Bacillus

Electrophoresis, 1998 Jun, 19(8-9), 1325 - 31
Natural resistance to intracellular parasites: a study by two-dimensional gel electrophoresis coupled with multivariate analysis; Kovarova H et al.; Natural resistance to Mycobacterium bovis bacillus Calmette-Guerin (BCG) is determined by the Bcg gene (Nramp1), which is exclusively expressed by mature macrophages . The Nramp1 gene is a dominant autosomal gene that has two allelic forms; r confers resistance and s confers susceptibility to infection with intracellular pathogen . Although the wide range of pleiotropic immunological effects of the Nramp1 gene has been described, the exact mechanism of its action remains elusive . In this study we searched for differentially expressed proteins that might provide clues in the studies on Nramp1 gene function . We performed two-dimensional gel electrophoresis of cellular proteins prepared from a B10R macrophage line derived from mice carrying the r allele of the Nramp1 gene, B10S macrophages carrying the s allele, and B10R-Rb macrophages transfected with Nramp1-ribozyme . The classification of protein patterns and selection of distinct proteins characteristic of r or s allele-carrying macrophages was performed using the principal component analysis . We found differential expression of four proteins with the following isoelectric point/molecular weight (pI/Mr) in B10R macrophages compared to B10S and B10R-Rb macrophages: 6.6/25, 7.0/22, 9.1/31.5, and 5.3/8.5 . The protein 7.0/22 has been identified as Mn-superoxide dismutase and the best candidate for protein p6.6/25 seems to be Bcl-2 according to the immunoblot analysis . When the splenic macrophages carrying the r or s allele were analyzed, the changes in relative abundance for proteins 6.6/25 and p7.0/22 were satisfactorily reproduced . Overall, the two identified proteins are important in the regulation of intracellular redox balance and the regulation of apoptosis in macrophages, respectively . Our findings may suggest their possible biological role in the innate immunity against intracellular pathogens.

Int J Biochem Cell Biol, 1998 May, 30(5), 579 - 95
Purification and partial characterization of a neutral protease from a virulent strain of Bacillus cereus; Sierecka JK; The factors involved in the pathogenesis of Bacillus cereus (B . cereus) in non-gastrointestinal diseases are poorly investigated . Some researchers suggest that B . cereus proteases may be involved in these illnesses . The aim of this work was to purify and characterize a protease isolated from a virulent strain of B . cereus to explain its assumptive damaging effect . The enzyme was purified in a four-step procedure involving ammonium sulfate fractionation, acetone precipitation, Bio-Gel filtration and column chromatography on DEAE-cellulose (DE-52 cellulose) . The enzyme appeared homogenous using disc electrophoresis . The specific activity of the protease was 72 U/mg of protein . The enzyme was shown to have a relative molecular mass of 29 kDa . The protease was most active at pH 7.0 and 40 degrees C with haemoglobin as the substrate . The enzyme was made completely inactive by ethylenediaminetetraacetic acid (EDTA), beta-mercaptoethanol, dithiothreitol (DTT) and benzamidine (at a concentration of 1 mM) and by diisopropylfluorophosphate (DIPF), L-cysteine, L-histidine, 1,10-phenanthroline (at a concentration of 10 mM) . Divalent cations, especially Ca2+ increased enzyme activity . The enzyme hydrolysed haemoglobin, albumin and casein as the substrates . With haemoglobin and albumin as the substrates Michaelis-Menten kinetics was observed . The obtained Km values were 86 +/- 40 microM (SD, n = 3) and 340 +/- 100 microM (SD, n = 3) for haemoglobin and albumin, respectively . The corresponding Vmax values were 1.26 +/- 0.1 (SD, n = 3) and 0.38 +/- 0.07 (SD, n = 3) mumol of tyrosine liberated per min, per ml, and per mg, while those for casein were not determined . It is concluded that this enzyme is a metal-chelator-sensitive, neutral protease damaging haemoglobin and albumin.

Biosci Biotechnol Biochem, 1998 Jun, 62(6), 1103 - 8
Phosphopentomutase of Bacillus stearothermophilus TH6-2: the enzyme and its gene ppm; Hamamoto T et al.; Phosphopentomutase catalyzes the transfer of an intramolecular phosphate on ribose or deoxyribose, and is involved in the salvage pathway of nucleoside synthesis . We identified a sequence 5'-upstream of the genes for the nucleoside phosphorylases of Bacillus stearothermophilus as the phosphopentomutase (ppm) gene . The novel gene corresponded to an open reading frame of 1,179 nucleotides that is translated into a putative 393-amino acid protein with a molecular weight of 43,735 . The gene product, partially purified from ppm-overexpressing Escherichia coli cells, was judged to be a monomer of a 44-kDa polypeptide . The phosphopentomutase was found to catalyze the phosphotransfer on not only ribose or deoxyribose but also arabinose or dideoxyribose.

Biosci Biotechnol Biochem, 1998 Jun, 62(6), 1093 - 102
Clustered proline residues around the active-site cleft in thermostable oligo-1,6-glucosidase of Bacillus flavocaldarius KP1228; Kashiwabara S et al.; The gene that coded for a cellular oligo-1,6-glucosidase (dextrin 6-alpha-D-glucanohydrolase, EC 3.2.1.10) in Bacillus flavocaldarius KP1228 (FERM-P9542) cells growing at 51-82 degrees C was expressed in Escherichia coli JM109 . The enzyme had a half-life of 10 min at 89.2 degrees C . Purification of the enzyme and its characterization showed that the enzyme was identical with the native one . Its primary structure of 529 residues with a molecular weight of 61,469 deduced from the gene was 40-42% identical to the sequences of less thermostable oligo-1,6-glucosidases from Bacillus cereus ATCC 7064, Bacillus coagulans ATCC 7050, and Bacillus thermoglucosidasius KP1006 . Sequence analysis showed that the B . flavocaldarius enzyme shared 14 proline residues at the same positions as in the three other enzymes, and that the B . flavocaldarius enzyme had 22 of 33 additional proline residues (cf . 1/5, 5/10, and 9/18 in the respective counterparts) in three long polypeptides constituting the active-site cleft, which connected the third, fourth, and eighth beta-strands to the corresponding third, fourth, and eighth alpha-helices in the (beta/alpha)8-barrel.

Appl Environ Microbiol, 1998 Aug, 64(8), 3036 - 41
A novel insecticidal toxin from photorhabdus luminescens, toxin complex a (Tca), and its histopathological effects on the midgut of manduca sexta
Blackburn M, Golubeva E, Bowen D, Ffrench-Constant RH.
Photorhabdus luminescens is a bacterium which is mutualistic with entomophagous nematodes and which secretes high-molecular-weight toxin complexes following its release into the insect hemocoel upon nematode invasion . Thus, unlike other protein toxins from Bacillus thuringiensis (delta-endotoxins and Vip's), P . luminescens toxin (Pht) normally acts from within the insect hemocoel . Unexpectedly, therefore, the toxin complex has both oral and injectable activities against a wide range of insects . We have recently fractionated the protein toxin and shown it to consist of several native complexes, the most abundant of which we have termed Toxin complex a (Tca) . This complex is highly active against the lepidopteran Manduca sexta . In view of the difference in the normal mode of delivery of P . luminescens toxin and the apparent communality in the histopathological effects of other gut-active toxins from B . thuringiensis, as well as cholesterol oxidase, we were interested in investigating the effects of purified Tca protein on larvae of M . sexta . Here we report that the histopathology of the M . sexta midgut is similar to that for other novel midgut-active toxins . Following oral ingestion of Tca by M . sexta, we observed an acceleration in the blebbing of the midgut epithelium into the gut lumen and eventual lysis of the epithelium . The midgut shows a similar histopathology following injection of Tca into the insect hemocoel . These results not only show that Tca is a highly active oral insecticide but also confirm the similar histopathologies of a range of very different gut-active toxins, despite presumed differences in modes of action and/or delivery . The implications for the mode of action of Tca are discussed.

Curr Microbiol, 1998 Sep, 37(3), 195 - 200
Distribution, serological identification, and PCR analysis of Bacillus thuringiensis isolated from soils of Korea; Kim HS et al.; A total, 58 strains of Bacillus thuringiensis were isolated from soils of various regions in Korea . Serological tests showed that B . thuringiensis isolates represented 10 H serotypes, indicating a varied flora of B . thuringiensis . But the H serotypes did not have a significantly uneven distribution, ranging from 1 to 11 isolates . In toxicity tests, 35% of all isolates were toxic to lepidoptera, 20% were toxic to diptera, and 9% were non-toxic isolates . Especially, a large number of lepidopteran/dipteran-active isolates (36%) were found . Forty all lepidopteran-active isolates produced typical rhomboidial inclusions, and the remainder, which belong to dipteran-active and non-toxic isolates, were spherical in shape . In addition, lepidopteran/dipteran-active isolates produced rhomboidal or spherical inclusions . PCR analysis using cryI, II, III, IV, and V gene-specific primers showed that the frequency of the cryIC gene (57%) predominated, followed by the cryIA(b) (45%) and cryIIA genes (34%) . But, the cryIE, cryIF, cryIII, cryIVC and cryV genes were not reactive . Several isolates had unusual PCR products and multiple insecticidal crystal protein genes . PCR results showed varied distribution of the cry-type gene . Seven isolates were selected for evaluation of novel activity according to the following criteria: flagellar serotypes, parasporal inclusion morphology, SDS-PAGE, plasmid DNA patterns, toxicity, and the cry-type gene in PCR analysis . Two isolates, named S333 (H7) and S225 (H7), among them synthesized PCR products of the cryIC gene, but the S333 isolate producing rhomboidal inclusion was toxic to both Plutella xylostella and Culex pipiens, whereas the S225 isolate having toxicity to only C . pipiens produced spherical inclusion.

Appl Environ Microbiol, 1998 Aug, 64(8), 2995 - 3003
Phage display of a biologically active Bacillus thuringiensis toxin; Kasman LM et al.; Activated forms of Bacillus thuringiensis insecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in Escherichia coli . Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in E . coli, since the molecular biology tools available for Bacillus are limited . To this end, we show that activated B . thuringiensis toxin (Cry1Ac) can be expressed in E . coli as a translational fusion with the minor phage coat protein of filamentous phage . Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac . Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage . Phage display of B . thuringiensis toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning.

Antimicrob Agents Chemother, 1998 Aug, 42(8), 2055 - 9
vanA gene cluster in a vancomycin-resistant clinical isolate of Bacillus circulans; Ligozzi M et al.; We report on the cloning and sequencing of the vanA gene cluster present in the glycopeptide-resistant clinical isolate Bacillus circulans VR0709 (R . Fontana, M . Ligozzi, C . Pedrotti, E . M . Padovani, and G . Cornaglia, Eur . J . Clin . Microbiol . Infect . Dis . 16:473-474, 1997) . The presence of a vanA-related gene in VR0709 was demonstrated in a PCR assay which permitted the specific amplification of an internal segment of vanA . Southern blotting suggested that the vanA gene was located in the chromosome in a 7 . 6-kb EcoRI fragment . DNA sequence analysis revealed the presence of all seven genes of the vanA cluster (vanR, vanS, vanH, vanA, vanX, vanY, and vanZ) . The degree of identity between homologous proteins encoded by Tn1546 and the chromosome of B . circulans VR0709 ranged from 87 to 95% . Neither PCR nor Southern blotting with specific primers and probes, respectively, showed the presence of open reading frames (ORFs) 1 and 2 which encode the transposase and the resolvase of Tn1546, respectively, the transposon found to carry the vanA gene cluster in enterococci . Determination of the sequences of the flanking regions of the van gene cluster of B . circulans revealed perfect inverted repeats of 10 bp which delineated a 9.2-kb region containing the van gene cluster and an ORF which encoded a putative protein (178 residues) which displayed a low level of identity (28%) to the resolvase of Tn1546 . These results suggest that glycopeptide resistance in B . circulans VR0709 is associated with the acquisition of a vanA gene cluster which shows a high degree of homology with that of enterococci . In B . circulans, however, the cluster is not carried by Tn1546 and is borne by the chromosome.

Acta Paediatr, 1998 Jun, 87(6), 702 - 4
Disseminated Bacillus Calmette-Guérin infection in a girl with hyperimmunoglobulin E syndrome; Pasic S et al.; Disseminated Bacillus Calmette-Guerin infection occurs in few well-defined immunodeficiencies, such as severe combined immunodeficiency, chronic granulomatous disease and paediatric acquired immunodeficiency syndrome . This severe complication of immunization against tuberculosis has been lethal in the majority of children who had primary immunodeficiency . Our patient, a 9-y-old girl with hyperimmunoglobulin E syndrome developed disseminated Bacillus Calmette-Guerin infection in infancy . Patients with hyperimmunoglobulin E syndrome (HIES) are susceptible to serious staphylococcal and fungal infections . Disseminated Bacillus Calmette-Guerin infection has not previously been reported in this rare immunodeficiency.

J Am Dent Assoc, 1998 Jul, 129(7), 985 - 91
How well does the Chemiclave sterilize handpieces?
Kolstad RA.
Using the Food and Drug Administration's protocol for testing health care sterilizers, the author investigated the ability of chemical vapor and steam to sterilize handpieces . Five internal sites of six high-speed handpiece models and four internal positions of one low-speed handpiece model were each inoculated with 10(6) Bacillus stearothermophilus spores . Half-cycle challenges were conducted with Chemiclave models EC 5500 and 8000 (Barnstead/Thermolyne) and with two autoclaves, Tuttnauer 2540M (large chamber) (Tuttnauer USA Co., Ltd.) and Statim Cassette (SciCan USA) . Experiments with spores either openly exposed or partially enclosed prove that chemical vapor is an excellent lethal agent, but strongly suggest penetration weakness {corrected}.

FEMS Immunol Med Microbiol, 1998 Jun, 21(2), 117 - 22
Effect of ribonuclease from Bacillus intermedius on human blood lymphocytes; Kurinenko BM et al.; By using a rosette formation test the effect of ribonuclease Bacillus intermedius (RNase Bi) on T- and B-lymphocytes in human peripheral blood has been studied in vitro . The RNase effect on T-lymphocytes depends on its concentration: low concentrations (10(-6)-10(-2) microg ml(-1)) stimulate E-rosette formation whereas high concentrations (10 microg ml(-1)) suppress it . The amount of B-lymphocytes decreases under RNase Bi influence in all concentrations tested . RNase Bi like thymus hormones influence immature lymphocytes (0-cells) by inducing the surface expression of E-receptors what leads to rosette formation and, thus, contributes to lymphocyte differentiation . The increase in the amount of active T-cells which represent the mature cell population also confirms the participation of RNase Bi in T-rank lymphocytes differentiation processes . The RNase Bi effect on the human lymphocytes depends on its catalytic activity.

Protein Sci, 1998 Jul, 7(7), 1538 - 44
Scan-rate dependence in protein calorimetry: the reversible transitions of Bacillus circulans xylanase and a disulfide-bridge mutant; Davoodi J et al.; The stabilities of Bacillus circulans xylanase and a disulfide-bridge-containing mutant (S100C/N148C) were investigated by differential scanning calorimetry (DSC) and thermal inactivation kinetics . The thermal denaturation of both proteins was found to be irreversible, and the apparent transition temperatures showed a considerable dependence upon scanning rate . In the presence of low (nondenaturing) concentrations of urea, calorimetric transitions were observed for both proteins in the second heating cycle, indicating reversible denaturation occurs under those conditions . However, even for these reversible processes, the DSC curves for the wild-type protein showed a scan-rate dependence that was similar to that in the absence of urea . Calorimetric thermograms for the disulfide mutant were significantly less scan-rate dependent in the presence of urea than in the urea-free buffer . The present data show that, just as for irreversible transitions, the apparent transition temperature for the reversible denaturation of proteins can be scan-rate dependent, confirming the prediction of Lepock et al . (Lepock JR, Rithcie KP, Kolios MC, Rodahl AM, Heinz KA, Kruuf J, 1992, Biochemistry 31:12706-12712) . The kinetic factors responsible for scan-rate dependence may lead to significant distortions and asymmetry of endotherms, especially at higher scanning rates . This points to the need to check for scan-rate dependence, even in the case of reversible denaturation, before any attempt is made to analyze asymmetric DSC curves by standard thermodynamic procedures . Experiments with the disulfide-bridge-containing mutant indicate that the introduction of the disulfide bond provides additional stabilization of xylanase by changing the rate-limiting step on the thermal denaturation pathway.

Appl Microbiol Biotechnol, 1998 Jun, 49(6), 737 - 42
Trans activation of the Escherichia coli ato structural genes by a regulatory protein from Bacillus megaterium: potential use in polyhydroxyalkanoate production
Pettinari MJ, Vazquez GJ, Kruger N, Vary PS, Steinbuchel A, Mendez BS.
A Bacillus megaterium genomic fragment, which encoded an activator homologous to sigma 54 regulators and which was capable of activating Escherichia coli ato genes in trans, was detected in a gene library of B . megaterium screened for beta-ketothiolase activity . The fragment presented only one complete open reading frame (ORF1), which encoded a protein of 398 amino acids . The recombinant plasmid complemented mutations in the Escherichia coli atoC regulatory gene . The constitutive expression of the E . coli ato operon mediated by ORF1 could be useful for the synthesis of polyhydroxyalkanoates with different flexibility properties by recombinant E . coli strains.

Vaccine, 1998 Jul, 16(11-12), 1166 - 71
Studies of vaccination of persons in close contact with leprosy patients in Argentina; Bottasso O et al.; A total of 670 adults living or working with leprosy patients, were examined for a BCG vaccination scar, and skin-tested with four new tuberculins . Based on the results 513 were vaccinated, 65 with Bacille de Calmette et Guerin (BCG) alone, 66 with BCG plus killed Mycobacterium vaccae and 382 with killed M . vaccae alone . Skin-testing was repeated 2-3 years later on 344 subjects, when all three vaccines were found to have been highly successful in increasing responses to Tuberculin and Leprosin A (p < 0.0005) with increased immune recognition of common and species-specific antigens . Mean diameters of induration to each skin-test were greatest in recipients of BCG alone (p < 0.05), which suggests that better immuno-regulation occurs after receiving vaccines that incorporate M . vaccae . The results suggest 10(8) M . vaccae alone might prove a valuable future vaccine, which would not require selective pre-vaccination procedures.

Eur Cytokine Netw, 1998 Jun, 9(2), 181 - 6
BCG-induced interleukin-6 upregulation and BCG internalization in well and poorly differentiated human bladder cancer cell lines; Bevers RF et al.; Intravesical bacillus Calmette-Guerin (BCG) is a successful therapy for superficial bladder cancer . However, the working mechanism of BCG after intravesical instillation is not completely understood . A functional role of urothelial (tumor) cells in the initiation of the BCG-induced immune reaction should be considered . Here, the possibility of a causal relationship between BCG-induced interleukin-6 (IL-6) synthesis and BCG internalization by urothelial tumor cells was examined in a series of human transitional bladder cancer (TCC) cell lines with different degrees of differentiation . The results showed that the well differentiated TCC cell lines, RT4, SBC-2, and SBC-7, did not possess the capacity to internalize BCG, which was associated with an inability to upregulate IL-6 synthesis when stimulated with BCG . Moreover, these cell lines expressed a low level of constitutive IL-6 synthesis . In contrast, the poorly differentiated TCC cells, T-24, TCC-SUP and J-82, were able to internalize BCG . In T24 and J82, but not in TCC-SUP cells, BCG internalization appeared to result in an upregulation of IL-6 synthesis . Constitutive IL-6 synthesis of the high grade cell lines was found to be cell line-dependent: both TCC-SUP and J82 cells exhibited a high level of constitutive IL-6 synthesis, whereas T24 cells exhibited a low level . The possible relationship between BCG internalization and IL-6 upregulation was studied in detail with the T24 cell line, which exhibited a low constitutive and high BCG-inducible IL-6 synthesis, using anti-BCG antibodies (alphaBCG) and Cytochalasin B as internalization inhibitors . Upregulation of IL-6 synthesis was significantly inhibited by alphaBCG or Cytochalasin B, indicating that internalization is a prerequisite for BCG-induced upregulation of IL-6 synthesis . In conclusion, upregulation of IL-6 production due to BCG internalization by poorly differentiated bladder carcinoma cells may be part of the mode of action of intravesical BCG therapy.

Extremophiles, 1997 Aug, 1(3), 151 - 6
Thermostable alkaline cellulase from an alkaliphilic isolate, Bacillus sp . KSM-S237; Hakamada Y et al.; Thermostable alkaline cellulase (endo-1,4-beta-glucanase, EC 3.2.1.4) activity was detected in the culture medium of a strictly alkaliphilic strain of Bacillus, designated KSM-S237 . This novel enzyme was purified to homogeneity by a two-step column-chromatographic procedure with high yield . The N-terminal amino acid sequence of the purified enzyme was Glu-Gly-Asn-Thr-Arg-Glu-Asp-Asn-Phe-Lys-His-Leu-Leu-Gly-Asn-Asp-Asn-Val- Lys-Arg . The enzyme had a molecular mass of approximately 86 kDa and an isoelectric point of pH 3.8 . The enzyme had a pH optimum of 8.6-9.0 and displayed maximum activity at 45 degrees C . The alkaline enzyme was stable up to 50 degrees C and more than 30% of the original activity was detectable after heating at 100 degrees C and at pH 9.0 for 10 min . The enzyme hydrolyzed carboxymethylcellulose, lichenan (beta-1,3;1,4-linkage), and p-nitrophenyl derivatives of cellotriose and cellotetraose . Crystalline forms of cellulose (Avicel and filter paper), H3PO4-swollen cellulose, NaOH-swollen cellulose, curdlan (beta-1,3-linkage), laminarin (beta-1,3;1,6-linkage), and xylan were barely hydrolyzed at all.

Extremophiles, 1997 Aug, 1(3), 135 - 41
K+/H+ antiporter in alkaliphilic Bacillus sp . no . 66 (JCM 9763); Kitada M et al.; A K+/H+ antiport system was detected for the first time in right-side-out membrane vesicles prepared from alkaliphilic Bacillus sp . no . 66 (JCM 9763) . An outwardly directed K+ gradient (intravesicular K+ concentration, K(in), 100 mM; extravesicular K+ concentration, K(out), 0.25 mM) stimulated uphill H+ influx into right-side-out vesicles and created the inside-acidic pH gradient (delta pH) . This H+ influx was pH-dependent and increased as the pH increased from 6.8 to 8.4 . Addition of 100 microM quinine inhibited the H+ influx by 75% . This exchange process was electroneutral, and the H+ influx was not stimulated by the imposition of the membrane potential (interior negative) . Addition of K+ at the point of maximum delta pH caused a rapid K+-dependent H+ efflux consistent with the inward exchange of external K+ for internal H+ by a K+/H+ antiporter . Rb+ and Cs+ could replace K+ but Na+ and Li+ could not . The H+ efflux rate was a hyperbolic function of K+ and increased with increasing extravesicular pH (pH(out)) from 7.5 to 8.5 . These findings were consistent with the presence of K+/H+ antiport activity in these membrane vesicles.

Extremophiles, 1997 May, 1(2), 61 - 6
Alkaline cellulases from alkaliphilic Bacillus: enzymatic properties, genetics, and application to detergents; Ito S; We have isolated a number of alkaliphilic Bacillus that produce alkaline exoenzymes and found a possible use for alkaline cellulase (carboxymethylcellulase) as an additive for improving the cleaning effect of detergents . Enzymatic properties of some candidate cellulases fulfilled the essential requirements for enzymes to be used practically in laundry detergents . Here I describe the properties and possible catalytic mechanism of the hydrolytic reaction and the gene for the industrial alkaline cellulase produced by one of the isolates, Bacillus sp . KSM-635.

Extremophiles, 1997 Nov, 1(4), 199 - 206
Isolation and characterization of bacteriophage BCJA1, a novel temperate bacteriophage active against the alkaliphilic bacterium, Bacillus clarkii; Jarrell KF et al.; The isolation and characterization of a novel bacteriophage active against the obligately alkaliphilic bacterium Bacillus clarkii is described . The bacteriophage, designated BCJA1 . is a member of the Siphoviridae family with a B1 morphology . It possesses an isometric head, which measures 65 nm between opposite apices, and a noncontractile tail of 195 nm length . It had a buoyant density of 1.518 g/ml and an estimated particle mass of 37 x 10(7) daltons . BCJA1 was stable over the pH range of 6-11 . A one-step growth experiment conducted at pH 10 demonstrated a latent period of about 40 min and a burst size of approximately 40 . The purified bacteriophage appeared to consist of 10 proteins with the major head and tail proteins likely to be of molecular weight 36500 and 28000, respectively . The genome size was estimated to be between 32.1 and 34.8 kb . The percent G + C content of purified bacteriophage DNA was 45.6 . The wildtype bacteriophage is temperate but a clear plaque mutant was isolated.

Extremophiles, 1997 Nov, 1(4), 163 - 9
Mechanisms of cytoplasmic pH regulation in alkaliphilic strains of Bacillus; Krulwich TA et al.; The central challenge for extremely alkaliphilic Bacillus species is the need to establish and sustain a cytoplasmic pH that is over two units lower than the highly alkaline medium . Its centrality is suggested by the strong correlation between the growth rate in the upper range of pH for growth, i.e., at values above pH 10.5, and the cytoplasmic pH . The diminishing growth rate at extremely high pH values correlates better with the rise in cytoplasmic pH than with other energetic parameters . There are also general adaptations of alkaliphiles that are crucial prerequisites for pH homeostasis as well as other cell functions, i.e., the reduced basic amino acid content of proteins or segments thereof that are exposed to the medium, and there are other challenges of alkaliphily that emerge from solution of the cytoplasmic pH problem, i.e., reduction of the chemiosmotic driving force . For cells growing on glucose, strong evidence exists for the importance of acidic cell wall components, teichuronic acid and teichuronopeptides, in alkaliphily . These wall macromolecules may provide a passive barrier to ion flux . For cells growing on fermentable carbon sources, this and other passive mechanisms may have a particularly substantial role, but for cells growing on both fermentable and nonfermentable substrates, an active Na+-dependent cycle is apparently required for alkaliphily and the alkaliphile's remarkable capacity for pH homeostasis . The active cycle involves primary establishment of an electrochemical gradient via proton extrusion, a secondary electrogenic Na+/H+ antiport to achieve net acidification of the cytoplasm relative to the outside pH, and mechanisms for Na+ re-entry . Recent work in several laboratories on the critical antiporters involved in this cycle has begun to clarify the number and characteristics of the porters that support active mechanisms of pH homeostasis.

J Urol, 1998 Aug, 160(2), 394 - 6
Treatment of recurrent penile condylomata acuminata with external application and intraurethral instillation of bacillus Calmette-Guerin; Bohle A et al.; PURPOSE: Condylomata acuminata are caused by human papillomavirus infection . Despite numerous treatment modalities these patients often demonstrate recurrent disease . We report initial experience with bacillus Calmette-Guerin (BCG) therapy in patients not responding to standard treatment . MATERIALS AND METHODS: Between October 1994 and March 1997, 6 men with rapidly recurrent external and intraurethral condylomata acuminata underwent BCG therapy after initial laser treatment . External application and intraurethral instillation of BCG were performed 6 times in weekly intervals . Followup studies included examination and endoscopic inspection of the urethra and bladder . RESULTS: Of the patients 3 completed 1 course of BCG and had no relapse of condylomata acuminata, 2 underwent a second course of BCG and 1 had relapse, and 1 had relapse after discontinuing therapy due to penile edema . The annual recurrence rate decreased from 3.2 before to 0.75 after BCG therapy (p < 0.05, test of equality of 2 percentages) . CONCLUSIONS: Immunotherapy with BCG is accepted treatment for superficial transitional cell carcinoma . The BCG induced immune response appears to reduce the recurrence rate in patients with condylomata acuminata.

Biochem Mol Biol Int, 1998 Jul, 45(3), 443 - 52
Production and purification of an extracellular cellulase from Bacillus brevis VS-1; Singh VK et al.; A bacteria identified as Bacillus brevis has been isolated from the soil . It has been found to secrete cellulase extracellularly whose production increased almost five times on addition of galactose in the culture medium . Production of cellulase has been found optimal at pH 5.5, 37 degrees C and 175 rpm speed using environmental orbital shaker . The cellulase has been purified using ultrafiltration and Sephadex G-200 column chromatography . The native molecular weight of the enzyme is found to be 33,000 +/- 2000 using Sephadex G-200 gel filtration chromatography . The subunit molecular weight (33,000 +/- 2,000) indicate monomeric nature of the enzyme . The enzyme showed Michaelis Menten kinetics exhibiting Km approximately 1.7 +/- 0.1 mg/ml for CMC . The enzyme activity got inhibited by heavy metals viz . Hg+2 and Ag+2.

J Biomol NMR, 1998 Feb, 11(2), 185 - 90
High-resolution detection of five frequencies in a single 3D spectrum: HNHCACO--a bidirectional coherence transfer experiment; Pang Y et al.; A new triple-resonance pulse sequence, 3D HNHCACO, is introduced and discussed, which identifies sequential correlations of the backbone nuclei (H alpha (i-1), C alpha (i-1), C(i-1), NH(i), N(i)) of doubly labeled proteins in H2O . The three-dimensional (3D) method utilizes a recording of 15N and 13C resonances in a single indirect time domain, the 13C' resonance in another indirect time domain, and detects both NH and H alpha protons . A bidirectional coherence transfer (NH(i) <--> N(i) <--> C(i-1) <--> C alpha (i-1) <--> H alpha (i-1)) is effectuated, resulting in a single high-resolution 3D spectrum that contains the frequencies of all five backbone nuclei . The experiment was applied to the 12.3 kDa ribonuclease from Bacillus intermedius (Binase).

