Protein Eng, 1997 Nov, 10(11), 1271 - 9 Subtilisin from psychrophilic antarctic bacteria: characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold; Narinx E et al.; A subtilisin excreted by the Antarctic Bacillus TA39 has been purified to homogeneity and characterised . Two independent genes subt1 and subt2 are present but only subt1 is expressed significantly in the culture medium . The enzyme displays the usual characteristics of cold enzymes i.e . a high catalytic efficiency at low and moderate temperatures and an increased thermosensitivity originating from a 3D structure probably more flexible than its mesophilic counterpart . This is corroborated by the analysis of the computerized structure which shows a significant decrease in the number and strength of intramolecular weak bonds such as salt bridges and aromatic interactions . The affinity for calcium is also almost three orders of magnitude lower than that of mesophilic subtilisin and the interactions with the solvent are significantly higher thanks to a large increase in the number of Asp residues in the loops connecting secondary structures . The relation between flexibility and activity was investigated by site-directed mutagenesis tending mainly to increase the rigidity of the molecular edifice through the incorporation of additional salt bridge, disulfide bridge, aromatic interaction and by increasing the affinity of the enzyme for calcium . An important stabilization of the molecular structure was achieved through a modification of a calcium ligand T85D . The thermostability of the mutated product expressed in a mesophilic Bacillus reaches that of mesophilic subtilisin . Most important is the fact that this mutation further enhances the specific activity by a factor close to 2 when compared to the wild type enzyme so that the overall activity of the mutated cold enzyme is about 20 times higher than that of mesophilic subtilisin, illustrating the fact that thermostability is not systematically inversely related to specific activity . This opens new perspectives in the use of cold enzymes in biotechnology. Protein Eng, 1997 Nov, 10(11), 1263 - 9 Improving the thermostability of Bacillus stearothermophilus neutral protease by introducing proline into the active site helix; Nakamura S et al.; A proline residue was introduced into the N-terminus (Ile140 and Asp141), the middle (Leu147) and the C-terminus (Asp153) of the active site helix of Bacillus stearothermophilus neutral protease for comparing the effects on the thermostability . Introduction of a proline residue into the N-terminus at sites 140 and 141 increased the half-survival temperature (HST) by 7.5 and 2.8 degrees C, respectively, from 68.3 degrees C of the wild-type enzyme . A proline residue at Leu147 decreased the HST by 10.2 degrees C, while no change was observed by introducing a proline residue in the C-terminus . These results were coincidental with the CD data which indicated increases in Tm values of 4.4 and 2.3 degrees C for I140P and D141P, respectively . Susceptibility to alpha-chymotrypsin hydrolysis markedly decreased in mutants I140P and D141P, while increasing in L147P . Molecular modeling suggested that glycine residues on the N-terminus side of proline residues in I140P and D141P relaxed the possible strain caused by proline introduction . The thermostability can, therefore, be explained based on changes in the molecular rigidity. Med Vet Entomol, 1998 Jan, 12(1), 98 - 102 Larvicidal toxicity of Japanese Bacillus thuringiensis against the mosquito Anopheles stephensi; Saitoh H et al.; Japanese isolates of Bacillus thuringiensis were screened for larvicidal activity against the mosquito Anopheles stephensi, the urban malaria vector of the Indian subcontinent . Among more than 30 strains identified, larvicidal activity causing > 80% mortality in 72 h was demonstrated for 41/1449 (2.8%) isolates . The majority of strains and isolates (97.2%) exhibited little or no larvicidal activity . Anopheles-active strains belonged to more than 12 H serotypes, especially H3ade (serovar fukuokaensis) and H44 (serovar higo) . SDS-PAGE profiles of inclusion proteins showed 4 distinct types among 6 active strains examined . The most active Japanese isolates were H20 strain 89-T-34-14 (LC50 4.4 micrograms/ml) and H44 serovar higo strain 74-E-45-24 (LC50 7.6 micrograms/ml), respectively, 13-fold and 23-fold less active than the international standard H14 serovar israelensis (LC50 0.33 microgram/ml). J Mol Biol, 1998 Feb 20, 276(2), 479 - 89 Cooperative folding of a protein mini domain: the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex; Spector S et al.; The peripheral subunit-binding domain from the dihydrolipoamide acetyltransferase (E2) component of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus is stably folded, despite its short sequence of only 43 amino acid residues . A 41 residue peptide derived from this domain, psbd41, undergoes a cooperative thermal unfolding transition with a tm of 54 degrees C . This three-helix protein is monomeric as judged by ultracentrifugation and concentration-dependent CD measurements . Peptides corresponding to the individual helices are largely unstructured both alone and in combination, indicating that the unusual stability of this protein does not arise solely from unusually stable alpha-helices . Chemical denaturation by guanidine hydrochloride is also cooperative with a delta GH2O of 3.1 kcal mol-1 at pH 8.0 and 25 degrees C . The chemical denaturation is broad with an m-value of 760 cal mol-1 M-1 . psbd41 contains a buried aspartate residue at position 34 that may provide stability and specificity to the fold . A mutant peptide, psbd41Asn was synthesized in which the buried aspartate residue was mutated to asparagine . This peptide still folds cooperatively and it is monomeric, but is much less thermostable than the wild-type with a tm of only 31 degrees C . Chemical denaturations at 4 degrees C give an m-value of 740 cal mol-1 M-1, similar to the wild-type, but the stability delta GH2O is only 1.4 kcal mol-1 . Both the wild-type and the mutant unfold at extremes of pH, but at 4 degrees C psbd41Asn is folded over a narrower pH range than the wild-type . Although the mutant unfolds cooperatively by thermal and by chemical denaturation, its NMR spectrum is significantly broader than that of the wild-type and it binds ANS . These results show that Asp34 is vital for the stability and specificity of this structure, the second smallest natural sequence known to fold in the absence of disulfide bonds or metal or ligand-binding sites. FEBS Lett, 1998 Feb 20, 423(2), 249 - 53 Identification of neutral and acidic sphingomyelinases in Helicobacter pylori; Lin YL et al.; We demonstrated for the first time the presence of sphingomyelinase (SMase) in Helicobacter pylori . Activation of SMase has been implicated as the cause of elevation of cellular ceramide levels and consequently of apoptosis . The data indicate that there are two classes of SMase, defined by their optimal pHs and cellular locations, existing in H . pylori . One is an Mg(2+)-dependent membrane-bound enzyme with an optimal activity at pH 7, and the other is an Mg(2+)-independent cytosolic enzyme with an optimal activity at pH 5 . Bisalumin, a bismuth salt, was found to inhibit the activities of both forms of SMase regardless of the presence of Mg2+ . By Western blot analysis, the membrane-bound SMases of H . pylori and Bacillus cereus were shown to be antigenically related and to have a similar denatured molecular mass of 28 kDa. Mikrobiol Z, 1997 Nov-Dec, 59(6), 43 - 9 {A comparative study of the biological properties of Biosporin and other commercial Bacillus-based preparations}; Sorokulova IB; A new probiotic Biosporin and other commercial biopreparations based on aerobic sporulating bacteria of the Bacillus genus have been comparatively studied for their specific activity and safety . It has been established that only Biosporin is characterized by expressed antagonistic activity in respect to a wide range of pathogenic and conditionally pathogenic microorganisms including those with multiple resistance to antibiotics . Biosporin is also characterized by the absence of any negative action on the organism of animals even in the doses considerably exceeding those recommended for use. J Formos Med Assoc, 1998 Feb, 97(2), 106 - 12 Clinical and microbiologic characteristics of Ochrobactrum anthropi bacteremia; Yu WL et al.; Ochrobactrum anthropi is an oxidase-positive, nonfermenting, gram-negative bacillus that is an emerging pathogen in immunocompromised patients, particularly in those with indwelling central venous catheters . Reports of clinical manifestation of O . anthropi bacteremia are rare . Herein, we report the clinical and microbiologic characteristics of O . anthropi bacteremia in 15 patients . There were eight males and seven females, whose ages ranged from 1 month to 84 years (mean, 34 years) . Ten infections were community acquired . All patients had severe underlying disease and manifested primary O . anthropi bacteremia with no obvious focus . Only three patients had central venous catheters in place at the onset of bacteremia, but none of these showed evidence of catheter-related infection . The mean duration of fever was 4 days (range 0-11) and the mean white blood cell count was 13 x 10(9)/L (range 5.5-28 x 10(9)) . All O . anthropi isolates tested were resistant to ampicillin, cephalothin, cefonicid, amoxicillin/clavulanic acid, piperacillin, aztreonam, and ceftazidime, and all were susceptible to gentamicin, amikacin, imipenem, ceftriaxone, and cefoperazone . Most were also susceptible to cefotaxime and moxalactam . Monotherapy with an aminoglycoside or an appropriate beta-lactam (such as cefotaxime or ceftriaxone) yielded good clinical response . None of the 15 patients died directly from O . anthropi bacteremia . Our findings indicate that O . anthropi is often community acquired and can be pathogenic in critically ill or immunocompromised patients with or without indwelling catheters . Although O . anthropi can produce clinically significant infections, the organism seems to be of relatively low virulence. J Invertebr Pathol, 1998 Mar, 71(2), 138 - 44 Isolation and activity of strains of bacillus thuringiensis toxic to larvae of the housefly (Diptera: muscidae) and tropical blowflies Johnson C, Bishop AH, Turner CL. Several natural isolates of Bacillus thuringiensis have been found which are active against larvae of Musca domestica, the housefly, as well against larvae of the family Calliphoridae . The toxicity is attributed to the Cry IB class of protoxin . Presolubilization of the toxin was not necessary to induce mortality in any of the insect species assayed . The addition of spores to the delta-endotoxin was, however, essential to inducing significant mortality in larvae of Chrysomya albiceps (Weid.) and enhanced mortality in the other species tested . J Invertebr Pathol, 1998 Mar, 71(2), 106 - 14 Molecular and phenotypic characterization of bacillus thuringiensis isolated from leaves and insects Hansen BM, Damgaard PH, Eilenberg J, Pedersen JC. Bacillus thuringiensis isolates from the phylloplane of organically cultivated cabbage were characterized using molecular and phenotypic methods . Of the 58 isolates under study, 31 belonged to serovar kurstaki, 16 did not react with any of the currently recognized antisera, 7 reacted with known antisera, and 4 could not be serotyped as they were nonmotile . Round crystals were found in 26 isolates, while bipyramidal crystals were found in the remaining 32 isolates, all of which had activity to lepidopteran larvae . Further, one isolate with unknown serotype and round crystals had lepidopteran activity . Colony hybridization was found to be a useful tool for screening the isolates for specific gene homologies and showed good correlation with the phenotypic observations . Polymerase chain reaction (PCR) was used for confirmation of the colony hybridization data, in most cases with concordant results . However, in one case some of the colony hybridization data could not be confirmed by PCR, due to DNA sequence variations in the binding area of one of the primers . The random amplified polymorphic DNA (RAPD) analysis showed that isolates otherwise indistinguishable could be distinguished by this method . However, the method was not able to distinguish the 31 kurstaki isolates . Further, the kurstaki isolates could not be distinguished from the B . thuringiensis serovar kurstaki HD-1 strain used in commercial products for lepidopteran control . One of the isolates was a serovar israelensis, but no genes encoding dipteran activity could be detected, and the RAPD analysis revealed that the DNA fingerprint of this israelensis isolate deviated from the israelensis ONR60A isolate used in commercial products . In conclusion we find that a molecular method like colony hybridization is suitable for screening large collections of bacteria . When colony hybridization data are combined with RAPD analyses isolates can be grouped based on genetic potential and DNA fingerprint, whereby further characterizations by PCR and the more labourious phenotypic methods can be performed more effectively . J AOAC Int, 1998 Jan-Feb, 81(1), 33 - 9 A bridging study between liquid chromatography and microbial inhibition assay methods for determining amoxicillin residues in catfish muscle; Ang CY et al.; A bridging study was conducted to establish the correlation between a liquid chromatographic (LC) method and a microbial inhibition (MI) method for analysis of amoxicillin residues in catfish muscle . The LC procedure involved precolumn derivatization with formaldehyde followed by LC separation with fluorescence detection . The MI procedure used Bacillus stearothermophilus as the test organism and was validated in this study before the bridging investigation . The 2 methods were compared for determination of both fortified and incurred samples . No significant