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 -