J Food Prot, 1998 Jul, 61(7), 921 - 3
Identification of Bacillus cereus by Fourier transform infrared spectroscopy (FTIR); Lin SF et al.; The objective of this study was to evaluate the potential of Fourier transform infrared spectroscopy (FTIR) for rapid identification of Bacillus cereus isolates . Ten B . cereus group isolates (comprising B . cereus, Bacillus mycoides, and Bacillus thuringiensis strains), five other Bacillus spp., and five non-Bacillus spp . were used . Two types of media, brain heart infusion (BHI) and Trypticase soy agar (TSA), were tested . The results indicated that all B . cereus group isolates produced characteristic absorbance peaks at wave numbers between 1738 and 1740 cm-1 . These peaks were not affected by the growth medium . None of the other bacteria tested showed a similar peak after growth on BHI or TSA . Absorbance peaks between 1800 and 1500 cm-1 of members of the B . cereus group had different shapes and sizes, suggesting that FTIR may be useful for rapid identification of species within the B . cereus group.

Biochem J, 1998 Aug 1, 333 ( Pt 3), 677 - 83
Bacillus thuringiensis Cry1Ac toxin interaction with Manduca sexta aminopeptidase N in a model membrane environment; Cooper MA et al.; The Bacillus thuringiensis Cry1Ac delta-endotoxin was shown to bind in a biphasic manner to Manduca sexta aminopeptidase N (APN) present in a novel model membrane . Surface plasmon resonance analysis allowed the quantification of toxin binding to M . sexta APN in a supported lipid monolayer . The initial binding was rapid and reversible, with an affinity constant of 110 nM . The second phase was slower and resulted in an overall affinity constant of 3.0 nM . Reagents used to disrupt protein-protein interactions did not dissociate the toxin after high-affinity binding was attained . The initial association between Cry1Ac and APN was inhibited by the sugar GalNAc, but the higher-affinity state was resistant to GalNAc-induced dissociation . The results suggest that after binding to M . sexta APN, the Cry1Ac toxin undergoes a rate-limiting step leading to a high-affinity state . A site-directed Cry1Ac mutant, N135Q, exhibited a similar initial binding affinity for APN but did not show the second slower phase . This inability to form an irreversible association with the APN-lipid monolayer helps explain the lack of toxicity of this protein towards M . sexta larvae and its deficient membrane-permeabilizing activity on M . sexta midgut brush border membrane vesicles.

J Nat Prod, 1998 Jul, 61(7), 939 - 41
Gibbilimbols A-D, cytotoxic and antibacterial alkenylphenols from Piper gibbilimbum; Orjala J et al.; Fractionation of the petroleum ether extract from the leaves of Piper gibbilimbum collected in Papua New Guinea afforded four new alkenylphenols, gibbilimbols A-D (1-4) . The structures of the isolates were elucidated by spectroscopic methods, mainly 1D- and 2D-NMR spectroscopy . Gibbilimbols A-D were found to be toxic to brine shrimp with an LC50 of approximately 5 microg/mL . Gibbilimbols A-D were further found to be cytotoxic toward KB nasopharyngal carcinoma cells (ED50 7.8-2.1 microg/mL) . All isolates also showed antibacterial activity toward Staphylococcus epidermidis and Bacillus cereus.

Orv Hetil, 1998 Jun 28, 139(26), 1563 - 70
{Results of the BCG vaccination in Hungary since 1929: evaluation of preventive and immunotherapeutic effectiveness}; Lugosi L; BACKGROUND: The BCG (Bacille Calmette-Guerin), a living attenuated bacterial vaccine with a characteristic residual virulence, has been used to prevent tuberculosis since 1921 (in Hungary non-systematically since 1929) and applied for immunostimulation in neoplasia since the 1960s . MEASURES: Considering the grave tuberculosis epidemiological situation in Hungary, the BCG revaccination became compulsory up to 20 years old tuberculin negatives since 1959 . The Pasteur P1173P2 BCG strain has been used for vaccine manufacturing with improved quality control methods according to the requirements of the WHO . With in systematic BCG primo and revaccination policy 8.1 million BCG vaccination from 1959 to 1983 then further 3.1 million between 1984 and 1996 have been performed . RESULTS: Linear regression analysis demonstrates that the decrease of the TB incidence in children was 3-5 times more rapid (annual average decrease was 25.5%) than in adult since 1959 . Multiple regression analysis indicates that the BCG is the strongest explanatory variable decreasing children TB incidence among other antituberculosis measures . The BCG vaccination efficacy ins demonstrated by 2 x 2 table analysis . The systematic BCG vaccination, the living and persisting BCG in the macrophages, confers acquired resistance against virulent TB infections . The immunostimulation in neoplasia has been applied with concentrated BCG developed in Hungary since 1979 . The adverse reactions are at accepted frequency . The number of BCG vaccinated subjects was estimated at 1.5 billion from 1948 to 1974 in the world . The yearly number of BCG vaccination in the WHOI-EPI System is estimated 50-100 million . CONCLUSION: The efficacy of the BCG vaccination can only be ensured if the vaccine is manufactured and controlled with standardized methods, and applied in a systematic vaccination programme . The effectiveness has to be evaluated in statistically valid biostatistical models.

FEMS Microbiol Lett, 1998 Jul 1, 164(1), 201 - 6
Discrimination among Bacillus cereus, B . mycoides and B . thuringiensis and some other species of the genus Bacillus by Fourier transform infrared spectroscopy; Beattie SH et al.; Fourier transform infrared spectroscopy (FTIR) in conjunction with canonical variate analysis was found to be effective in discriminating among spectra of 9 representative strains of Bacillus spp., including B . cereus, B . mycoides and B . thuringiensis . The method was also able to discriminate according to species among spectra of 14 other non-type strains of B . cereus, 12 of B . mycoides and 12 of B . thuringiensis with a success rate of > 95%, even without using a prior classification of the groups by species . FTIR spectroscopy can be used for the rapid and accurate differentiation of species in the genus Bacillus that are of importance to the food and dairy industry.

Curr Opin Pulm Med, 1998 May, 4(3), 154 - 61
Examining the biology of tuberculosis; Gonzalez-Rothi RJ; The task of critically reviewing current advances in a scientifically burgeoning topic such as tuberculosis is daunting . From 2965 published articles on tuberculosis in 1997 cited in MEDLINE, 180 were selected, and approximately one third of those are included in this review . This paper highlights how molecular biology has greatly advanced our understanding of the epidemiology and transmission of tuberculosis worldwide . In addition, recent data on the dynamics of transmission between close contacts are provided that may challenge conventional views . A global view of drug-resistant tuberculosis is presented, and new data on the prevalence of tuberculosis in diabetics, bone marrow transplant recipients, patients with renal disease, and HIV-infected persons are highlighted . Recent studies on the genetic susceptibility of tuberculosis are outlined, and we review immunotherapy and the impact of tuberculosis on the natural course of HIV infection . Chemoprevention, duration of therapy, and bacille Calmette-Guerin vaccination in the setting of HIV are discussed . The value and limitation of nucleic acid amplification tests in rapid diagnosis is addressed, and old diagnostic tests are revisited with new data . Novel mycobacterial culture systems are discussed and screening-for-infection approaches are revisited, including tuberculin skin testing in bacille Calmette-Guerin vaccinees . Therapeutic issues addressing antituberculous drug absorption in HIV-infected patients, the role of pharmacokinetics in predicting clinical outcomes, and important drug-drug interactions are discussed.

Biochim Biophys Acta, 1998 Jul 28, 1386(1), 199 - 210
Catalytic activity of thermolysin under extremes of pressure and temperature: modulation by metal ions; Kudryashova EV et al.; The catalytic activity of thermolysin (TL), a Zn-dependent neutral protease from Bacillus thermoproteolyticus, has been studied over a wide interval of pressures (1 bar to 4 kbar) and temperatures (20 degreesC to 80 degreesC) by monitoring hydrolysis of a low-molecular-mass substrate, 3-(2-furylacryloyl)-glycyl-L-leucine amide . This reaction shows a very large negative value for the activation volume and, because of that, simultaneous increase in temperature and pressure leads to a significant (up to 40-fold) acceleration of the reaction . At pressures higher than 2-2.5 kbar, the reaction rate starts to decrease due to disactivation of TL . This disactivation is explained in part by pressure-promoted dissociation of zinc ion from the active site and can be inhibited by adding exogenous zinc . Thus, this thermostable protease does not specifically show a higher stability at high pressure in comparison with small mesophilic proteases.

Biophys J, 1998 Aug, 75(2), 1010 - 5
Obfuscation of allosteric structure-function relationships by enthalpy-entropy compensation; Tlapak-Simmons VL et al.; The pH and temperature dependence of the allosteric properties of phosphofructokinase (PFK) from Bacillus stearothermophilus have been studied from 5 to 9 and 6 to 40 degrees C, respectively . Throughout this pH and temperature range the allosteric ligands MgADP and phospho(enol)pyruvate (PEP) have no effect on kcat . The dissociation constants of the substrate, fructose 6-phosphate, and the allosteric ligands, as well as the absolute value of the coupling free energies between these ligands, all increase when the pH is raised, indicating that the inhibition by PEP and the activation by MgADP increase despite each ligand's somewhat lower affinity . However, the constituent coupling enthalpies and entropies substantially diminish in absolute value as pH is increased, suggesting that the magnitudes of molecular perturbations engendered by the binding of allosteric ligands do not correlate with the magnitudes of the functional consequences of those perturbations . Temperature and pH exert their influence on the observed allosteric behavior by changing the relative contributions made by the largely compensating DeltaH and TDeltaS terms to the coupling free energy.

Biochem Biophys Res Commun, 1998 Jul 20, 248(2), 372 - 7
Improved thermostability of a Bacillus alpha-amylase by deletion of an arginine-glycine residue is caused by enhanced calcium binding; Igarashi K et al.; alpha-Amylase from alkaliphilic Bacillus KSM-1378 (LAMY) is a novel semi-alkaline enzyme which has a high specific activity, a value 5-fold higher than that of a Bacillus licheniformis enzyme at alkaline pH . Thermostability of this enzyme could be improved by deletion of the Arg181-Gly182 residue by means of site-directed mutagenesis . The wild-type and engineered LAMYs were very similar with respect to specific activity, pH-activity curve, temperature-activity curve, susceptibility to inhibitors, and pattern of hydrolysis products from soluble starch and maltooligosaccharides . However, the engineered enzyme also acquired increased pH stability and resistance to sodium dodecyl sulfate and especially chelating reagents, such as ethylenediaminetetraacetate and ethyleneglycol-bis (beta-aminoethylether)tetraacetate . This is the first report that thermostability of alpha-amylase is improved by enhanced calcium binding to the enzyme molecule .

Medicina (B Aires), 1997, 57(5), 581 - 6
Cross-reactivity of anti-10kD heat shock protein antibodies in leprosy and tuberculosis patients; Rojas RE et al.; The response to recombinant 10-kD heat shock protein (HSP) of Mycobacterium leprae (rML10) was evaluated by indirect ELISA in sera from leprosy patients, household contacts, tuberculosis patients and healthy controls in a leprosy-endemic area in the North East of Argentina . Some technical parameters were analyzed: within-assay and between-assay variability, dose-response curves and detectability indexes (specificity and sensitivity) of ELISA applied to measure anti-10 kDa antibodies . High levels of these antibodies have already been reported in positive bacilloscopy patients; herein we have also demonstrated that tuberculosis patients sera cross-react with this M . leprae antigen . This test seems to have a low sensitivity and specificity for leprosy detection; it confirms that antibodies against highly conserved HSP antigens are important in the polyclonal response against mycobacterial epitopes in leprosy as well as in tuberculosis.

Lett Appl Microbiol, 1998 May, 26(5), 387 - 90
Isolation of a strain of Bacillus thuringiensis ssp . kurstaki HD-1 encoding delta-endotoxin Cry1E; Chang JH et al.; A strain of Bacillus thuringiensis, STB-1, toxic against Spodoptera exigua, was isolated . Bacillus thuringiensis STB-1 produced bipyramidal inclusions and reacted with the H antiserum of B . thuringiensis ssp . kurstaki . The plasmid and protein profiles of B . thuringiensis STB-1 were compared with those of its reference strains, ssp . kurstaki and ssp . kenyae . To verify the gene type of B . thuringiensis STB-1, PCR analysis was performed with Spodoptera-specific cry gene primers . The result showed that B . thuringiensis STB-1, unlike its reference strains, had crylAa, crylAb, crylAc and crylE, suggesting that B . thuringiensis STB-1 was a unique strain with respect to gene type . In addition, B . thuringiensis STB-1 showed a high level of toxicity against both S . exigua and Bombyx mori, whereas B . thuringiensis ssp . kurstaki HD-1 or ssp . kenyae showed a high level of toxicity against only Bombyx mori or S . exigua, respectively.

J Appl Microbiol, 1998 May, 84(5), 883 - 8
Characterization of mosquito larvicidal parasporal inclusions of a Bacillus thuringiensis serovar higo strain; Saitoh H et al.; The parasporal inclusion proteins of the type strain of Bacillus thuringiensis serovar higo (H44), that have moderate mosquitocidal activity, were characterized . The purified parasporal inclusions, spherical in shape, were examined for activity against the two mosquito species, Culex pipiens molestus and Anopheles stephensi and the moth-fly, Telmatoscopus albipunctatus . The LC50 values of the inclusion for the two mosquitoes were 3.41 and 0.15 microgram.ml-1, respectively . No mortality was shown for T . albipunctatus larvae by the inclusions at concentrations up to 1 mg ml-1 . Solubilized parasporal inclusions exhibited no haemolytic activity against sheep erythrocytes . Parasporal inclusions consisted of eight proteins with molecular masses of 98, 91, 71, 63, 59, 50, 44 and 27 kDa . Of these, the 50 and 44 kDa proteins were the major components . Analysis with immunoblotting revealed that, among several inclusion proteins of B . thuringiensis serovar israelensis, only two proteins of 130 kDa and 110 kDa reacted weakly with antibodies against higo proteins . N-terminal amino acid sequences of the 98, 91, and 71 kDa proteins showed 85-100% identity to those of the two established Cry protein classes, Cry4A and Cry10A.

J Appl Microbiol, 1998 May, 84(5), 791 - 801
Bacillus isolates from the spermosphere of peas and dwarf French beans with antifungal activity against Botrytis cinerea and Pythium species; Walker R et al.; A range of isolation procedures including washing, sonication and incubation in nutrient broth were used separately and in combination to obtain potential bacterial antagonists to Botrytis cinerea and Pythium mamillatum from the testae and cotyledons of peas and dwarf French beans . Heat treatment was also used to bias this selection towards spore-forming bacteria . Ninety-two bacterial isolates were obtained, 72 of which were provisionally characterized as species of Bacillus . Four of these Bacillus isolates (B3, C1, D4 and J7) displayed distinct antagonism in vitro against Botrytis cinerea and P . mamillatum when screened using dual culture analysis . Further characterization of these antagonists using API 50CHB biochemical profiling identified isolate D4 as Bacillus polymyxa and isolates B3, C1 and J7 as strains of B . subtilis . In vitro screening techniques, using cell-free and heat-killed extracts of liquid cultures against Botrytis cinerea, demonstrated the production of antifungal compounds by these four Bacillus antagonists . With each isolate the antifungal activity was found not to be either exclusively spore-bound nor released entirely into the medium but present in both fractions . The antifungal compounds produced by these isolates were shown to be heat-stable . Their identification, production and release require further study for exploitation as biocontrol systems.

J Am Mosq Control Assoc, 1998 Jun, 14(2), 183 - 5
Laboratory and field evaluation of efficacy of VectoBac 12AS against Culex sitiens (Diptera: Culicidae) larvae; Brown MD et al.; Laboratory bioassay studies of the efficacy of VectoBac 12AS (active ingredient: 1,200 International Toxic Units {ITU}/mg Bacillus thuringiensis var . israelensis) against field-collected late 3rd/early 4th-instar larvae of Culex sitiens indicated excellent control potential . A 95% lethal concentration (LC95) value of 1.381 x 10(7) ITU was calculated, which equated to a dosage of 0.011 liters/ha . This dosage represented 1.8% of the recommended lowest dosage rate for the product . A field trial of VectoBac 12AS against late 3rd/early 4th-instar field specimens of Cx . sitiens in floating mesh cylinders was then conducted in salt-marsh pools near Coomera Marina, southeast Queensland, Australia . At a rate of 0.5 liters/ha, 100% mortality of Cx . sitiens larvae was recorded at 24 h posttreatment.

Infect Immun, 1998 Aug, 66(8), 3643 - 8
An epitope delivery system for use with recombinant mycobacteria; Hetzel C et al.; We have developed a novel epitope delivery system based on the insertion of peptides within a permissive loop of a bacterial superoxide dismutase molecule . This system allowed high-level expression of heterologous peptides in two mycobacterial vaccine strains, Mycobacterium bovis bacille Calmette-Guerin (BCG) and Mycobacterium vaccae . The broader application of the system was analyzed by preparation of constructs containing peptide epitopes from a range of infectious agents and allergens . We report detailed characterization of the immunogenicity of one such construct, in which an epitope from the Der p1 house dust mite allergen was expressed in M . vaccae . The construct was able to stimulate T-cell hybridomas specific for Der p1, and it induced peptide-specific gamma interferon responses when used to immunize naive mice . This novel expression system demonstrates new possibilities for the use of mycobacteria as vaccine delivery vehicles.

Rays, 1998 Jan-Mar, 23(1), 225 - 30
BCG and prospects for new vaccines against tuberculosis; Ortona L et al.; Seventy-five years have elapsed since its introduction and a renewed interest has arisen in the vaccination with bacillus Calmette-Guerin (BCG) for the prevention of tuberculosis . This interest has been motivated by the increase in tuberculosis, especially in multidrug-resistant tuberculosis . The efficacy of BCG has been questioned for decades, however, new epidemiological studies have shown a protective effect in some populations and categories at risk . Protection is more evident in the populations with a high incidence of the disease, especially against disseminated and invasive disease . The use of this vaccination is advised for specific populations based on the risk of infection and disease . However, BCG has a limited benefit . New agents produced with methods of molecular biology are supplying encouraging results in the animal model.

Rays, 1998 Jan-Mar, 23(1), 9 - 14
General epidemiology of tuberculosis; Ortona L et al.; An outline of the history of tuberculosis is offered for consideration including the present estimated incidence world-wide . The epidemiology of the disease with respect to its etiology is described focusing on the risk of infection, the development of clinical manifestations subsequent to the exposure to the bacillus, and the risk of reactivation . The situation of the epidemiology of tuberculosis in Italy is analyzed based on available information after the closure of TB dispensaries following the introduction of National Health Service in 1978 . In last years over 5000 cases of tuberculosis per year have been notified; however what percentage of the real incidence of TB this data represents, is unknown.

FEMS Microbiol Lett, 1998 Jun 15, 163(2), 229 - 36
16S-23S rRNA internal transcribed spacers as molecular markers for the species of the 16S rRNA group I of the genus Bacillus; Daffonchio D et al.; The internal transcribed spacers between the 16S and the 23S ribosomal RNA genes were used to discriminate species of the 16S rRNA group I of the genus Bacillus by PCR . The spacer-PCR fingerprints clearly discriminated the different species, except those closely related like the members of the 'B . cereus group' (B . cereus, B . thuringiensis and B . mycoides) and the species of the 'B . subtilis group' (B . amyloliquefaciens and B . licheniformis) . Examining in more detail the shortest internal transcribed spacers, B . subtilis group species were distinguished by single-strand conformation polymorphism analysis, whereas B . mycoides was differentiated from B . cereus/B . thuringiensis by restriction analysis.

Extremophiles, 1998 May, 2(2), 83 - 92
Studies on the respiratory system in alkaliphilic Bacillus; a proposed new respiratory mechanism; Higashibata A et al.; Respiratory electron transfer systems in two alkaliphilic Bacillus species, YN-1 and YN-2000, were investigated In the cyanide-sensitive pathway of the obligate alkaliphilic Bacillus YN-1, the terminal enzyme was a caa3-type cytochrome c oxidase constituting up to just 10% of the total oxygen-reducing activity, while 90% of the respiratory activity was due to cyanide-insensitive, nonproteinaceous material with a molecular weight of 662 . These results were consistent with the cyanide-tolerant growth of the bacterium . The molecular and catalytic properties of the nonproteinaceous material were not identical with those of menaquinones extracted from the bacterium . Furthermore, the nonproteinaceous material was also found in the facultative alkaliphilic Bacillus YN-2000, when that bacterium was cultivated in alkaline conditions . A new respiratory oxygen-reducing mechanism comprising a nonproteinaceous component and a catalase is proposed for these alkaliphilic Bacillus species.

Arch Virol, 1997, 142(9), 1933 - 6
Oleavirus, a new genus in the family Bromoviridae; Martelli GP et al.; Oleavirus is a monotypic genus having olive latent virus 2 (OLV-2) as the type species . OLV-2 is transmitted by inoculation of sap but not by aphids . Virus particles have different shape and size, ranging from quasi spherical to bacilliform with length of 37, 43, 48, and 55 nm, respectively, and a diameter of ca . 18 nm . Virions do not contain lipids or carbohydrates and possess a single coat protein species with molecular mass of ca . 24 kDa, which is not required for infectivity . Individual particles contain a single molecule of linear, positive sense ssRNA, constituting ca . 19% of their weight . The genome consists of three functional non polyadenylated, capped, positive sense, single-stranded RNA molecules occurring as three functional species of 3126 nt (RNA1, monocistronic), 2734 nt (RNA2, monocistronic), and 2438 nt (RNA3, bicistronic) . Virions encapsidate a fourth RNA species 2078 nt in size (RNA4) with no apparent messenger activity . Virus replication is thought to occur in the cytoplasm possibly in connection with vesicular structures . The strategy of replication encompasses proteolytic processing and subgenomic RNA production . Oleavirus does not have a complete straightforward relationship with any of the current genera in the Bromoviridae, but shows homologies in diverging directions with one genus of the family or another.

Am J Gastroenterol, 1998 Jul, 93(7), 1055 - 9
The immunohistological diagnosis of E . coli O157:H7 colitis: possible association with colonic ischemia; Su C et al.; OBJECTIVE: E . coli O157:H7 may cause hemorrhagic colitis resembling ischemic colitis . Diagnosis is usually made by finding sorbitol-negative colonies on MacConkey agar that react with O157 and H7 antisera . Most ischemic colitis is idiopathic, but some may be caused by E . coli O157:H7, inasmuch as this organism can produce fibrin thrombi in colon vasculature . The objectives of this study were to determine whether E . coli O157:H7 infection can be diagnosed retrospectively from paraffin blocks of colon sections and whether an association exists between E . coli O157:H7 infection and colonic ischemia . METHODS: Paraffin-embedded sections of normal colon (n = 2) and various colitides {ischemic (n = 11), E . coli O157:H7 (n = 2), IBD (n = 8) and pseudomembranous (n = 3)} were used . Sections were deparaffinized, rehydrated, incubated with 3% peroxide in methanol, rinsed, and incubated with peroxidase-labeled antibody isolated from goats immunized with whole E . coli O157:H7 . Sections were stained with peroxidase chromagen reagent and counterstained with hematoxylin . Coarse, granular, orange-brown staining was considered positive . To determine the localization of the chromagen deposits, three cases that stained positive, including one of the culture-proved E . coli O157:H7 colitis and two of colonic ischemia, were processed for electron microscopy . RESULTS: Both cases (100%) of E . coli O157:H7 colitis and three of 11 (27.3%) cases of ischemic colitis stained positive by light microscopy . In one culture-proved case, electron microscopy demonstrated staining of bacillary structures; in two cases of colonic ischemia, extensive deposits of chromagen material were present that were associated neither with inflammatory cells nor with bacterial forms . CONCLUSIONS: Immunoperoxidase staining of archival sections may be used to diagnose E . coli O157:H7 infection . An etiological role for this organism is possible in some cases of colonic ischemia.

J Immunol, 1998 Jul 15, 161(2), 1045 - 54
Bacille Calmette-Guérin vaccination enhances human gamma delta T cell responsiveness to mycobacteria suggestive of a memory-like phenotype; Hoft DF et al.; Bacille Calmette-Guerin (BCG) immunity can be studied as one experimental model for mycobacterial protective immunity . We have used flow cytometry to investigate human T cell subsets induced by BCG vaccination . PBMC harvested from BCG-vaccinated individuals and controls were stimulated with mycobacterial Ags, and the T cell subsets present after 7 days of in vitro expansion were characterized . The most dramatic expansions induced by mycobacterial Ags were detected in gamma delta T cells . The gamma delta T cell expansions measured after in vitro stimulation with mycobacterial Ags were significantly greater in BCG responders compared with nonsensitized controls, indicating that BCG vaccination induced gamma delta T cell activation associated with enhanced secondary responses . The majority of gamma delta T cells induced by BCG vaccination were gamma 9+ delta 2+ T cells reactive with isoprenyl pyrophosphates . Coculture with CD4+ T cells induced optimal gamma delta T cell expansion, although IL-2 alone could provide this helper function in the absence of CD4+ T cells . Gamma delta T cells were found to provide helper functions for mycobacterial specific CD4+ and CD8+ T cells as well, demonstrating reciprocal stimulatory interactions between gamma delta T cells and other T cell subsets . Finally, prominent mycobacterial specific gamma delta T cell expansions were detected in a subset of unvaccinated controls with evidence for prior sensitization to mycobacterial lysates (elevated mycobacterial specific lymphoproliferative responses) . These latter findings are consistent with the hypothesis that exposure to atypical mycobacteria or related environmental Ags may induce gamma delta T cells cross-reactive with Ags present in the Mycobacterium tuberculosis complex . Our results suggest that gamma delta T cells may be capable of developing a memory immune-like phenotype, and therefore might be important targets for new vaccines.

Microbios, 1998, 93(374), 43 - 54
Comparative antibacterial and antifungal effects of some phenolic compounds; Aziz NH et al.; The antimicrobial potential of eight phenolic compounds isolated from olive cake was tested against the growth of Escherichia coli, Klebsiella pneumoniae, Bacillus cereus, Aspergillus flavus and Aspergillus parasiticus . The phenolic compounds included p-hydroxy benzoic, vanillic, caffeic, protocatechuic, syringic, and p-coumaric acids, oleuropein and quercetin . Caffeic and protocatechuic acids (0.3 mg/ml) inhibited the growth of E . coli and K . pneumoniae . The same compounds apart from syringic acid (0.5 mg/ml) completely inhibited the growth of B . cereus . Oleuropein, and p-hydroxy benzoic, vanillic and p-coumaric acids (0.4 mg/ml) completely inhibited the growth of E . coli, K . pneumoniae and B . cereus . Vanillic and caffeic acids (0.2 mg/ml) completely inhibited the growth and aflatoxin production by both A . flavus and A . parasiticus, whereas the complete inhibition of the moulds was attained with 0.3 mg/ml p-hydroxy benzoic, protocatechuic, syringic, and p-coumaric acids and quercetin.

Int J Immunopharmacol, 1997 Nov-Dec, 19(11-12), 629 - 44
The immunopharmacology of head and neck cancer: an update; Hadden JW; Patients with head and neck squamous cell cancer have cell-mediated immune defects and anergy, which progress with disease . T-lymphocytopenia and dysfunction, monocyte dysfunction, prostaglandins, antigen-antibody complexes, serum and cell suppressive factors, radiation therapy and poor nutrition with zinc deficiency all contribute . Nevertheless, cell-mediated immunoreactivity to tumor is manifest in the majority of the patient's blood and regional nodes, and in the tumor itself by tumor-infiltrating lymphocytes . Lymphocytes from these sources cloned in the presence of interleukin-2 +/- tumor extracts show relatively specific cytotoxicity against squamous cell cancer . Humoral immunity is intact, and increased IgA and IgE levels and antibodies reactive to tumor antigens are common . Tumor-associated antigens detected in serum and tumor include carcinoembryonic antigen, tumor polypeptide antigen, squamous cell cancer antigens, tumor antigen-4 and various mucin antigens . The mucin antigens, in particular, can elicit T-cell responses . Humoral reactivity to such antigens is manifest in circulating immune complexes and immunoglobulin coating of tumor surfaces . Immunotherapeutic efforts in head and neck squamous cell cancer should logically employ T-cell adjuvants, contrasuppression and immunorestoration . Non-specific stimulation with bacille Calmette-Guerin (BCG), levamisole and other agents has not been successful . Encouraging results have been observed in limited trials with indomethacin and plasmapheresis . Early trials with local administration of low dosages of interferon-alpha, natural interleukin-2 and a natural interleukin mixture have produced partial and complete regressions with no toxicity and with intense leukocyte infiltration indicating cellular immunity . Efforts are needed to define the mechanisms and the antigens involved in these reactions . On the contrary, treatments with high dosages of recombinant interferon-alpha and interleukin-2 have yielded few responses and considerable toxicity . Combination strategies are discussed which may improve upon these initial immunotherapeutic effects of these low dose trials.