differences were found between the methods when all data were included in statistical computations . The linear correlation of LC means versus MI means had a slope of 0.972 and a negligible intercept (1.0 ng/g), with a correlation coefficient of 0.9962 . LC was more specific and showed better sensitivity than MI for amoxicillin residues at < or = 10 ng/g . For practical purposes, values obtained by the 2 methods can be considered equivalent. Arch Bronconeumol, 1997 Dec, 33(11), 566 - 71 {Microepidemics of tuberculosis; apropos of 2 school outbreaks in the area 15 of the Valencia community}; Calpe JL et al.; Schools are settings with high concentrations of young people with little exposure to Mycobacterium tuberculosis and greater risk of developing disease when infection occurs as the result of sporadic localized outbreaks . We studied two outbreaks in two elementary schools (A and B) after two cases of bacilliferous pulmonary tuberculosis were detected in teachers in 1990 and 1994 . Contacts were trace din school A by the primary care physician and in school B by the pneumologist and public health authorities . Contacts were classified as belonging to the risk group (RG) or the low risk group (LRG) . The RG was composed of 187 contacts in school A and 59 in school B . Individuals in the LRG numbered 429 and 116 respectively . Mantoux positives numbered 108 in the RG and 45 in the LRG in school A (p < 0.001) . In school B 50 RG individuals and 29 LRG individuals were positive (p < 0.001) . The proportion of Mantoux positives was greater in the RG of school B than in the RG of school A (p < 0.01), probably owing to longer time of evolution of disease and possible laryngeal involvement in the index case . Likewise, tuberculin positives were fewer in the LRG of school A than in the LRG of school B (p < 0.001), owing to the small size of the LRG in school A . Thirteen cases of tuberculosis were seen in school A, six of which called for drug prophylaxis after contacts were traced . The nature of the index case and the conditions of exposure are both important in such outbreaks, demonstrating the need to act appropriately to trace contacts, preferably under the supervision of a pneumologists. J Gen Intern Med, 1998 Feb, 13(2), 131 - 6 Diagnosing HIV-related disease: using the CD4 count as a guide; Jung AC et al.; OBJECTIVE: To summarize current information on the relation between CD4 counts and the risk of different HIV-related diseases . MEASUREMENTS AND MAIN RESULTS: MEDLINE search of English language articles between 1985 and 1996 using the medical subject heading (MeSH) term "CD4 lymphocyte count" and searches using key words of multiple HIV-related diseases were conducted . Some HIV-related diseases can be stratified to different CD4 count levels . Regardless of their CD4 count, HIV-infected patients are susceptible to sinusitis, Kaposi's sarcoma, community-acquired pneumonia, and oral hairy leukoplakia . In advanced HIV, when CD4 is below 200/mm3, Pneumocystis carinii pneumonia, toxoplasmosis, progressive multifocal leukoencephalopathy, Mycobacterium avium complex, molluscum contagiosum, and bacillary angiomatosis all increase in incidence . In very advanced HIV disease, when CD4 counts are below 50/mm3, patients are at risk of pseudomonas pneumonia, cytomegalovirus retinitis, central nervous system lymphoma, aspergillosis, and disseminated histoplasmosis. Eur J Clin Invest, 1998 Jan, 28(1), 1 - 12 Immunological relatedness of the protective mechanisms against tuberculosis and cancer; Cocito C et al.; BACKGROUND: Bacille Calmette-Guerin (BCG), an attenuated strain of tuberculous bacillus, is the source of vaccines providing unclear and variable protection against tuberculosis (TB) and cancer . Thermostable macromolecular antigens (TMAs) are major mycobacterial complexes immunodominant in disease . A60 (TMA complex of BCG) protects mice against TB development, via T lymphocyte (TL)-mediated macrophage (Mphi) activation, halting intracellular mycobacterial replication . In most A60-primed mice, cytolytic TLs and Mphi infiltrate cancer tissue, resulting in 80-100% rejection . Adoptive TL transfer is indispensable for Mphi-dependent tumour cell inactivation via oxygen and nitrogen radicals . Neoplasm development induces immune anergy with depletion ofA60-specific TL and activated Mphi . A60 protects mice against TB and cancer by inducing the synthesis of three lymphokines: interleukin 2 (IL-2), interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) . Tumour cells prevent A60-dependent synthesis of these lymphokines in vivo and in vitro . CONCLUSION: These data provide some clues to immune surveillance and tumour escape mechanisms, as well as to the antituberculous and antineoplastic BCG action. Biosci Biotechnol Biochem, 1998 Jan, 62(1), 167 - 9 Escherichia coli transformant expressing the glucose dehydrogenase gene from Bacillus megaterium as a cofactor regenerator in a chiral alcohol production system; Kataoka M et al.; Escherichia coli JM109 (pGDA2) overexpressing the glucose dehydrogenase (GDH) gene from Bacillus megaterium IWG3 was examined for use as a cofactor regenerator . In the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate by E . coli JM109 (pKAR) which is an aldehyde reductase-overproducing transformant, E . coli JM109 (pGDA2) can act as an NADPH regenerator with NADP+ and glucose, similarly to commercially available GDH. Biosci Biotechnol Biochem, 1998 Jan, 62(1), 66 - 71 Gene cloning and characterization of thermostable lipase from Bacillus stearothermophilus L1; Kim HK et al.; The gene coding for an extracellular lipase of Bacillus stearothermophilus L1 was cloned in Escherichia coli . Sequence analysis showed an open reading frame of 1254 bp, which encodes a polypeptide of 417 amino acid residues . The polypeptide was composed of a signal sequence (29 amino acids) and a mature protein of 388 amino acids . An alanine replaces the first glycine in the conserved pentapeptide (Gly-X-Ser-X-Gly) around the active site serine . The expressed lipase was purified by hydrophobic interaction and ion exchange chromatography using buffers containing 0.02% (v/v) Triton X-100 . The lipase was most active at 60-65 degrees C and in alkaline conditions around pH 9-10 . The lipase had highest activity toward p-nitrophenyl caprylate among the synthetic substrates and tripropionin among the triglycerides . It hydrolyzed beef tallow and palm oil more rapidly than olive oil at 50 degrees C. Appl Environ Microbiol, 1998 Mar, 64(3), 1075 - 8 Influence of structural properties and kinetic constraints on Bacillus cereus growth; Stecchini ML et al.; The influence of structural properties and kinetic constraints on the behavior of Bacillus cereus was investigated on agar media . Dimensional criteria were used to study the growth in bacterial colonies . The architecture of the agar gel as modified by the agar content was found to influence the colony size, and smaller colonies were observed on media containing 50 to 70 g of agar liter-1 . Except at low nutrient levels, colonies responded to nutrient gradients by decreasing in size the farther away they were from the nutrient source, and the decrease in colony size was influenced by the agar content . The diffusivities of glucose and a protein (insulin-like growth factor) were not affected by the gel architecture, suggesting that other factors, such as mechanical factors, could influence microbial growth in the agar systems used . Increasing the viscosity of the liquid phase of the agar media by adding polyvinylpyrrolidone resulted in a reduction in colony size . When the agar concentration was increased, the colony areas were not influenced by the viscosity of the system. Appl Environ Microbiol, 1998 Mar, 64(3), 1059 - 65 Utilization of oligopeptides by Listeria monocytogenes Scott A; Verheul A et al.; For effective utilization of peptides, Listeria monocytogenes possesses two different peptide transport systems . The first one is the previously described proton motive force (PMF)-driven di- and tripeptide transport system (A . Verheul, A . Hagting, M.-R . Amezaga, I . R . Booth, F . M . Rombouts, and T . Abee, Appl . Environ . Microbiol, 61:226-233, 1995) . The present results reveal that L . monocytogenes possesses an oligopeptide transport system, presumably requiring ATP rather than the PMF as the driving force for translocation . Experiments to determine growth in a defined medium containing peptides of various lengths suggested that the oligopeptide permease transports peptides of up to 8 amino acid residues . Peptidase activities towards several oligopeptides were demonstrated in cell extract from L . monocytogenes, which indicates that upon internalization, the oligopeptides are hydrolyzed to serve as sources of amino acids for growth . The peptide transporters of the nonproteolytic L . monocytogenes might play an important role in foods that harbor indigenous proteinases and/or proteolytic microorganisms, since Pseudomonas fragi as well as Bacillus cereus was found to enhance the growth of L . monocytogenes to a large extent in a medium in which the milk protein casein was the sole source of nitrogen . In addition, growth stimulation was elicited in this medium when casein was hydrolyzed by using purified protease from Bacillus licheniformis . The possible contribution of the oligopeptide transport system in the establishment of high numbers of L . monocytogenes cells in fermented milk products is discussed. Appl Environ Microbiol, 1998 Mar, 64(3), 1018 - 23 Enzyme characteristics of beta-D-galactosidase- and beta-D-glucuronidase-positive bacteria and their interference in rapid methods for detection of waterborne coliforms and Escherichia coli; Tryland I et al.; Bacteria which were beta-D-galactosidase and beta-D-glucuronidase positive or expressed only one of these enzymes were isolated from environmental water samples . The enzymatic activity of these bacteria was measured in 25-min assays by using the fluorogenic substrates 4-methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl-beta-D-glucuronide . The enzyme activity, enzyme induction, and enzyme temperature characteristics of target and nontarget bacteria in assays aimed at detecting coliform bacteria and Escherichia coli were investigated . The potential interference of false-positive bacteria was evaluated . Several of the beta-D-galactosidase-positive nontarget bacteria but none of the beta-D-glucuronidase-positive nontarget bacteria contained unstable enzyme at 44.5 degrees C . The activity of target bacteria was highly inducible . Nontarget bacteria were induced much less or were not induced by the inducers used . The results revealed large variations in the enzyme levels of different beta-D-galactosidase- and beta-D-glucuronidase-positive bacteria . The induced and noninduced beta-D-glucuronidase activities of Bacillus spp . and Aerococcus viridans were approximately the same as the activities of induced E . coli . Except for some isolates identified as Aeromonas spp., all of the induced and noninduced beta-D-galactosidase-positive, noncoliform isolates exhibited at least 2 log units less mean beta-D-galactosidase activity than induced E . coli . The noncoliform bacteria must be present in correspondingly higher concentrations than those of target bacteria to interfere in the rapid assay for detection of coliform bacteria. Appl Environ Microbiol, 1998 Mar, 64(3), 871 - 9 Phylogeny of the main bacterial 16S rRNA sequences in Drentse A grassland soils (The Netherlands); Felske A et al.; The main bacteria in peaty, acid grassland soils in the Netherlands were investigated by ribosome isolation, temperature gradient gel electrophoresis, hybridization, cloning, and sequencing . Instead of using only 16S rDNA to determine the sequences present, we focused on rRNA to classify and quantify the most active bacteria . After direct ribosome isolation from soil, a partial amplicon of bacterial 16S rRNA was generated by reverse transcription-PCR . The sequence-specific separation by temperature gradient gel electrophoresis yielded soil-specific fingerprints, which were compared to signals from a clone library of genes coding for 16S rRNA . Cloned 16S rDNA sequences matching with intense bands in the fingerprint were sequenced . The relationships of the sequences to those of cultured organisms of known phylogeny were determined . Most of the amplicons originated from organisms closely related to Bacillus species . Such sequences were also detected by direct dot blot hybridization on soil rRNA: a probe specific for Firmicutes with low G+C content counted for about 50% of all bacterial rRNA . The bacterial activity in Drentse A grassland soil could be estimated by direct dot blot hybridization and sequencing of clones; it was found that about 65% of all the bacterial ribosomes originated from Firmicutes . The most active bacteria apparently were Bacillus species, from which about half of the sequences derived . Other sequences similar to those of gram-positive bacteria were only remotely related to known Firmicutes with a high G+C content . Other sequences were related to Proteobacteria, mainly the alpha subclass. Insect Biochem Mol Biol, 1997 Nov, 27(11), 887 - 900 Insect chitinases: molecular biology and potential use as biopesticides; Kramer KJ et al.; Chitin, an insoluble structural polysaccharide that occurs in the exoskeletal and gut linings of insects, is a metabolic target of selective pest control agents . One potential biopesticide is the insect molting enzyme, chitinase, which degrades chitin to low molecular weight, soluble and insoluble oligosaccharides . For several years, our laboratories have been characterizing this enzyme and its gene . Most recently, we have been developing chitinase for use as a biopesticide to control insect and also fungal pests . Chitinases have been isolated from the tobacco hornworm, Manduca sexta, and several other insect species, and some of their chemical, physical, and kinetic properties