific antibodies in mice injected with pSO2C1 was high and durable as significant antibody titers were observed up to 119 wk after injection of the plasmid . This persistent immune response is likely owing to the existence of a protein and/or DNA depot in the organism, which serves to maintain the immune response, acting as a secondary or booster immunization.

Mol Biotechnol, 2004 Jun, 27(2), 89 - 100
Effect of specific mutations in helix alpha7 of domain I on the stability and crystallization of Cry3A in Bacillus thuringiensis; Park HW et al.; Insecticidal crystal (Cry) proteins of Bacillus thuringiensis crystallize after synthesis forming large inclusions that stabilize these toxins in the environment after cell lysis until eaten by an insect . Despite the biological importance of crystallization, little is known about the structural elements of Cry molecules that facilitate this process . We identified subdomains that affect Cry3A structure possibly through improper folding by chimeric-scanning mutagenesis, substituting short peptides of a truncated 70-kDa Cry1C molecule that does not crystallize into Cry3A, a wild-type 70-kDa molecule that crystallizes readily . Cry3A consists of three domains that contain five different blocks of conserved amino acids . Domain substitution and mutagenesis within these blocks suggested that the specific structure of block 2, which spans the junction between domains I and II, was important to the relative stability of Cry3A and subsequent crystallization . Amino acid sequences of particular importance to stability in Cry3A block 2 were identified using three substitution mutants, each spanning about a third of this block . One that consisted of Cry1C helix alpha7 yielded no detectable protein, whereas the other two produced characteristic Cry3A crystals . Specific mutations in this region showed tyrosine 268 was critical to normal stability of Cry3A and subsequent crystallization in that a mutant, Y268L, was less stable than wild-type Cry3A and failed to form a characteristic Cry3A crystal . Circular dichroism analysis showed a decrease in this mutant's alpha-helicity, indicating the importance of tyrosine 268 to the specific conformation of helix alpha7 that facilitates stability and normal crystallization.

Eur J Histochem, 2004 Apr-Jun, 48(2), 185 - 90
Adhesion of ectomycorrhizal bacteria to plant cells: an in vitro evidence; Citterio B et al.; In this study we have investigated, by combining microbial and microscopical techniques, the adhesion ability of bacteria present in Tuber borchii ectomycorrhizosphere . Our data demonstrate that a common pool of bacteria - Pseudomonas, Bacillus, Micrococcus and Moraxella - occurs in all ectomycorrhizal homogenates and that most of these bacteria are able to attach in vitro to plant cells.

Tuberculosis (Edinb), 2004, 84(5), 317 - 26
Strain-related virulence of the dominant Mycobacterium tuberculosis strain in the Canadian province of Manitoba; Petrelli D et al.; SETTING: The Canadian province of Manitoba . OBJECTIVE: To confirm the putative hypervirulence observed in Mycobacterium tuberculosis Type1 strain and further characterize the progression and manifestation of pulmonary tuberculosis caused by this strain in comparison to other common clinical isolates from Manitoba . DESIGN: C3H and BALB/c mice were exposed to aerosols either of Type1, Type2, Type5, Type72 or H37Rv strain to study their respective survival profiles . Additionally, bacillary loads and lung histology were examined at 15 days post-exposure . RESULTS: In both mouse models, Type-1 infected mice succumbed to disease significantly earlier than other strains (p < or =0.0002) . Differences between average log(10) CFU values between clinical isolates were less than 1log(10) difference . In C3H mice, the amount of granulomatous inflammation was highest in Type1 infected mice but not significantly different than all clinical strains . In contrast, BALB/c mice infected with Type1 induced the lowest amount of granulomatous inflammation compared to other clinical isolates . CONCLUSION: Our results indicate that although mice infected with the Type1 strain died significantly earlier than mice infected with other clinical strains in both C3H and BALB/c mice, the hypervirulence of the Type1 strain is not attributed to the growth rate of the organism, as differences in growth between clinical M . tuberculosis isolates were insignificant.

Water Res, 2004 Jun, 38(11), 2667 - 76
Characterization of degradation process of cyanobacterial hepatotoxins by a gram-negative aerobic bacterium; Ishii H et al.; A bacterium termed 7CY, capable of decomposing cyanobacterial toxins, was isolated from surface water sample of Lake Suwa and degradation of microcystin-RR and nodularin-Har was investigated . The isolated 7CY was a gram-negative, aerobic bacillus, and a member of a genus Sphingomonas . The strain degraded microcystin-LY, -LW, and -LF completely as well as microcystin-LR within 4 days after their addition (6 microg/ml) whereas degradation of nodularin-Har did not occur at all during experiment . On the contrary, the strain was capable of degrading nodularin-Har in the presence of microcystin-RR and both toxins were completely decomposed within 6 days . The strain scarcely degraded nodularin-Har in the presence of microcystin-RR when glucose and ammonium chloride were added to the medium . The degradation of nodularin-Har did not occur in the medium from which bacterial cells had been removed after degradation of microcystin-RR . Furthermore, when microcystin-RR and nodularin-Har were added to the cytoplasm fraction of 7CY cells, microcystin-RR was rapidly degraded within 18 h, but nodularin-Har was not . The strain 7CY may require an enzyme(s) induced during the degradation of microcystin-RR in order to utilize nodularin-Har as nutrition.

Tuberculosis (Edinb), 2004, 84(3-4), 188 - 96
Comparative expression studies of a complex phenotype: cord formation in Mycobacterium tuberculosis; Gao Q et al.; The aggregation of mycobacteria into structures known as cords is an intrinsic property of the human tubercle bacillus . This property is thought to be determined by the lipid composition of the bacterial cell surface and may contribute to the virulence of the organism . Using microarray technology, we compared the pattern of gene expression of H37Rv, a virulent, cording strain of Mycobacterium tuberculosis, with H37Ra, an avirulent, non-cording strain derived from the same original patient isolate, under five different nutrient combinations and growth conditions . Under all of these conditions, H37Rv formed cords and H37Ra did not . By focusing our analysis only on genes that were differentially expressed under all conditions, we hoped to enrich the resulting gene list for genes associated with cording . We identified 22 genes that were consistently expressed at higher levels in H37Rv than in H37Ra under all conditions tested . Genes involved in lipid metabolism and the cell membrane were significantly enriched in our gene list, indicating that the cell wall and the cell membrane may be the major sites of difference between these two strains . This work represents a new strategy for enriching gene lists for relevant genes, which may also be applicable for other types of problems.

Arch Microbiol, 2004 Sep, 182(1), 90 - 5 Epub 2004 Jun 16.
Anaerobiosis and low specific growth rates enhance hemolysin BL production by Bacillus cereus F4430/73; Duport C et al.; Bacillus cereus F4430/73 produced the highest levels of hemolysin BL (HBL) when grown under anaerobiosis in MOD medium . Anaerobic cells grown in a chemostat at low specific growth rate (0.1-0.2 h(-1)) expressed up to sevenfold more HBL than did cells held at a faster growth rate . At 0.2 h(-1), the presence of 90 mM glucose resulted in inhibition of HBL production . Glucose was found to repress HBL induction at the mRNA level, indicating the potential involvement of catabolite repression in the regulation of HBL . Based on these data, it is suggested that growth rate could be an effector of catabolite regulation of HBL.

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