J Biol Chem, 1998 Jul 24, 273(30), 19097 - 101
Genetically engineered zinc-chelating adenylate kinase from Escherichia coli with enhanced thermal stability; Perrier V et al.; In contrast with adenylate kinase from Gram-negative bacteria, the enzyme from Gram-positive organisms harbors a structural Zn2+ bound to 3 or 4 Cys residues in the structural motif Cys-X2-Cys-X16-Cys-X2-Cys/Asp . Site-directed mutagenesis of His126, Ser129, Asp146, and Thr149 (corresponding to Cys130, Cys133, Cys150, and Cys153 in adenylate kinase from Bacillus stearothermophilus) in Escherichia coli adenylate kinase was undertaken for determining whether the presence of Cys residues is the only prerequisite to bind zinc or (possible) other cations . A number of variants of adenylate kinase from E . coli, containing 1-4 Cys residues were obtained, purified, and analyzed for metal content, structural integrity, activity, and thermodynamic stability . All mutants bearing 3 or 4 cysteine residues acquired zinc binding properties . Moreover, the quadruple mutant exhibited a remarkably high thermal stability as compared with the wild-type form with preservation of the kinetic parameters of the parent enzyme.

Tuber Lung Dis, 1997, 78(1), 57 - 66
Progression of chronic pulmonary tuberculosis in mice aerogenically infected with virulent Mycobacterium tuberculosis; Rhoades ER et al.; There are several critical differences in the pulmonary granulomatous response to Mycobacterium tuberculosis between the mouse and other animal models such as the guinea pig or rabbit . One key difference is a conspicuous lack of central caseating necrosis in pulmonary lesions of immunologically intact mice . To determine whether normal mice could develop such pathology in response to highly virulent clinical isolates of M . tuberculosis, C57BL/6 mice were infected aerogenically with varying doses of three different strains, and the development of a granulomatous response was followed for as long as a year . Whereas such conditions failed to induce caseating necrosis in the lungs of these mice, all of the infections induced a granulomatous response which progressed similarly . We present here a descriptive report of the gross pathological progression of tuberculosis in the lungs of the mice . In each case, the disease progressed in five discrete stages, which were delineated on the basis of several criteria including the extent of granulomatous involvement, the cell types present, the degree of lymphocyte organization, and the presence of destructive sequelae such as airway epithelium erosion and airway debris . Quicker progression of disease along these five stages was induced by increasing the size of the inoculum or by the more virulent mycobacterial strains . The infections with the virulent strains were not resolved, and the later stages of the granulomatous response coincided with an increasing bacillary load and a loss of organized lymphocytes in the infected lungs which ultimately resulted in the death of the host . These results indicate that although C57BL/6 mice do not manifest a caseating form of pulmonary tuberculosis, they manifest an equally pathogenic granulomatous response which appears as a chronic interstitial fibrosing response that fails to contain the infection at a time that organized lymphocyte involvement wanes in the lung.

Tuber Lung Dis, 1997, 78(1), 47 - 55
Effect of cytokine modulation by thalidomide on the granulomatous response in murine tuberculosis; Moreira AL et al.; SETTING: Experimental murine tuberculosis . OBJECTIVE: To evaluate the effect of cytokine modulation by thalidomide on the progression of the lung granulomatous response following aerosol tuberculosis infection in mice . DESIGN: Mice infected by the respiratory route with 200-500 viable Mycobacterium tuberculosis Erdman were treated with daily subcutaneous injections of thalidomide (30 mg/kg) or saline for 4 weeks . The bacillary load, granulomatous response and cytokine production in the lungs were evaluated . RESULTS: Aerosol M . tuberculosis infection resulted in a progressive granulomatous response in the lungs . At 28 days after infection, large granulomata with central necrosis and no apoptosis were observed . The infection induced high serum and lung cytokine mRNA levels . Thalidomide treatment resulted in a significant reduction in tumor necrosis factor-alpha, interleukin 6 (IL-6) and IL-10 protein levels (blood) and mRNA expression (lungs) . IL-12 and interferon-gamma were unaffected . The lungs of thalidomide-treated mice had smaller granulomata with apoptotic cells and no necrosis . Thalidomide treatment did not change the bacillary load . CONCLUSION: Thalidomide immunomodulation reduces inflammatory cytokines and concomitant lung pathology following acute aerosol M . tuberculosis infection, without increasing the bacillary load.

Tuber Lung Dis, 1997, 78(1), 13 - 9
A novel repeat sequence specific to Mycobacterium tuberculosis complex and its implications; Lee TY et al.; Restriction fragment length polymorphism analysis of Korean clinical isolates of Mycobacterium tuberculosis using a 245 bp fragment of IS6110 revealed a conserved 3.5 kb Pvull fragment . Attempts to clone this 3.5 kb fragment resulted in the serendipitous discovery of a novel repeat sequence present within a separate 3.5 kb Pvull genomic fragment . Nucleotide sequencing of a 823 bp region containing the putative repeat sequence revealed the presence of three small direct repeats, three palindromes and a 453 bp region that was analogous to 455 bp of a M . tuberculosis sequence previously reported . The presence of this 453 bp repeat sequence was demonstrated in standard mycobacterial strains belonging to the M . tuberculosis complex, including the H37Rv, H37Ra, Erdman, and Canetti strains and M . bovis and M . bovis bacille Calmette-Guerin (BCG) . Other mycobacterial species (M . kansasii, M . smegmatis, M . simiae, M . fortuitum, M . scrofulaceum, M . intracellulare, M . avium, and M . haemophilum) did not contain this sequence, suggesting that the 453 bp repeat sequence was specific to the M . tuberculosis complex . Of the 13 Korean and 12 other clinical isolates of M . tuberculosis tested, all contained three to four copies of the repeat sequence . The Southern blot patterns of the various M . tuberculosis strains allowed classification into five different groups . The most frequent pattern was the 'BCG-type' (4.7, 3.5, and 2.4 kb bands); the second most frequent pattern was the '4-band-type' (13, 4.7, 3.5, and 2.4 kb), observed only in the Korean clinical isolates, and the third most common pattern was the M . tuberculosis H37Rv/H37Ra/M . bovis-type (13, 4.7, and 3.5 kb bands) . Upstream sequences indicate proximity to the rhamnose biosynthesis (rfb) cluster of M . tuberculosis . Our results indicate that the repeat sequence may be useful for the design of probe and polymerase chain reaction primers for the identification and epidemiological testing of members of the M . tuberculosis complex.

Biochemistry, 1998 Jul 14, 37(28), 10173 - 80
Spectroscopic characterization of a binuclear metal site in Bacillus cereus beta-lactamase II; Orellano EG et al.; The zinc metalloenzyme beta-lactamase II (betaLII) from Bacillus cereus has been overexpressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and the metal binding properties of recombinant betaLII toward Zn(II) and Co(II) have been studied by fluorescence and activity measurements . The apoenzyme is able to bind two metal ion equivalents, which confer on betaLII its maximum enzymatic efficiency . The enzyme is partially active with one metal ion equivalent . The diCo(II) and a mixed Zn(II)Co(II) derivative of betaLII were obtained and probed by electronic and paramagnetic NMR spectroscopy . In the high-affinity site, the metal is bound to three His residues and a solvent molecule, adopting a tetrahedral geometry . A Cys, a His, and an Asp residue are coordinated to the low-affinity metal site, together with two or three solvent molecules . This coordination polyhedron resembles the binuclear metal site of the Bacteroides fragilis beta-lactamase {Concha, N., Rasmussen, B . A., Bush, K., and Herzberg, O . (1996) Structure 4, 823-836; Carfi, A., Duee, E., Paul-Soto, R., Galleni, M., Frere, J . M., and Dideberg, O . (1998) Acta Crystallogr . D54, 47-57} but differs from that resulting from the X-ray study of betaLII {Carfi, A., Pares, S., Duee, E., Galleni, M., Duez, C., Frere, J . M., and Dideberg, O . (1995) EMBO J . 14, 4914-4921} . These results suggest that this binuclear metal site may be a general feature of metallo-beta-lactamases.

Nat Struct Biol, 1998 Jul, 5(7), 585 - 92
The X-ray structure of a cobalamin biosynthetic enzyme, cobalt-precorrin-4 methyltransferase; Schubert HL et al.; Biosynthesis of the corrin ring of vitamin B12 requires the action of six S-adenosyl-L-methionine (AdoMet) dependent transmethylases, closely related in sequence . The first X-ray structure of one of these, cobalt-precorrin-4 transmethylase, CbiF, from Bacillus megaterium has been determined to a resolution of 2.4 A . CbiF contains two alphabeta domains forming a trough in which S-adenosyl-L-homocysteine (AdoHcy) binds . The location of AdoHcy and a number of conserved residues, helps define the precorrin binding site . A second crystal form determined at 3.1 A resolution highlights the flexibility of two loops around this site . CbiF employs a unique mode of AdoHcy binding and represents a new class of transmethylase.

Curr Microbiol, 1998 Aug, 37(2), 80 - 7
Genetic diversity of Bacillus cereus/B . thuringiensis isolates from natural sources; Helgason E et al.; The genetic diversity and relationships among 154 Bacillus cereus/B . thuringiensis isolates recovered from soil samples from five geographic areas in Norway were investigated with multilocus enzyme electrophoresis (MEE) . Cluster analysis revealed two major groups (designated cluster I and cluster II) separated at genetic distance greater than 0.55 . Cluster I included 62 electrophoretic types (ETs) originating from all five locations, whereas, in cluster II, all but one isolate were from the same location . The isolates were also serotyped with B . thuringiensis flagellar antisera, and 28 distinct serotypes were identified . In general, serotyping did not show correlation to the genetic diversity of the isolates . The presence of IS231- and IS240-like transposable elements was detected in 14% of the strains of cluster II only . Parasporal crystals were observed in three strains; ten other strains were toxic to Trichoplusia ni . We conclude that B . cereus/B . thuringiensis from soil exhibit a high degree of recombination.

Int J Tuberc Lung Dis, 1998 Jul, 2(7), 575 - 9
Hepatosplenic tuberculosis: a cause of persistent fever during recovery from prolonged neutropenia; Chakrabarti S et al.; SETTING: Hepatosplenic abscesses in neutropenic patients, especially during the recovery phase, are almost always attributed to fungal infections . We report similar lesions due to Mycobacterium tuberculosis in neutropenic patients in a tertiary care centre in India . OBJECTIVE: To characterize the features of hepatosplenic tuberculosis in neutropenic patients . DESIGN: Retrospective comparison of disease pattern and response to treatment of hepatosplenic tuberculosis in febrile neutropenia patients (four of 30 with severe prolonged neutropenia) and in non neutropenic patients diagnosed during the same 12-month period (n = 4, control group) . RESULTS: The disease in the neutropenic patients typically presented during the recovery phase of neutropenia, with ultrasonic abnormalities similar to those seen in hepatosplenic fungal infections . In contrast to the marked organomegaly and typical granulomatous response found in the control group, the disease in the neutropenic patients was characterised by an absence of organomegaly, non-involvement of other sites, poor inflammatory response and a high bacillary load . The initial response to therapy was satisfactory in both groups . CONCLUSION: Tuberculosis needs to be considered in the diagnostic work-up of hepatosplenic abscesses that occur during the recovery phase of neutropenia.

J Biol Chem, 1998 Jul 17, 273(29), 18052 - 9
Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus unfolds through a monomeric intermediate . A quantitative analysis under equilibrium conditions; Park YC et al.; Tyrosyl-tRNA synthetase from Bacillus stearothermophilus comprises an N-terminal domain (residues 1-319), which is dimeric and forms tyrosyladenylate, and a C-terminal domain (residues 320-419), which binds the anticodon arm of tRNATyr . The N-terminal domain has the characteristic fold of the class I aminoacyl-tRNA synthetases . The unfolding of the N-terminal domain by urea at 25 degreesC under equilibrium conditions was monitored by its intensities of light emission at 330 and 350 nm, the ratio of these intensities, its ellipticity at 229 nm, and its partition coefficient, in spectrofluorometry, circular dichroism, and size-exclusion chromatography experiments, respectively . These experiments showed the existence of an equilibrium between the native dimeric state of the N-terminal domain, a monomeric intermediate state, and the unfolded state . The intermediate was compact and had secondary structure, and its tryptophan residues were partially buried . These properties of the intermediate and its inability to bind 1-anilino-8-naphthalenesulfonate showed that it was not in a molten globular state . The variation of free energy deltaG(H2O) and its coefficient m of dependence on the concentration of urea were, respectively, 13.8 +/- 0.2 kcal.mol-1 and 0.9 +/- 0.1 kcal.mol-1.M-1 for the dissociation of the native dimer and 13.9 +/- 0.6 kcal.mol-1 and 2.5 +/- 0.1 kcal.mol-1.M-1 for the unfolding of the monomeric intermediate.

J R Soc Med, 1998 Mar, 91(3), 133 - 4
Exacerbation of atopic dermatitis after bacillus Calmette-Guérin vaccination; Dalton SJ et al.; In two children with atopic dermatitis, routine vaccination with bacillus Calmette-Guerin (BCG) was followed by severe exacerbation of skin disease . If the sequence is cause and effect, a possible mechanism is stimulation of a Th2 lymphocyte cytokine profile by the vaccine, with migration of activated lymphocytes to inflamed skin . In children with active atopic dermatitis, BCG vaccination is best deferred until remission.

Plasmid, 1998 Jul, 40(1), 30 - 43
Kinetics of conjugative transfer: a study of the plasmid pXO16 from Bacillus thuringiensis subsp . israelensis; Andrup L et al.; The aggregation-mediated conjugation system of Bacillus thuringiensis subsp . israelensis, encoded by the 200-kb plasmid pXO16, is highly potent in transferring itself and efficient in mobilizing other nonconjugative plasmids . The present study reveals some salient features of this conjugation system . Our observations can be summarized as follows: (i) The conjugative transfer takes about 3(1/2) to 4 min . For a 200-kb plasmid this corresponds to about 1 kb per second . (ii) The ability to transfer the plasmid seems to be evenly distributed among the donors . (iii) Functionally, the mating complex was found to consist of one donor and one recipient cell, even though aggregates comprising thousands of interconnected cells are formed . (iv) Having donated the plasmid, the donor needs a "period of recovery" of about 10 min before it can redonate the plasmid . (v) Secondary transfer, i.e., transfer from newly formed transconjugants, is delayed about 40 min . This maturation time exceeds the generation time, and it may indicate that to display donor activity, a surface protein (the aggregation substance) has to be uniformly incorporated into the cell wall . Lastly, we found that when the experiments were sufficiently short and when the recipient cells were in excess compared with the donors, the process of conjugation could be reasonably described by a kinetic model analogous to the Michaelis-Menten model for enzyme catalysis . This allowed us to estimate (vi) the maximal conjugation rate to be about 0.05 transconjugant per donor per minute, and (vii) the Km value, i.e., the concentration of recipient that results in half of the maximal conjugation rate, to be about 4 x 10(6) recipients/ml .

Arch Esp Urol, 1998 May, 51(4), 389 - 90
{Hematuria, dysuria, pollakiuria and general malaise, in a patient treated with BCG instillations}; Medina Perez M et al.; OBJECTIVE: To report a case of granulomatous cystitis in a patient receiving bacillus Calmette-Guerin intravesical therapy for urothelial carcinoma in situ . METHODS: A 58-year-old man undergoing BCG intravesical therapy for urothelial carcinoma in situ presented symptoms of intense cystitis . Cystoscopy was performed and several bladder cold biopsies were obtained . RESULTS: Histopathological analysis demonstrated epithelioid granulomas . CONCLUSION: Cystitis arising from BCG therapy is defined as drug-induced or BCG-induced cystitis . Intense cystitis and malaise are a serious complication since it is not possible to distinguish patients with a simple uncomplicated local reaction from those who will develop progressive systemic infection . Cystoscopy and biopsy can be helpful in determining the nature of the condition and are recommended.

Eur J Biochem, 1998 Apr 15, 253(2), 480 - 4
Pyrrole-2-carboxylate decarboxylase from Bacillus megaterium PYR2910, an organic-acid-requiring enzyme; Omura H et al.; Inducible pyrrole-2-carboxylate decarboxylase, which catalyzes the decarboxylation of pyrrole-2-carboxylate to pyrrole and CO2 in stoichiometric amounts, was purified from Bacillus megaterium PYR2910 . The purity of the enzyme was shown by SDS/PAGE and gel-permeation HLPC . The enzyme has a molecular mass of approximately 98 kDa and consists of two identical subunits . It is highly specific for pyrrole-2-carboxylate, and also catalyzes the reverse reaction, the carboxylation of pyrrole . A unique feature of this enzyme is its requirement of an organic acid, such as acetate, propionate, butyrate or pimelate . A possible catalytic mechanism including a cofactor function of organic acid is discussed.

J Infect Dis, 1998 Jul, 178(1), 138 - 46
Cellular immune responses to four doses of percutaneous bacille Calmette-Guérin in healthy adults; Lowry PW et al.; To explore the hypothesis that low-dose immunization might induce preferential Th1 cell immunity, 76 adults were vaccinated with one of four doses of bacille Calmette-Guerin (BCG): The doses contained very low (1.6 x 10(5) cfu), low (3.2 x 10(6) cfu), standard (1.6 x 10(8) cfu), or high (3.2 x 10(8) cfu) levels of BCG . Delayed-type hypersensitivity responses occurred 8 weeks after vaccination in 10% of persons given very low or low doses of BCG, compared with 95% and 100% of persons given standard or high doses, respectively . Lymphoproliferative responses, which were increased only for high-dose vaccinees, peaked 2 weeks after vaccination and were directed chiefly against Mycobacterium tuberculosis-secreted proteins, particularly the antigen 85 complex . Significant increases in mycobacteria-specific interferon-gamma expression were present 16 weeks after vaccination only for persons given standard or high doses of BCG . Percutaneous BCG appears capable of inducing a temporary Th1-like immune response, but standard or higher dosages are required.

J Clin Microbiol, 1998 Jul, 36(7), 2138 - 9
Bacillus thuringiensis subsp . konkukian (serotype H34) superinfection: case report and experimental evidence of pathogenicity in immunosuppressed mice; Hernandez E et al.; We present a case of severe war wounds infected by Bacillus thuringiensis serotype H34 and describe the experimental protocol used to demonstrate its ability to infect mice after cutaneous inoculation . This case is interesting because B . thuringiensis is considered to be a contaminant in laboratories and receives inadequate attention.

Nucleic Acids Res, 1998 Jul 15, 26(14), 3348 - 9
BfiI, a restriction endonuclease from Bacillus firmus S8120, which recognizes the novel non-palindromic sequence 5'-ACTGGG(N)5/4-3'; Vitkute J et al.; A new type IIS restriction endonuclease Bfi I hasbeen partially purified from Bacillus firmus S8120 . Bfi I recognizes the non-palindromic hexanucleotide sequence 5'-ACTGGG(N)5/4-3' and makes a staggered cut at the fifth base pair downstream of the recognition sequence on the upper strand, producing a single base 3' protruding end.

Carbohydr Res, 1997 Dec, 305(3-4), 561 - 8
Transfer reactions catalyzed by cyclodextrin glucosyltransferase using 4-thiomaltosyl and C-maltosyl fluorides as artificial donors; Bornaghi L et al.; Cyclodextrin glycosyltransferase enzyme from Bacillus circulans catalyzed the effective conversion of 4-thio-alpha-maltosyl fluoride into cyclo-alpha-(1-->4(2))-thiomalto -tetraoside, -pentaoside, -hexaoside and linear hemithiomaltooligosaccharides . However, under the same conditions, C-maltosyl fluoride afforded only linear modified maltotetraose, maltohexaose and maltooctaose in moderate yield.

Carbohydr Res, 1997 Dec, 305(3-4), 393 - 400
Enzymatic synthesis, isolation, and analysis of novel alpha- and beta-galactosyl-cycloisomalto-octaoses; Koizumi K et al.; Novel branched cycloisomalto-octaoses (CI8s) were enzymatically synthesized by transgalactosylation with alpha-galactosidase from coffee bean and beta-galactosidase preparations from Penicillium multicolor and Bacillus circulans, using melibiose and lactose as donor substrates, and CI8 which is a cyclic homogeneous oligosaccharide composed of eight glucose units bound by alpha-(1-->6)-linkages, as an acceptor . alpha-Galactosyl-CI8s and beta-galactosyl-CI8s obtained were isolated and purified by HPLC . Their structures were elucidated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOFMS) and NMR spectroscopy.

Biosci Biotechnol Biochem, 1998 May, 62(5), 1028 - 30
Thermostable neutral protease resembling thermolysin derived from Bacillus brevis MIB001; Takii Y et al.; A microbe producing a protease with strong thermostability that was released extracellularly was isolated from soil . The isolate, MIB001, grew at from 15 to 51 degrees C and pH 5.1-8.8 and was tentatively identified as a strain of Bacillus brevis . Rabbit antisera raised against a pure preparation of the protease did not cross-react with thermolysin or neutral metalloprotease from Bacillus stearothermophilus KP1236.

Appl Environ Microbiol, 1998 Jul, 64(7), 2723 - 5
Insecticidal activity of Bacillus laterosporus; Orlova MV et al.; The Bacillus laterosporus strains 921 and 615 were shown to have toxicity for larvae of the mosquitoes Aedes aegypti, Anopheles stephensi, and Culex pipiens . The larvicidal activity of B . laterosporus was associated with spores and crystalline inclusions . Purified B . laterosporus 615 crystals were highly toxic for Aedes aegypti and Anopheles stephensi.

Appl Environ Microbiol, 1998 Jul, 64(7), 2497 - 502
Development of a streptavidin-conjugated single-chain antibody that binds Bacillus cereus spores; Koo K et al.; Control of microorganisms such as Bacillus cereus spores is critical to ensure the safety and a long shelf life of foods . A bifunctional single chain antibody has been developed for detection and binding of B . cereus T spores . The genes that encode B . cereus T spore single-chain antibody and streptavidin were connected for use in immunoassays and immobilization of the recombinant antibodies . A truncated streptavidin, which is smaller than but has biotin binding ability similar to that of streptavidin, was used as the affinity domain because of its high and specific affinity with biotin . The fusion protein gene was expressed in Escherichia coli BL21 (DE3) with the T7 RNA polymerase-T7 promoter expression system . Immunoblotting revealed an antigen specificity similar to that of its parent native monoclonal antibody . The single-chain antibody-streptavidin fusion protein can be used in an immunoassay of B . cereus spores by applying a biotinylated enzyme detection system . The recombinant antibodies were immobilized on biotinylated magnetic beads by taking advantage of the strong biotin-streptavidin affinity . Various liquids were artificially contaminated with 5 x 10(4) B . cereus spores per ml . Greater than 90% of the B . cereus spores in phosphate buffer or 37% of the spores in whole milk were tightly bound and removed from the liquid phase by the immunomagnetic beads.

Appl Environ Microbiol, 1998 Jul, 64(7), 2490 - 6
Construction and expression of a bifunctional single-chain antibody against Bacillus cereus p6ores; Koo K et al.; The variable-region genes of monoclonal antibody against Bacillus cereus spores were cloned from mouse hybridoma cells by reverse transcription-PCR . The heavy- and light-chain variable-region genes were connected by a 45-base linker DNA to allow folding of the fusion protein into a functional tertiary structure . For detection of protein expression, a 10-amino-acid strep tag (biotin-like peptide) was attached to the C terminus of recombinant antibody as the reporter peptide . The single-chain antibody construct was inserted into the expression vector and expressed in Escherichia coli under the control of the T7 RNA polymerase-T7 promoter expression system . The expressed single-chain antibody was detected on Western blots by using a streptavidin-conjugated enzyme system . This small recombinant antibody fragment (ca . 28,000 Da by calculation) had B . cereus spore binding ability and antigen specificity similar to those of its parent native monoclonal antibody.

Appl Environ Microbiol, 1998 Jul, 64(7), 2479 - 84
A two-component monooxygenase catalyzes both the hydroxylation of p-nitrophenol and the oxidative release of nitrite from 4-nitrocatechol in Bacillus sphaericus JS905; Kadiyala V et al.; Bacteria that metabolize p-nitrophenol (PNP) oxidize the substrate to 3-ketoadipic acid via either hydroquinone or 1,2,4-trihydroxybenzene (THB); however, initial steps in the pathway for PNP biodegradation via THB are unclear . The product of initial hydroxylation of PNP could be either 4-nitrocatechol or 4-nitroresorcinol . Here we describe the complete pathway for aerobic PNP degradation by Bacillus sphaericus JS905 that was isolated by selective enrichment from an agricultural soil in India . Washed cells of PNP-grown JS905 released nitrite in stoichiometric amounts from PNP and 4-nitrocatechol . Experiments with extracts obtained from PNP-grown cells revealed that the initial reaction is a hydroxylation of PNP to yield 4-nitrocatechol . 4-Nitrocatechol is subsequently oxidized to THB with the concomitant removal of the nitro group as nitrite . The enzyme that catalyzed the two sequential monooxygenations of PNP was partially purified and separated into two components by anion-exchange chromatography and size exclusion chromatography . Both components were required for NADH-dependent oxidative release of nitrite from PNP or 4-nitrocatechol . One of the components was identified as a reductase based on its ability to catalyze the NAD(P)H-dependent reduction of 2,6-dichlorophenolindophenol and nitroblue tetrazolium . Nitrite release from either PNP or 4-nitrocatechol was inhibited by the flavoprotein inhibitor methimazole . Our results indicate that the two monooxygenations of PNP to THB are catalyzed by a single two-component enzyme system comprising a flavoprotein reductase and an oxygenase.

Biochem Biophys Res Commun, 1998 Jun 29, 247(3), 659 - 62
Peroxide reductase activity of NADH dehydrogenase of an alkaliphilic Bacillus in the presence of a 22-kDa protein component from Amphibacillus xylanus; Koyama N et al.; The NADH oxidase of Amphibacillus xylanus shows high NADH-peroxide reductase activity for hydrogen peroxide and alkyl hydroperoxides in the presence of a 22-kDa disulfide-containing protein component (Y . Niimura, L . B . Poole, and V . Massey, J . Biol.Chem . 270, 25645-25650, 1995) . It was found that the membrane-bound NADH dehydrogenase of an alkaliphilic Bacillus (YN-1) involved in the respiratory chain also exhibits reductase activity for hydrogen peroxide and cumene hydroperoxide in the presence of the 22-kDa component from Amphibacillus xylanus . Vmax values for these substrates were as high as those of the NADH oxidase of A . xylanus . Although the 38-kDa protein produced by trypsin treatment of NADH dehydrogenase retains NADH dehydrogenase activity, it exhibited no peroxide reductase activity in the presence of the 22-kDa component from A . xylanus . The NADH dehydrogenase of YN-1 might not only catalyze electron flow from NADH to the respiratory chain, but also function for scavenging peroxide.

J Invertebr Pathol, 1998 Jul, 72(1), 73 - 81
Processing of delta-endotoxin of Bacillus thuringiensis subsp . kurstaki HD-1 in Heliothis armigera midgut juice and the effects of protease inhibitors; Shao Z et al.; Bombyx mori was found to be more sensitive to the protoxins of HD-1 than Heliothis armigera . SDS-PAGE analysis showed that a large amount of activated toxin was yielded from protoxin by B . mori gut juice while little was yielded by H . armigera . Further degradation of activated toxin was observed in H . armigera midgut juice detected by SDS-PAGE . pH influenced the proteolytic activity of the midgut juice significantly, but there was no obvious effect of pH on the degradation of activated toxin . Specific inhibitor study revealed the presence of trypsin, chymotrypsin, and elastase in the midgut juice . TLCK, TPCK, elastatinal and some general serine protease inhibitors successfully prevented the excessive degradation of protoxin in H . armigera midgut juice . Chymotrypsin inhibitors showed strong inhibitory effects against the further degradation of activated toxin, indicating that chymotrypsin played a major role in the process . It was presumed that the excessive degradation of protoxin in H . armigera midgut juice was responsible for the low sensitivity of the insect to Bt . Further study demonstrated that the excessive degradation in vitro was triggered by SDS treatment . However, all of the tested serine protease inhibitors expressed synergism with protoxin against H . armigera larvae, suggesting that the excessive degradation of protoxin may occur in vivo to some extent and may be triggered by receptor binding of activated toxin .

J Invertebr Pathol, 1998 Jul, 72(1), 9 - 20
Histopathological effects of Bacillus thuringiensis on the alimentary canal of the sheep louse, Bovicola ovis; Hill CA et al.; Sequential observations were made of the ultrastructural effects of Bacillus thuringiensis (Bt) subsp . kurstaki strain WB3S16 on midgut epithelial cells of the sheep biting louse, Bovicola ovis, after the lice were fed, ad libitum, a powdered preparation of Bt spores, delta-endotoxin crystals, and lysed cellular components . Light microscope observations revealed cytopathological changes to the midgut epithelial cells 4 h postfeeding . Transmission electron micrographs showed that the microvilli of the midgut epithelial cells became disrupted 4-8 h postfeeding, after which the cells became vacuolated and swollen, and the cell organelles lost definition and disappeared . Paralysis and death of B . ovis occurred between 8 and 12 h postfeeding, coincident with midgut cells lysis and release of cellular contents into the midgut lumen . The histopathological effects reported here are similar to those reported in lepidopteran and coleopteran larvae affected by the delta-endotoxin crystal proteins . The constituent fractions of the Bt preparation were tested for toxicity to B . ovis using a feeding bioassay . Native delta-endotoxin crystals were not toxic to B . ovis and remained intact in the midgut of the insect . There was no evidence that the native Bt crystal was involved in the cytopathology and death of the lice . However, in vitro solubilized delta-endotoxin crystal proteins were significantly toxic to B . ovis . In addition, a louse active toxin was associated with the Bt membranes and culture supernatant .