have been determined . Also, cDNA and genomic clones for the chitinase from the hornworm have been isolated and characterized . Transgenic plants that express hornworm chitinase constitutively have been generated and found to exhibit host plant resistance . A transformed entomopathogenic virus that produces the enzyme displayed enhanced insecticidal activity . Chitinase also potentiated the efficacy of the toxin from the microbial insecticide, Bacillus thuringiensis . Insect chitinase and its gene are now available for biopesticidal applications in integrated pest management programs . Current knowledge regarding the molecular biology and biopesticidal action of insect and several other types of chitinases is described in this mini-review. Rev Sci Tech, 1997 Aug, 16(2), 441 - 51 Milk pasteurisation and safety: a brief history and update; Holsinger VH et al.; A brief history of the development of milk pasteurisation is presented and updated . Concerns about the margin of safety provided by current pasteurisation standards in terms of milk-borne pathogens such as mycobacteria (in particular Mycobacterium paratuberculosis) and other emerging pathogens such as Listeria monocytogenes and Escherichia coli O157:H7 are discussed . With the exception of the endospores of Bacillus cereus, current standards appear to be adequate for public health assurance of milk safety provided good manufacturing practices are followed. Rev Sci Tech, 1997 Aug, 16(2), 391 - 4 Escherichia coli O157:H7 in livestock in Japan; Sekiya J; The largest ever outbreak in Japan of Escherichia coli O157 infection in humans occurred in 1996 . As a result, surveys were conducted later the same year to evaluate the sources and pathogenesis of this bacillus in livestock animals at the farm and in abattoirs . One of the surveys resulted in the isolation of E . coli O157 in 0.62% of cattle on randomly selected farms . Although no confirmation has been made with regard to the source of E . coli O157 infection and its pathogenicity in livestock animals, preventive measures are being applied at both farm and processing levels. Berl Munch Tierarztl Wochenschr, 1998 Jan, 111(1), 9 - 12 {Combined effect of temperature a(W) and pH on proteases from Pseudomonas and Bacillus spp.}; Klug C et al.; The factors affecting protease activity were investigated on certain media . Five strains of Bacillus (B.) cereus, four strains of B . subtilis, five strains of Pseudomonas (Ps.) aeruginosa and two strains of Ps . fluorescens produced the proteases that were tested . The enzyme activity was evaluated under 32 different combinations of temperature (2 and 7 degrees C), water activity (aw) (0.80-0.98) and pH (4.0-7.3) . A decrease in water activity and pH and/or temperature caused a decrease in protease activity . It appeared that the combined effect of temperature, aw and pH influences enzyme production significantly compared with the influence of single environmental factors . Temperature plays the crucial role in these reactions . Enzyme activities could be observed even at low temperature . Although the exoprotease of Ps . spp . showed intense activity at refrigeration temperature, low pH and aw, Bacillus proteases were comparably less active under the same conditions, but nevertheless proved to be significant. Nucleic Acids Res, 1998 Feb 15, 26(4), 1084 - 91 Cloning and analysis of the four genes coding for Bpu10I restriction-modification enzymes; Stankevicius K et al.; The Bpu 10I R-M system from Bacillus pumilus 10, which recognizes the asymmetric 5'-CCTNAGC sequence, has been cloned, sequenced and expressed in Escherichia coli . The system comprises four adjacent, similarly oriented genes encoding two m5C MTases and two subunits of Bpu 10I ENase (34.5 and 34 kDa) . Both bpu10IR genes either in cis or trans are needed for the manifestation of R . Bpu 10I activity . Subunits of R . Bpu 10I, purified to apparent homogeneity, are both required for cleavage activity . This heterosubunit structure distinguishes the Bpu 10I restriction endonuclease from all other type II restriction enzymes described previously . The subunits reveal 25% amino acid identity . Significant similarity was also identified between a 43 amino acid region of R . Dde I and one of the regions of higher identity shared between the Bpu 10I subunits, a region that could possibly include the catalytic/Mg2+binding center . The similarity between Bpu 10I and Dde I MTases is not limited to the conserved motifs (CM) typical for m5C MTases . It extends into the variable region that lies between CMs VIII and IX . Duplication of a progenitor gene, encoding an enzyme recognizing a symmetric nucleotide sequence, followed by concerted divergent evolution, may provide a possible scenario leading to the emergence of the Bpu 10I ENase, which recognizes an overall asymmetric sequence and cleaves within it symmetrically. Ann Emerg Med, 1998 Mar, 31(3), 370 - 5 Tuberculosis exposure and control in an urban emergency department; Behrman AJ et al.; STUDY OBJECTIVE: To measure tuberculosis (TB) conversion rates among staff of an urban emergency department compared with other hospital employees (OHEs) and to evaluate the effectiveness of new TB control measures . METHODS: A prospective interventional cohort study was performed in an academic adult 1,000-bed urban hospital, with more than 6,000 employees, and an annual ED census of 43,000 . As part of a hospital-wide program, all employees were screened for tuberculin reactivity (Siebert purified protein derivative {PPD} of tuberculin) annually during a 3 1/2-year period . Additional information collected on each employee included country of birth, ethnicity, history of bacille Calmette-Guerin (BCG) vaccination, length and site of employment, and age . At the end of the second year, TB control measures including construction of a new ED facility with TB respiratory isolation rooms, nonrecirculated air, and droplet shields for registrars were implemented . Relative risk (RR) and 95% confidence intervals (CIs) were calculated . RESULTS: During the first screening cycle, PPD status was obtained on 5,697 hospital employees, 88 of whom worked primarily in the ED . Baseline status was 81% PPD- (induration <5 mm), 9% PPD+ (induration > or =10 mm), and 10% refused skin testing . ED staff did not differ from OHEs with regard to PPD status, age, ethnicity, BCG history, foreign birth, residing in a county with high TB prevalence, or length of employment . During the second cycle, 6 of 50 (12%) previously PPD- ED staff and 51 of 2,514 (2%) previously PPD OHEs converted to PPD+ status (RR=5.9; 95% CI 2.7,13.1) . After implementing TB control measures, the conversion rate during the third year dropped to 0 for ED staff, whereas the OHEs had a conversion rate of 1.2% during that cycle . CONCLUSION: TB conversion rates were 5.9 times greater in ED staff members than OHEs before engineering controls were implemented . The ED TB conversion rate fell dramatically in the year following new TB controls . Many EDs are high-risk sites for TB exposure and may benefit from similar measures. Int J Food Microbiol, 1997 Sep 16, 38(2-3), 229 - 34 Modelling Bacillus cereus growth; Chorin E et al.; The aim of this study was to model the growth, in a model system, of toxigenic strains of Bacillus cereus as a function of temperature, pH and water activity . Optical density (OD) values were transformed into numbers of colony-forming units (CFU) by the use of a 'calibrating' relation . The growth curves were then fitted to the Gompertz function which allowed the estimation of the lag-time (lambda) and the growth rate (mu) . These two parameters were then modelled according to the controlling factors which were pH and water activity at 20 and 30 degrees C. Ukr Biokhim Zh, 1997 May-Jun, 69(3), 28 - 35 Bacterial proteases: production, isolation and utilization in animal nutrition; Michalik I et al.; Under conditions of submerged fermentation of Bacillus licheniformis strain L-3 in 15-L MBR-Schulzer bioreactor, the maximum production of proteolytic enzymes was achieved in the nutrient medium which contained 1% milk powder, 0.3% yeast autolysate, 0.5% corn starch and malt (20 ml per 100 ml of the medium) . The preparation obtained of extracellular alkaline bacterial Ser-protease of the subtilisin type is characterized by optimum pH 9.8-10.2 good up to a temperature stability (65 degrees C) and has molecular weight cca of 26 kDa . The use of chemical mutagens (HNO2, 5-bromouracil) has enabled to select new strains (L-3N and L-3U) whose protease activity is 1.8-2.2 times higher as compared to the original Bacillus licheniformis strain L-3 . The addition of these enzymes to the fodder has a positive effect on the retention of nitrogen substances. Int J Food Microbiol, 1997 Aug 19, 38(1), 25 - 30 Survival of heated Bacillus coagulans spores in a medium acidified with lactic or citric acid; Palop A et al.; The influence of the intensity of heat treatments on the capacity of citric or lactic acid to prevent growth of survivors of Bacillus coagulans spores after 10 days storage at 35 degrees C was studied . In most cases, the number of survivors during storage decreased . The extent of this spore inactivation depended on the intensity of previous heat treatment and the pH of the medium and the acidulant used . The inactivating effect of storage was pronounced even at pH values less acidic than those used by the canning industry . Citric acid was more effective than lactic acid on spores given only low heat treatments, but lactic was more effective against those given more severe heat treatments . The severity of heat treatment required for lactic to be more effective than citric acid increased with pH of the medium . Heat treatment also required increased pH for heated spores to grow . pH 4.6, regardless of acidulant used, was unable to prevent growth of unheated spores but a less acidic pH (pH 5.2) did prevent growth even when spores had been given only mild heat treatments (10 s at 100 degrees C). Br J Nutr, 1997 Dec, 78(6), 1015 - 29 Dose-response effects of raw potato starch on small-intestinal escape, large-bowel fermentation and gut transit time in the rat; Mathers JC et al.; This study was designed to quantify starch digestion within the small and large bowels separately when raw potato starch (RPS) was included at 0-240 g/kg in diets fed to growing male Wistar rats . RPS was incorporated in the diets at the expense of maize starch which was expected to be almost completely digested in the small bowel . The digestibility of the maize starch was 0.99 but only 0.28 of the RPS was digested before the terminal ileum so that with increasing intakes of RPS there was a progressive increase in starch supply to the large bowel (LB) . Of this starch 0.77, 0.72 and 0.73 was fermented in the large bowel when RPS constituted 80, 160 and 240 g/kg diet respectively . With increasing RPS intake, there was a curvilinear response in molar proportion of butyrate in caecal contents with a maximum value at about 80 g RPS/kg diet . The molar proportion of acetate increased linearly, that of propionate was unchanged, whilst proportions of the minor short-chain fatty acids all declined markedly with increasing RPS intake . The novel marker Bacillus stearothermophilus spores (BSS) was compared with CrEDTA in estimation of whole-gut mean transit time (MTT) when given together in a single test meal . Whilst estimates of MTT for the two markers were strongly correlated within individual rats (r2 0.72), BSS produced estimates that were 13 h longer than those based on CrEDTA . Neither marker detected a change in MTT with increasing RPS intake but, with both, the rate constant (k1) for the 'largest mixing pool' declined significantly (P < 0.001) as dietary RPS concentration was changed from 0-240 g/kg. Rev Mal Respir, 1997 Dec, 14 Suppl 5, S60 - 71 {Pulmonary manifestations of tuberculosis in children}; Olivier C; The occurrence of tuberculosis in children is dependent on a contagious bacillus carrying adult . Among 500 cases notified annually, perhaps 5 or 6% of the total infectious reservoir in France, 75% have parenchymal pulmonary disease and/or lymph nodes . These tuberculous diseases only represent 10% of the pulmonary disorders: 90% remain primary infections (PI active) or latent infections . These are most often asymptomatic (PI Latent) or of low grade activity (PI active) . The CT scanner and fibreoptic bronchoscopy are indispensable complementary investigations in tuberculous disease . Whatever the clinical picture the diagnosis rests on bacteriological confirmation (but only 30% of cultures are positive) and most often rests on a body of evidence: for example a contagious adult living in proximity or a contagious family, or other risk factors are present . The evidence of a child with whatever form of pulmonary tuberculosis, even a latent primary infection, requires treatment which is adapted in such a way to enable a cure and to protect against subsequent endogenous re-activation . A coherent system of co-operation between the hospital and community service and between paediatricians and adult physicians is indispensable to find the index adult case to break the chain of contagion . There are two specific aspects in children, first congenital tuberculosis when a diagnosis is difficult and secondly tuberculosis in a child who is HIV positive when the management can be delicate. FEMS Microbiol Lett, 1998 Mar 1, 160(1), 55 - 60 Prevalence of beta-exotoxin, diarrhoeal toxin and specific delta-endotoxin in natural isolates of Bacillus thuringiensis; Perani M et al.; Of newly isolated colonies with the appearance of Bacillus thuringiensis, 47.5% were found to produce the parasporal crystals characteristic of this species . These positive isolates were screened using the polymerase chain reaction for their possession of a gene encoding a specific protoxin type, CryIB . Strains with and without this gene were screened for their ability to produce beta-exotoxin