Eur J Immunol, 1998 Jun, 28(6), 1762 - 72
Failure of P strain mice to respond to vaccination against schistosomiasis correlates with impaired production of IL-12 and up-regulation of Th2 cytokines that inhibit macrophage activation; Oswald IP et al.; In contrast to most inbred strains, P mice fail to develop significant resistance to Schistosoma mansoni infection as a result of vaccination with either radiation-attenuated cercariae or schistosome antigens plus Bacillus Calmette Guerin, and this failure correlates with defects in macrophage larvicidal activity . Supernatant fluids from antigen-treated in vitro cultures of splenocytes from vaccinated P mice demonstrate less macrophage stimulatory activity than do supernatants from cells of vaccine-responsive strains such as C57BL/6 . This is not due either to diminished production of the macrophage-activating cytokine IFN-gamma by P mice, or to a lesser responsiveness of macrophages from P mice to activation by IFN-gamma . Rather, P splenocytes produce two-to threefold higher amounts of IL-4 and IL-10, cytokines which down-regulate the cytotoxic potential of IFN-gamma-treated macrophages . Thus, the macrophage-activating potential of cytokine preparations from vaccinated P mice can be completely recovered by in vitro treatment with antibodies to IL-4 or IL-10 . Moreover, lower levels of IL-12, a cytokine involved in promoting development of Th1 responses, are produced by splenocytes from P mice as compared to C57BL/6 counterparts . These studies indicate that a genetic predisposition toward an impaired production of IL-12 and an increased production of down-regulatory Th2 cytokines correlate with low response to vaccination against S . mansoni.

FEBS Lett, 1998 May 22, 428(1-2), 57 - 8
Contribution of arginine-82 and arginine-86 to catalysis of RNases from Bacillus intermedius (binase); Yakovlev GI et al.; To elucidate the functional role of Arg82 and Arg86 in the enzyme activity of binase, the extracellular ribonuclease of Bacillus intermedius, we used site-directed mutagenesis . On cleavage of various substrates the catalytic activity of binase mutant Arg86 Ala is 2.7 x 10(3) - 7.7 x 10(3) times less than that of the native enzyme . The decrease in activity is determined preferentially by the decrease in the molecular rate constant kcat with a relatively small change of enzyme-substrate affinity, characterized by Km . This is the expected result if Arg86 acts to lower the energy of a transition state of the reaction . The replacement of Arg82 by Ala causes a 5-19-fold activity decrease, depending on the substrate . We propose that this residue does not have a direct catalytic function in the molecular mechanism of the binase action and that the activity decrease of binase on the replacement of Arg82 by alanine is mediated by the effect of Arg82 on the pK of catalytic residues.

J Biochem (Tokyo), 1998 Jul, 124(1), 45 - 50
Lysyl-tRNA synthetase from Bacillus stearothermophilus . Stopped-flow kinetic analysis of enzyme.lysyladenylate formation; Takita T et al.; Amino acid activation reaction of the lysyl-tRNA synthetase {L-lysine:tRNALys ligase (AMP forming); EC 6.1.1.6} from Bacillus stearothermophilus was studied fluorometrically by the stopped-flow method . The addition of L-lysine to the enzyme solution caused quenching of the protein fluorescence and the subsequent addition of ATP restored the quenched fluorescence {Takita et al . (1996) J . Biochem . 119, 680-689; Takita et al . (1997) 121, 244-250} . In the stopped-flow analysis, however, the former fluorescence change (quenching) could not be detected, while the latter change (restoration) was detectable . The L-lysine binding process was suggested to be much faster than the ATP binding process, being completed within the dead-time of the apparatus, ca . 3 ms . The hyperbolic dependence of kapp on the initial ATP concentration suggested that the ATP binding to the enzyme.L-lysine complex followed a two-step mechanism . Two L-lysine analogues that exhibit the qualitatively similar behavior to L-lysine in the fluorometric titration, L-lysine hydroxamate and L-lysine amide, were examined similarly . The two-step process was also suggested for these analogues, and the forward rate constant in the rate-determining step for L-lysine amide (221+/-7 s-1) was significantly larger than those for L-lysine (45.7+/-4.6 s-1) and L-lysine hydroxamate (14 . 5+/-1.7 s-1) at pH 8.0, 30 degrees C.

Commun Dis Public Health, 1998 Jun, 1(2), 84 - 8
Complications of bacille Calmette-Guérin (BCG) vaccination and immunotherapy and their management; Grange JM; Complications of bacille Calmette-Guerin (BCG) vaccination are uncommon . Fewer than one in 1000 people vaccinated develop significant local reactions, and serious disseminated disease develops in fewer than one in a million . Localised complications--which include hypersensitivity reactions, abscesses at the injection site, and localised lymphadenopathy--are usually self limiting . They usually result from faulty technique, including the accidental intracutaneous injection of the stronger percutaneous vaccine, or poor selection of subjects for vaccination . Abscesses at the injection site usually respond to drainage and chemotherapy with isoniazid or erythromycin . Lymphadenopathy responds poorly to antimicrobial treatment and surgery may be needed for suppurating or discharging lesions to hasten recovery and give a good cosmetic result . Disseminated disease usually occurs in people with impaired immunity, in whom it is often fatal . BCG should never be given to people who are known to be infected with HIV, but the risk of complications in children born to HIV infected mothers is low . Disseminated disease can also result from intravesical instillation of BCG to treat bladder cancer, but this responds to antituberculosis chemotherapy.

Eur Urol, 1998, 33(5), 457 - 63
Primary bladder carcinoma in situ: assessment of early BCG response as a prognostic factor; Orsola A et al.; OBJECTIVES: To evaluate the prognosis of primary bladder carcinoma in situ (CIS) according to the response to bacillus Calmette-Guerin (BCG) . Patients and METHODS: Twenty-six cases of primary CIS were treated with BCG . Mean, median and minimum follow-up periods were 47, 56 and 24 months . At 6 months, the patients were evaluated endoscopically and the response was classified as complete, partial or failure . RESULTS: Twenty-one patients (80.8%) showed complete response to BCG, 3 did so after a second course; 28.5% relapsed or progressed at a mean of 44 months . Five patients (19.2%) did not respond initially and all progressed in a period of 6 months . Early response to BCG was the only significant prognostic factor (p < 0.005) . CONCLUSIONS: A high- and a low-risk group of bladder CIS can be differentiated according to the response to BCG . CIS of the bladder has a poor prognosis, and the number of patients who developed progressive disease is significantly higher among the nonresponders.

J Nat Prod, 1998 Jun 26, 61(6), 729 - 33
Enhancement of NO production in activated macrophages in vivo by an antimalarial crude drug, Dichroa febrifuga; Murata K et al.; The effect of an antimalarial crude drug, Dichroafebrifuga Lour . on nitric oxide (NO) production in bacillus Calmette Guerin-induced mouse peritoneal macrophages activated by lipopolysaccharide was investigated . The NO production was significantly enhanced by an oral administration of a MeOH extract of D . febrifuga . Febrifugine (1) was isolated as the main active compound, and the activation was dose-dependent in the dosage range of 0.1-1 mg/kg/day.

Semin Respir Infect, 1998 Jun, 13(2), 100 - 8
Immunologic response and pathophysiology of Legionella infection; Friedman H et al.; Legionella pneumophila, the causative agent of legionnaires' disease, is a gram-negative pleomorphic bacillus and fastidious in its growth in artificial medium . These bacteria grow readily intracellularly, including growth in macrophages and other phagocytic cells . Humoral antibodies develop readily to these bacteria not only in infected patients, but also in persons who have had subclinical exposure . High-levels of serum antibodies may also occur in individuals who recover from infection . However, cell-mediated immunity based on lymphocytes reacting with the organisms and cytokines produced by such lymphocytes are important in resistance . Vaccines prepared from killed Legionella or their components readily induce cell-mediated immunity . Immune resistance to disease depends on lymphocyte-based immunity, activating cytokine formation, some of which activate macrophages to resist infection . Resistance to Legionella infection by experimental animals such as mice correlates with activation of macrophages, which can inhibit replication of the bacteria . Much recent experimental work has involved studies using inbred animals, including inbred mice genetically resistant to Legionella versus mice genetically susceptible . Detailed studies show that regulation of macrophage resistance versus susceptibility to infection is mediated by specific genetic mechanisms . Induction of cytokines by Legionella can activate immune cells, especially helper T cells . Th 1 type helper cells that produce type 1 class cytokines, such as interferon gamma and interleukin-2 (IL-2), are known to be important in cellular immunity to Legionella as well as to other opportunistic intracellular bacteria . In contrast, Th 2 type helper cells, which secrete type 2 class cytokines such as IL-4, IL-5, and IL-6, activate B lymphocytes to produce humoral antibodies important in resistance to extracellular bacteria which secrete toxins and extracellular factors as compared to intracellular bacteria such as Legionella . Although Legionella, similar to other ubiquitous opportunistic pathogens, can cause serious infection in immunocompromised individuals, these bacteria have many distinguishing characteristics, such as very rapid replication in macrophages from susceptible individuals . However, activated macrophages restrict the growth of these bacteria . Infection by Legionella, if recognized clinically, can be readily treated with appropriate antibiotics . Currently, many studies are in progress concerning the mechanism of pathogenicity and assessment of the molecular biologic mechanisms of protective immune responses to this bacterium, which causes serious infection in immunocompromised individuals.

Science, 1998 Jun 26, 280(5372), 2129 - 32
Insecticidal toxins from the bacterium Photorhabdus luminescens; Bowen D et al.; Transgenic plants expressing Bacillus thuringiensis (Bt) toxins are currently being deployed for insect control . In response to concerns about Bt resistance, we investigated a toxin secreted by a different bacterium Photorhabdus luminescens, which lives in the gut of entomophagous nematodes . In insects infected by the nematode, the bacteria are released into the insect hemocoel; the insect dies and the nematodes and bacteria replicate in the cadaver . The toxin consists of a series of four native complexes encoded by toxin complex loci tca, tcb, tcc, and tcd . Both tca and tcd encode complexes with high oral toxicity to Manduca sexta and therefore they represent potential alternatives to Bt for transgenic deployment.

Immunology, 1998 Mar, 93(3), 307 - 13
Inhibition of an established allergic response to ovalbumin in BALB/c mice by killed Mycobacterium vaccae; Wang CC et al.; Allergic disorders are mediated by T lymphocytes secreting T helper 2 (Th2) cytokines, interleukin-4 (IL-4) and interleukin-5 (IL-5), resulting in high levels of serum immunoglobulin E (IgE) and recruitment of eosinophils . One of the treatment strategies is to downregulate the Th2 component by inducing a T helper 1 (Th1) response to the relevant allergen, because Th1 and Th2 cytokines are thought to be mutually antagonistic . In this study, we examined the effects of Mycobacterium vaccae, a potent inducer of Th1 immunity, on allergic responses in a murine model . A single injection of M . vaccae into ovalbumin (OVA)-preimmunized BALB/c mice suppressed serum IgE over a wide dose range (10(7), 10(8) or 10(9) M . vaccae) . Further experiments, using 10(7) M . vaccae injected twice, showed that this treatment inhibited not only serum IgE, but also the potential for ovalbumin-induced IL-5 production by spleen cells . This non-specific ability of a mycobacterium to decrease Th2 activity, even when not presented together with the allergen, is in agreement with recent epidemiological studies on the impact of bacillus Calmette-Guerin (BCG) vaccination, and of other potent Th1 stimuli, on the incidence of atopy . The suppression of serum IgE and allergen-specific IL-5 synthesis by M . vaccae suggest that this organism is likely to have clinical application in the immunotherapy of allergy.

Wei Sheng Wu Xue Bao, 1996 Aug, 36(4), 303 - 6
{Characterization of insecticidal crystal proteins of Bacillus thuringiensis subsp . chinensis CT-43}; Sun M et al.; Bacillus thuringiensis subsp . chinensis CT-43, no flagellum, produces various shaped parasporal crystals, which consist of 140000, 130000, and 65000 proteins . Based on two kinds of mutant, 140000 and 130000 crystal protein individually forms bipyramidal crystal and the 65000 protein forms cubidal crystal, and that the 140000 and 130000 protein is activated by trypsin into 55000 and 66000 proteins and 60000 protein, respectively . Bioassay were conducted to 3rd instar Plutella xylostella larvae with crystals, soluble crystal proteins, and activated crystal proteins, respectively, and it indicated that high toxic mutants can be obtained by curing low toxic crystal genes, and that the toxicity of crystals can be improved 16.3 to 58.4 times after solubilization.

Wei Sheng Wu Xue Bao, 1996 Aug, 36(4), 295 - 302
{Distribution of Bacillus thuringiensis in soils of north and south of China}; Dai S et al.; 221 isolates of Bacillus thuringiensis were isolated in 1491 soil samples from North and South of China . H-serotypes and larvicidal characters of all Bt isolates have been identified . The rate of Bt-harbouring soil sample and the rate of Bt isolates in Northeast and Neimeng were in 12.6% and 17.2% respectively . Predominant serotypes were H4, H10, H3, H13, H5 and H29 . The most fertile Bt-harbouring area was the Heilongjiang Province with rate of Bt-harbouring sample of 21.4% and rate of Bt isolate of 29.4% . Rate of Bt-harbouring sample and rate of Bt isolate in Northwest area were 6.6% and 7.1% respectively . Main serotypes were H4, H5, H19, H10 and H3 . In four provinces of Southern China, both rates above were 18.6% and 29.5%, but frequency of Bt distribution was varied seriously in different distinct . Predominant serotypes in soils from Southern China were H3 and H5 . Results of bioassay showed that the percentage of strains high active to Heliothis armigera and Plogioidera versicolora were 1.6% and 1.1% in soils from North of China . In contrast to North of China, Bt strains active to H . armigera were 5.3% and none of Bt was effective to P . versicolora in South of China . A strain H27-05 was high toxic to H . armigera and showed temperate toxicity to P . versicolora.

Curr Microbiol, 1998 Jul, 37(1), 52 - 7
Biological, immunological, and genetic analysis of Bacillus thuringiensis isolated from granary in Korea; Kim HS et al.; To isolate a naturally occurring novel Bacillus thuringiensis strain, we investigated the distribution, toxicity, morphology, H serotype, and gene type of B . thuringiensis from residue samples of granary in Korea . A total of 163 B . thuringiensis isolates out of 411 samples producing spore and crystal were obtained . In toxicity tests, 80% of all isolates were toxic to lepidoptera, and 12% were not toxic to any of tested insects . And dipteran-active and lepidopteran/dipteran-active isolates were rare (2% and 6%, respectively) . 152 B . thuringiensis isolates produced typical rhomboidal crystals, and the remainder produced parasporal inclusions with various morphologies . Serological test showed that B . thuringiensis isolates in granary represented 12 H serotypes, indicating varied distribution of B . thuringiensis . Of these, the serotype 3ab predominated, followed by the serotype 7 and 4ac . B . thuringiensis isolates of the serotype 3ab, 4ac, 5ab, 7, 8ab, 9, and 23 were toxic to lepidoptera, and the serotype 8bd, 12, 18, and 20ac were nontoxic, while 14 isolates were untypable by 33 B . thuringiensis H antisera . The frequency of toxicity against lepidoptera and diptera was primarily highly toxic . PCR analysis using cryI gene type-specific primers showed that cryIA(b) genes are frequently found and cryIE gene exists in only one isolate . Analysis of B . thuringiensis crystals and plasmid DNAs indicated a diversity of crystal and gene types.

Curr Microbiol, 1998 Jul, 37(1), 6 - 11
Target range of zwittermicin A, an aminopolyol antibiotic from Bacillus cereus; Silo-Suh LA et al.; Zwittermicin A is a novel antibiotic produced by Bacillus cereus UW85, which suppresses certain plant diseases in the laboratory and in the field . We developed a rapid method for large-scale purification of zwittermicin A and then studied the in vitro activity of zwittermicin A against bacteria, fungi, and protists . Zwittermicin A was highly active against the Oomycetes and their relatives, the algal protists, and had moderate activity against diverse Gram-negative bacteria and certain Gram-positive bacteria as well as against a wide range of plant pathogenic fungi . Zwittermicin A was more active against bacteria and fungi at pH 7-8 than at pH 5-6 . When zwittermicin A was combined with kanosamine, another antibiotic produced by B . cereus, the two acted synergistically against Escherichia coli and additively against Phytophthora medicaginis, an Oomycete . The results indicate that there are diverse potential applications of this new class of antibiotic.

Am J Infect Control, 1998 Jun, 26(3), 232 - 8
An effectiveness and cost analysis of presumptive treatment for Mycobacterium tuberculosis; Brewer TF et al.; BACKGROUND: Delay in treatment of tuberculosis has contributed to both the spread of tuberculosis and its case fatality rate . METHODS: Decision analysis was used to examine the effectiveness and cost of presumptive treatment in patients evaluated for tuberculosis . RESULTS: Over a range of assumptions, empiric antituberculous therapy for acid-fast bacillus smear-positive persons lowers mortality and cost per person evaluated when available rapid diagnostic laboratory methods for tuberculosis are used . In contrast, the average cost per life saved by giving presumptive treatment to all acid-fast bacillus smear- and HIV-negative patients exceeds . $1 million . Empiric treatment for HIV-infected patients with acid-fast bacillus-negative smears decreases average mortality by 2% at an additional cost of $8000 per life saved . When the prevalence of multiple-drug resistance exceeds 9.6%, presumptive drug-resistant therapy for acid-fast bacillus smear-positive patients, rather than the initial four-drug regimen recommended for much of the United States, minimizes both mortality and costs . CONCLUSIONS: Empiric antituberculous therapy often minimizes average mortality and cost for patients evaluated for tuberculosis when rapid diagnostic methods are used.

Kekkaku, 1998 May, 73(5), 339 - 47
{Development of the intratracheal infection model of experimental murine mycobacteriosis: comparison with the intravenous infection model}; Doi N; An intratracheal infection method of experimental murine mycobacteriosis was developed for an in vivo study of antimycobacterial agents . Two models of intratracheal (IT) and intravenous (i.v.) routes of infection with mycobacteria of the same inoculum dose were compared in terms of the mean survival days of mice or bacterial loads in organs during the course of infection . IT model with either of M . bovis Ravenel, M . tuberculosis Kurono, M . tuberculosis H37Rv or M . intracellular N-256 exhibited a much more distinct lung-specific infection than i.v . model with the same dose of respective mycobacterial strains . The intratracheal infection method presented in this report does not require any special equipment and is a much safer method for the researcher than airborne infection . In this model, following slight anesthetizing of mice, bacillary suspension was injected quantitatively into lungs through the mouth and trachea by using a specially modified needle set with a short fine polyethylene tube . This IT model may be useful not only for the in vivo assessment of anti-mycobacterial agents but also for the comparison of virulence among various mycobacterial strains.

Kekkaku, 1998 May, 73(5), 329 - 37
{Laboratory media for the cultivation of tubercle bacillus}; Saito H; A variety of different media for the cultivation of mycobacteria have been described but a few of them are in use today . Those currently used can be characterized by three basic types . The first is egg-based media represented by Ogawa and Lowenstein-Jensen . The second type is agar-based media; the most common one are Middlebrook 7H10 and 7H11 . The third type is liquid media such as Middlebrook 7H9 . Several weeks of incubation may be required for the isolation of M . tuberculosis on solid media . Substantial improvement in the time to detection and the recovery rate was realized by using broth-based culture system such as the BACTEC 460TB, Septi-Chek AFB, MGIT and BACTEC 9000 . In the BACTEC 460TB system, the mycobacteria is detected radiometrically . The processed specimen is added to a modified 7H9 medium (BACTEC 12B) containing 14C-labeled palmitic acid and an antibiotic complex, PANTA . Mycobacterial growth can be ascertained by the liberation of 14CO2 and detected by BACTEC 460TB instrument . The Septi-Chek AFB is a biphasic medium which combines broth and solid media . The liquid medium is a modified Middlebrook 7H9 in a carbon-dioxide-enriched culture bottle . After inoculation of the sample, the bottle is capped with a slide consisting of three solid media; a non-selective Middlebrook 7H11 agar, an egg-based medium, and chocolate agar . A novel system is the MGIT, which is a nonradiometric broth method for the detection of mycobacteria from clinical specimens . The MGIT consists of a modified Middlebrook 7H9 broth and a sensor embedded in silicone on the bottom of a tube . The appearance of orange-colored fluorescence in the sensor when excited indicates the growth of mycobacteria . MB Redox is a modified, serum-supplemented Kirchner medium containing p-indonitrotetrazolium violet (INT) as an indicator of microbial growth . The INT is reduced by the redox system of the mycobacteria to deep violet-colored formazan . This substance is water insoluble and is reduced to the cell surface, by which bacterial clamps can be easily detected by their violet color . At present, the egg-based media are the first choice for the culture of clinical samples . However, there are advantages to each type of medium and not all strains of mycobacteria can be recovered on a single medium . Therefore, it is recommended that one representative of each type of medium be used for primary isolation; one example in Japan may be Ogawa egg medium in combination with Middlebrook 7H11 and MGIT.

Arch Biochem Biophys, 1998 Jun 15, 354(2), 263 - 9
Reasoning enantioselectivity and kinetics of seleno-subtilisin from the subtilisin template; Haring D et al.; The active-site serine (Ser221) of subtilisin Carlsberg(from Bacillus licheniformis) and subtilisin BPN' (fromBacillus amyloliquefaciens) was chemically converted into a selenocystein . Contrary to subtilisin's protease activity the semisynthetic seleno-subtilisin catalyzed the reduction of hydroperoxides . Enantioselectivity and kinetics of this reaction were studied by kinetic resolution of five racemic alkyl aryl hydroperoxides catalyzed by the seleno-subtilisin variants . Due to the identical tertiary structure of subtilisin and seleno-subtilisin, the enzymes have comparable substrate binding properties . Thus, a rational screening for suitable peroxidase substrates featuring structural characteristics of known subtilisin substrates was enabled . The enantioselective recognition of (S)-configured alkyl aryl hydroperoxides by seleno-subtilisin was comprehensible by subtilisin's preference for comparable (S)-alkyl aryl amines or alcohols . The analysis of chiral products by multidimensional gas chromatography revealed enantiomeric excesses up to 98% . Kinetics of seleno-subtilisin were rationalized on the basis of the established substrate-catalyst interactions of the subtilisin framework . The Carlsberg and BPN' peroxidase variants revealed typical differences in turnover numbers (kcat) and Michaelis-Menten affinity constants (Km) already known from subtilisin variants . Turnover numbers of seleno-subtilisin BPN' were lower and Km values were higher in comparison to Carlsberg variant . Substrate affinity of several substituted 1-arylethyl hydroperoxides to seleno-subtilisin was reasonable in comparison to corresponding aryl boronic acid inhibitors of subtilisin .

J Immunol, 1998 Jun 15, 160(12), 6112 - 20
Altered intestinal immune system but normal antibacterial resistance in the absence of P-selectin and ICAM-1; Steinhoff U et al.; ICAM-1 and P-selectin are adhesion molecules that regulate leukocyte migration, extravasation to inflammatory sites, and other immune cell interactions . T cell-mediated resistance against acute infection with Listeria monocytogenes and chronic infection with Mycobacterium bovis Calmette-Guerin bacillus was investigated in mutant mice lacking P-selectin and/or ICAM-1 . Mice deficient in P-selectin (Psel-/-), ICAM-1 (ICAM-/-), or the combination of both (Psel-/- x ICAM-/-) showed normal bacterial clearance, comparable delayed-type hypersensitivity reactions, and equivalent memory T cell responses . Additionally, the distribution of alpahbeta vs gammadelta T lymphocyte populations was examined . Normal lymphocyte distributions were noted in thymus, spleen, and blood, whereas mutant mice showed marked alterations in the intestinal intraepithelial (i-IEL) and lamina propria lymphocytes . Differences in i-IEL populations were reflected functionally by differential lytic activities and cytokine productions of i-IEL populations from mutant mice . Despite these changes within the mucosal immune system of mutant mice, their resistance against oral infection with L . monocytogenes was apparently unimpaired . These findings demonstrate that P-selectin and ICAM-1 are critically involved in the shaping of lymphocyte populations of the gut but have only a minor influence on systemic and regional host defense against intracellular bacteria.

Int J Immunopharmacol, 1997 Sep-Oct, 19(9-10), 463 - 8
Down-regulation of tumor necrosis factor-alpha, moderate reduction of interleukin-1beta, but not interleukin-6 or interleukin-10, by glucan immunomodulators curdlan sulfate and lentinan; Masihi KN et al.; The effects of glucan-based immunomodulators curdlan sulfate (CRDS) and lentinan on cytokine production stimulated by lipopolysaccharide (LPS) in bacillus Calmette-Guerin (BCG)-primed mice were investigated . Pretreatment with CRDS or lentinan before LPS administration induced a striking inhibition of up to 89% of circulating tumor necrosis factor-alpha (TNF), a moderate reduction of 25% of interleukin (IL)-1beta, no significant differences in IL-6 or IL-10 levels, and a marked depression of chemiluminescence activity . Animals receiving CRDS prior to infection with alpha-hemolysin positive Escherichia coli inhibited measurable TNF production by 63% . The ability of CRDS and lentinan to significantly reduce the TNF production in vivo indicates the potential of glucans in possible therapeutic strategies that are based on down-regulation of TNF.

Clin Infect Dis, 1998 Jun, 26(6), 1296 - 9
Characterization of Bartonella henselae isolated from bacillary angiomatosis lesions in a human immunodeficiency virus-infected patient in Germany; Arvand M et al.; Infections with Bartonella (Rochalimaea) henselae can result in a variety of clinical entities, including bacillary angiomatosis in immunocompromised hosts . The fastidious nature of this bacterium has so far prevented the culture of many clinical isolates . We report the recovery of the first European B . henselae isolate associated with bacillary angiomatosis . The isolate was cultured in a frozen skin biopsy specimen from a human immunodeficiency virus (HIV)-infected patient and was characterized by means of biochemical, bacteriologic, immunologic, and molecular biological methods including pulsed-field gel electrophoresis . This strain was compared with two B . henselae strains isolated in the United States to determine the relationship between the isolates . We found that it was phenotypically and genotypically indiscernible from B . henselae Houston-1, a blood culture isolate from an HIV-infected patient in Houston . These data suggest that one B . henselae clone is associated with human infections in Europe and the United States.

Biotechnology (N Y), 1996 Feb, 14(2), 171 - 6
Transgenic Indica rice breeding line IR58 expressing a synthetic cryIA(b) gene from Bacillus thuringiensis provides effective insect pest control; Wunn J et al.; The Indica rice breeding line IR58 was transformed by particle bombardment with a truncated version of a synthetic cryIA(b) gene from Bacillus thuringiensis . This gene is expressed under control of the CaMV 35S promoter and allows efficient production of the lepidopteran specific delta-endotoxin . R0, R1 and R2 generation plants displayed a significant insecticidal effect on several lepidopterous insect pests . Feeding studies showed mortality rates of up to 100% for two of the most destructive insect pests of rice in Asia, the yellow stem borer (Scirpophaga incertulas) and the striped stem borer (Chilo suppressalis), and feeding inhibition of the two leaffolder species Cnaphalocrocis medinalis and Marasmia patnalis . Introduction of stem borer resistance into the germplasm of an Indica rice breeding line now makes this agronomically important trait available for conventional rice breeding programs.

Biochemistry, 1998 Jun 23, 37(25), 9001 - 8
The 18 kDa cytochrome c553 from Heliobacterium gestii: gene sequence and characterization of the mature protein; Albert I et al.; The 18 kDa cytochrome c553 is the dominant c-type cytochrome in cell membranes of Heliobacterium gestii . After solubilization, this cytochrome was purified in three steps as a complex with two other proteins of 32 and 42 kDa . The redox midpoint potential of the cytochrome c553 was determined to be +215 mV . The EPR spectra clearly show the presence of an ascorbate-reducible low-spin heme with gz = 3.048 and gy = 2.238 . The gx = trough could not be detected . In addition, a Cu(II) signal with g = 2.058 was observed, indicating that one component of the cytochrome c553 complex contains a bound copper ion . The gene for the 18 kDa cytochrome c553, cyhA, consists of 429 bp coding for a protein of 142 amino acids . The association of the cytochrome with the cytoplasmic membrane is mediated by two fatty acid molecules, one palmitate and one stearate, that could be identified by mass spectrometry . Both fatty acids are most likely bound to the cysteine residue of the N-terminally processed protein via a glycerol moiety . The amino acid sequence deduced from the DNA sequence exhibits partial identity to the membrane-bound cytochrome c551 from Bacillus PS3 {Fujiwara, Y., Oka, M., Hamamoto, T., and Sone, N . (1993) Biochem . Biophys . Res . Commun . 1144, 213-219} and to the cytochrome c subunit (NorC) of the nitrous reductase from Pseudomonas stutzeri {Zumft, W . G., Braun, C., and Cuypers, H . (1994) Eur . J . Biochem . 219, 481-490}.

Biotechnology (N Y), 1995 Apr, 13(4), 362 - 5
Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco; McBride KE et al.; The Bacillus thuringiensis (Bt) crystal toxins are safe biological insecticides, but have short persistance and are poorly effective against pests that feed inside plant tissues . Production of effective levels of these proteins in plants has required resynthesis of the genes encoding them . We report that amplification of an unmodified crylA(c) coding sequence in chloroplasts up to approximately 10,000 copies per cell resulted in the accumulation of an unprecedented 3-5% of the soluble protein in tobacco leaves as protoxin . The plants were extremely toxic to larvae of Heliothis virescens, Helicoverpa zea, and Spodoptera exigua . Since the plastid transgenes are not transmitted by pollen, this report has implications for containment of Bt genes in crop plants . Furthermore, accumulation of insecticidal protein at a high level will facilitate improvement in the management of Bt resistant insect populations.

Biophys J, 1998 Jun, 74(6), 3165 - 72
Tyrosine quenching of tryptophan phosphorescence in glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus; Strambini GB et al.; Tyrosine is known to quench the phosphorescence of free tryptophan derivatives in solution, but the interaction between tryptophan residues in proteins and neighboring tyrosine side chains has not yet been demonstrated . This report examines the potential role of Y283 in quenching the phosphorescence emission of W310 of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus by comparing the phosphorescence characteristics of the wild-type enzyme to that of appositely designed mutants in which either the second tryptophan residue, W84, is replaced with phenylalanine or Y283 is replaced by valine . Phosphorescence spectra and lifetimes in polyol/buffer low-temperature glasses demonstrate that W310, in both wild-type and W84F (Trp84-->Phe) mutant proteins, is already quenched in viscous low-temperature solutions, before the onset of major structural fluctuations in the macromolecule, an anomalous quenching that is abolished with the mutation Y283V (Tyr283-->Val) . In buffer at ambient temperature, the effect of replacing Y283 with valine on the phosphorescence of W310 is to lengthen its lifetime from 50 micros to 2.5 ms, a 50-fold enhancement that again emphasizes how W310 emission is dominated by the local interaction with Y283 . Tyr quenching of W310 exhibits a strong temperature dependence, with a rate constant kq = 0.1 s(-1) at 140 K and 2 x 10(4) s(-1) at 293 K . Comparison between thermal quenching profiles of the W84F mutant in solution and in the dry state, where protein flexibility is drastically reduced, shows that the activation energy of the quenching reaction is rather small, Ea < or = 0.17 kcal mol(-1), and that, on the contrary, structural fluctuations play an important role on the effectiveness of Tyr quenching . Various putative quenching mechanisms are examined, and the conclusion, based on the present results as well as on the phosphorescence characteristics of other protein systems, is that Tyr quenching occurs through the formation of an excited-state triplet exciplex.

Nature, 1998 Jun 11, 393(6685), 537 - 44
Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence; Cole ST et al.; Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus . The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding of the biology of this slow-growing pathogen and to help the conception of new prophylactic and therapeutic interventions . The genome comprises 4,411,529 base pairs, contains around 4,000 genes, and has a very high guanine + cytosine content that is reflected in the blased amino-acid content of the proteins . M . tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation.

J Appl Microbiol, 1998 Apr, 84(4), 501 - 8
delta-Endotoxin proteins associated with spherical parasporal inclusions of the four Lepidoptera-specific Bacillus thuringiensis strains; Wasano N et al.; Four Lepidoptera-specific reference strains of Bacillus thuringiensis, belonging to serovars sumiyoshiensis (H3a:3d), fukuokaensis (H3a:3d:3e), darmstadiensis (H10a:10b) and japonensis (H23), which produce spherical parasporal inclusions, were examined for comparative characterization of delta-endotoxins . SDS-PAGE profiles of the alkali-solubilized parasporal inclusions revealed the presence of single major protein bands of 130 kDa in the four strains . Chymotrypsin and trypsin treatment of the proteins gave profiles different from those of the strains HD-1 (serovar kurstaki, H3a:3b:3c) and T84 A1 (serovar sotto, H4a:4b) . Also, minor variations were observed in proteolysis profiles among the four strains . The LC50 values of purified parasporal inclusions for the silkworm (Bombyx mori) larvae were 7.35, 6.45, 3.08 and 2.63 micrograms g-1 diet, respectively, showing that their toxicity levels were 5-15 times lower than that of the strain HD-1 (0.49 microgram g-1 diet) . Analysis by immunodiffusion and immunoblotting with polyclonal antisera revealed that parasporal inclusion proteins of the four strains are highly related, whereas they shared few or no common antigens with those of the strains HD-1, T84 A1 and Buibui (serovar japonensis).

Arch Biochem Biophys, 1998 Jun 1, 354(1), 31 - 9
Polysaccharide lyase: molecular cloning of gellan lyase gene and formation of the lyase from a huge precursor protein in Bacillus sp . GL1; Hashimoto W et al.; A bacterium, Bacillus sp . GL1, produced constitutively the extracellular polysaccharide-degrading enzyme (gellan lyase) with a molecular mass of 140 kDa . A genomic DNA library of the bacterium was constructed in Escherichia coli using the cosmid vector, Charomid 9-36 . The gene encoding the lyase was cloned by screening for a gellan-degrading phenotype in E . coli cells and the nucleotide sequence of the gene was determined . The gene contained an open reading frame consisting of 7425 base pairs coding a polypeptide with a molecular mass of 263 kDa . The polypeptide contained the same amino acid sequence as N-terminal amino acid sequence of the enzyme and exhibited no homology with any previously published protein sequences . E . coli cells transformed with the gene exhibited gellan lyase activity and produced a protein with a molecular mass of about 260 kDa intracellularly . The protein was purified and shown to have the closely similar enzymatic properties to those of the native enzyme from Bacillus sp . GL1 with respect to optimal pH and temperature for activity, substrate specificity, and the mode of enzyme action . These results suggest that, in Bacillus sp . GL1, gellan lyase is first produced as a huge precursor protein (263 kDa) and then the protein is posttranslationally processed into extracellular mature form (140 kDa) through excising C-terminal peptide of about 120 kDa.

Biochemistry, 1998 May 26, 37(21), 7664 - 9
Pre-steady state kinetic analysis of an enzymatic reaction monitored by time-resolved electrospray ionization mass spectrometry; Zechel DL et al.; For the first time, the new technique of time-resolved electrospray ionization mass spectrometry (ESI-MS) has been used to accurately measure the pre-steady state kinetics of an enzymatic reaction by monitoring a transient enzyme intermediate . The enzyme used to illustrate this approach, Bacillus circulans xylanase, is a retaining glycosidase that hydrolyzes xylan or beta-xylobiosides through a double-displacement mechanism involving a covalent xylobiosyl-enzyme intermediate . A low steady state level of this intermediate formed during the hydrolysis of 2,5-dinitrophenyl beta-d-xylobioside was detected by time-resolved ESI-MS . The low concentration of this intermediate and its rate of formation did not permit pre-steady state kinetic analysis . By contrast, the covalent intermediate accumulates fully when the Tyr80Phe mutant hydrolyzes the same substrate . Using time-resolved ESI-MS, the pre-steady state kinetic parameters for the formation of the covalent intermediate in the mutant xylanase have been determined . The kinetic data are in agreement with those determined by monitoring the release of 2, 5-dinitrophenol with stopped-flow UV-vis spectroscopy . This demonstrates that time-resolved ESI-MS can be used to accurately monitor the pre-steady state kinetics of enzymatic reactions, with the advantage of identifying transient enzyme intermediates by their mass.

Aust N Z J Surg, 1998 May, 68(5), 340 - 4
Bacillus Calmette-Guerin (BCG) immunotherapy for bladder cancer: review of complications and their treatment; Paterson DL et al.; BACKGROUND: Intravesical bacillus Calmette-Guerin (BCG) is widely used in the management of bladder cancer but because it is a living organism, local and disseminated infection may result . METHODS: A prospective assessment of complications of this therapy in 200 patients in Queensland was performed . A review of management of complications of intravesical BCG was also carried out . RESULTS: Major side effects were rare . Cystitis was the most common side effect, being seen to some degree in all patients, although only forcing cessation of BCG therapy in two patients . Two patients developed persistent cystitis necessitating institution of isoniazid and rifampicin . Two patients had culture-proven bladder infection that presented several months after the BCG treatment . These patients also responded to two-drug antituberculous therapy . While low-grade fever is very common with this therapy, seven patients (3.5%) had fevers of > 39 degrees C within 48 h of receiving BCG . Fevers may be an indication of severe disseminated mycobacterial infection, which has a high mortality, so it needs to be treated aggressively . Alternatively bacterial sepsis with gram-negative bacterial pathogens or a hypersensitivity reaction to BCG may cause this degree of fever, and cannot be rapidly distinguished from fulminant mycobacterial infection . One patient in the present series developed pneumonia attributed to mycobacterial dissemination . CONCLUSIONS: The key to appropriate management of complications of BCG therapy is awareness of their possibility, even months or years after the therapy has been given . Appropriate empirical therapy in acute situations and mycobacterial culture in chronic situations can then be performed.

Oncol Rep, 1998 Jul-Aug, 5(4), 823 - 6
Immunisation of colorectal cancer patients with autologous tumour cells; Diederichsen AC et al.; Patients with colorectal cancer were entered into a clinical phase I trial of immunotherapy with an autologous tumour cell/bacillus Calmette-Guerin (BCG) vaccine . We attempted to describe the possible effects and side effects of the immunisation, and further to investigate whether expression of immune-response-related surface molecules on the tumour cells in the vaccine correlated with survival . The first and second vaccine comprised of 107 irradiated tumour cells mixed with BCG, the third of irradiated tumour cells only . Thirty-nine patients were considered, but only 6 patients fulfilled the criteria for inclusion . No serious side effects were observed . With three years of observation time, two patients are healthy, while the rest have had recurrence, and two of them have died . In all vaccines, all tumour cells expressed HLA class I, some expressed HLA class II and none expressed CD80 . There was an inverse relation between survival and HLA class II expression . This highlights an essential problem, in the absence of CD80 expression the expression of HLA class II may induce anergy . In future attempts to develop improved vaccines this problem should be addressed.

J Biochem (Tokyo), 1998 May, 123(5), 932 - 6
An assay method for glycogen debranching enzyme using new fluorogenic substrates and its application to detection of the enzyme in mouse brain; Omichi K et al.; An assay method for glycogen debranching enzyme involving fluorogenic dextrins as substrates was developed . Two dextrins were prepared from 6-O-alpha-D-glucosyl-alpha-cyclodextrin and glucose by taking advantage of the action of Bacillus macerans cyclodextrin glucanotransferase, and converted by pyridylamination to fluorogenic derivatives . Structural analysis of the fluorogenic dextrins by FAB-MS, partial acid hydrolysis, and glucoamylase digestion revealed that they were Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glcalpha1- 4Glcalpha1-4Glc-PA (FD6) and Glcalpha1-4Glcalpha1-4(Glcalpha1-6)Glcalpha1- 4Glcalpha1-4Glcalpha1-4G lc-PA (FD7) . Using the glycogen debranching enzyme from rabbit muscle, FD6 and FD7 were, respectively, hydrolyzed to PA-maltopentaose and PA-maltohexaose, in addition to glucose, showing that these two fluorogenic dextrins are suitable substrates for assaying the glycogen debranching enzyme . An assay method involving the separation and quantification by HPLC of the characteristic fluorogenic products was successfully applied to determination of the distribution of the enzyme activity in mouse cerebrum.

Biol Chem, 1998 Apr-May, 379(4-5), 569 - 71
A pair of single-strand and double-strand DNA cytosine-N4 methyltransferases from Bacillus centrosporus; Merkiene E et al.; Sequence analysis of the BcnI restriction-modification system revealed the presence of an open reading frame encoding a second cytosine-N4 methyltransferase, M.BcnIA, in the vicinity of the genes specifying the previously characterized cytosine-N4 methyltransferase M.BcnIB and restriction endonuclease R.BcnI . Both methyltransferases were purified from the E . coli cells expressing the individual genes, and their enzymatic efficiencies in vitro were compared with a variety of DNA substrates . Both enzymes act on 5'-CC(C/G)GG-3' sites in double-stranded DNA, however, M.BcnIA can also, with a comparable efficiency, modify the specific targets in single-stranded DNA . The biological significance of the presence of the tandem methyltransferases in the BcnI system is discussed.

Acta Paediatr, 1998 Apr, 87(4), 458 - 9
Routine vaccination and vaccine-preventable infections in children born to human immunodeficiency virus-infected mothers . European Collaborative Study; Dunn DT et al.; Information on vaccinations and vaccine-preventable infections collected in a prospective study of children born to human immunodeficiency virus (HIV)-infected mothers was analysed for reports of adverse reactions and to estimate the clinical efficacy of vaccines . No vaccinated, HIV-infected child developed measles (56 child-years' follow-up), mumps (33), rubella (33) or pertussis (239), and only one adverse reaction - to Bacillus Calmette-Guerin (BCG) - was reported . These findings provide limited evidence of the safety and efficacy of routine vaccination of HIV-infected children.

Appl Biochem Biotechnol, 1998 Spring, 70-72, 207 - 13
Isolation, identification, and keratinolytic activity of several feather-degrading bacterial isolates; Zaghloul TI et al.; Several feather-degrading bacterial isolates were isolated from Egyptian soil . These isolates were able to degrade chicken feather, when grown on basal medium containing 1% native feather as a source of energy, carbon, and nitrogen . Feather waste, generated in large quantities as a byproduct of commercial poultry processing, is nearly pure keratin, which is not easily degradable by common proteolytic enzymes . The isolates were identified according to the morphological characteristics, biochemical tests, and API 50 CHB Bacillus system . Proteolytic and keratinolytic activities of these isolates were monitored throughout the cultivation of the bacterial isolates on feather . Resulting soluble proteins, which were released as a result of the biodegradation of feather, were demonstrated by SDS-PAGE.

Scand J Immunol, 1998 May, 47(5), 453 - 8
Internalization of Mycobacterium bovis Bacillus Calmette-Guérin into osteoblast-like MC3T3-E1 cells and bone resorptive responses of the cells against the infection; Hotokezaka H et al.; Mycobacterium bovis BCG (BCG) is a live vaccine used worldwide against tuberculosis . However, it has unfavourable side effects such as osteitis or osteomyelitis, and these sometimes lead to vertebral caries in some patients as a result of bone resorption . Osteoblasts might play a role in the bone resorption caused by BCG infection, because they are central cells in bone metabolism . Cultured osteoblast-like cell lines (MC3T3-E1) derived from C57BL mice susceptible to BCG infection cells were infected with BCG at several doses . Interestingly, internalization of BCG-enveloped phagosome-like membrane in osteoblast-like cells were observed by transmission electron microscopy (TEM) . Owing to infection, the proliferation and alkaline phosphatase activity of the osteoblast-like cells were reduced in a dose-dependent manner . On the other hand, interleukin (IL)-6 production was considerably enhanced by infection . These results suggest that BCG infects osteoblasts, suppressing their proliferation and differentiation and inducing bone resorption, which may be related to osteitis/osteomyelitis and bone caries caused by BCG infection.

FEMS Immunol Med Microbiol, 1998 Apr, 20(4), 311 - 8
Affinity purification of recombinant cholera toxin B subunit oligomer expressed in Bacillus brevis for potential human use as a mucosal adjuvant; Yasuda Y et al.; For use as a mucosal adjuvant for human vaccines, a simple method has been developed for the affinity purification of recombinant cholera toxin B subunit which had been expressed in a safe host, Bacillus brevis . Recombinant cholera toxin B subunit, adsorbed quantitatively to a D-galactose-agarose column, was eluted with an 0.1-0.4 M D-galactose gradient with a yield of > 90% . The cholera toxin B subunit preparation was similar to the native cholera toxin B subunit with respect to GM1 binding ability, remarkable stability of the pentamer, and the dissociation-reassociation property by shifting pHs . Cross-linking experiments with glutaraldehyde demonstrated that the pentameric form was predominant; tetrameric, trimeric, dimeric and monomeric forms were detected to a lesser extent, and additionally 10- and 15-mers were observed depending on the concentration of the cholera toxin B subunit.

J Virol, 1998 Jul, 72(7), 6024 - 33
Particle polymorphism caused by deletion of a peptide molecular switch in a quasiequivalent icosahedral virus; Dong XF et al.; The capsid of flock house virus is composed of 180 copies of a single type of coat protein which forms a T=3 icosahedral shell . High-resolution structural analysis has shown that the protein subunits, although chemically identical, form different contacts across the twofold axes of the virus particle . Subunits that are related by icosahedral twofold symmetry form flat contacts, whereas subunits that are related by quasi-twofold symmetry form bent contacts . The flat contacts are due to the presence of ordered genomic RNA and an ordered peptide arm which is inserted in the groove between the subunits and prevents them from forming the dihedral angle observed at the bent quasi-twofold contacts . We hypothesized that by deleting the residues that constitute the ordered peptide arm, formation of flat contacts should be impossible and therefore result in assembly of particles with only bent contacts . Such particles would have T=1 symmetry . To test this hypothesis we generated two deletion mutants in which either 50 or 31 residues were eliminated from the N terminus of the coat protein . We found that in the absence of residues 1 to 50, assembly was completely inhibited, presumably because the mutation removed a cluster of positively charged amino acids required for neutralization of encapsidated RNA . When the deletion was restricted to residues 1 to 31, assembly occurred, but the products were highly heterogeneous . Small bacilliform-like structures and irregular structures as well as wild-type-like T=3 particles were detected . The anticipated T=1 particles, on the other hand, were not observed . We conclude that residues 20 to 30 are not critical for formation of flat protein contacts and formation of T=3 particles . However, the N terminus of the coat protein appears to play an essential role in regulating assembly such that only one product, T=3 particles, is synthesized.

Biochemistry, 1998 May 5, 37(18), 6513 - 22
Phosphatidylcholine activation of bacterial phosphatidylinositol-specific phospholipase C toward PI vesicles; Qian X et al.; The effect of different phospholipids on the kinetic behavior of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis toward PI vesicles has been investigated . Cosonicated PC/PI vesicles displayed enhanced hydrolysis of PI when less than 0 . 20 mole fraction PC was incorporated into the vesicle; higher mole fractions of PC led to a decrease from the maximum activity mimicking surface dilution of substrate . Since the PC could affect PI-PLC binding to vesicles, the effect of separate PC vesicles on enzymatic hydrolysis of PI vesicles was examined . Separate phosphatidylcholine vesicles were found to activate PI-PLC-catalyzed cleavage of PI vesicles up to 7-fold . The activation was completely abolished when the PC vesicle was composed of cross-linked molecules . In the absence of enzyme, fluorescence resonance energy transfer studies did not detect any fusion between PI and PC vesicles if the total lipid concentration was below 2 mM . Higher total lipid concentrations (>20 mM) increased PC transfer between PC and PI vesicles, producing a PI vesicle population with small amounts of PC in the outer monolayer . This suggested that the activation of PI-PLC toward PI vesicles reflects the time scale of transfer of PC from PC vesicles to PI vesicles . Cosonicated PC/PI vesicles provide a measure of enzyme activity versus mole fraction of PC that can be used to estimate the extent of vesicle exchange or fusion between separate vesicle pools . The effects of other phospholipid vesicles on PI-PLC hydrolysis of PI were also examined; zwitterionic lipids were activators while anionic phospholipids inhibited activity . The results indicated that PC molecules in the PI interface allosterically bind to PI-PLC and help anchor enzyme in a more active conformation to the PI interface.

Cell Mol Biol (Noisy-le-grand), 1998 May, 44(3), 557 - 61
An improvement in the dissociating properties of sodium dodecyl sulfate-containing sample buffers used in polyacrylamide gel electrophoresis; Garcia-Patrone M et al.; Protein complexes present different degrees of stability . We have previously described a glycoprotein from Bacillus thuringiensis that appeared as a multimer unable to be dissociated by the usual SDS-containing sample buffers of pH 6.8 . In order to dissociate the complex, a SDS-containing sample buffer of pH 9 was described . In the present report three additional protein complexes with different degrees of stability and the effect of that dissociating sample buffer are described . The study of SDS critical micellar concentration values as a function of pH explains the improvement of dissociating properties at pH 9.

Biochem Biophys Res Commun, 1998 May 29, 246(3), 933 - 4
Volume 244, Number 3 (1998), in Article No . RC988170, "Induction of CYP102 (Cytochrome P450BM-3) in Bacillus megaterium by 17 beta-Estradiol and 4-sec-Butylphenol," by Nancy Eddy Hopkins, Neil English, Valerie Hughes, Christopher W . Rowley, C . Roland Wolf, and William L . Alworth, pages 868-872:
Escherichia coli ribosomal protein L3 stimulates the helicase activity of the Bacillus stearothermophilus PcrA helicase.
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UKEscherichia coli ribosomal protein L3 stimulates the in vitro helicase activity of Bacillus stearothermophilus PcrA helicase upon a variety of different substrates . L3 has no intrinsic helicase or ATPase activity nor is it able to stimulate the ATPase activity of PcrA . Gel mobility shift assays revealed that the affinity of PcrA for a variety of different DNA species (single-stranded, nicked and 3'-tailed) was enhanced in the presence of L3 . We suggest that the stimulatory effect of L3 upon the helicase activity of PcrA is mediated via a protein-protein interaction which promotes cooperative binding of PcrA to its DNA substrate . This activity of L3 appears to be specific for PcrA helicase.

Hum Exp Toxicol, 1998 Apr, 17(4), 231 - 8
Biovolatilization of antimony and sudden infant death syndrome (SIDS); Jenkins RO et al.; 1 . The aerobic filamentous fungus S . brevicaulis IMI 17297 methylated antimony from Sb2O3 substrate, with the formation of gaseous trimethylantimony (TMA) . No evidence was found for the generation of other gaseous antimony compounds by this organism . 2 . Biovolatilization of inorganic antimony was greatest during cultivation of the fungus on solid media at 25 degrees C, and occurred more readily from antimony (III) substrates than from antimony (V) substrates . 3 . Under simulated cot environment conditions (CO2 enriched atmosphere, 33 degrees C) the fungus exhibited an altered morphology and a reduced capability to volatilize inorganic antimony from the pure compound . 4 . No evidence of antimony biovolatilization from cot mattress PVC was found, unless antimony was released from PVC by heat treatment (at 80 or 100 degrees C) . 5 . These data suggest that normal cot environment conditions are non-optimal for volatilization of antimony by S . brevicaulis, and that Sb2O3 in cot mattress PVC is not bioavailable . 6 . Cot mattress isolates of S . brevicaulis also volatilized antimony (not encapsulated by PVC), whereas those of other filamentous fungi (Penicillium spp., Aspergillus niger, Aspergillus fumigatus, Alternaria sp.) and of bacteria (Bacillus spp.) did not . 7 . The oxidation products of TMA may be the true determinants of toxicity for biogenic antimony gases produced in an aerobic environment.

Biosci Biotechnol Biochem, 1998 Apr, 62(4), 727 - 34
Cloning, sequencing, and expression of the Bombyx mori receptor for Bacillus thuringiensis insecticidal CryIA(a) toxin; Nagamatsu Y et al.; Bacillus thuringiensis strains produce insect-specific Bt toxins . Bt CryIA(a) toxin binds to a 175-kDa glycoprotein (BtR175) on the microvillus membranes of columnar cells in the Bombyx mori midgut and causes lysis of the cells . BtR175 was purified, and its cDNA was cloned . The cDNA encodes a newly identified 193.3-kDa preproprotein form of BtR175 that includes nine extracellular cadherin repeats, a 23.5-kDa membrane-proximal domain, a membrane-spanning region, and a 13.6-kDa cytoplasmic domain . Spodoptera frugiperda cells transfected with a recombinant baculovirus DNA carrying the cDNA produced a 175-kDa protein that reacted with anti-BtR antibodies and the Bt CryIA(a) toxin.

Folia Microbiol (Praha), 1998, 43(1), 23 - 6
Degradation of fenitrothion by Bacillus stearothermophilus adhering to silica; Kumari B et al.; B . stearothermophilus strain AG-49, when cultivated in mineral medium in the presence of silica (SA), adhered to SA . Adhesion depended on age of culture, contact time and glucose concentration of the culture medium . Mid-exponential phase culture (5 h) required minimum contact time (30 min) for maximum adhesion . 0.6% glucose concentration was optimum . Quantitative variation in protein and saccharide extractable in sodium chloride and sodium dodecyl sulfate (SDS) was observed . Five % degradation of fenitrothion by adherent B . stearothermophilus could be achieved in 4 d.

Ultrastruct Pathol, 1998 Mar-Apr, 22(2), 127 - 33
Fibrous long-spacing collagen in bacillary angiomatosis; Borczuk AC et al.; Fibrous long-spacing (FLS) collagen is a distinct ultrastructural form of collagen present in normal tissue, various tumors, and tissues degraded by bacterial collagenases in vivo and in vitro . An association between FLS collagen and bacillary angiomatosis has not been previously described . Six cases of bacillary angiomatosis, including one autopsy case with disseminated disease, were examined ultrastructurally . In addition, Kaposi sarcoma (3), pyogenic granuloma (3), capillary hemangioma (3), and cavernous hemangioma (2) were examined for comparison . A vascular proliferation in a lymph node from a patient with AIDS (1) and a case of pulmonary capillary hemangiomatosis (1), also in an AIDS patient, were studied . Abundant FLS collagen was identified in 4 of 6 cases of bacillary angiomatosis, in close association with the organisms . FLS collagen was not seen beyond the immediate vicinity of the organisms . The FLS collagen in bacillary angiomatosis was seen in skin biopsies and in lung and skeletal muscle in the autopsy case; in the latter case, as well as in the two AIDS-associated, nonbacillary angiomatosis, non-Kaposi sarcoma vascular proliferations, there was a striking distribution of FLS collagen around small blood vessels . Occasional FLS collagen was observed in all three pyogenic granuloma . When present in pyogenic granuloma, FLS collagen was intermixed with subendothelial collagen . Abundant FLS collagen was identified in close association with the organisms of bacillary angiomatosis in four cases; this morphologic alteration was seen in skin as well as lung and skeletal muscle . An association between FLS collagen and endothelial cells in normal tissue (Descemet's membrane) and in certain vascular proliferations appears to exist.

Klin Khir, 1997, (11-12), 69 - 71
{Immune reaction to the heterologic test-antigen in mice with anti-cancer vaccine}; Potebnia GP et al.; The stimulated influence of the antitumoral vaccine, prepared with the help of filtrate of the Bacillus mesentericus AB-56 or the antibiotic, extracted from it, on the immune answer to heterologic test-antigen (ram erythrocytes) was established . While the vaccine injection to laboratory animals with developing tumor their capacity to resist to the tumors was rising up.

J Mol Biol, 1998 May 15, 278(4), 871 - 8
On the global architecture of initiation factor IF3: a comparative study of the linker regions from the Escherichia coli protein and the Bacillus stearothermophilus protein; Hua Y et al.; Initiation factor IF3 is a protein involved in the initiation stage of protein synthesis . It consists of two global domains linked by a 20 residue long, solvent-exposed linker . Recently, the structure of the N and C-terminal domains of the Bacillus stearothermophilus protein have been solved by X-ray crystallography and the structure of the intact Escherichia coli protein has been studied by NMR . These two studies have led to apparently contradictory models for the domain organization of IF3 . The NMR study of the E . coli protein indicates that the linker region is flexible, while the studies of the isolated N and C-terminal domains of the B . stearothermophilus protein suggest that the linker forms a rigid helical rod . In order to resolve this discrepancy, a set of peptides corresponding to the linker regions of the B . stearothermophilus and the E . coli protein were synthesized . Circular dichroism and NMR spectroscopy were used to study the helical content as a function of pH, temperature, peptide concentration and ionic strength . Both peptides are monomeric . The estimated helical content of the linker fragment from B . stearothermophilus is 68% at high pH and 1 degree C . The measured helicity decreases to 53% at pH 7.0 and 1 degree C . In contrast, the peptide corresponding to the E . coli IF3 linker region is largely unstructured with a maximum helical content of 15% at high pH and only 8% at pH 7.0, 1 degree C . These results suggest that the different structures observed for the two intact proteins may be due to the different intrinsic stability of the two linker peptides . The helical content of the two linker peptides is, however, much closer when the peptides are compared at the respective temperatures of optimum growth for E . coli and B . stearothermophilus (3% versus 17%) . The pH and ionic strength dependence of the helical content of the B . stearothermophilus peptide demonstrates that side-chain/side-chain interactions play an important role in stabilizing the helical structure . In addition, studies with mutant peptides show that the first Asp residue in the linker sequence helps to stabilize the helix via an N- capping interaction.

Biosci Biotechnol Biochem, 1998 Apr, 62(4), 795 - 7
Purification and properties of acetylacetoin synthase from Bacillus sp . YUF-4; Ui S et al.; In Bacillus sp . YUF-4, acetylacetoin synthase was induced by acetoin, while glucose inhibited the induction . The enzyme was purified 111-fold by 6 purification steps, and a further purification followed, by HPLC using a TSK gel, Phenyl-5PW RP . The resulting enzyme gave a single band with a molecular mass of 62 kDa by SDS-PAGE and 220 kDa by gel filtration . Some enzymic characteristics were studied.