and Bacillus cereus-type enterotoxin . It was found that 35% of the isolates possessed the cryIB gene; of these 83% also produced enterotoxin and 58% produced beta-exotoxin . No statistical significance was found for linkage between any of these characteristics . The probability, therefore, of isolating a strain of B . thuringiensis which specifically possessed the cryIB gene but did not produce either of the other, undesired, toxins, from the soil sample used, was 1.2%. Infect Dis Clin North Am, 1998 Mar, 12(1), 137 - 55 Bartonella-associated infections; Spach DH et al.; Bartonella-associated infections occur in immunocompetent and immunocompromised patients . The spectrum of diseases caused by Bartonella species has expanded and now includes cat-scratch disease, bacillary angiomatosis, bacillary peliosis, bacteremia, endocarditis, and trench fever . Most Bartonella-associated infections that occur in North America and Europe are caused by B . henselae or B . quintana . The domestic cat serves as the major reservoir for B . henselae; the reservoir for the modern day B . quintana infection remains unknown . Methods used to diagnose Bartonella-associated infections include histopathologic analysis of biopsy specimens, culture of tissue samples, blood culture, and serology . Available data on treatment of Bartonella-associated infections remain relatively sparse but would suggest that erythromycin or doxycycline provide the best responses. Chemosphere, 1998 Mar, 36(6), 1291 - 304 Asking for the indicator function of bioassays evaluating soil contamination: are bioassay results reasonable surrogates of effects on soil microflora? Ronnpagel K, Janssen E, Ahlf W. In evaluating the biological effect of solid materials like soil a bacterial contact assay often shows higher sensitivity than elutriate testing . Results of the Bacillus cereus contact assay for some environmental important toxicants are presented in this article . A comparison with another heterotrophic soil bacterium, Arthrobacter globiformis, shows comparable sensitivity . In a bioassay approach organisms at the level of individuals or populations are exposed to soil material to determine the significance of contaminants . An investigation that incorporates community level processes in comparison with toxicity test results provides a better understanding of the indicator function of bioassays . Comparison of soil bioassays (aqueous and solid phase) with ecological parameters demonstrates the problems in predicting ecological effects of soil contamination. Int J Biol Macromol, 1997 Dec, 21(4), 307 - 17 Use of 3-D computer modelling and kinetic studies to analyse grapefruit pyrophosphate-dependent phosphofructokinase; Van Praag E; The glycolytic reaction of grapefruit PPi-dependent phosphofructokinase (PFP) depends on the presence of Fru-2,6-P2 (Ka = 6.7 nM) . This molecule was further demonstrated in grapefruit juice sac cells . Citrate, alpha-ketoglutarate and isocitrate competitively inhibited the binding of Fru-2,6-P2 to PFP . The affinity for Fru-6-P (Km = 159 microM) and PPi (Km = 33 microM) were not affected by the addition of these molecules . In the gluconeogenic reaction, the presence of Fru-2,6-P2 did not affect the Km of Fru-1,6-P2 (61 microM) in contrast to orange fruit PFP . These results led to the building of a computer model of PFP, based on the known structure of Bacillus stearothermophilus ATP-dependent phosphofructokinase (ATP-PFK) . The results show that catalysis of Fru-6-P in the alpha chain is most unlikely, due to amino-acid substitutions and that Fru-2,6-P2 can bind between the alpha and beta subunits. Immunol Cell Biol, 1997 Dec, 75(6), 595 - 603 Immunological requirements for a subunit vaccine against tuberculosis; Elhay MJ et al.; Tuberculosis remains one of the most important threats to world health . Current vaccination and prevention strategies are inadequate and there is an urgent need for a new vaccine . The current vaccine bacille Calmette-Guerin (BCG), is unable to protect against re-activation of disease in later life and its efficacy varies tremendously in different human populations . An ideal replacement would be a non-living subunit vaccine that could impart protective efficacy greater than BCG but without its drawbacks . Before such a goal is achieved, however, there are many parameters that need to be examined in experimental systems . Such studies have revealed that apart from the selection of immunologically relevant antigens, dosage of antigen and type of adjuvant need to be chosen carefully . These parameters need to be examined in the context of the complex biology of the disease and, despite recent progress in defining host/pathogen interactions, experimental vaccines tested so far have fallen short of the protective efficacy of BCG . A coordinated approach, stimulating the various facets of cell-mediated immunity will probably be essential for development of protective immunity through subunit vaccination. Immunol Cell Biol, 1997 Dec, 75(6), 591 - 4 DNA vaccines against tuberculosis; Lowrie DB et al.; This edited transcript of a presentation at the 'Vaccines Beyond 2000' conference describes a series of investigations by the authors throwing light on the mechanisms of protective immunity against tuberculosis in mice and raising hope for a new kind of vaccine to replace bacille Calmette-Guerin (BCG) . DNA encoding only one or a few protein antigens was found capable of conferring persistent protection equal to the effect of BCG . The essential features seem to be an endogenous origin of the antigen within transfected mouse cells which favours the development of CD8+/CD44hi/IFN-gamma-producing T cells with antigen-specific cytotoxicity . Such cells were the most efficient in adoptive transfer of protection from infected or DNA-vaccinated mice to naive mice. Immunol Cell Biol, 1997 Dec, 75(6), 554 - 60 Cellular responses and Mycobacterium bovis BCG growth inhibition by bovine lymphocytes; Carpenter E et al.; Cellular responses of a group of cattle immunized subcutaneously with a low dose of Mycobacterium bovis bacille Calmette-Guerin vaccine (BCG) were measured in vitro and compared with nonimmunized control animals . PBMC taken from immunized animals proliferated and produced IFN-gamma in the presence of M . bovis BCG culture filtrate proteins . The addition of PBMC from immunized animals to M . bovis BCG-infected autologous macrophages also resulted in secretion of IFN-gamma . In contrast, the responses of PBMC from control animals were comparatively low over the period of study . In experiments to study the interaction of non-adherent lymphocytes with infected macrophages, M . bovis BCG growth was inhibited in cultures containing autologous PBMC from immunized and non-immunized control animals . The degree of inhibition was related to lymphocyte concentration but did not correlate with IFN-gamma production . Treatment of macrophages with recombinant IFN-gamma prior to, or postinfection did not alter the intracellular growth kinetics of mycobacteria . It appears, therefore, that although M . bovis BCG immunization of cattle stimulates the generation of a T cell-mediated immune response to M . bovis BCG, the cattle may already possess a high level of innate resistance to M . bovis BCG that requires the presence of lymphocytes. J Vector Ecol, 1997 Dec, 22(2), 122 - 4 Field evaluation of Vectobac G, Vectobac 12AS and Bactimos WP against the dengue vector Aedes albopictus in tires; Sulaiman S et al.; The efficacy of three formulations of Bacillus thuringiensis var . israelensis was studied against Aedes albopictus in discarded tires . The formulations were: Vectobac G (corn cob formulation), Vectobac 12AS (aqueous suspension), and Bactimos WP (wettable powder formulation) . Both Vectobac G and Vectobac 12AS were effective for 24 hr with more than 80% mortality . Both Vectobac formulations were significantly more effective than Bactimos WP for 24 hr after treatment (P < 0.0005) . A week after treatment, Vectobac 12AS was significantly different than Bactimos WP (P < 0.05) . However, Vectobac G did not differ significantly from Bactimos WP (P > 0.05); two weeks after spraying there was no significant difference among the various formulations (P > 0.05). Proteins, 1998 Feb 1, 30(2), 155 - 67 Crystal structures and properties of de novo circularly permuted 1,3-1,4-beta-glucanases; Ay J et al.; The 1,3-1,4-beta-glucanases from Bacillus macerans and Bacillus licheniformis, as well as related hybrid enzymes, are stable proteins comprised of one compact jellyroll domain . Their structures are studied in an effort to reveal the degree of redundancy to which the three-dimensional structure of protein domains is encoded by the amino acid sequence . For the hybrid 1,3-1,4-beta-glucanase H(A16-M), it could be shown recently that a circular permutation of the sequence giving rise to the variant cpA16M-59 is compatible with wildtype-like enzymatic activity and tertiary structure (Hahn et al., Proc . Natl . Acad . Sci . USA 91:10417-10421, 1994) . Since the circular permutation yielding cpA16M-59 mimicks that found in the homologous enzyme from Fibrobacter succinogenes, the question arose whether de novo circular permutations, not guided by molecular evolution of the 1,3-1,4-beta-glucanases, could also produce proteins with native-like fold . The circularly permuted variants cpA16M-84, cpA16M-127, and cpA16M-154 were generated by PCR mutagenesis of the gene encoding H(A16-M), synthesized in Escherichia coli and shown to be active in beta-glucan hydrolysis . CpA16M-84 and cpA16M-127 were crystallized in space groups P2(1) and P1, respectively, and their crystal structures were determined at 1.80 and 2.07 A resolution . In both proteins the main parts of the beta-sheet structure remain unaffected by the circular permutation as is evident from a root-mean-square deviation of main chain atoms from the reference structure within the experimental error . The only major structural perturbation occurs near the novel chain termini in a surface loop of cpA16M-84, which becomes destabilized and rearranged . The results of this study are interpreted to show that: (1) several circular permutations in the compact jellyroll domain of the 1,3-1,4-beta-glucanases are tolerated without radical change of enzymatic activity or tertiary structure, (2) the three-dimensional structures of simple domains are encoded by the amino acid sequence with sufficient redundancy to tolerate a change in the sequential order of secondary structure elements along the sequence, and (3) the native N-terminal region is not needed to guide the folding polypeptide chain toward its native conformation. Bioorg Khim, 1997 Oct, 23(10), 783 - 94 {Secondary structure of binase in solution by 1H NMR}; Reibarkh MYa et al.; Nearly all resonances were assigned in the two-dimensional 1H NMR spectra of binase, guanylospecific ribonuclease from Bacillus intermedius containing 109 amino acid residues . The exchange rates of amide protons with the solvent deuterium were measured in 2H2O at pH 6.7 and 30 degrees C . Coupling constants 3J of H-NC alpha-H, NOE contacts, solvent exchange rates of amide protons, and indices of C alpha H chemical shifts were measured, and the binase secondary structure was deduced from these data . It involves three alpha-helices in the N-terminal part (the 6-16, 26-31, and 41-45 segments) and a beta-sheet formed by five antiparallel beta-strands (51-55, 71-75, 86-90, 95-99, and 104-108 segments) . The binase secondary structure was compared with that of its closest homologue, barnase from B . amyloliquefaciens. J Biol Chem, 1998 Feb 6, 273(6), 3438 - 46 Crystal structure of barley 1,3-1,4-beta-glucanase at 2.0-A resolution and comparison with Bacillus 1,3-1,4-beta-glucanase; Muller JJ et al.; Both plants and bacteria produce enzymes capable of degrading the mixed-linked beta-glucan of the endosperm cell walls of cereal grains . The enzymes share the specificity for beta-1,4 glycosyl bonds of O-3-substituted glucose units in linear polysaccharides and a similar cleavage mechanism but are unrelated in sequence and tertiary structure . The three-dimensional structure of the 1,3-1, 4-beta-glucanase isoenzyme EII from barley was determined from monoclinic crystals at a resolution of 2.0 A . The protein is folded into a betaalpha8 barrel structure as has been shown previously (Varghese, J . N., Garrett, T . P . J., Colman, P . M., Chen, L., Hoj, P . B., and Fincher, G . B . (1994) Proc . Natl . Acad . Sci . U.S.A . 91, 2785-2789) by diffraction analysis at lower resolution of tetragonal crystals . It contains one N-glycosylation site which is described in detail with the sugar moieties attached to residue Asn190 . The geometry and hydration of the barley 1,3-1,4-beta-glucanase is analyzed; a model beta-glucan fragment is placed into the binding site by molecular dynamics simulation, and the beta-glucan binding grooves of the plant and bacterial enzymes are compared . Their active sites are shown to have a small number of common features in generally dissimilar geometries that serve to explain both the identical substrate specificity and the observed differences in inhibitor binding. Chem Biol, 1997 Dec, 4(12), 927 - 37 The bacitracin biosynthesis operon of Bacillus licheniformis ATCC 10716: molecular characterization of three multi-modular peptide synthetases; Konz D et al.; BACKGROUND: The branched cyclic dodecylpeptide antibiotic bacitracin, produced by special strains of Bacillus, is synthesized nonribosomally by a large multienzyme complex composed of the three bacitracin synthetases BA1, BA2 and BA3 . These enzymes activate and incorporate the constituent amino acids of bacitracin by a thiotemplate mechanism in a pathway driven by a protein template . The biochemical features of these enzymes have been studied intensively but little is known about the molecular organization of their genes . RESULTS: The entire bacitracin synthetase operon containing the genes bacA-bacC was cloned and sequenced, identifying a modular structure typical of peptide synthetases . The bacA gene product (BA1, 598kDa) contains five modules, with an internal epimerization domain attached to the fourth; bacB encodes BA2 (297kDa), and has two modules and a carboxy-terminal epimerization domain; bacC encodes BA3, five modules (723kDa) with additional internal epimerization domains attached to the second and fourth . A carboxy-terminal putative thioesterase domain was also detected in BA3 . A putative cyclization domain was found in BA1 that may be involved in thiazoline ring formation . The adenylation/thioester-binding domains of the first two BA1 modules were overproduced and the detected amino-acid specificity coincides with the first two amino acids in bacitracin . Disruption of chromosomal bacB resulted in a bacitracin-deficient mutant . CONCLUSIONS: The genes encoding the bacitracin synthetases BA1, BA2 and BA3 are organized in an operon, the structure of which reflects the modular architecture expected of peptide synthetases . In addition, a putative thiazoline ring formation domain was identified in the BA1 gene. J Endod, 1998 Jan, 24(1), 51 - 3 An evaluation of sterilization of endodontic instruments in artificial sponges; Velez AE et al.; The ability to sterilize endodontic files inserted into synthetic sponges was tested . Sponges were subjected to 5 cycles of either dry heat (Driclave) or steam under pressure (autoclave) sterilization . Sterilization was corroborated by microbiological tests . The sponges and files were pre-sterilized separately using steam under pressure . One hundred eighty files contaminated with Bacillus stearothermophilus spores (experimental and positive control) and 60 noncontaminated files (negative control), were inserted into 60 sponges . After each cycle, each file and a portion of sponge surrounding the file were transferred aseptically to tubes containing trypticase soy broth culture medium for bacteriological analysis . None of the tubes containing files and portions of sponges that were subjected to autoclave grew Bacillus stearothermophilus spores . Two of 60 (3.33%) of the tubes that were subjected to sterilization by Driclave demonstrated bacterial growth . Although the sponges tolerated the dry heat cycles well physically, sterilization was achieved in only 96.67% of the cases. Clin Exp Immunol, 1998 Feb, 111(2), 286 - 92 Monocytes modulate enhancement of HIV-1 replication by Mycobacterium tuberculosis; De Haas CJ et al.; To investigate the effects of Mycobacterium tuberculosis on HIV-1 replication, peripheral blood mononuclear cells (PBMC) of bacille Calmette-Guerin (BCG)-vaccinated donors and non-BCG-vaccinated donors were infected in vitro with a lymphotropic isolate of HIV-1 and cultured in the presence of purified protein derivative (PPD) . Addition of PPD resulted in enhanced HIV-1 replication and lymphoproliferation in BCG-vaccinated donor PBMC, while PPD had no such effects in control PBMC . HIV-1 replication increased even more when monocytes were removed from PBMC, while lymphoproliferation was decreased . High percentages of monocytes were associated with a decreased HIV-1 replication and proliferation that could not be reversed by addition of antibodies against the cytokines IL-1, transforming growth factor-beta (TGF-beta) or indomethacin . PPD stimulates PBMC to release IL-10, a cytokine known to down-regulate proliferation and HIV-1 replication . PPD-induced effects on proliferation as well as HIV-1 replication could be partially blocked by adding a monoclonal antibody against MHC class II molecules, suggesting that part of the mechanism of PPD-induced enhancement is T memory cell activation. FEMS Microbiol Lett, 1998 Feb 1, 159(1), 113 - 20 Endogenous protease-activated 66-kDa toxin from Bacillus thuringiensis subsp . kurstaki active against Spodoptera littoralis; Kumar NS et al.; The anti-lepidopteran toxin from sporulated Bacillus thuringiensis subsp . kurstaki cells, generated by the proteolytic action of endogenous protease(s) on the protoxin, was purified and studied to identify the effect of such proteolysis on the biochemical nature of the toxin . The active toxin was purified employing anion-exchange chromatography to absolute homogeneity, as indicated by SDS-PAGE and Western blotting . Antisera to the purified toxin (66 kDa) crossreacted with the protoxin (132 kDa) confirming its origin from protoxin . The purified toxin with a pI of 7.95 was derived from the N-terminal region of the protoxin (pI 7.6) . Circular dichroism data revealed that the toxin has significant secondary structure and it undergoes pH dependent conformational change . Unlike the toxin generated by exogenous proteases such as trypsin, etc., the endogenous protease(s) activated toxin is highly lethal to a tolerant insect variety of the lepidopteran order, Spodoptera littoralis. FEMS Microbiol Lett, 1998 Feb 1, 159(1), 47 - 58 Bacillus licheniformis MC14 alkaline phosphatase I gene with an extended COOH-terminus; Kim JW et al.; Bacterial alkaline phosphatases (APases), except those isolated from Bacillus licheniformis, are approximately 45-kDa proteins while eucaryotic alkaline phosphatases are 60 kDa . To answer the question of whether the apparent 60-kDa alkaline phosphatase from Bacillus licheniformis accurately reflected the size of the protein, the entire gene was analyzed . DNA sequence analysis of the alkaline phosphatase I (APaseI) gene of B . licheniformis MC14 indicated that the gene could code for a 60-kDa protein of 553 amino acids . The deduced protein sequence of APaseI showed about 32% identity to those of B . subtilis APase III and IV and had apparent sequence homologies in the core structure and active sites that are conserved among APases of various sources . The extra carboxy-terminal sequence of APaseI, which made the enzyme bigger than other procaryotic APases, was not homologous to those of eucaryotic APases . The amino acid composition of APaseI was most similar to that of salt-dependent APase among the isozymes of B . licheniformis MC14 . Another open reading frame of 261 amino acids was present 142 nucleotide upstream of the APaseI gene and its predicted amino acid sequence showed 68% identity to that of glucose dehydrogenase of B . megaterium. Biochemistry, 1998 Feb 17, 37(7), 1926 - 32 Structure of the Bacillus agaradherans family 5 endoglucanase at 1.6 A and its cellobiose complex at 2.0 A resolution; Davies GJ et al.; The enzymatic degradation of cellulose, by cellulases, is not only industrially important in the food, paper, and textile industries but also a potentially useful method for the environmentally friendly recycling of municipal waste . An understanding of the structural and mechanistic requirements for the hydrolysis of the beta-1,4 glycosidic bonds of cellulose is an essential prerequisite for beneficial engineering of cellulases for these processes . Cellulases have been classified into 13 of the 62 glycoside hydrolase families {Henrissat, B., and Bairoch, A . (1996) Biochem J . 316, 695-696} . The structure of the catalytic core of the family 5 endoglucanase, Ce15A, from the alkalophilic Bacillus agaradherans has been solved by multiple isomorphous replacement at 1.6 A resolution . Ce15A has the (alpha/beta)8 barrel structure and signature structural features typical of the grouping of glycoside hydrolase families known as clan GH-A, with the catalytic acid/base Glu 139 and nucleophile Glu 228 on barrel strands beta 4 and beta 7 as expected . In addition to the native enzyme, the 2.0 A resolution structure of the cellobiose-bound form of the enzyme has also been determined . Cellobiose binds preferentially in the -2 and -3 subsites of the enzyme . Kinetic studies on the isolated catalytic core domain of Ce15A, using a series of reduced cellodextrins as substrates, suggest approximately five to six binding sites, consistent with the shape and size of the cleft observed by crystallography. Biochemistry, 1998 Feb 17, 37(7), 1810 - 8 Characterization of a buried neutral histidine in Bacillus circulans xylanase: internal dynamics and interaction with a bound water molecule; Connelly GP et al.; NMR spectroscopy was used to characterize the dynamic behavior of His149 in Bacillus circulans xylanase (BCX) and its interaction with an internal water molecule . Rate constants for the specific acid- and base-catalyzed exchange following bimolecular kinetics (EX2) of the nitrogen-bonded H epsilon 2 of this buried, neutral histidine were determined . At pDmin 7.0 and 30 degrees C, the lifetime for this proton is 9.9 h, corresponding to a protection factor of approximately 10(7) relative to that predicted for an exposed histidine . The apparent activation energies measured for specific acid and base catalysis (7.0 and 17.4 kcal/mol) indicate that exchange occurs via local structural fluctuations . Consistent with its buried environment, the N epsilon 2-H bond vector of His149 shows restricted mobility, as evidenced by an order parameter S2 = 0.83 determined from 15N relaxation measurements . The crystal structure of BCX reveals that a conserved, buried water hydrogen-bonds to the H epsilon 2 of His149 . Strong support for this interaction in solution is provided by the observation of a negative nuclear Overhauser effect (NOE) and positive rotating-frame Overhauser effect (ROE) between His149 H epsilon 2 and a water molecule with the same chemical shift as the bulk solvent . However, the chemical shift of H epsilon 2 (12.2 ppm) and a D/H fractionation factor close to unity (0.89 +/- 0.02) indicate that this is not a so-called low-barrier hydrogen bond . Lower and upper bounds on the lifetime of the internal water are estimated to be 10(-8) and 10(-3) s . Therefore the chemical exchange of solvent protons with those of His149 H epsilon 2 and the diffusion or physical exchange of the internal water to which the histidine is hydrogen-bonded differ in rate by over 7 orders of magnitude. Virus Genes, 1997, 15(3), 195 - 201 Genetic variation of rice tungro bacilliform virus in the Philippines; Villegas LC et al.; Restriction fragment length polymorphisms (RFLPs) were found in 27 full genome length clones from a glasshouse isolate of rice tungro bacilliform pararetrovirus (RTBV) from the International Rice Research Institute (IRRI), the Philippines and from clones from 5 field isolates from different parts of the Philippines . There was much less variation between the IRRI clones than between the field isolate clones . The RFLPs were due to single base changes and represented about 10% of the potential sites . Sequencing across the region between nt 7772 and 7989 confirmed that the field isolates differed from the published sequence more than did the IRRI clones . The most common substitutions were G > A, A > G and T > C . Sequence heterogeneity was also noted in PCR products from RTBV DNA from the isolates . These observations are discussed in relation to the quasispecies population concept of viruses. Nagoya J Med Sci, 1997 Nov, 60(3-4), 101 - 8 Cytokines and local cellular immunity; Shimokata K; The characteristics and function of human lymphocytes at a morbid site were studied . Exudative-sensitized lymphocytes in tuberculous pleural fluid reacted to the specific antigen more effectively and produced higher titers of lymphokines including interferon gamma (IFN-gamma) and interleukin 2 (IL-2) than circulating lymphocytes . CD4+/CD8- T-cell subset is responsible for the antigen-specific IFN-gamma production in pleural T-lymphocytes of patients with tuberculous pleurisy . Thus, activated T-lymphocytes involve the production of lymphokines at the morbid site and they effectively exert local cellular immunity through the action of such lymphokines . Immunofluorescence study showed an increased production of inducible nitric oxide synthase (iNOS) and peroxynitrite in bacille de Calmette-Guerin (BCG)-inoculated human alveolar macrophages (AM) . Reverse transcriptase-polymerase chain reaction methods also revealed the higher expression of iNOS-coding mRNA . Colony assay demonstrated that human AM effectively killed BCG in their cytoplasm . However, treatment of AM with NG-monomethyl-L-arginine monoacetate resulted in markedly reduced killing activity . These results clearly show that BCG-induced nitric oxide (NO) and its reactive product with the oxygen radical, peroxynitrite, could play an important role in BCG killing in human AM. Mikrobiol Z, 1997 Sep-Oct, 59(5), 7 - 13 {The carbohydrate specificity of the extracellular lectins in bacteria of the genus Bacillus}; Kovalenko EA; The paper presents the study of interaction of extracellular sialo-specific lectins of Bacillus genus bacteria with different types of erythrocytes and affinity of these biologically active substances to derivatives of N-acetylneuramine acid and natural sialo and asialo conjugates . It is established that lectins under study possess a very narrow carbohydrate specificity and distinguish strictly definite carbohydrate structures . The identification processes first of all require the availability of N-glycolylneuraminic acid in the structure of receptors, and to the less extent of alpha-isomer of N-acetylneuraminic acid in O-acetylated form added to terminal galactose alpha 2-6-or alpha 2-3-bonds. Mikrobiol Z, 1997 Sep-Oct, 59(5), 3 - 6 {The amino acid and monosaccharide composition of the extracellular sialo-specific lectins in bacteria of the genus Bacillus}; Kovalenko EA; Chemical composition of extracellular sialo-specific lectins of Bacillus genus bacteria has been studied . It is shown that these biopolymers are glycoproteins distinguished by the quantitative and qualitative composition of proteins and hydrocarbons . It is established that the given bacteria can synthesize one or two extracellular lectins one of which represents N-asparagine-bound glycoprotein, while another one belongs to O-serine/threonine-bound glycoproteins. Arch Environ Contam Toxicol, 1998 Jan, 34(1), 6 - 11 Liquid-gas partitioning