Biosci Biotechnol Biochem, 1998 Apr, 62(4), 718 - 26
Identification of Bombyx mori midgut receptor for Bacillus thuringiensis insecticidal CryIA(a) toxin; Nagamatsu Y et al.; As part of a study of the mechanism by which Bacillus thuringiensis insecticidal crystal protein acts, a Bombyx mori receptor to the CryIA(a) toxin specific for lepidopterans was examined . Histological examination showed that the toxin acted on the brush-border membrane of the midgut columnar cells and broke its infolding structure, causing cell lysis . The membrane vesicles were purified, and a 175-kDa protein binding the toxin was found that accounted for some 0.015% of membrane proteins . The protein, designated BtR175, was a glycoprotein that reacted with concanavalin A . Anti-BtR antibodies inhibited the binding of toxin to membrane vesicles in vitro and decreased the effect of the toxin to silkworms in vivo . BtR175, although found in the gut, was not found in fat bodies, integument, or silk glands . These results indicated that BtR175 was the receptor protein for the insecticidal toxin . Proteins (137 and 107 kDa) binding the CryIA(a) toxin also were found in the gut membranes of Tenebrio moritor larvae, a coleopteran not sensitive to the toxin . The specificity of the toxin could not be explained only in term of the existence of its binding protein.

Biochemistry, 1998 May 12, 37(19), 6958 - 66
Discrepancies between the NMR and X-ray structures of uncomplexed barstar: analysis suggests that packing densities of protein structures determined by NMR are unreliable; Ratnaparkhi GS et al.; The crystal structure of the C82A mutant of barstar, the intracellular inhibitor of the Bacillus amyloliquefaciens ribonuclease barnase, has been solved to a resolution of 2.8 A . The molecule crystallizes in the space group I41 with a dimer in the asymmetric unit . An identical barstar dimer is also found in the crystal structure of the barnase-barstar complex . This structure of uncomplexed barstar is compared to the structure of barstar bound to barnase and also to the structure of barstar solved using NMR . The free structure is similar to the bound state, and there are no significant main-chain differences in the 27-44 region involved in barstar binding to barnase . The C82A structure shows significant differences from the average NMR structure, both overall and in the binding region . In contrast to the crystal structure, the NMR structure shows an unusually high packing value based on the occluded surface algorithm, indicating errors in the packing of the structure . We show that the NMR structures of homologous proteins generally show large differences in packing value, while the crystal structures of such proteins have very similar packing values, suggesting that protein packing density is not well determined by NMR.

Proc Natl Acad Sci U S A, 1998 Apr 28, 95(9), 5299 - 304
Mycobacterium bovis Bacille Calmette-Guérin strains secreting listeriolysin of Listeria monocytogenes; Hess J et al.; Recombinant (r) Mycobacterium bovis strains were constructed that secrete biologically active listeriolysin (Hly) fusion protein of Listeria monocytogenes . The r-BCG strains pAT261:Hly or pMV306:Hly expressed plasmid multicopies or chromosomal single copies of the hly gene, respectively . Human and murine macrophage-like cell lines were infected with r-BCG pAT261:Hly and pMV306:Hly strains . Interestingly, intracellular persistence of both r-BCG strains was reduced in macrophages as compared with the parental BCG strain . By immunogold labeling Hly was detected in membrane structures and within the phagosomal space of macrophages . In addition, Hly was localized within cytoplasmic vacuoles outside the mycobacteria-containing phagosome of host cells infected with r-BCG pAT261:Hly or r-BCG pMV306:Hly . Hly fusions consistently colocalized with a lysosome-associated membrane glycoprotein, suggesting that membrane-attack conformation of Hly was not altered . Although r-BCG pAT261:Hly and r-BCG pMV306:Hly microorganims apparently did not egress into the cytoplasmic compartment of host cells, they both improved major histocompatibility complex class I presentation of cophagocytosed soluble protein as compared with wild-type BCG microbes . These data suggest that Hly secretion endows BCG with an improved capacity to stimulate CD8 T cells . Because CD8 T cells play a major role in protection against tuberculosis such Hly secreting r-BCG constructs are antituberculosis vaccine candidates.

Parazitologiia, 1998 Jan-Feb, 32(1), 11 - 20
{The relationship of the larval behavioral traits of the malarial mosquito Anopheles messeae (Diptera: Culicidae) to its sensitivity to the entomopathogenic bacterium Bacillus thuringiensis subspecies israelensis}; Burlak VA; Behavioural actions of the malarial mosquito Anopheles messeae larvae and their correlations with the susceptibility to the entomopathogenic bacterium Bacillus thuringiensis israelensis (Bti) were studied . The larvae were differentiated according to their feeding mode ("filter-feeders" and "scraper-feeders") and to their avoidance reaction on a concussion of water surface by water drops ("divers" and "undivers") . The "scraper-feeders" and "divers" showed a tendency of decreased susceptibility to Bti, while the "filter-feeders" and "undivers" did not . The presence of the white pigment (striped genotypes) on a dorsum of the larvae correlated with the reaction of avoidance and phyllotaxis but not with the susceptibility to Bti.

Mol Gen Mikrobiol Virusol, 1998, (2), 32 - 5
{Isolation and specificity of novel restriction endonucleases Bsp40091 and AsiI, isoschizomers of BamHI}; Korolev SV et al.; New type II restriction endonucleases AsiI and Bsp40091 are detected in Azotobacter species N55 and Bacillus species 4009, respectively . Purified preparations of the restriction enzymes free from interfering nucleases and phosphatases were obtained by column chromatography on phosphocellulose and heparin-sepharose (Asil) and phosphocellulose and DEAE-cellulose (Bsp40091) . The yield of purified AsiI and Bsp40091 was 16 x 10(3) and 8 x 10(3) units per g of wet cells, respectively . The above restriction endonucleases recognize the 5'-G decreases GATCC-3' sequence on double-stranded DNA and cleave it as shown, thus being true isoschizomers of BamHI restriction endonuclease.

Gene, 1998 Jun 8, 212(2), 167 - 77
The Bacillus stearothermophilus argCJBD operon harbours a strong promoter as evaluated in Escherichia coli cells; Savchenko A et al.; We have shown that the B . stearothermophilus argCJBD genes form a single operon . In B . stearothermophilus, a specific repressor governs operon expression by binding to the argCo operator site overlapping the Parg promoter sequence (Dion et al., 1997) . Therefore, the enzymatic and transcriptional analyses performed in this work did not reflect the potential strength of Parg in the native host . For evaluation of the Parg promoter strength, E . coli was used as a host since its own ArgR repressor does not interact with the B . stearothermophilus heterologous operator . Parg-promoted argC gene expression dramatically increased, reaching up to 38% of the total protein in E . coli cells . An AT-rich sequence upstream of a -35 site of Parg was found to be indispensable for the promoter strength . Plasmids carrying the B . stearothermophilus argCJBD operon linked with its Parg/argCo region were unstable in E . coli . Stabilization of plasmids was achieved by repression of B . stearothermophilus arg genes through the action of the B . subtilis AhrC repressor.

PDA J Pharm Sci Technol, 1998 Mar-Apr, 52(2), 60 - 5
Selection of biological indicator for validating microwave heating sterilization; Sasaki K et al.; For the purpose of selecting an appropriate biological indicator for evaluation of the effects of microwave heating sterilization, we examined aerobic bacterial spores to determine whether microwaves have non-thermal sterilization effects . After microwave irradiation on dry bacterial spores (three species), none of the bacterial spores were killed . The survival rate of the spores after microwave irradiation of spore suspensions (twelve species) was compared with that after heating by a conventional method . The order of heat resistance in the bacterial species was similar between the two heating methods . Bacillus stearothermophilus spores were the most heat-resistant . These results suggest that microwaves have no non-thermal sterilization effects on bacterial spores, the specific resistant spores to microwave heating, and microwave heating sterilization can be evaluated in the same way as for conventional heating sterilization . As a biological indicator for evaluation of overkill sterilization, B . stearothermophilus spores may be appropriate for microwave heating sterilization as well as steam sterilization.

Appl Environ Microbiol, 1998 Jun, 64(6), 2158 - 65
Effects of midgut-protein-preparative and ligand binding procedures on the toxin binding characteristics of BT-R1, a common high-affinity receptor in Manduca sexta for Cry1A Bacillus thuringiensis toxins; Keeton TP et al.; The identity of the physiologically important Cry1A receptor protein(s) in the lepidopteran Manduca sexta has been a matter of dispute due to the multiple proteins which bind the Cry1Ac toxin . Cry1Aa, Cry1Ab, and Cry1Ac exhibit essentially identical toxicities toward M . sexta larvae and show a high degree of sequence and presumed structural identities . These similarities make it likely that there is a common mechanism of toxicity in these lepidopteran-specific toxins in terms of both mode of action and the receptor proteins through which these toxins exert their lepidopteran-specific toxicity . Investigators in our laboratory previously demonstrated that the cloned 210-kDa glycoprotein BT-R1 binds all three Cry1A toxins (T . P . Keeton and L . A . Bulla, Jr., Appl . Environ . Microbiol . 63:3419-3425, 1997) . This protein remains a common binding protein even after being subjected to various midgut membrane preparation and processing protocols . The method used to isolate proteins from the M . sexta larval midgut in no significant way affects the results of ligand binding and vacuum blotting experiments, and we have been unable to detect specific, high-affinity binding of any Cry1A toxin to Cry1Ac binding proteins other than BT-R1 . Alterations in blot substrate and blocking, hybridization, and washing buffers support these conclusions . Collectively, these results indicate that in M . sexta the cadherin-like BT-R1 protein is a common high-affinity receptor protein for the Cry1A family of toxins.

Microbiologia, 1997 Dec, 13(4), 453 - 62
Gram-positive bacteria of marine origin: a numerical taxonomic study on Mediterranean isolates; Ortigosa M et al.; A numerical taxonomic study was performed on 65 Gram-positive wild strains of heterotrophic, aerobic, marine bacteria, and 9 reference strains . The isolates were obtained from oysters and seawater sampled monthly over one year, by direct plating on Marine Agar . The strains were characterized by 96 morphological, biochemical, physiological and nutritional tests . Clustering yielded 13 phena at 0.62 similarity level (Sl coefficient) . Only one of the seven phena containing wild isolates could be identified (Bacillus marinus) . A pronounced salt requirement was found in most isolates.

Appl Environ Microbiol, 1998 Jun, 64(6), 2187 - 91
Structure of the beta-galactosidase gene from Thermus sp . strain T2: expression in Escherichia coli and purification in a single step of an active fusion protein; Vian A et al.; The nucleotide sequence of both the bgaA gene, coding for a thermostable beta-galactosidase of Thermus sp . strain T2, and its flanking regions was determined . The deduced amino acid sequence of the enzyme predicts a polypeptide of 645 amino acids (Mr, 73,595) . Comparative analysis of the open reading frames located in the flanking regions of the bgaA gene revealed that they might encode proteins involved in the transport and hydrolysis of sugars . The observed homology between the deduced amino acid sequences of BgaA and the beta-galactosidase of Bacillus stearothermophilus allows us to classify the new enzyme within family 42 of glycosyl hydrolases . BgaA was overexpressed in its active form in Escherichia coli, but more interestingly, an active chimeric beta-galactosidase was constructed by fusing the BgaA protein to the choline-binding domain of the major pneumococcal autolysin . This chimera illustrates a novel approach for producing an active and thermostable hybrid enzyme that can be purified in a single step by affinity chromatography on DEAE-cellulose, retaining the catalytic properties of the native enzyme . The chimeric enzyme showed a specific activity of 191,000 U/mg at 70 degrees C and a Km value of 1.6 mM with o-nitrophenyl-beta-D-galactopyranoside as a substrate, and it retained 50% of its initial activity after 1 h of incubation at 70 degrees C.

Vaccine, 1998 Jan-Feb, 16(2-3), 150 - 5
Recombinant cholera toxin B subunit acts as an adjuvant for the mucosal and systemic responses of mice to mucosally co-administered bovine serum albumin; Tochikubo K et al.; We examined the mucosal adjuvant activity of recombinant cholera toxin B subunit (rCTB) produced by Bacillus brevis carrying pNU212-CTB by intranasal or oral co-administration of bovine serum albumin (BSA) . Intranasal administration stimulated a high level of BSA-specific serum IgG antibody response and BSA-specific IgA antibody responses in the nasal and pulmonary lavages . Oral administration induced a moderate level of BSA-specific serum IgG antibody and a low level of BSA-specific IgA antibody in the large intestinal washes . These results show that CTB alone can act as an intranasal or oral delivery carrier; it also has strong adjuvant properties for stimulating serum IgG and mucosal IgA immune responses to unrelated, non-coupled antigens after intranasal or oral co-immunization.

Arch Biochem Biophys, 1998 May 15, 353(2), 221 - 7
Analysis of the gene encoding cyclomaltodextrinase from alkalophilic Bacillus sp . I-5 and characterization of enzymatic properties; Kim TJ et al.; The gene encoding cyclomaltodextrinase (CDase) was cloned from alkalophilic Bacillus sp . I-5 . The nucleotide sequence of the gene was determined and the physicochemical properties of the enzyme were investigated . The gene had an open reading frame of 559 amino acids with a predicted molecular weight of 64,884 . The enzyme was purified to near homogeneity from Escherichia coli cells carrying a recombinant plasmid that contained the CDase gene . The enzyme hydrolyzed cyclomaltoheptaose (beta-CD) 13 times better than starch and 33 times better than pullulan, and it had transglycosylation activity . The enzyme also hydrolyzed acarbose, a pseudotetrasaccharide inhibitor of glucosidases . The enzyme was stabilized by Ca2+ and the activity was increased more than twofold in the presence of 5 mM EDTA . The optimum temperature of the enzyme was elevated from 40 to 50 degrees C by Ca2+ ion and the thermal activity was maintained more than 80% at 60 degrees C in the presence of Ca2+ . Comparison of known amino acid sequences of several amylolytic enzymes with cyclomaltodextrinase activity, site-directed mutagenesis of the enzyme, and substrate specificity of the enzyme imply that the region between the third and the fourth conserved regions of the enzyme may play an important role in binding and degradation of cyclomaltodextrin.

J Biochem (Tokyo), 1998 Mar, 123(3), 508 - 15
Chemo-enzymatic synthesis of galactosylmaltooligosaccharidonolactone as a substrate analogue inhibitor for mammalian alpha-amylase; Takada M et al.; We performed chemo-enzymatic transformation of maltooligosaccharides into both end-modified oligosaccharidonolactones of potential use as substrate analogue inhibitors for mammalian alpha-amylases . Enzymatic modification of the non-reducing end glucosyl residue of the maltooligosaccharide was first performed by transglycosylation with beta-D-galactosidase from Bacillus circulans . When maltotriose and maltotetraose were the acceptors, the enzyme regioselectively synthesized 4(3)-O-beta-D-galactosyl maltotriose (LG3) and 4(4)-O-beta-D-galactosyl maltotetraose (LG4) from lactose as a donor . LG4 was further selectively hydrolyzed with a specific alpha-amylase to afford 4(2)-O-beta-D-galactosyl maltose (LG2) . The anomer hydroxyl groups of LG2 and LG3 were chemically oxidized to give the corresponding lactones, 4(2)-O-beta-D-galactosyl maltobionolactone (LG2O) and 4(3)-O-beta-D-galactosyl maltotrionolactone (LG3O), respectively . LG2O and LG3O, which are competitive inhibitors for mammalian alpha-amylases, exhibited Ki values of the order of 2.8-18.0 microM, with p-nitrophenyl alpha-maltopentaoside (G5P) as the substrate . On 1H-NMR analysis, these oligosaccharidonolactones were shown to be transformed into the corresponding aldonic acid forms with time in an aqueous solution . In this case, the lactone form was essential for the occurrence of the alpha-amylase inhibitor.

Microbiol Res, 1998 Apr, 153(1), 23 - 7
Purification and some properties of IMP dehydrogenase of Bacillus cereus; Miyamoto T et al.; IMP dehydrogenase was purified from a crude extract of B, cereus cells . The molecular mass of the purified enzyme was estimated to be 56 kDa by SDS-PAGE and 225 kDa by gel filtration . The optimum pH of the enzyme was about 9.5 . The first seven residues at N-terminus of the enzyme was determined to be Met-Trp-Glu-Ser-Lys-Phe-Val . The enzyme showed a significant specificity for inosine nucleotides among 15 purines and pyrimidines tested, but not acted on other purines and pyrimidines including inosine . Among 11 metal ions and 3 enzyme inhibitors tested, Al3+ activated the IMP dehydrogenase . The enzyme activity was strongly inhibited by Zn2+ and Fe3+.

J Exp Biol, 1998 May 21, 201 (Pt 12), 1851 - 8
Cry1Ac, a bacillus thuringiensis toxin, triggers extracellular Ca2+ influx and Ca2+ release from intracellular stores in Cf1 cells
Potvin L, Laprade R, Schwartz JL.
Intracellular Ca2+ concentration was measured in single Cf1 cells (Choristoneura fumiferana, spruce budworm) loaded with Fura-2, a Ca2+-sensitive fluorescent probe . Cf1 cells displayed Ca2+ surges in response to Cry1Ac and Cry1C proteins, two Cf1-toxic Bacillus thuringiensis products, but not to Cry1Aa and Cry3A, which are not toxic to Cf1 cells . In the presence of extracellular Ca2+, the toxin-induced Ca2+ response was insensitive to methoxyverapamil, a voltage-dependent Ca2+ channel blocker, but was abolished by lanthanum, a general inhibitor of Ca2+ transport . In the absence of external Ca2+, Cry1Ac induced a small intracellular Ca2+ transient which was inhibited by TMB-8, a blocker of Ca2+ release from inositol-1,4,5-trisphosphate-sensitive pools . Under these conditions, thapsigargin, which inhibits intracellular Ca2+-ATPases, elicited a Ca2+ surge when applied alone . However, subsequent addition of Cry1Ac failed to induce a Ca2+ signal, indicating a depletion of intracellular Ca2+ pools . In Cf1 cells, therefore, bioactive B . thuringiensis toxins triggered intracellular Ca2+ surges which were mainly due to the influx of extracellular Ca2+ through toxin-made pores, as confirmed by planar lipid bilayer experiments . Furthermore, TMB-8- and thapsigargin-sensitive Ca2+ stores contributed to the Cry1Ac-induced Ca2+ signal.

J Pharm Pharmacol, 1998 Mar, 50(3), 285 - 90
Study of the effects of dispase in the chemotherapy of multicellular tumour spheroids of small-cell lung carcinoma in man; Matsuoka H et al.; Multicellular tumour spheroids (MTS), diameter 650 microm, from PC-6, SBC-1 and NCL-H60 small-cell lung carcinoma cell-lines in man were prepared by the liquid overlay culture method and used to study the influence of treatment with dispase (bacterial neutral protease from Bacillus polymyxa, 1000 units mL(-1)) on the effectiveness of carboplatin, as determined by colony-forming assay . When carboplatin alone was used on monolayers the curve of survival fraction against concentration was exponential in shape, indicating that the drug was active against the monolayer . When MTS were treated with medium concentrations (10(-5) and 10(-4) M) of carboplatin alone the survival fraction-concentration curve showed that the effectiveness of the treatment was less than that against the monolayer . On treatment of MTS with carboplatin and dispase the survival fraction-concentration curve was similar to that obtained for the monolayer and the survival fraction of the core of the MTS was also less than when carboplatin alone was used . These results imply that dispase dissolves the intercellular matrix of the MTS enabling enhanced infiltration of carboplatin into the core of the MTS . Dispase thus indirectly increases the effectiveness of carboplatin.

J Am Mosq Control Assoc, 1998 Mar, 14(1), 69 - 71
Laboratory bioassay to compare susceptibilities of Aedes aegypti and Anopheles albimanus to Bacillus thuringiensis var . israelensis as affected by their feeding rates; Mahmood F; This study presents the effect of differences in the feeding rates of Aedes aegypti and Anopheles albimanus on their susceptibilities to Bacillus thuringiensis israelensis . Aedes aegypti was more susceptible than An . albimanus because of its faster rate of feeding . Aedes aegypti ingested 11.5 times more spores than did An . albimanus, resulting in lower LT50 values . Anopheles albimanus larvae fed at a slower rate and required fewer spores than Ae . aegypti to induce 50% mortality . These findings support earlier reports of much higher concentrations of B . thuringiensis required to kill various anopheline species.

J Am Mosq Control Assoc, 1998 Mar, 14(1), 33 - 9
Impact of treatments with Bacillus sphaericus on Anopheles populations and the transmission of malaria in Maroua, a large city in a savannah region of Cameroon; Barbazan P et al.; Simultaneously with a control of breeding sites primarily for Culex quinquefasciatus and secondarily for anophelines with Bacillus sphaericus in the town of Maroua (120,000 inhabitants) in North Cameroon, a survey of anopheline populations and of transmission rates of malaria was performed . Monthly night catches in 8 districts of the town emphasized the relation between the biting rate by Anopheles in the districts and two main factors . One factor was the distance of a district from the breeding sites, i.e., natural flooded areas along the periphery of the town or artificial breeding sites (ditches, puddles) filled with rain water during the rainy season and with water from the water network throughout the year . The second factor was the density of the habitation that reduced dispersal of female mosquitoes from the breeding sites and the risk for inhabitants to be injected because of scattered bites . The treatment with B . sphaericus was followed by a delay (2 months) in the beginning of the transmission period and a decrease in the incidence of malaria cases studied in a health facility of the town . It thus seems to be possible to reduce malaria transmission by applying B . sphaericus to the breeding sites, but this requires a good knowledge of the location and dynamics of breeding sites and an improved formulation of the pesticide.

J Urol, 1998 Jun, 159(6), 1885 - 91
Superficial bladder tumors and increased reactivity against mycobacterial antigens before bacillus Calmette-Guerin therapy; Zlotta AR et al.; PURPOSE: The precise mechanism of action of bacillus Calmette-Guerin (BCG) in bladder cancer treatment remains poorly understood . Whether bladder tumor cells are destroyed by nonspecific mechanisms or targeted by specifically activated lymphocytes recognizing cognate antigens is unclear . To investigate a possible cross-reactivity between BCG and bladder cell tumors, we tested before BCG treatment the lymphoproliferation of peripheral blood lymphocytes against several mycobacterial antigens, including the secreted fibronectin binding antigen 85 complex from BCG (AG 85) in patients with superficial bladder tumors compared to control matched patients . MATERIALS AND METHODS: Using a whole blood assay, T cell response against purified protein derivative, BCG extract, whole BCG, purified AG 85, and the nonspecific mitogens pokeweed and phytohemagglutinin was investigated in 79 patients with superficial bladder tumors before BCG and in 39 control subjects without malignancy matched for age and sex . Neither group had a history of tuberculosis . Lymphoproliferation was measured with a tritiated thymidine uptake assay on day 7 of culture . RESULTS: Of the 79 patients with superficial transitional cell carcinoma, a significant lymphoproliferative response before BCG against PPD, BCG extract, whole BCG and AG 85 was observed in 65 (82.2%), 67 (84.81%), 30 (37.97%) and 49 (62.02%) patients, respectively . Of the 39 controls only 26 (64.1%), 23 (58.9%), 3 (7.7%) and 3 (7.7%) patients, respectively, had a significant lymphoproliferation against PPD, BCG extract, BCG and AG 85 (p >0.05, p = 0.004, p = 0.00001 and p = 0.00001, respectively) . In terms of lymphoproliferative levels, patients with superficial transitional cell carcinoma also showed a significantly higher response against PPD (p = 0.000012), BCG extract (p = 0.000001), AG 85 (p = 0.000001), whole BCG (p = 0.00001) and pokeweed (p = 0.01) than controls but not against phytohemagglutinin . CONCLUSIONS: Patients with superficial transitional cell carcinoma demonstrate an increased lymphoproliferation against mycobacterial antigens before BCG compared to control subjects . Although a nonspecific activation of the immune system cannot be excluded at this stage, our data may suggest the possible existence of bladder cancer antigens cross-reactive with mycobacterial antigens responsible for boosting precursor cells witnessing previous contacts with mycobacteria . The implication of these findings in the antitumoral mechanism of action of BCG are under investigation.

J Urol, 1998 Jun, 159(6), 1793 - 801
Superficial bladder cancer: the role of interferon-alpha; Belldegrun AS et al.; PURPOSE: We evaluate the clinical experience with recombinant interferon-alpha in superficial transitional cell carcinoma and discuss the most rational use of recombinant interferon-alpha in the context of current treatment options . MATERIALS AND METHODS: The available data were reviewed and discussed at a consensus conference in August 1996 . The conclusions and recommendations are those of the authors based on the consensus reached at that meeting . RESULTS: While bacillus Calmette-Guerin (BCG) is recognized as the most efficacious intravesical agent in the prophylaxis and treatment of superficial transitional cell carcinoma, it is associated with significant toxicities and a 20 to 40% relapse rate . Interferons, particularly recombinant interferon-alpha, have demonstrated efficacy against primary and recurrent papillary transitional cell carcinoma and carcinoma in situ with minimal toxicity, although the response and relapse rates are inferior to BCG . Intravesical recombinant interferon-alpha therapy has also produced responses in patients who failed to respond or were refractory to BCG or chemotherapy . CONCLUSIONS: The clinical experience suggests that recombinant interferon-alpha has an important role in the treatment of superficial transitional cell carcinoma, particularly as second line therapy following failure of BCG or chemotherapy, and it may have synergistic effects when combined with chemotherapy or BCG . We propose a prospective randomized study comparing the efficacy of recombinant interferon-alpha, BCG and BCG plus recombinant interferon-alpha as maintenance following complete response to primary BCG therapy . The proposed study would also investigate the efficacy of BCG plus recombinant interferon-alpha as second line therapy following BCG failure . This study will be important to determine the most effective strategy to integrate recombinant interferon-alpha into current treatment options for superficial bladder cancer.

Zhonghua Jie He He Hu Xi Za Zhi, 1996 Dec, 19(6), 350 - 2
{Cloning and identification of the Mycobacterium bovis BCG gene for signal peptide of antigen 85-B}; Zeng X et al.; OBJECTIVE: To clone and identify the Mycobacterium bovis BCG gene for signal peptide of antigen 85-B . METHODS: Using a polymerase chain reaction technique, the signal peptide sequence (position 94 to 211) of antigen 85-B was technique, which is one of the major protein secreted by Mycobacterium bovis BCG (Bacille-Calmette-Guerin) . The gene of 117bp fragment was cloned into the SacI and EcoRI sites of pBluecript SK, and its sequence was determined by using T7DNA polymerase kit . RESULTS: The sequence analysis showed that the sequence of 117bp signal peptide was not misincorporated and in 3' end understream of signal peptide the 12 multi cloning sites for foreign gene could be provided by the recombinant plasmid . CONCLUSIONS: The signal sequence of BCG antigen 85-B was cloned into the pBluescript plasmid . The recombinant plasmid was called BCG-S01 . According to the genetic information a secreted protein from M . bovis BCG could be utilized in making a useful vaccine vehicle to produce and secrete a vaccinal protein from M . bovis BCG living cells.

Biochim Biophys Acta, 1998 Apr 10, 1380(2), 268 - 74
Distribution of glycoproteins with beta-N-acetylgalactosaminylated N-linked sugar chains among bovine tissues; Sakiyama T et al.; Only a small number of glycoproteins has been reported to contain N-linked sugar chains with GalNAcbeta1-->4GlcNAc structure . Our previous studies showed that most glycoproteins from bovine milk fat globule membranes contain beta-N-acetylgalactosaminylated N-linked sugar chains {Sato et al., J . Biochem . 114 (1993) 890-900} . In order to study how widely this glycosylation occurs, lectin blot analysis of membrane glycoproteins from 12 bovine tissues was performed using Wistaria floribunda agglutinin (WFA), which interacts with oligosaccharides terminating with N-acetylgalactosamine . The WFA-positive bands were detected in samples from most tissues except for intestine although the number and reactivity of bands to lectin varied among the tissues . Upon pretreatment of blotted filters with Bacillus beta-N-acetylgalactosaminidase or N-glycanase, no lectin binding was observed . WFA-agarose column chromatography of oligosaccharides released by hydrazinolysis from membrane glycoproteins of bovine tissues except for intestine revealed that a few to 18% of the released oligosaccharides bind and are eluted from the column with 100 mM N-acetylgalactosamine . These results indicate that many glycoproteins from a variety of bovine tissues contain N-linked sugar chains with GalNAcbeta1-->4GlcNAc structure, suggesting a wider occurrence of this glycosylation in bovine tissues .

Biochim Biophys Acta, 1998 Apr 10, 1380(2), 223 - 31
Separation and characterization of three beta-galactosidases from Bacillus circulans; Vetere A et al.; Crude preparation of Bacillus circulans beta-galactosidase is known to have a good transglycolytic activity . Two isoforms of the enzyme have been described so far in the literature . Aiming at separating these two forms to assess their relative contribution to the regioselectivity of transglycosylation, we observed the presence of a third isoform never described before . This paper deals with the isolation procedures of the three enzymes and a re-consideration of their properties . The estimated molecular weight for the isoforms were 212 kDa (I), 145 kDa (II) and 86 kDa (III), respectively . Kinetic parameters were determined towards the hydrolysis of o-nitrophenyl-beta-d-galactopyranoside (ONPG) and lactose . For ONPG the following values of Km were found: 3.6, 5.0 and 3.3 mM for I, II and III, respectively, whereas for lactose the values were 3.7, 2.94 and 2.71 mM, respectively .