of the gasoline oxygenate methyl tert-butyl ether (MTBE) under laboratory conditions and its effect on growth of selected algae; Rousch JM et al.; The partitioning of the widely used gasoline additive methyl tert-butyl ether (MTBE) between liquid growth media and gaseous phase was measured daily under laboratory conditions to determine how closely dissolved MTBE concentrations matched nominal concentrations . Total (gaseous and dissolved) MTBE averaged across 6 days for 29.6, 503.2, and 1005.7 mg L-1 MTBE treatments were 89.9, 90.3, and 73.0% of nominal, respectively, and mean dissolved MTBE in these same treatments were 74.6, 73.8, and 69.6% of total MTBE, respectively . This suggests that dissolved MTBE concentrations can vary substantially from nominal . The effect of MTBE on the growth of selected algae was also evaluated under laboratory conditions . Three unicellular algae, Selenastrum capricornutum (Chlorophyta), Navicula pelliculosa (Bacillariophyta), and Synechococcus leopoliensis (= Anacystic nidulans, Cyanophyta = Cyanobacteria), representative of three taxonomic groups, were used as test organisms . Toxicity tests were acute and increase in cell number was used as an indicator of growth . Algal species were exposed by injection of MTBE into sealed vessels containing defined liquid growth media . The growth of N . pelliculosa and S . leopoliensis was negatively affected at nominal 2400 mg L-1 MTBE, whereas the growth of S . capricornutum was negatively affected at nominal 4800 mg L-1 MTBE and positively affected at nominal 600 mg L-1 MTBE . The differential sensitivity of the growth of these representative species suggests that MTBE may alter algal community composition in the natural environment. Int J Urol, 1997 Nov, 4(6), 552 - 6 Prognostic markers of intravesical bacillus Calmette-Guérin therapy for multiple, high-grade, stage T1 bladder cancers; Lee E et al.; BACKGROUND: Prediction of a response to intravesical bacillus Calmette-Guerin (BCG) therapy for bladder cancer is clinically important . We determined whether several molecular markers have prognostic value in intravesical BCG therapy for multiple, high-grade, stage T1 bladder cancers . METHODS: The expressions of p53 (clone D07), bcl-2 (100-D5), cathepsin-D (C5), c-myc(9E11), c-erbB-2 (CB11) and Ki-67 (MM1) were determined by immunohistochemistry in paraffin-embedded tissues from 32 multiple, T1, grade II-III bladder cancer patients (15 BCG responders, 17 nonresponders) who had undergone a single course of BCG therapy (Pasteur strain, 5 x 10(8) CFU weekly for 6 weeks) after complete removal of the tumors . The association between the expression of these markers and the response to BCG was assessed by univariate and multivariate analyses . RESULTS: There was no difference in patient and tumor characteristics between the 2 groups . Using multivariate analysis, the only useful marker was p53, with the overexpression of the p53 protein inversely related to the response to BCG therapy (P = 0.0182) . CONCLUSION: Our results suggest that the status of p53 expression offers significant clinical information and may be a useful tool in the selection of suitable candidates for BCG therapy in multiple, high-grade stage T1 bladder cancer patients. Mol Biochem Parasitol, 1997 Dec 15, 90(2), 423 - 32 Mutagenesis study of the glycosylphosphatidylinositol phospholipase C of Trypanosoma brucei; Carnall N et al.; The glycosylphosphatidylinositol phospholipase C (GPI-PLC) from Trypanosoma brucei is particularly effective in hydrolysing the GPI-anchors of some proteins . The enzyme is inhibited by Zn2+ and p-chloromercurylphenylsulphonic acid, both of which can act as sulphydryl reagents, suggesting that a cysteine residue may be important in catalysis . Single cysteine to serine mutants have been produced for all eight cysteines in GPI-PLC; all the mutants were fully active in vitro and were still susceptible to p-chloromercurylphenylsulphonic acid inhibition . In contrast, a single histidine 34 to glutamine mutation totally inactivated GPI-PLC . The histidine was chosen after a sequence alignment with the Bacillus cereus phosphatidylinositol phospholipase C (PI-PLC) suggested a conservation of active site residues, including histidine 34 which is central to the proposed reaction mechanism (Heinz D.W., Ryan M., Bullock T.L., Griffith O.H . EMBO J 1995;14:3855-3863) . The results suggest that the GPI-PLC and bacterial PI-PLCs have conserved active sites and that the inhibition of GPI-PLC by sulphydryl reagents can occur through more than one residue. Genetics, 1998 Jan, 148(1), 13 - 8 The effect of mismatch repair and heteroduplex formation on sexual isolation in Bacillus; Majewski J et al.; In Bacillus transformation, sexual isolation is known to be an exponential function of the sequence divergence between donor and recipient . Here, we have investigated the mechanism under which sequence divergence results in sexual isolation . We tested the effect of mismatch repair by comparing a wild-type strain and an isogenic mismatch-repair mutant for the relationship between sexual isolation and sequence divergence . Mismatch repair was shown to contribute to sexual isolation but was responsible for only a small fraction of the sexual isolation observed . Another possible mechanism of sexual isolation is that more divergent recipient and donor DNA strands have greater difficulty forming a heteroduplex because a region of perfect identity between donor and recipient is required for initiation of the heteroduplex . A mathematical model showed that this heteroduplex-resistance mechanism yields an exponential relationship between sexual isolation and sequence divergence . Moreover, this model yields an estimate of the size of the region of perfect identity that is comparable to independent estimates for Escherichia coli . For these reasons, and because all other mechanisms of sexual isolation may be ruled out, we conclude that resistance to heteroduplex formation is predominantly responsible for the exponential relationship between sexual isolation and sequence divergence in Bacillus transformation. J Am Mosq Control Assoc, 1997 Dec, 13(4), 384 - 8 Performance of ULV formulations (Pesguard 102/Vectobac 12AS) against three mosquito species; Yap HH et al.; Adulticidal and larvicidal performances of a water-based pyrethroid microemulsion Pesguard PS 102 (AI d-allethrin and d-phenothrin, both at 5.0% w/w) and Vectobac 12AS, an aqua-suspension Bacillus thuringiensis israelensis (B.t.i.) formulation (AI 1,200 ITU/mg) were assessed against mosquitoes Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus using a Leco ULV Fog Generator Model 1600 and a Scorpion 20 ULV AirBlast Sprayer . Laboratory-cultured mosquito adults and larvae were used for efficacy assessment . For trials using Leco, both pyrethroid and bacterial formulations were dispersed both singly and in combination with Pesguard PS 102 at a dosage of 0.2 liters/ha and B.t.i . at a dosage of 1.0 liter/ha . Similar trials with the Scorpion were also conducted with Pesguard PS 102 at a dosage of 0.2 liters/ha and a higher dosage of B.t.i . (1.5 liters/ha) . Experiments were conducted in a football field (200 x 100 m) where five check points at 10, 25, 50, 75, and 100 m downwind from the spray nozzle were chosen for efficacy assessments . Knockdown and mortality were scored at 1 and 24 h postspraying . Results from both trials showed that mortality values varied with distance from spray nozzle . For trials with Leco, fogging with the combination of Pesguard PS 102 and B.t.i . provided larvicidal mortality of > 80% for both Aedes species and of > 60% for Cx . quinquefasciatus larvae at several check points, depending on wind conditions . Complete mortality of adult Aedes mosquitoes at 24 h posttreatment was also achieved, while mortality values for Culex adults reached > 90% under strong wind conditions . As for trials with the Scorpion 20, high adult and larval mortalities were also achieved, with > 90% mortality at some check points . The above study demonstrated the possibility of achieving both larvicidal and adulticidal effects when using a combination of B.t.i . and Pesguard PS 102 in ULV space spray. J Am Mosq Control Assoc, 1997 Dec, 13(4), 356 - 60 Study on the flying height of Aedes caspius and Culex pipiens females in the Po Delta area, Italy; Bellini R et al.; We have studied the vertical distribution of Culicidae in the "Ancona di Bellocchio" protected area (Regional Park of the Po Delta, Emilia-Romagna, Italy) by means of nonilluminated CDC traps baited with CO2 . Traps were placed at heights of 1.5, 3, 4, and 5 m from the ground in open areas and at 1.5, 3, 4, 5, 6, and 7 m in wooded areas . We calculated the average flying height of the species caught, i.e., in order of decreasing flying height, Culex pipiens Linnaeus s.l., Culex modestus Ficalbi, Coquillettidia richiardii (Ficalbi), Aedes detritus (Haliday), Aedes caspius (Pallas) . We also calculated the linear regression lines for both sites and found that 90% of Ae . caspius flew within a height of 1.64 m from the ground level, 95% within 2.68 m, and 99% at a height not exceeding 4 m; whereas 90% of Cx . pipiens moved within 3.45 m from the ground level, 95% within 4.02 m, and 99% within 4.76 m . The vertical distributions of Ae . caspius and Cx . pipiens did not vary significantly over the seasons and were not affected by the presence of trees or variations in temperature and wind velocity within the range of measurements obtained . The data obtained provide useful information for planning Ae . caspius control measures based on a mechanical barrier capable of preventing mosquitoes from moving toward residential settlements and tourist resorts bordering on the protected area as an alternative to aerial treatment with the larvicide Bacillus thuringiensis subsp . israelensis. J Am Mosq Control Assoc, 1997 Dec, 13(4), 305 - 10 Field trials of VectoLex CG, a Bacillus sphaericus larvicide, in Illinois waste tires and catch basins; Siegel JP et al.; The susceptibilities of Aedes triseriatus (Say), Anopheles punctipennis (Say), Culex restuans (Theobald), and Culex pipiens (L.) larvae to VectoLex CG were determined . VectoLex, formulated on corncob granules (10-14 mesh) was applied to 2 tire dumps and numerous catch basins located in east-central Illinois . VectoLex, formulated as effervescent tablets, was also applied to catch basins . In a sunlit dump, there was a 99.6% reduction in Ae . triseriatus, Cx . restuans, and Cx . pipiens larvae as long as 32 days after treatment, and no pupae were recovered during this interval . There was still a 71.6% larval reduction 74 days after treatment . There was an overall reduction of 22% for An . punctipennis larvae . In a shaded dump, no larval Ae . triseriatus, Cx . restuans, or Cx . pipiens were recovered for 25 days after treatment, and no pupae were recovered 25-67 days after treatment . There was still a 68.7% larval reduction 74 days after treatment . An . punctipennis was unaffected . In one catch basin study, VectoLex was comparable to Altosid (average of 46 days vs . 50 days until larvae were recovered from catch basins) . As the summer progressed, the duration of VectoLex control was reduced to 30 days . VectoLex effervescent tablets (evaluated mid-August through September) gave 18-day control . VectoLex was effective against Ae . triseriatus, Cx . restuans, and Cx . pipiens, but was not effective against An . punctipennis in waste tires . VectoLex was effective against Cx . restuans and Cx . pipiens in catch basins. J Am Mosq Control Assoc, 1997 Dec, 13(4), 297 - 304 Field trials with Bacillus sphaericus formulations against polluted water mosquitoes in a suburban area of Bangkok, Thailand; Mulla MS et al.; Two newly developed Bacillus sphaericus larvicidal formulations, VectoLex CG (corncob granules) and VectoLex WDG (water dispersible granules), were tested against Culex quinquefasciatus larvae in 4 highly polluted breeding sites in Thailand . VectoLex CG, applied at rates of 0.5-2 g/m2, gave satisfactory to complete control of late-instar larvae and pupae for up to 4 wk after treatment . The VectoLex WDG, which had higher potency and was applied at rates of 0.1-0.5 g/m2, gave satisfactory control for 1-4 wk after treatment . Among the factors influencing longevity of control were dosage of a given formulation, precipitation, and flooding of the treated sites; the latter had the greatest impact . Presence of larvivorous fish did not seem to influence larval populations because there were heavy populations of mosquito larvae present in the test sites in the presence of moderate numbers of fish before the application of B . sphaericus treatments. J Urol, 1998 Mar, 159(3), 1079 - 84 p53 status does not predict initial clinical response to bacillus Calmette-Guerin intravesical therapy in T1 bladder tumors; Pages F et al.; PURPOSE: In superficial urothelial tumors of the bladder, p53 status is currently the most informative pretreatment parameter to define a population at higher risk for invasive carcinoma . Also, in T1 tumors, occurrence of muscular invasion is often related to an early relapse following BCG therapy . With the knowledge of biological parameters able to identify the group of initial BCG therapy non-responders, it would be possible to offer earlier treatment to the patients who need a more aggressive mode of therapy . The aim of this work was to study the predictive value of the p53 tumor status on the early BCG therapy response . MATERIALS AND METHODS: The population included a selected group of 43 patients presenting T1 bladder tumors with no carcinoma in situ (Tis), treated by transurethral resection (TUR) followed by intravesical BCG therapy . Clinical outcome was analyzed in relation to usual clinical and histopathological parameters, and pretreatment p53 tumor status was assayed by an immunohistochemical technique using DO7 monoclonal antibody . For 16 specimens, p53 gene was investigated using a Single Strand