Biochim Biophys Acta, 1998 Apr 10, 1380(2), 183 - 97
In vivo roles of Bm3R1 repressor in the barbiturate-mediated induction of the cytochrome P450 genes (P450(BM-3) and P450(BM-)1) of Bacillus megaterium; Liang Q et al.; We previously showed {Q . Liang, A.J . Fulco, J . Biol . Chem., 270 (1995) 18606-18614) that the binding of Bm3R1 repressor to Barbie box elements and operator sites in the 5'-flanking regions of the P450BM-3 and P450BM-1 (CYP102 and CYP106) genes in Bacillus megaterium was a critical factor in their regulation at the level of transcription . We now describe experiments that delineate specific roles for Bm3R1 in the barbiturate-mediated induction of these genes . We directly demonstrate the interaction of Bm3R1 with Barbie box and operator sequences and show that high in vivo levels of Bm3R1 prevent putative positive factors from binding to Barbie box elements, strongly inhibit the expression of the P450 genes, prolong the lag phase of growth in Bacillus megaterium cultures and increase the sensitivity of the cells to the growth-inhibitory effects of barbiturates . Finally, our data suggest that there may be two forms of Bm3R1, either of which can interact with OIII, the bicistronic operator sequence .

Biochemistry, 1998 Apr 28, 37(17), 5968 - 73
Site-directed mutagenesis combined with chemical modification as a strategy for altering the specificity of the S1 and S1' pockets of subtilisin Bacillus lentus; DeSantis G et al.; By combining site-directed mutagenesis with chemical modification, we have altered the S1 and S1' pocket specificity of subtilisin Bacillus lentus (SBL) through the incorporation of unnatural amino acid moieties, in the following manner: WT --> Cysmutant + H3CSO2SR --> Cys-SR, where R may be infinitely variable . A paradigm between extent of activity changes and surface exposure of the modified residue has emerged . Modification of M222C, a buried residue in the S1' pocket of SBL, caused dramatic changes in kcat/KM, of an up to 122-fold decrease, while modification of S166C, which is located at the bottom of the S1 pocket and is partially surface exposed, effected more modest activity changes . Introduction of a positive charge at S166C does not alter kcat/KM, whereas the introduction of a negative charge results in lowered activity, possibly due to electrostatic interference with oxyanion stabilization . Activity is virtually unaltered upon modification of S156C, which is located toward the bottom of the S1 pocket and surface exposed and whose side chain is solvated . An unexpected structure-activity relationship was revealed for S166C-SR enzymes in that the pattern of activity changes observed with increasing steric size of R was not monotonic . Molecular modeling analysis was used to analyze this unprecedented structure-activity relationship and revealed that the position of the beta-carbon of Cys166 modulates binding of the P1 residue of the AAPF product inhibitor.

J Clin Microbiol, 1998 Apr, 36(4), 1028 - 31
Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by ligase chain reaction; Moore DF et al.; Sputum specimens received for the diagnosis of tuberculosis or other mycobacterial infections were tested by a ligase chain reaction (LCR)-based assay and acid-fast stain and culture techniques . Results from the LCR assay (Abbott LCx Mycobacterium tuberculosis {MTB} Assay) were compared to results from standard culture techniques held for 6 weeks . Four hundred ninety-three specimens from 205 patients suspected of pulmonary tuberculosis were included in the prospective study . Thirty-four (6.9%) of the specimens were culture positive for M . tuberculosis, and 13 (38%) of these were also fluorochrome stain positive . LCR sensitivities and specificities compared to culture were 74 and 98%, respectively . LCR sensitivity was 100% for fluorochrome stain-positive specimens and 57% for fluorochrome stain-negative specimens . Nine LCR-negative, culture-positive specimens were the result of low concentrations of M . tuberculosis . No inhibitors were detected in any of these specimens . Of the eight LCR-positive, culture-negative specimens, five were from patients with active tuberculosis . With these considered culture misses, final LCR sensitivity, specificity, positive predictive value, and negative predictive value were 77, 99, 91, and 98%, respectively . The same performance values for the fluorochrome acid-fast bacillus smear were 33, 98, 62, and 94%, respectively . After normal laboratory sputum processing, the Abbott LCx MTB Assay can be completed in 6 h . Thus, it is possible to have results available within 8 h of specimen submission.

Allergy, 1998 Mar, 53(3), 255 - 65
Aeroallergens and viable microbes in sandstorm dust . Potential triggers of allergic and nonallergic respiratory ailments; Kwaasi AA et al.; Aeroallergens and antigens in sandstorm dust, extracts of which were skin prick test (SPT) positive in allergic patients, were detected by rocket immunoelectrophoresis and ELISA . Fungi and bacteria isolated by agar settle plates and soil dilution and soil washing methods were enumerated and identified . Cat dander, Acacia, Alternaria, Aspergillus, Chenopodium, Cladosporium, Bermuda grass, Pithecellobium, Prosopis, Rumex, cultivated rye, and Washingtonia palm allergens were detected by both methods . Viable microbes including 1892 +/- 325 colony-forming units (cfu) of bacteria, and 869 +/- 75 cfu of fungi were isolated per gram of dust by the soil dilution method . Randomly selected microbial colonies on streaking and subculture were found to consist of between two and seven mixed colonies . Fungi including Alternaria, Aspergillus, Botrytis, Cladosporium, Mortierella, Mucor, Mycelia sterilia, Penicillium, Pythium, Ulocladium, Verticillium, and some yeasts were isolated . Actinomyces, Bacillus, Pseudomonas, and mostly coagulase-negative Staphylococcus species were identified, but the bulk of unidentified bacterial isolates were mainly mixed colonies of rods, cocci, coccobacilli, and some filamentous types . Six-hour agar settle-plate counts during sandstorms were 100 and 40% higher for bacteria and fungi, respectively, than without sandstorms . The most abundant aeroallergens were those of Acacia, Alternaria, Aspergillus, Bermuda grass, Cladosporium, cultivated rye, Prosopis, and cat dander . Pithecellobium dulce, Rumex crispus, and Washingtonia palm allergens were detectable for the first time in Riyadh . IgE reactivities of the dust in man were demonstrated by ELISA using sera from atopic, exposed, and normal subjects . These results indicate that sandstorm dust is a prolific source of potential triggers of allergic and nonallergic respiratory ailments, and the methods mentioned here should be routinely used for quick sampling of the environment.

Chest, 1998 May, 113(5), 1190 - 4
Rapid diagnosis of tuberculosis in various biopsy and body fluid specimens by the AMPLICOR Mycobacterium tuberculosis polymerase chain reaction test; Shah S et al.; STUDY OBJECTIVES: This study was undertaken to determine the usefulness of the AMPLICOR Mycobacterium tuberculosis (MTB) polymerase chain reaction (PCR) test (Roche Diagnostic Systems, Inc; Branchburg, NJ) in diagnosing TB in tissue and body fluid specimens other than respiratory secretions . DESIGN AND SETTING: Prospective analysis of clinical and laboratory data in patients with suspected TB at the four divisional hospitals of Catholic Medical Center, located in New York . PATIENTS AND MEASUREMENTS: A total of 1,090 tissue and body fluid specimens from 1,032 patients with suspected TB were subjected to acid-fast bacillus (AFB) smear, culture, and the AMPLICOR MTB PCR test . RESULTS: Of the 1,090 specimens, 32 grew M tuberculosis complex and 8 specimens grew isolates belonging to the Mycobacterium avium complex (MAC) . The AMPLICOR MTB PCR test was positive for 24 of the 32 specimens that grew M tuberculosis . It was also positive for four additional specimens that were culture-negative for M tuberculosis or MAC . Two of these specimens were from patients with a previously recorded positive sputum culture for M tuberculosis . The AMPLICOR test was negative for all eight specimens that yielded MAC only . When AMPLICOR MTB PCR test results were compared with the confirmed clinical diagnosis of TB, the sensitivity, specificity, positive predictive value, and negative predictive value for the AMPLICOR MTB PCR test were 76.4%, 99.8%, 92.8%, and 99.2%, respectively . PCR results were available within 6.5 hours, compared with an average of 3 weeks for culture of M tuberculosis . CONCLUSIONS: These data establish the utility of the AMPLICOR MTB PCR test for the rapid detection of M tuberculosis in tissue and body fluid specimens other than respiratory secretions.

FEMS Microbiol Lett, 1998 May 1, 162(1), 185 - 91
Cloning of the thermostable phytase gene (phy) from Bacillus sp . DS11 and its overexpression in Escherichia coli; Kim YO et al.; Phytase hydrolyzes phytate to release inorganic phosphate, which would decrease the addition of phosphorus to feedstuffs for monogastric animals and thus reduce environmental pollution . The gene encoding phytase from Bacillus sp . DS11 was cloned in Escherichia coli and its sequence determined . A 560-bp DNA fragment was used as a probe to screen the genomic library . It was obtained through PCR of Bacillus sp . DS11 chromosomal DNA and two oligonucleotide primers based on N-terminal amino acid sequences of the purified protein and the cyanogen bromide-cleaved 21-kDa fragment . The phy cloned was encoded by a 2.2-kb fragment . This gene comprises 1152 nucleotides and encodes a polypeptide of 383 amino acids with a deduced molecular mass of 41,808 Da . Phytase was produced to 20% content of total soluble proteins in E . coli BL21 (DE3) using the pET22b(+) vector with the inducible T7 promoter . This is the first nucleic sequence report on phytase from a bacterial strain.

Dis Esophagus, 1998 Jan, 11(1), 72 - 4
Tuberculosis of the esophagus; Perdomo JA et al.; We report a case of a patient with esophageal tuberculosis, a very uncommon form of extrapulmonar tuberculosis . Initially, because of constitutional symptomatology and radiological findings of mediastinal lymph node enlargement, lymphoma was considered . However, the endoscopic findings of ulcerative masses and a sinus tract revealed by esophagram were suspicious of tuberculous origin . Diagnosis was achieved after bacterial examination of smear samples from esophageal ulcers that revealed bacillus tuberculous and histological demonstration of caseating granulomas in cervical lymph nodes . Tuberculous mediastinal lymphadenitis was thought to be source of the spread to esophagus . The patient was successfully treated with a three antituberculous drugs regimen . In spite of its rarity, even in patients without risk factors, the diagnosis would be considered in the differential diagnosis of uncertain esophageal lesions.

J Endod, 1997 Jan, 23(1), 32 - 4
Cleaning of endodontic files, Part I: The effect of bioburden on the sterilization of endodontic files; Johnson MA et al.; Ninety-two new endodontic files were randomly assigned to five groups with varying parameters of contamination, cleaning method, and sterilization (steam or chemical) . Files were instrumented in bovine teeth to accumulate debris and a known contaminant, Bacillus stearothermophilus . Positive controls produced growth on both T-soy agar plates and in T-soy broth . Negative controls and experimental files (some with heavy debris) failed to produce growth . The results showed that there was no significant difference between contaminated files that were not cleaned before sterilization and contaminated files that were cleaned before sterilization . Bioburden present on endodontic files does not appear to affect the sterilization process.

Prog Nucleic Acid Res Mol Biol, 1998, 60, 1 - 27
Bacillus thuringiensis insecticidal proteins: molecular mode of action; Rajamohan F et al.; Growing interest in biorational pesticides has placed the Bacillus thuringiensis insecticidal crystal proteins at the forefront of pesticides for plant genetic engineering . The development of improvement pesticides, both in enhanced activity and broader host range, depends on an understanding of its mechanism of action . This review presents a complete overview of the bacterium and the group of insecticidal proteins known as Cry proteins or delta-endotoxins . The molecular mode of action is described in detail, including the mapping of receptor binding sites by site-directed mutagenesis, the known receptors, and the ion-channel activity of the toxins.

J Infect Dis, 1998 May, 177(5), 1352 - 7
Field trial of a vaccine against New World cutaneous leishmaniasis in an at-risk child population: safety, immunogenicity, and efficacy during the first 12 months of follow-up; Armijos RX et al.; The safety, immunogenicity, and efficacy of a vaccine against cutaneous leishmaniasis in rural Ecuadorian children was assessed in a randomized, controlled, double-blinded study . Vaccine group subjects received 2 intradermal doses of a whole, killed promastigote vaccine cocktail plus bacille Calmette-Guerin (BCG) adjuvant . Control subjects got 2 doses of BCG only . The subjects who received both vaccination doses, 438 in the vaccine group (79.3%) and 406 in the control group (83.4%), were followed for 12 months . No serious adverse side effects were identified in either group . Significantly more vaccine group subjects than controls converted to a positive Montenegro skin test (85.1% vs . 20.1%; chi2 = 279; P < .001) . The incidence of cutaneous leishmaniasis was significantly reduced in the vaccine compared with the control group (2.1% vs . 7.6%; chi2 = 8.95; P < .003) . The protective efficacy of the vaccine was 72.9% (95% confidence interval = 36.1%-88.5%).

Nucleic Acids Res, 1998 Jun 1, 26(11), 2686 - 93
Characterisation of Bacillus stearothermophilus PcrA helicase: evidence against an active rolling mechanism; Bird LE et al.; PcrA from Bacillus stearothermophilus is a DNA helicase for which, despite the availability of a crystal structure, there is very little biochemical information . We show that the enzyme has a broad nucleotide specificity, even being able to hydrolyse ethenonucleotides, and is able to couple the hydrolysis to unwinding of DNA substrates . In common with the Escherichia coli helicases Rep and UvrD, PcrA is a 3'-5' helicase but at high protein concentrations it can also displace a substrate with a 5' tail . However, in contrast to Rep and UvrD, we do not see any evidence for dimerisation of the protein even in the presence of DNA . The enzyme shows a specificity for the DNA substrate in gel mobility assays, with the preferred substrate being one with both single and double stranded regions of DNA . We propose that these data, together with existing structural evidence, support an inchworm rather than a rolling model for 3'-5' helicase activity.

J Biochem (Tokyo), 1998 Apr, 123(4), 564 - 7
Guanidine hydrochloride-induced changes of the E2 inner core of the Bacillus stearothermophilus pyruvate dehydrogenase complex; Hiromasa Y et al.; The limited proteolysis of the Bacillus stearothermophilus pyruvate dehydrogenase complex by V8 protease yields its core structure solely composed of lipoate acetyltransferase (E2) fragments . The changes in the core with guanidine hydrochloride (GdnHCl) were biphasic: below 0.8 M (first) and above 1.0 M (second) GdnHCl . The changes in the first phase were slight but significant: decreases in ellipticity and light scattering, and an increase in E2 activity . Insignificant changes in the molecular shape and size of the core were detected on fluorescence spectroscopy, ultracentrifugation, gel filtration, and electron microscopy . On the other hand, the changes in the second phase were drastic; the core was disassembled and denatured.

J Antibiot (Tokyo), 1998 Mar, 51(3), 261 - 6
UCH9, a new antitumor antibiotic produced by Streptomyces: I . Producing organism, fermentation, isolation and biological activities; Ogawa H et al.; We developed a microbial prescreen using Bacillus stearothermophilus NUB3620 and bacteriophage TP-68 to detect potential antitumor compounds acting on DNA or topoisomerases . During the course of screening microbial cultures for their antibacteriophage activities, we found that Streptomyces sp . isolated from a soil sample collected in Iwakuni city, Yamaguchi prefecture, Japan, produced a new antitumor antibiotic, UCH9 . UCH9 was isolated from culture broth by a combination of EtOAc extraction and column chromatography . UCH9 has a new structure related to the antitumor antibiotic chromomycins . It exhibited antimicrobial activity against Gram-positive organisms . UCH9 also showed cytotoxic activity against HeLa S3 cells with an IC50 value of 13 nM and exhibited antitumor activity in vivo against mouse leukemia P388.

Biol Pharm Bull, 1998 Apr, 21(4), 311 - 4
Use of hemoglobin as an iron source by Bacillus cereus; Sato N et al.; The hemoglobin binding activity of Bacillus cereus cells was measured with fluoresceinisothiocyanate (FITC)-conjugated hemoglobin using flow cytometry . Growth of B . cereus was markedly inhibited by the addition of apo-transferrin . B . cereus could not use transferrin-bound iron as an iron source in serum . The growth inhibition was reversed by the addition of a FeCl3 solution, erythrocytes or hemoglobin . B . cereus released hemolysin; these findings suggested that the hemoglobin released from erythrocytes by B . cereus hemolysin binds to B . cereus and is thus used as an iron source.

Drugs, 1998 May, 55(5), 699 - 704
Diagnosis and treatment of Whipple's disease; Singer R; Whipple's disease is a rare systemic infectious disease . To date, it has neither been possible to culture the bacillus Tropheryma whippelii, nor to infect other individuals with the pathogen . Today the diagnosis is confirmed by means of polymerase chain reaction (PCR) technology . Typically, the material for the PCR analysis comes from the duodenum . The diagnosis can also be established in this way on the basis of other tissue, or the cerebrospinal fluid . Treatment should only be carried out with antibiotics which cross into the cerebrospinal fluid, since there can also be an unrecognised involvement of the CNS . At present, the favoured method of treatment is the daily parenteral administration of 1.2 million units of benzylpenicillin (penicillin G) and streptomycin 1 g for a period of 2 weeks . This is followed by treatment with cotrimoxazole (trimethoprim 160 mg and sulfamethoxazole 800 mg) twice daily for 1 to 2 years . The treatment should begin and end with a PCR analysis of cerebrospinal fluid, in order to definitively diagnose infection of the CNS with Whipple's disease and to document the disappearance of the bacillus from the CNS.

Biochem Mol Biol Int, 1998 Apr, 44(4), 825 - 32
Effects on larvicidal activity of single proline substitutions in alpha3 or alpha4 of the Bacillus thuringiensis Cry4B toxin; Uawithya P et al.; The possible role of alpha-helices 3 and 4 in toxicity of the dipteran-active Bacillus thuringiensis Cry4B delta-endotoxin was investigated by employing proline substitutions via site-directed mutagenesis . Similar to the wild-type Cry4B, the mutant toxins were over-expressed in Escherichia coli as cytoplasmic inclusions and were structurally stable upon solubilization and trypsin activation . The substitution of glutamine 149 by proline in the center of helix 4 (Q149P) resulted in a nearly complete loss of toxicity against Aedes aegypti mosquito-larvae . However, single proline replacements near the center of helix 3 (V119P) and at the N-terminus of helix 4 (Q140P) did not decrease larvicidal activity . The toxicity of E . coli cells expressing the wild-type toxin was significantly reduced by two-hour preincubation with the non-toxic mutant (Q149P), thus indicating that the primary binding step was not affected by the proline substitution in helix 4 . The results therefore reveal a crucial role for helix 4 of the Cry4B toxin in toxicity, possibly in membrane insertion and pore formation rather than in receptor recognition.

Am J Infect Control, 1998 Apr, 26(2), 143 - 5
Levels of microbial contamination on surgical instruments; Rutala WA et al.; OBJECTIVE: To ascertain the microbial load and type of organisms on used surgical instruments following standard cleaning, which consisted of the use of a washer sterilizer followed by sonic cleaning . DESIGN: In this prospective experimental study, used surgical instruments were immersed in Peptamin Tween broth, the broth agitated, and then filtered through a 0.45 microm filter . Quantitative cultures were performed, and all microbes were identified by using standard techniques . SETTING: This study was conducted at a 660-bed university hospital . RESULTS: The microbial load remaining on used surgical instruments after cleaning was as follows: 36 (72%) instruments 0 to 10 colony-forming units (CFU), 7 (14%) instruments 11 to 100 CFU, and 7 (14%) instruments > 100 CFU . Organisms contaminating the instruments included coagulase-negative staphylococcus (56%) followed by Bacillus (22%) and diphtheroids (14%) . No other microbes were isolated from more than 4% of the instruments . CONCLUSION: Most used nonlumen surgical instruments contain less than 100 CFU of relatively nonpathogenic microorganisms after cleaning . This suggests that new low-temperature sterilization technologies are likely to be highly effective in preventing cross-transmission of infection via nonlumen medical instruments.

J Pathol, 1998 Jan, 184(1), 96 - 102
Differentiation of BCG-induced lymphadenitis from tuberculosis in lymph node biopsy specimens by molecular analyses of pncA and oxyR; Yan JJ et al.; Without culture, differentiation of bacille Calmette-Guerin-induced lymphadenitis (BCG-LA) from tuberculosis (TB) is sometimes difficult by histology, but is important because of different treatment schemes . The purpose of this study was to investigate the feasibility of differentiating BCG-LA from TB in lymph nodes (LNs) by molecular analyses of two recently identified genes, pncA and oxyR . In both genes, a single tuberculosis difference exists between Mycobacterium bovis and M . tuberculosis . M tuberculosis complex (MTC) DNA was first detected in nine of ten formalin-fixed, paraffin-embedded LNs from patients aged under 20 years with suspected mycobacterial infections, using polymerase chain reaction (PCR) for IS6110, an insertion sequence specific for MTC species . PCR, together with direct DNA sequencing and PCR-restriction fragment length polymorphism (RFLP) assay, was then performed to identify polymorphic nucleotide in pncA and oxyR, respectively . For comparison, 37 adult cases of tuberculous lymphadenitis were also analysed by PCR-single strand conformation polymorphism (SSCP) assay for pncA and by PCR-RFLP for oxyR . The results revealed that five of the nine IS6110-positive child cases had a G residue at nucleotide 169 in pncA, and also had a three-band pattern after digesting the amplified oxyR segment with AluI, suggesting BCG-LA . The remaining four child cases, as well as all adult cases with detectable IS6110, showed no motility shift in pncA PCR-SSCP and had the same one-band pattern as M . tuberculosis in oxyR PCR-RFLP, suggesting TB lymphadenitis . The data from molecular analyses showed a good correlation with the vaccination history and clinicopathological findings, except for one case . This study indicates that molecular assay of either oxyR or pncA could be a rapid and useful tool to distinguish BCG-LA from TB.

Transplantation, 1998 Apr 15, 65(7), 1000 - 3
Peloisis hepatis due to Bartonella henselae in transplantation: a hemato-hepato-renal syndrome; Ahsan N et al.; BACKGROUND: Bacillary peliosis hepatis is an uncommon but well recognized disease due to disseminated Bartonella infections occurring predominantly in immunocompromised individuals infected with human immunodeficiency virus, type 1 . A similar condition in the absence of Bartonella infection when described in organ transplant patients was felt to be secondary to azathioprine and/or cyclosporine . METHODS: Herein, we report the first case of bacillary peliosis hepatis due to systemic Bartonella henselae infection in a patient after kidney transplant . The patient presented with severe anemia, persistent thrombocytopenia, and hepato-renal syndrome . DNA-based polymerase chain reactions (PCR), which allowed direct detection of both B henselae and quintana DNA in patient's peripheral blood and liver tissue, were used . Indirect immunofluorescence assay for Bartonella serology was performed on peripheral blood . RESULTS: Histopathology of the liver biopsy demonstrated peliosis hepatis . Indirect immunofluorescence assay for Bartonella serology was positive, and B henselae DNA was identified by PCR in the peripheral blood and liver tissue . Treatment with a 3-month course of oral erythromycin resulted in an excellent clinical response . CONCLUSIONS: The present case suggests that although various anti-rejection therapies and opportunistic infections are associated with hepatic and renal dysfunction along with bone marrow suppression, the diagnostic evaluation in this situation should include liver biopsy and a careful search for evidence of systemic Bartonella infection, e.g., exposure to cats, Bartonella serology, and Bartonella DNA by PCR . A reduction in immunosuppression and prolonged therapy with antibiotics such as erythromycin will often result in early recovery.

Transplantation, 1998 Apr 15, 65(7), 966 - 70
Microbiological evaluation of glycerolized cadaveric donor skin; van Baare J et al.; BACKGROUND: Human cadaveric donor skin is commonly used for the treatment of extensive burns . To minimize the risk of transfer of bacteria, viruses, and prions to the recipient, the donor and cadaver skin are screened according to standard transplantation protocols . METHODS: Since 1984, glycerol in a concentration of 85% has been used as a preservative of cadaver skin; here, data on bacteriological contamination of cadaver skin of 1929 skin donors are reviewed . RESULTS: Results show a reduction of contamination with 70% when antibiotics were used during the processing procedure . Overall, 10.1+/-4.1% of the cadaver skin showed initial bacterial contamination, but after prolonged storage all skin eventually showed no bacterial growth . The most commonly detected bacteria species was Staphylococcus epidermidis (76.7+/-7.0%) . The spore-forming Bacillus species was most resistant to inactivation by glycerol, but eventually also this species was no longer detected . CONCLUSIONS: In conclusion, preservation of skin in 85% glycerol reduces the risk of bacterial transfer to the recipient and allows an increase in yield of cadaver skin of approximately 10%.

Biochemistry, 1998 Apr 21, 37(16), 5755 - 60
General base catalysis by the phosphatidylcholine-preferring phospholipase C from Bacillus cereus: the role of Glu4 and Asp55; Martin SF et al.; To assess what roles the active site residues Glu4 and Asp55 of the phosphatidylcholine-preferring phospholipase C of Bacillus cereus (PLCBc) might play in binding and catalysis, selected mutants were prepared through site-directed mutagenesis of the plc gene . The mutants were then expressed in Escherichia coli and purified as fusion proteins with the maltose binding protein (MBP) . Kinetic analysis showed that mutations at Glu4 had only modest effects on the catalytic activity, whereas those at Asp55 led to proteins whose values for kcat/KM were 10(4)-10(6) times less than that of the wild-type enzyme . The modest decrease in catalytic activity and the pH-dependent profile of the E4L mutant strongly suggest that glutamic acid at position 4 is not the general base in the PLCBc-catalyzed reaction . Rather, the results support the hypothesis that Glu4 is primarily involved in substrate binding, perhaps by electrostatic stabilization of the positive charge of the choline moiety of the phosphatidylcholine substrate . Examination of X-ray crystallographic data of PLCBc and its various complexes reveals that the carboxylate side chain of Asp55 is positioned such that it could activate a water for nucleophilic attack on the substrate or serve as a ligand for Zn1 . However, the involvement of the side chain of Asp55 as an important Zn1 ligand is not consistent with the atomic absorption and thermostability data obtained for the D55L mutant, which are virtually identical with that of the wild-type enzyme . The large reduction in the measured kcat/KM of the D55E, D55N, and D55L mutants of PLCBc indicates that Asp55 plays a critical role in catalysis and likely serves as the general base in the hydrolysis of phosphatidylcholine by PLCBc.

Biochemistry, 1998 Apr 14, 37(15), 5312 - 9
A single calcium binding site is crucial for the calcium-dependent thermal stability of thermolysin-like proteases; Veltman OR et al.; Thermostable thermolysin-like proteases (TLPs), such as the TLP of Bacillus stearothermophilus CU-21 (TLP-ste), bind calcium in one double (Ca1,2) and two single (Ca3, Ca4) calcium binding sites . The single sites are absent in thermolabile TLPs, suggesting that they are determinants of (variation in) TLP stability . Mutations in the Ca3 and Ca4 sites of TLP-ste indeed reduced thermal stability, but only mutations in the Ca3 site affected the calcium-dependence of stability . The predominant effect of the Ca3 site results from the fact that the Ca3 site is part of a region of TLP-ste, which unfolding is crucial for thermal inactivation . Thermal inactivation is not caused by the absence of calcium from the Ca3 site per se, but rather by unfolding of a region of TLP-ste for which stability depends on the occupancy of the Ca3 site . In accordance with this concept is the observation that the effects of mutations in the Ca3 site could be compensated by stabilizing mutations near this site . In addition, it was observed that the contribution of calcium binding to the Ca3 was substantially reduced in extremely stable TLP-ste variants containing multiple stabilizing mutations in the Ca3 region . Apparently, in these latter variants, unfolding of the Ca3 region contributes little to the overall process of thermal inactivation.

Biochemistry, 1998 Apr 14, 37(15), 5305 - 11
Probing catalytic hinge bending motions in thermolysin-like proteases by glycine --> alanine mutations; Veltman OR et al.; The active site of thermolysin-like proteases (TLPs) is located at the bottom of a cleft between the N- and C-terminal domains . Crystallographic studies have shown that the active-site cleft is more closed in ligand-binding TLPs than in ligand-free TLPs . Accordingly, it has been proposed that TLPs undergo a hinge-bending motion during catalysis resulting in "closure" and "opening" of the active-site cleft . Two hinge regions have been proposed . One is located around a conserved glycine 78; the second involves residues 135 and 136 . The importance of conserved glycine residues in these hinge regions was studied experimentally by analyzing the effects of Gly --> Ala mutations on catalytic activity . Eight such mutations were made in the TLP of Bacillus stearothermophilus (TLP-ste) and their effects on activity toward casein and various peptide substrates were determined . Only the Gly78Ala, Gly136Ala, and Gly135Ala + Gly136Ala mutants decreased catalytic activity significantly . These mutants displayed a reduction in kcat/Km for 3-(2-furylacryloyl)-L-glycyl-L-leucine amide of 73%, 62%, and 96%, respectively . Comparisons of effects on kcat/Km for various substrates with effects on the Ki for phosphoramidon suggested that the mutation at position 78 primarily had an effect on substrate binding, whereas the mutations at positions 135 and 136 primarily influence kcat . The apparent importance of conserved glycine residues in proposed hinge-bending regions for TLP activity supports the idea that hinge-bending is an essential part of catalysis.