Conformation Polymorphism (SSCP) analysis and sequence determination . RESULTS: p53 anomalies were strongly correlated to smoking behavior (p = 0.003) and tumoral grade (p = 0.025) . Univariate analysis revealed an absence of correlation between p53 immunostaining and initial, one and two years response-rate to BCG therapy . However, longterm followup revealed a trend between positive staining and disease progression . The p53 molecular study validated the use of DO7 immunostaining in detection of p53 anomalies . CONCLUSIONS: In T1 bladder tumors, pretreatment p53 determination was not useful to define a group of early BCG non-responders . Thus, p53 status and immunological response induced by BCG endovesical therapy are two independent events. J Urol, 1998 Mar, 159(3), 788 - 91 Correlation between p53 over expression and response to bacillus Calmette-Guerin therapy in a high risk select population of patients with T1G3 bladder cancer; Lebret T et al.; PURPOSE: The aim of this study was to determine if p53 status, assessed before intravesical bacillus Calmette-Guerin (BCG) therapy, can predict clinical outcome in a high risk population of patients with stage T1, grade G3 bladder cancer and if it can be used to select patients responsive to therapy . MATERIAL AND METHODS: After complete transurethral resection 35 patients with T1G3 bladder carcinoma received 6 weekly instillations of BCG and nonresponsive patients received a second course . After treatment cystoscopy and randomized biopsies of the bladder mucosa were performed . Pathologists had sufficient material to perform immunomarking in 25 cases using the peroxidase-antiperoxidase technique with antiprotein monoclonal antibody p53 . The results were expressed in percentage of marked nuclei . We established 5% increment thresholds from 0 to 60% . Contingent tables were established, and chi-square and Fisher's exact test were performed for each 5% threshold . RESULTS: Median followup was 51.3 months (range 25 to 144) . Of the 25 patients 8 (32%) did not respond to BCG therapy and 17 (68%) did . Immunomarkings were not statistically different between BCG responsive and nonresponsive patients for 0, 5, 10, 20, 35, 40, 45, 55 and 65 thresholds . Chi-square and Fisher's exact test were 0.91 and 0.83, 0.40 and 0.20, 0.58 and 0.29, 0.96 and 0.81, 0.80 and 0.88, 0.67 and 0.73, 0.91 and 0.83, 0.80 and 0.38, 0.69 and 0.32, respectively . CONCLUSIONS: Our results indicate that the percentage of p53 immunomarked cell cannot currently be used to predict clinical response to BCG therapy and, therefore, p53 over expression is not a viable indicator of T1G3 recurrence when using this treatment. J Can Dent Assoc, 1998 Jan, 64(1), 42 - 7 Elimination of microorganisms from dental operatory compressed air; De Ciccio A et al.; Compressed air is used to power high-speed handpieces, as well as to dry and clean surfaces in the oral cavity during patient treatment, in all dental operatories . The compressed air used in the dental operatories located in large institutions such as universities or hospitals is generally obtained from a central source, and is produced by continually running compressors . In operatories located in private practice settings, compressed air is obtained from small on-site air compressors, which may be run less frequently . A survey was made of operatories in the Montreal area to determine the microbial load of the compressed air produced by air compressors . An air sampler was used to collect compressed air and impinge it on a rotating agar medium surface . Compared to the air produced from compressors in constant use, the air collected from compressors that ran intermittently had a very high microbial load . The efficacy of an apparatus designed to sterilize the contaminated air produced by small, on-site compressors was tested . Called a Purilair, this device heats every particle of inflowing compressed air to 250 degrees C and then forces it through a fine-pore ceramic filter . In three private practice operatories, an in-line Purilair effectively sterilized the air being delivered by small compressors . The same result was obtained in the laboratory when lyophilized spores and cells of Bacillus stearothermophilus and conidia of Penicillium notatum and Aspergillus niger were sprayed into the intake line of the apparatus. Appl Environ Microbiol, 1997 Dec, 63(12), 4948 - 51 Comparative study of the toxic actions of 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane and 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene on the growth and respiratory activity of a microorganism used as a model; Donato MM et al.; A strain of Bacillus stearothermophilus was used as a model for a comparative study of the toxic effect of 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane and 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene . Bacterial growth, the O2 consumption rate, and respiration-related enzymatic activities provided quantitative data in agreement with results reported for other systems . The use of this bacterium for screening for chemical toxicity is discussed. J Cell Sci, 1997 Dec, 110 ( Pt 24), 3099 - 104 The Bacillus thuringiensis Cry1Ac toxin-induced permeability change in Manduca sexta midgut brush border membrane vesicles proceeds by more than one mechanism; Carroll J et al.; Aminopeptidase N purified from whole Manduca sexta midgut binds the Cry1Ac insecticidal toxin from Bacillus thuringiensis and this binding is inhibited by N-acetylgalactosamine (GalNAc) . We have examined the membrane permeabilising activity of the Cry1Ac toxin using brush border membrane vesicles (BBMV) prepared from the anterior (A-BBMV) and posterior (P-BBMV) subregions of the M . sexta midgut . A toxin mixing assay demonstrated a faster rate of toxin activity on P-BBMV than on A-BBMV . In the presence of GalNAc this rapid activity on P-BBMV was reduced to the rate seen with A-BBMV . GalNAc had no effect on the rate of A-BBMV permeabilisation by Cry1Ac . Aminopeptidase N assays of A- and P-BBMV demonstrated that this Cry1Ac binding protein is concentrated in the posterior midgut region of M . sexta . It therefore appears that there are two mechanisms by which Cry1Ac permeabilises the M . sexta midgut membrane: a GalNAc-sensitive mechanism restricted to the posterior midgut region, probably involving aminopeptidase N binding, and a previously undetected mechanism found in both the posterior and anterior regions. J Immunol, 1998 Feb 15, 160(4), 1796 - 803 Effects of aminoguanidine on latent murine tuberculosis; Flynn JL et al.; A unique feature of Mycobacterium tuberculosis is its ability to establish latent infection in the human host, which can reactivate to cause disease years later . In the present study, the mechanisms involved in the control of latent tuberculous infection were examined using two murine experimental tuberculosis models . Analysis of the model involving infection of mice with a relatively low inoculum of the virulent Erdman strain of M . tuberculosis indicated that in vivo inhibition of reactive nitrogen intermediate (RNI) production by the nitric oxide synthase inhibitor aminoguanidine resulted in reactivation . This reactivation was evidenced by hepatosplenomegaly, a robust tissue granulomatous reaction, and increased bacillary load . IFN-gamma, TNF-alpha, and inducible nitric oxide synthase were all expressed throughout the latent phase of infection . Reactivation of latent tuberculous infection by aminoguanidine treatment was confirmed using a second murine tuberculosis model based on treatment with antimycobacterial drugs . Results obtained using this drug-based model also suggested the existence of an RNI-independent antimycobacterial mechanism(s) operative in the latent phase of infection . Together, these data suggest that both RNI-dependent and -independent mechanisms contribute to the prevention of tuberculous reactivation. Acta Microbiol Immunol Hung, 1997, 44(3), 281 - 9 Thermostable, high salt-tolerant amylase from Bacillus megaterium VUMB-109; Jana M et al.; A bacterial strain, Bacillus megaterium VUMB-109, has been isolated and identified which produces salt-tolerant, thermostable amylase (16 U/ml culture filtrate) . Cultural conditions such as carbon and nitrogen sources, metal ions, temperature and pH have been optimized for enzyme production . The partially purified enzyme was active over a wide range of pH (4.5-10) and exhibited maximum activity at 95 degrees C, retaining 90% original activity at 100 degrees C . Partially purified enzyme was stable at 70 degrees C for 60 min . The enzyme was stable in NaCl up to 5m over 24 h without losing its original activity. J Mol Biol, 1998 Jan 23, 275(3), 491 - 502 Crystal structure of beta-glucosidase A from Bacillus polymyxa: insights into the catalytic activity in family 1 glycosyl hydrolases; Sanz-Aparicio J et al.; Family 1 glycosyl hydrolases are a very relevant group of enzymes because of the diversity of biological roles in which they are involved, and their generalized occurrence in all sorts of living organisms . The biological plasticity of these enzymes is a consequence of the variety of beta-glycosidic substrates that they can hydrolyze: disaccharides such as cellobiose and lactose, phosphorylated disaccharides, cyanogenic glycosides, etc . The crystal structure of BglA, a member of the family, has been determined in the native state and complexed with gluconate ligand, at 2.4 A and 2.3 A resolution, respectively . The subunits of the octameric enzyme display the (alpha/beta)8 barrel structural fold previously reported for other family 1 enzymes . However, significant structural differences have been encountered in the loops surrounding the active-center cavity . These differences make a wide and extended cavity in BglA, which seems to be able to accommodate substrates longer than cellobiose, its natural substrate . Furthermore, a third sub-site is encountered, which might have some connection with the transglycosylating activity associated to this enzyme and its certain activity against beta-1,4 oligosaccharides composed of more than two units of glucose . The particular geometry of the cavity which contains the active center of BglA must therefore account for both, hydrolytic and transglycosylating activities . A potent and well known inhibitor of different glycosidases, D-glucono-1,5-lactone, was used in an attempt to define interactions of the substrate with specific protein residues . Although the lactone has transformed into gluconate under crystallizing conditions, the open species still binds the enzyme, the conformation of its chain mimicking the true inhibitor . From the analysis of the enzyme-ligand hydrogen bonding interactions, a detailed picture of the active center can be drawn, for a family 1 enzyme . In this way, Gln20, His121, Tyr296, Glu405 and Trp406 are identified as determinant residues in the recognition of the substrate . In particular, two bidentate hydrogen bonds made by Gln20 and Glu405, could conform the structural explanation for the ability of most members of the family for displaying both, glucosidase and galactosidase activity. J Mol Biol, 1998 Jan 23, 275(3), 453 - 64 Protein and Mg(2+)-induced conformational changes in the S15 binding site of 16 S ribosomal RNA; Orr JW et al.; The Bacillus stearothermophilus ribosomal protein S15 binds to the central domain of the 16 S rRNA inducing a conformational change in a three-way helical junction . To understand the nature of this conformational change, extended-helical junctions were prepared to examine the effects of S15 or Mg2+ binding on the relative helical orientation using native gel electrophoretic mobility and transient electric birefringence . The free junction is planar with approximately 120 degrees interhelical angles, whereas S15 and Mg2+ yield a junction conformation that remains planar in which two helices, 21 and 22, become colinear and the third, helix 20, forms a 60 degrees angle with respect to helix 22 . This conformational change is thought to be important for directing the assembly of the central domain of the 30 S ribosomal subunit. J Clin Microbiol, 1998 Feb, 36(2), 467 - 9 Value of examining multiple sputum specimens in the diagnosis of pulmonary tuberculosis; Nelson SM et al.; To objectively assess the value of examining multiple sputum specimens in maximizing the sensitivity of detection of Mycobacterium tuberculosis, we retrospectively reviewed the acid-fast bacillus smear and culture results of patients diagnosed with culture-proven pulmonary tuberculosis (TB) at Hennepin County Medical Center between 1986 and 1996 . Two hundred and forty six persons were diagnosed with pulmonary TB in the time period analyzed . In 93% of these cases (229 of 246) the laboratory diagnosis was made by detection of M . tuberculosis in sputum specimens; however, only 52% (120 of 229) of these patients had at least three sputum specimens submitted to the laboratory at the time of diagnosis . Of the patients from whom at least three specimens were collected, 47% (56 of 120) had at least one smear-positive specimen; the third or later specimen submitted was the first smear-positive specimen for 13% (7 of 56) of these persons but was the first culture-positive specimen for only 7% (4 of 56) . Of the 64 patients with smear-negative specimens, for only 5% (3 of 64) was the third or subsequent specimen submitted the first from which M . tuberculosis was recovered . This data indicates that, in our institution, the overwhelming majority of culture-proven pulmonary TB cases are diagnosed from the first or second sputum specimen submitted to the laboratory and that only rarely is a third specimen of diagnostic value. Zentralbl Veterinarmed A, 1997 Dec, 44(9-10), 551 - 8 Diagnostic studies of abortion in Danish dairy herds; Agerholm JS et al.; Diagnostic findings in 218 aborted bovine foetuses are reported . The materials were examined in a matched case-control study of 69 Danish dairy herds with a sudden increase in the number of abortions and a corresponding 69 control herds . Foetuses aborted during the subsequent 6-month period were examined to identify the cause of abortion if possible . A total of 186 specimens were submitted from case herds and 32 from control herds . A likely cause of abortion was diagnosed in 73 foetuses . The most common cause was bovine viral diarrhoea virus (BVDV: 13%) followed by Neospora caninum infection (10%), mycosis (5%) and Bacillus licheniformis infection (4%) . Foetal and/or placental lesions were found in a further 27 cases . Only BVDV infection and neosporosis were diagnosed in more than one foetus per herd and only protozoal associated abortions occurred significantly more frequently in the case, rather than in the control, herds. Appl Environ Microbiol, 1998 Feb, 64(2), 756 - 9 Functional complementation of nontoxic mutant binary toxins of Bacillus sphaericus 1593M generated by site-directed mutagenesis; Shanmugavelu M et al.; Alanine residues were substituted by site-directed mutagenesis at selected sites of the N- and C-terminal regions of the binary toxin (51- and 42-kDa peptides) of B . sphaericus 1593M, and the mutant toxins were cloned and expressed in Escherichia coli . Bioassays with mosquito larvae, using binary toxins derived from individual mutants, showed that the substitution of alanine at some sites in both the 51-kDa and the 42-kDa peptides resulted in a total loss of activity . Surprisingly, after mixing two nontoxic derivatives of the same peptide, i.e., one mutated at the N-terminal end and the other mutated at the C-terminal end of either the 51-kDa or the 42-kDa peptide, the toxicity was restored . This result indicates that the altered binary toxins can functionally complement each other by forming oligomers. Trans R Soc Trop Med Hyg, 1997 Sep-Oct, 91(5), 544 - 6 An outbreak of bartonellosis in Zamora Chinchipe Province in Ecuador; Cooper P et al.; We report an outbreak of human bartonellosis in Zamora Chinchipe Province in Ecuador, which occurred in 1995-1996 . Nineteen cases were seen, of which 18 presented with classical oroya fever (fever and profound anaemia) and one with verruga peruana; 11 of the cases (58%) had positive blood films containing Bartonella bacilliformis . The houses of cases and neighbouring controls were visited; blood samples for thin films and cultures were collected from members of each house and a questionnaire was administered to investigate possible risk factors for disease transmission . In none of those sampled was B . bacilliformis bacteriologically demonstrable . All case houses were located in isolated areas at the margin of forest and the presence of dead rodents was reported only in case houses (P < 0.05) . We suggest that human bartonellosis is a zoonosis with a natural rodent reservoir and that migrant humans infected in this way may become a temporary reservoir host in populated areas. Antibiot Khimioter, 1997, 42(11), 15 - 21 {Actinomycin D influence on biosynthesis of extracellular ribonucleases by sporulating bacteria}; Gabdarakhmanova LA et al.; The influence of actinomycin D on the synthesis of extracellular ribonucleases by "Bacillus intermedius" (binase), B.pumilus (RNAse Bp) and B.amyloliquefaciens (barnase) was studied comparatively . When added during the active synthesis of the enzymes actinomycin D stimulated the biosynthesis of binase and RNAse Bp and had no influence on the barnase biosynthesis . The response of the bacillary RNAse biosynthesis to the added actinomycin D correlated with the differences in the nucleotide sequences of the genes encoding the enzymes . The Escherichia coli SURE recombinant strains carrying the plasmids with the genes of binase, RNAse Bp and barnase under different regulatory sequences, as well as the E.coli MC4100 recombinant strains carrying the plasmids with the beta-galactosidase gene under the promoters of the bacillary RNAse were isolated . However, the expression of the bacillary ribonuclease genes in the E.coli recombinant strains carrying the plasmids with the genes of the enzymes, as well as the expression of the beta-galactosidase gene from the promotors of the bacillary RNAses was not stimulated by actinomycin D irrespective of the dose and addition time. Biochim Biophys Acta, 1998 Jan 5, 1368(1), 27 - 34 Video imaging analysis of the plasma membrane permeabilizing effects of Bacillus thuringiensis insecticidal toxins in Sf9 cells; Villalon M et al.; The size and ionic selectivity of the pores formed by the insecticidal crystal protein Cry1C from Bacillus thuringiensis in the plasma membrane of Sf9 cells, an established cell line derived from the fall armyworm Spodoptera frugiperda, were analyzed with a video imaging technique . Changes in the permeability of the membrane were estimated from the rate of osmotic swelling of the cells . In the presence of Cry1C, which is toxic to Sf9 cells, the permeability of the cell membrane to KCl and glucose increased in a dose-dependent manner . In contrast, Cry1Aa, Cry1Ab and Cry1Ac, toxins to which Sf9 cells are not susceptible, had no detectable effect . Pores formed by Cry1C allowed the diffusion of sucrose, but were impermeable to the trisaccharide raffinose . On the basis of the hydrodynamic radii of these substances, the diameter of the pores was estimated to be 1.0-1.2 nm . In the presence of salts, the rate of swelling of cells exposed to Cry1C was about equally influenced by the size of the anion as by that of the cation, indicating that the ionic selectivity of the pores is low. Int J Tuberc Lung Dis, 1997 Aug, 1(4), 377 - 83 Intraduodenal vaccination of brushtail possums with bacille Calmette-Guérin enhances immune responses and protection against Mycobacterium bovis infection; Buddle BM et al.; SETTING: An effective oral bacille Calmette-Guerin (BCG) vaccine would have advantages for use in humans and as an oral bait vaccine for protecting wild-life against bovine tuberculosis . OBJECTIVE: To compare the level of protection against tuberculosis in intraduodenally BCG-vaccinated possums with those vaccinated intragastrically in order to determine whether degradation of BCG in the stomach lowers vaccine efficacy . DESIGN: Three groups of five possums were vaccinated with BCG by the intraduodenal, intragastric or subcutaneous routes, with a fourth group serving as unvaccinated controls . The animals were later challenged intratracheally with a low dose of virulent Mycobacterium bovis . RESULTS: Possums vaccinated intraduodenally with BCG had significantly greater lymphocyte blastogenic responses to bovine purified protein derivative (PPD) and lower lung bacterial counts in comparison with intragastrically vaccinated animals . In comparison with unvaccinated animals, all of the BCG-vaccinated groups had significant protection against M . bovis infection as assessed by changes in body weight, lung weight and reduction in numbers of mycobacteria and granulomas in the spleen . CONCLUSION: The enhanced immune responses and protection against bovine tuberculosis observed in the intraduodenally BCG-vaccinated possums indicated that if BCG vaccine is protected from degradation in the stomach its efficacy should improve. Parazitologiia, 1997 Jan-Feb, 31(1), 45 - 52 {Distribution of natural parasites--fungi and microsporidia--in a population of malarial mosquito larva (Diptera: Culicidae) before and after infection by the entomopathogenic bacteria Bacillus thuringiensis israelensis}; Burlak VA et al.; The distribution specificity of fungi and microsporidies in natural population of Anopheles messeae Fall . and A . beklemishevi Stegny et Kab . and those which survived after treatment them by Bacillus thuringiensis israelensis (Bti) were observed . Parasitic fungus nonselectively affected individuals of both species and all inversion genotypes of A . messeae . Microsporidia Parathelohania messeae affected males and it has not species and genotypic specificity . The 4-th instar larvae of both species infected by parasitic fungus after treatment them by Bti did not survive . The level of microsporidian infection of A . messeae and A . beklemishevi after Bti treatment was reduced from 1.1 +/- 0.5 to 0.5 +/- 0.3% and from 1.3 +/- 1.3 to 0.7 +/- 0.5% accordingly . A . beklemishevi was registered as Parathelohania messeae host for the first time . The harmonious relationships between malaria mosquitoes and their parasites in natural populations may be destructed by the Bti treatments. J Clin Invest, 1997 Dec 1, 100(11), 2658 - 64 Partial interferon-gamma receptor 1 deficiency in a child with tuberculoid bacillus Calmette-Guérin infection and a sibling with clinical tuberculosis; Jouanguy E et al.; Complete interferon-gamma receptor 1 (IFNgammaR1) deficiency has been identified previously as a cause of fatal bacillus Calmette-Guerin (BCG) infection with lepromatoid granulomas, and of disseminated nontuberculous mycobacterial (NTM) infection in children who had not been inoculated with BCG . We report here a kindred with partial IFNgammaR1 deficiency: one child afflicted by disseminated BCG infection with tuberculoid granulomas, and a sibling, who had not been inoculated previously with BCG, with clinical tuberculosis . Both responded to antimicrobials and are currently well without prophylactic therapy . Impaired response to IFN-gamma was documented in B cells by signal transducer and activator of transcription 1 nuclear translocation, in fibroblasts by cell surface HLA class II induction, and in monocytes by cell surface CD64 induction and TNF-alpha secretion . Whereas cells from healthy children responded to even low IFN-gamma concentrations (10 IU/ml), and cells from a child with complete IFNgammaR1 deficiency did not respond to even high IFN-gamma concentrations (10,000 IU/ml), cells from the two siblings did not respond to low or intermediate concentrations, yet responded to high IFN-gamma concentrations . A homozygous missense IFNgR1 mutation was identified, and its pathogenic role was ascertained by molecular complementation . Thus, whereas complete IFNgammaR1 deficiency in previously identified kindreds caused fatal lepromatoid BCG infection and disseminated NTM infection, partial IFNgammaR1 deficiency in this kindred caused curable tuberculoid BCG infection and clinical tuberculosis. J Am Mosq Control Assoc, 1997 Sep, 13(3), 263 - 9 Control of Culex quinquefasciatus (Diptera: Culicidae) with Bacillus sphaericus in Maroua, Cameroon; Barbazan P et al.; Two strategies were tested to control Culex quinquefasciatus with Bacillus sphaericus in Maroua (population 130,000), Cameroon . The treatment of all potential breeding sites (27,000) with B . sphaericus during the dry season caused up to a 90% reduction in the adult biting rate . Because of the short persistence of B . sphaericus and the occurrence of new breeding sites, unacceptable levels of adult biting rates were reached again in 5 months . In the second strategy, two treatments per year of the most productive breeding sites (10,000) stopped the biting rate increase during the rainy season . The results were only partially successful because of variations in B . sphaericus toxicity . The first treatment required 1,200 man-days of work vs . 200 for the simplified treatments . The density of breeding sites depends on the rainfall and the presence of a tap-water network . A sustained control program of Cx . quinquefasciatus will depend upon the dynamics of the principal breeding sites and an improved formulation of B . sphaericus. J Am Mosq Control Assoc, 1997 Sep, 13(3), 218 - 26 International indoor and outdoor evaluation of Bacillus sphaericus products: complexity of standardizing outdoor protocols; Thiery I et al.; Only one Bacillus sphaericus strain, strain 2362, is currently used commercially to control Culex larval populations . A reliable methodology, easily used, was developed to identify new strains for field application . Larvicidal activities of 3 highly mosquitocidal strains, strains C3-41, Mal, and LB24, previously selected in the laboratory, were compared with that of strain 2362 in tropical and European countries . The following steps were performed: production and titration of acetonic powders from these 4 strains on local Culex species, survey of initial and residual activity under standardized indoor and outdoor conditions, and evaluation of the efficacy of liquid formulations of the 4 strains in natural breeding sites of Culex . In indoor conditions, strain C3-41 showed the highest activity on both Culex pipiens and Culex quinquefasciatus; strain Mal was the least active . The residual activity causing 80% mortality differed from 20 to 90 days according to the strains and the country . Outdoor experiments with powders (0.02-1.6 mg/liter) were performed and the initial toxicities were similar in all cases . Residual activities were very different, from 6 to 95 days posttreatment . Liquid formulations were applied to larval habitats (from 0.1 to 10 g/m2) . In tropical countries, larval recolonization in cesspits or ponds occurred after 10-35 days . In Europe, higher doses were needed in polluted water than in clear water (from 3 to 10 liter/ha) for the same control, and the time before 80% residual activity was reached was less than 9-12 days . However, in cesspits, residual activity could be observed for 12 days to 5 mo . A strain 3-5 times more active than the others in bioassays is not significantly detectable from those strains in field trials. Ned Tijdschr Geneeskd, 1997 Jun 14, 141(24), 1199 - 203 {The history of healing; beriberi: 'kind of paralysis'}; de Knecht-van Eekelen A; Beriberi began to attract increasing attention in the second half of the 19th century, mostly due to the large numbers of victims among Dutch military personnel in the Netherlands East Indies . A study, carried out in Atjeh in 1886 by the Japanese Sugenoya and F . J . Cornelissen, led to the conclusion that there existed a beriberi bacillus which they could make visible in stained microscopical prepar