Biochim Biophys Acta, 1998 Mar 13, 1370(2), 280 - 8
Self-assembled alpha-hemolysin pores in an S-layer-supported lipid bilayer; Schuster B et al.; The effects of a supporting proteinaceous surface-layer (S-layer) from Bacillus coagulans E38-66 on a 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC) bilayer were investigated . Comparative voltage clamp studies on plain and S-layer supported DPhPC bilayers revealed no significant difference in the capacitance . The conductance of the composite membrane decreased slightly upon recrystallization of the S-layer . Thus, the attached S-layer lattice did not interpenetrate or rupture the DPhPC bilayer . The self-assembly of a pore-forming protein into the S-layer supported lipid bilayer was examined . Staphylococcal alpha-hemolysin formed lytic pores when added to the lipid-exposed side . The assembly was slow compared to unsupported membranes, perhaps due to an altered fluidity of the lipid bilayer . No assembly could be detected upon adding alpha-hemolysin monomers to the S-layer-faced side of the composite membrane . Therefore, the intrinsic molecular sieving properties of the S-layer lattice do not allow passage of alpha-hemolysin monomers through the S-layer pores to the lipid bilayer . In comparison to plain lipid bilayers, the S-layer supported lipid membrane had a decreased tendency to rupture in the presence of alpha-hemolysin .

Biochemistry, 1998 Apr 7, 37(14), 4958 - 67
Crystallographic study of steps along the reaction pathway of D-amino acid aminotransferase; Peisach D et al.; The three-dimensional structures of two forms of the D-amino acid aminotransferase (D-aAT) from Bacillus sp . YM-1 have been determined crystallographically: the pyridoxal phosphate (PLP) form and a complex with the reduced analogue of the external aldimine, N-(5'-phosphopyridoxyl)-d-alanine (PPDA) . Together with the previously reported pyridoxamine phosphate form of the enzyme {Sugio et al . (1995) Biochemistry 34, 9661}, these structures allow us to describe the pathway of the enzymatic reaction in structural terms . A major determinant of the enzyme's stereospecificity for D-amino acids is a group of three residues (Tyr30, Arg98, and His100, with the latter two contributed by the neighboring subunit) forming four hydrogen bonds to the substrate alpha-carboxyl group . The replacement by hydrophobic groups of the homologous residues of the branched chain L-amino acid aminotransferase (which has a similar fold) could explain its opposite stereospecificity . As in L-aspartate aminotransferase (L-AspAT), the cofactor in D-aAT tilts (around its phosphate group and N1 as pivots) away from the catalytic lysine 145 and the protein face in the course of the reaction . Unlike L-AspAT, D-aAT shows no other significant conformational changes during the reaction.

J Biol Chem, 1998 Apr 10, 273(15), 9292 - 6
Role of DNA in the activation of the Cry1A insecticidal crystal protein from Bacillus thuringiensis; Clairmont FR et al.; The Cry1A insecticidal crystal protein (protoxin) from six subspecies of Bacillus thuringiensis as well as the Cry1Aa, Cry1Ab, and Cry1Ac proteins cloned in Escherichia coli was found to contain 20-kilobase pair DNA . Only the N-terminal toxic moiety of the protoxin was found to interact with the DNA . Analysis of the crystal gave approximately 3 base pairs of DNA per molecule of protoxin, indicating that only a small region of the N-terminal toxic moiety interacts with the DNA . It is proposed that the DNA-protoxin complex is virus-like in structure with a central DNA core surrounded by protein interacting with the DNA with the peripheral ends of the C-terminal region extending outward . It is shown that this structure accounts for the unusual proteolysis observed in the generation of toxin in which it appears that peptides are removed by obligatory sequential cleavages starting from the C terminus of the protoxin . Activation of the protoxin by spruce budworm (Choristoneura fumiferana) gut juice is shown to proceed through intermediates consisting of protein-DNA complexes . Larval trypsin initially converts the 20-kilobase pair DNA-protoxin complex to a 20-kilobase pair DNA-toxin complex, which is subsequently converted to a 100-base pair DNA-toxin complex by a gut nuclease and ultimately to the DNA-free toxin.

Biochem J, 1998 Apr 1, 331 ( Pt 1), 251 - 6
Utilization of phosphatidylcholine and production of diradylglycerol as a consequence of sphingomyelin synthesis; Sillence DJ et al.; 1 . After the degradation of cell-surface sphingomyelin (SM) by exogenous sphingomyelinase (SMase), the resynthesis of SM by baby-hamster kidney (BHK) and human leukaemia-60 (HL-60) cells was examined in relation to utilization of substrate phosphatidylcholine (PtdCho) and generation of the expected product, diradylglycerol (DRG) . Using {3H}choline-labelled BHK cells incubated in non-radioactive medium, SMase caused a release of phosphocholine, which was derived approximately equally from SM and PtdCho, consistent with the anticipated resynthesis of SM at the expense of PtdCho . However, with choline-labelled cells incubated in radioactive medium or {14C}acetate-labelled cells treated with SMase, no loss of radioactivity from PtdCho or accumulation of labelled DRG was observed, suggesting that any DRG produced as a consequence of SM synthesis must have been rapidly converted back into PtdCho . In contrast, SMase treatment of HL-60 cells caused more than a doubling of DRG levels at the expense of PtdCho, and this appears to be the first demonstration of a rise in DRG related to the synthesis of SM . The DRG produced consisted of about 80% 1,2-diacylglycerol and 18% 1-O-alkyl-2-acylglycerol species, a similar composition to that of the DRG backbone of total cell PtdCho . 2 . The requirement for cell-surface PtdCho in the biosynthesis of SM by BHK cells was also investigated . Treatment of {3H}choline-labelled BHK cells with Bacillus cereus PtdCho-specific phospholipase C (PLC) rapidly degraded about 6% of the total PtdCho, which was assumed to represent the cell-surface pool . This did not appear to be the pool of PtdCho required for SM synthesis, since (a) the released phosphocholine was additional to that derived from PtdCho in cells treated with SMase and (b) treatment with PLC did not affect SM synthesis, either de novo or in response to degradation of cell-surface SM by SMase . These findings suggest either that there is no SM synthase in the plasma membrane or, if it is present, then it does not utilize cell-surface PtdCho as a substrate.

New Microbiol, 1998 Apr, 21(2), 183 - 96
Studies on the phytoplankton populations and physico-chemical conditions of treated sewage discharged into Lake Manzala in Egypt; el-Naggar ME et al.; Over a full year, the phytoplankton populations and physico-chemical conditions of treated sewage discharged into Lake Manzala in Egypt were investigated . Sixty-seven species of algae were identified, 18 Cyanophyta (Cyanobacteria), 19 Chlorophyta, 21 Bacillariophyta, 6 Euglenophyta, 2 Cryptophyta and one species Pyrrhophyta . Nitzschia (6 spp.), Scenedesmus (6 spp.), Navicula (4 spp.), Oscillatoria (4 spp.) and Euglena (4 spp.) were the most common genera . A remarkable seasonal variation in species composition and standing crop of the phytoplankton populations was noted during the study . The total phytoplankton standing crop appeared to be mainly dependent on the growth of certain species viz., Oscillatoria chalybea, O . princepes, O . tenuis, Microcystis aeruginosa, Anabaena constricta (Cyanophyta), Nitzschia obtusa, Bacillaria paradoxa, Cocconeis placentula, Cyclotella meneghiniana (Bacillariophyta), Pandorina morum, Volvox sp . (Chlorophyta) and Phacus curvicauda (Euglenophyta) . The continuous presence of Anabaena constricta and Nitzschia palea was recorded in the treated sewage . The least represented algal divisions were Pyrrhophyta and Cryptophyta, both in terms of quality and quantity . The data indicate that the secondary effluents were unstable in their chemical features and grossly polluted . Therefore, the treatment systems must treat the discharged sewage to a tertiary level before discharging into Lake Manzala.

Eur J Biochem, 1998 Apr 1, 253(1), 251 - 62
Molecular and enzymatic characterization of a maltogenic amylase that hydrolyzes and transglycosylates acarbose; Cha HJ et al.; A gene encoding a maltogenic amylase of Bacillus stearothermophilus ET1 was cloned and expressed in Escherichia coli . DNA sequence analysis indicated that the gene could encode a 69,627-Da protein containing 590 amino acids . The predicted amino acid sequence of the enzyme shared 47-70% identity with the sequences of maltogenic amylase from Bacillus licheniformis, neopullulanase from B . stearothermophilus, and cyclodextrin hydrolase (CDase) 1-5 from an alkalophilic Bacillus 1-5 strain . In addition to starch, pullulan and cyclodextrin, B . stearothermophilus could hydrolyze isopanose, but not panose, to glucose and maltose . Maltogenic amylase hydrolyzed acarbose, a competitive inhibitor of amylases, to glucose and a trisaccharide . When acarbose was incubated with 10% glucose, isoacarbose, containing an alpha-1,6-glucosidic linkage was produced as an acceptor reaction product . B . stearothermophilus maltogenic amylase shared four highly similar regions of amino acids with several amylolytic enzymes . The beta-cyclodextrin-hydrolyzing activity of maltogenic amylase was enhanced to a level equivalent to the activity of CDase when its amino acid sequence between the third and the fourth conserved regions was made more hydrophobic by site-directed mutagenesis . Enhanced transglycosylation activity was observed in most of the mutants . This result suggested that the members of a subfamily of amylolytic enzymes, including maltogenic amylase and CDase, could share similar substrate specificities, enzymatic mechanisms and structure/function relationships.

Biochem J, 1998 May 15, 332 ( Pt 1), 101 - 9
Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5'-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer; Lehto MT et al.; Many hydrolytic enzymes are attached to the extracellular face of the plasma membrane of eukaryotic cells by a glycosylphosphatidylinositol (GPI) anchor . Little is currently known about the consequences for enzyme function of anchor cleavage by phosphatidylinositol-specific phospholipase C . We have examined this question for the GPI-anchored protein 5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5), both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid bilayer vesicles, using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC) . Membrane-bound, detergent-solubilized and cleaved 5'-nucleotidase all obeyed Michaelis-Menten kinetics, with a Km for 5'-AMP in the range 11-16 microM . The GPI anchor was removed from essentially all 5'-nucleotidase molecules, indicating that there is no phospholipase-resistant pool of enzyme . However, the phospholipase was much less efficient at cleaving the GPI anchor when 5'-nucleotidase was present in detergent solution, dimyristoyl phosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared with the native plasma membrane, egg phosphatidylcholine and a sphingolipid/cholesterol-rich mixture . Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor . Catalytic activation, characterized by an increase in Vmax, was observed following PI-PLC cleavage of reconstituted 5'-nucleotidase from vesicles of several different lipids . The highest degree of activation was noted for 5'-nucleotidase in egg phosphatidylethanolamine . An increase in Vmax was also noted for a sphingolipid/cholesterol-rich mixture, the native plasma membrane and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl phosphatidylcholine showed little activation . Km generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-rich mixture . Insertion of the GPI anchor into a lipid bilayer appears to reduce the catalytic efficiency of 5'-nucleotidase, possibly via a conformational change in the enzyme, and activity is restored on release from the membrane.

J Basic Microbiol, 1998, 38(1), 33 - 9
Unique appendages associated with spores of Bacillus cereus isolates; Mizuki E et al.; Electron microscopic observations revealed the presence of a new type of large appendage on the spores of two Bacillus cereus strains isolated from phylloplanes . The appendages were thin and sword-like in shape, having the sizes of 1.5 to 2.8 microns in length and 0.03 to 0.6 micron in width . There were no core or sheath structures in these appendages . The number of appendages on a spore ranged from three to more than twenty, radiating from the swelling on one end of the exosporium . These appendages gave a unique octopus- or jellyfish-like feature to the spores.

Infect Immun, 1998 May, 66(5), 2122 - 7
A tumor necrosis factor mimetic peptide activates a murine macrophage cell line to inhibit mycobacterial growth in a nitric oxide-dependent fashion; Britton WJ et al.; The control of mycobacterial infections depends on the cytokine-mediated activation of mononuclear phagocytes to inhibit the growth of intracellular mycobacteria . Optimal activation requires the presence of T-cell-derived gamma interferon (IFN-gamma) and other signals, including tumor necrosis factor (TNF) . Recently, an 11-mer peptide based on amino acids 70 to 80 of the human TNF sequence, TNF(70-80), was found to have TNF mimetic properties, which include the activation of human and mouse neutrophils to kill Plasmodia spp . Therefore, we investigated the capacity of TNF(70-80) to activate the murine macrophage cell line RAW264.7 infected with the vaccine strain Mycobacterium bovis bacillus Calmette-Guerin (BCG) . When RAW264.7 cells were pretreated with human TNF or TNF(70-80) in the presence of IFN-gamma, there was a dose-dependent reduction in the replication of BCG as measured by the uptake of 3H-labeled uracil and a concomitant release of nitric oxide as measured by the nitrite in the culture supernatants . TNF- or TNF(70-80)-induced macrophage activation was dependent on IFN-gamma and was inhibited by neutralizing monoclonal antibody to human TNF and by anti-IFN-gamma antisera . Both nitrite release and BCG growth inhibition were abrogated by competitive inhibitors of L-arginine, which blocked the activation of inducible nitric oxide synthase . A soluble form of the Type 1 TNF receptor blocked the activation of BCG-infected macrophages by human TNF and TNF(70-80), demonstrating that the effect of TNF(70-80) is dependent on signaling through TNF receptor I . The mimetic effects of TNF(70-80) on macrophage activation in vitro suggest that treatment with TNF(70-80) may modulate mycobacterial infections in vivo.

Appl Environ Microbiol, 1998 May, 64(5), 1750 - 8
Germination, growth, and sporulation of Bacillus thuringiensis subsp . israelensis in excreted food vacuoles of the protozoan Tetrahymena pyriformis; Manasherob R et al.; Spores of Bacillus thuringiensis subsp . israelensis and their toxic crystals are bioencapsulated in the protozoan Tetrahymena pyriformis, in which the toxin remains stable . Each T . pyriformis cell concentrates the spores and crystals in its food vacuoles, thus delivering them to mosquito larvae, which rapidly die . Vacuoles containing undigested material are later excreted from the cells . The fate of spores and toxin inside the food vacuoles was determined at various times after excretion by phase-contrast and electron microscopy as well as by viable-cell counting . Excreted food vacuoles gradually aggregated, and vegetative growth of B . thuringiensis subsp . israelensis was observed after 7 h as filaments that stemmed from the aggregates . The outgrown cells sporulated between 27 and 42 h . The spore multiplication values in this system are low compared to those obtained in carcasses of B . thuringiensis subsp . israelensis-killed larvae and pupae, but this bioencapsulation represents a new possible mode of B . thuringiensis subsp . israelensis recycling in nontarget organisms.

J Biol Chem, 1998 Apr 3, 273(14), 7996 - 8002
Evidence against the Bm1P1 protein as a positive transcription factor for barbiturate-mediated induction of cytochrome P450BM-1 in bacillus megaterium; Shaw GC et al.; The Bm1P1 protein was previously proposed to act as a positive transcription factor involved in barbiturate-mediated induction of cytochrome P450BM-1 in Bacillus megaterium . We now report that the bm1P1 gene encodes a protein of 217 amino acids, rather than the 98 amino acids as reported previously . In vitro gel shift assays indicate that the Bm1P1 protein did not interact with probes comprising the regulatory regions of the P450BM-1 gene . Moreover, disruption of the bm1P1 gene did not markedly affect barbiturate induction of P450BM-1 expression . A multicopy plasmid harboring only the P450BM-1 promoter region could increase expression of the chromosome-encoded P450BM-1 . The level of expression is comparable with that shown by a multicopy plasmid harboring the P450BM-1 promoter region along with the bm1P1 gene . These results strongly suggest that the Bm1P1 protein is unlikely to act as a positive regulator for barbiturate induction of P450BM-1 expression . Finally, deletion of the Barbie box did not markedly diminish the effect of pentobarbital on expression of a reporter gene transcriptionally fused to the P450BM-1 promoter . This suggests that the Barbie box is unlikely to be a key element in barbiturate-mediated induction of P450BM-1.

J Biotechnol, 1998 Feb 5, 60(1-2), 15 - 22
Development of ELISA and enzyme-linked immunofiltration assay (ELIFA) methods for monitoring cyclodextrin glycosyltransferase (CGTase) production and bacterial growth in Bacillus macerans batch cultures; Nogrady N et al.; Immunochemical methods were developed for monitoring cyclodextrin (CD) glycosyltransferase (CGTase) production and growth of an industrial CD-producing Bacillus macerans strain . Extracellular concentrations of CGTase released into a non-transparent culture medium during a 44 h long fermentation were detected by an indirect antigen inhibition enzyme-linked immunosorbent assay (ELISA) . The ELISA was sensitive (minimal detection level 6 ng ml-1) and highly reproducible (coefficients of variation < or = 1.2 and 5.9%, within-runs and between-runs, respectively) compared to assays of CGTase activity (coefficients of variation < or = 4.2 and 7.0%, respectively) . The ELISA, in combination with enzyme activity measurements, was useful to detect the decrease in the specific CGTase activities after 36 h of incubation, which was clearly indicative of the proteolytic degradation of CGTase . B . macerans cell numbers were estimated using an enzyme-linked immunofilter assay (ELIFA) . The assay took less than 1 h and the coefficients of variation within and between-runs (2.9-6.4%) were considerably less than for viable counting (10.6-15.4%) . In the exponential phase of growth, ELIFA results correlated more closely with the cell counting based on total protein than with viable counts . Nevertheless, in the phase of cell lysis, the bacterial cell number was systematically underestimated by ELIFA in comparison to both viable cell number and total protein determinations . Thus cell antigens detected with immunological procedures might be lost during the transition from vegetative cells to spores . On the other hand, the ELIFA procedure was specific for B . macerans cells and was a better indicator of the onset of the different growth phases than the cell numbers calculated from the protein assay.

Biosci Biotechnol Biochem, 1998 Mar, 62(3), 514 - 20
Cloning, sequence analysis, and expression in Escherichia coli of a gene coding for an enzyme from Bacillus circulans K-1 that degrades guar gum; Yoshida S et al.; A 2,048-bp nucleotide sequence containing a gene coding for an enzyme that degraded guar gum from Bacillus circulans K-1 was identified by polymerase chain reaction walking . This G-gene consisted of 1,551 nucleotides coding for a protein with Mr 55,242 . The enzyme was overexpressed in Escherichia coli JM109 cells by the cloning the G-gene downstream of the lac Z promoter of pUC19 . The molecular mass of recombinant G-enzyme estimated by SDS-PAGE was 62 KDa, close to that from strain K-1 . Analysis of the recombinant enzyme showed GalNAc, Xyl, GlcNAc, Man, Glc, and Gal to account for 1.7%, 14.4%, 6.1%, 3.2%, 54.2%, and 10.4%, respectively, of the total monosaccharides . Polyacrylamide gel electrophoresis of this enzyme with staining gave a red band . The results suggested that the sugars accounted for the differences in the molecular masses . The recombinant enzyme had two kinds of N-terminal sequences, Thr-Met-Ile-Thr-Pro-Ser-Phe-Ala-Ser-Gly-Phe-Tyr-Val-Ile and Ile-Thr-Pro-Ser-Phe-Ala-Ser-Gly-Phe-Tyr-Val-Ile-Gly-Thr . Comparison of these sequences with the deduced N-terminal sequence coded for the G-gene showed that the amino acid, first Met, of the lac Z gene or the next residues Thr-Met in the recombinant enzyme were absent in the native enzyme . Methionines near and at the N-terminus of the mature protein probably were digested by methionine aminopeptidases of E . coli after translation . The properties of recombinant G-enzyme were similar to those of the enzyme from K-1 cells.

J Invertebr Pathol, 1998 May, 71(3), 263 - 7
Environmental persistence of Bacillus thuringiensis spores following aerial application; Smith RA et al.; Soil and leaf populations of Bacillus thuringiensis (Bt) were monitored following aerial application of commercial Bt formulations at the rate of 72 billion international units per acre per year during a 5-year period . Data from soil sample spore counts suggested that Bt spores persisted in Wasatch forest soils for up to 2 years but they did not proliferate . Bt isolates were recovered from leaf samples 12 months post application from sprayed, previously sprayed and from nonsprayed areas . The frequency and diversity of Bt isolates recovered from leaves was independent of sample area spray history . In accordance with U.S . Forest Service criteria, aerial application of Bt during a 5-year period resulted in the eradication of gypsy moth (Lymantria dispar, L) from the Wasatch Front region of the Wasatch Mountain Range, Utah .

Curr Opin Ophthalmol, 1998 Jun, 9(3), 59 - 65
Endophthalmitis following open-globe injuries; Duch-Samper AM et al.; Endophthalmitis following open-globe injuries is caused by a specific range of microorganisms, of which Bacillus sp . and coagulase-negative Staphylococcus are the most frequent . Risk factors include the presence of an intraocular foreign body, injury inflicted by organic material, delay in surgery, and the type of wound involved . Despite important advances in medical and surgical management, this type of endophthalmitis continues to pose a poor prognosis . In this sense, we consider prevention to be the best approach . We report our protocols for the prevention and treatment of endophthalmitis following open-globe injuries, based on recent experimental studies on the ocular pharmacokinetics of antibiotics and on multicenter studies of the treatment of endophthalmitis.

Lett Appl Microbiol, 1998 Feb, 26(2), 161 - 5
Diarrhoeal enterotoxin production by psychrotrophic Baccillus cereus present in reconstituted milk-based infant formulae (MIF); Rowan NJ et al.; One hundred reconstituted milk-based infant formulae (MIF) representative of 10 leading brands available in many European Economic Community countries were examined for psychrotrophic Bacillus cereus and for the presence of diarrhoeal enterotoxin . Of the 38 B . cereus isolates recovered from MIF, one, four and 16 strains grew at 4, 6 and 8 degrees C after 15 d . One (2.6%), two (5.3%) and six (15.8%) of the isolates were identified as potential psychrotrophic food poisoning strains as they were both enterotoxigenic and exhibited good growth at 4, 6 and 8 degrees C, respectively . Enterotoxin was not detected in MIF in which less than 5.36 log10 cfu of B . cereus ml-1 had grown . While psychrotrophic enterotoxigenic B . cereus strains occur occasionally in MIF, brief storage of reconstituted MIF at the recommended refrigeration temperature of 4 degrees C will allow this product to remain safe for consumption.

Insect Biochem Mol Biol, 1997 Dec, 27(12), 1027 - 37
cDNAs for a chymotrypsinogen-like protein from two strains of Plodia interpunctella; Zhu YC et al.; Gut proteinases are involved in the solubilization and activation of insecticidal toxins produced by Bacillus thuringiensis and may also be involved in resistance development . Approximately threefold lower chymotrypsin-like enzyme activity was observed in a Bt(entomocidus)-resistant strain of the Indianmeal moth, Plodia interpunctella, than that in the Bt-susceptible strain . Because chymotrypsin-like proteinases are involved in Bt protoxin activation in P . interpunctella, we compared cDNA sequences, mRNA expression levels, and genomic DNA for chymotrypsin-like enzymes in Bt-susceptible and Bt-resistant strains of P . interpunctella . To isolate cDNA coding for chymotrypsinogen-like proteinases, a probe was developed using polymerase chain reaction (PCR) amplification of a cDNA library from the Bt-susceptible strain using a vector primer and a degenerate primer corresponding to a conserved sequence in the active site of serine proteinases . This probe was used to screen cDNA libraries from resistant and susceptible strains . Predicted amino acid sequences from cDNA clones of each strain share similarity with sequences of chymotrypsin-like proteinases and are most similar to a chymotrypsin-like proteinase from the tobacco hornworm, Manduca sexta . cDNAs for putative chymotrypsinogen-like proteins, from both Bt-susceptible and Bt-resistant strains of P . interpunctella share an identical open reading frame of 846 nucleotides . The encoded proteins contain amino acid sequence motifs of serine proteinase active sites, disulfide-bridge cysteine residues, and both zymogen activation and signal peptides . A difference between these cDNAs was observed only in the untranslated region where a substitution of guanine for adenine occurred in the Bt-resistant strain . Southern and Northern blotting analyses indicated that there are no major differences in chymotrypsinogen-like genomic organization and mRNA expression in the two strains . These data suggest that chymotrypsinogen-like proteinase genes and their transcription are similar in the Bt-susceptible and Bt-resistant strains of P . interpunctella.

Rhinology, 1998 Mar, 36(1), 43 - 5
Association of rhinoscleroma with rhinosporidiosis; al-Serhani AM et al.; Rhinoscleroma caused by the bacillus Klebsiella rhinoscleromatis and rhinosporidiosis caused by the fungus Rhinosporidium seebri are rare, specific nasal infections, both of which have a certain geographical distribution . To the best of our knowledge no association between them has been reported in the international literature . We have documented such an association in two male Indian patients aged-32 and 27 years, respectively-both presenting with unilateral blood-stained discharge and nasal blockage . They showed strawberry-like polypoidal masses, and histological examination confirmed the diagnosis . Klebsiella rhinoscleromatis was cultured twice in the first case . The patients were treated with complete excision and a long course of septrin, for which Klebsiella rhinoscleromatis is sensitive . The purpose of this paper is to report the first association of these two granulomatous infections, to show the impact of immigration on the differential diagnosis, and to review the relevant literature.

Int Urol Nephrol, 1998, 30(1), 41 - 4
Prospective randomized comparison of intravesical BCG therapy with standard dose versus low doses in superficial bladder cancer; Yalcinkaya F et al.; In this study, we evaluated low dose intravesical bacillus Calmette-Guerin (BCG) therapy following transurethral resection (TUR) in 80 patients with superficial bladder cancer . The patients were divided into two groups . Of the Connaught BCG strain 81 mg was given to 40 patients in Group 1 and 54 mg to the remainder of 40 patients in Group 2 . BCG was introduced once a week for 6 weeks . Tumour recurrence was seen in 6 patients in Group 1 and in 10 patients in Group 2 . Recurrence rates per month were 0.71 and 1.49, respectively . There was no significant difference in complication rates . These data suggest that while the standard dose (81 mg) intravesical therapy of BCG is more effective than the low dose, there was no significant difference in side effects between the two groups.

J Gen Virol, 1998 Apr, 79 ( Pt 4), 925 - 9
Tubules containing virions are present in plant tissues infected with Commelina yellow mottle badnavirus; Cheng CP et al.; Tubular structures containing bacilliform virions were observed in cell-free extracts of Commelina diffusa infected with Commelina yellow mottle badnavirus (CoYMV) . The exterior of the tubule reacted with antibodies to CoYMV movement protein, but not with antibodies to virus coat protein . Similar tubular structures containing bacilliform particles were also observed in ultrathin sections of CoYMV-infected C . diffusa . These tubular structures traversed the cell wall at points where this was thickened or protruded . No similar structures were observed in healthy C . diffusa . These observations support the hypothesis that the virion-containing tubular structures observed in cell-free extracts are the same as those observed in situ, that these structures are composed, at least in part, of virus movement protein, and that they play a role in the cell-to-cell trafficking of virions of CoYMV.

Indian J Exp Biol, 1997 Nov, 35(11), 1191 - 3
Effect of insecticidal crystal proteins of Bacillus thuringiensis var . israelensis on the enzymes of rat intestinal brush border membrane vesicles; Rani SS et al.; In vivo treatment of intestinal brush border membrane vesicles with solubilized insecticidal crystal proteins (ICP) from the two strains of B . thuringiensis var . israelensis (VCRC B17 and VCRC MB24) caused no adverse effect on gamma glutamyl transpeptidase, Na+K+ATPase, sucrase and lactase enzymes . But, exposure of membrane vesicles to solubilized ICP's in vitro, lead to significant reduction in the activity of Na+K+ATPase, sucrase and lactase enzymes.

Prikl Biokhim Mikrobiol, 1998 Mar-Apr, 34(2), 175 - 9
{Study of cytokinins produced by rhizospheric microorganisms}; Veselov SIu et al.; A high-molecular-weight complex of a polysaccharide and biologically active cytokinins was found in the culture liquid of rhizosphere microorganisms of the genus Bacillus . Enzyme immunoassay and thin-layer chromatography showed that zeatin riboside and a hormone nucleotide were the main cytokinins observed . Components of this complex were linked by a noncovalent bond.

Int J Syst Bacteriol, 1998 Jan, 48 Pt 1, 107 - 16
PCR fingerprinting of whole genomes: the spacers between the 16S and 23S rRNA genes and of intergenic tRNA gene regions reveal a different intraspecific genomic variability of Bacillus cereus and Bacillus licheniformis {corrected}; Daffonchio D et al.; Genomic diversity in 21 strains of Bacillus cereus and 10 strains of Bacillus licheniformis was investigated by random amplified polymorphic DNA (RAPD) analysis, which samples the whole genome, and by two PCR fingerprinting techniques sampling the hypervariable spacers between the conserved 16S and 23S rRNA genes of the rRNA gene operon (ITS-PCR) and regions between tRNA genes (tDNA-PCR) . RAPD analysis showed a remarkable diversity among strains of B . cereus that was not observed with the rRNA and tRNA intergenic-spacer-targeted PCR, where all the strains showed practically identical fingerprints . A wide variability among the B . cereus strains was also observed in the plasmid profiles, suggesting that the genetic diversity within B . cereus species can arise from plasmid transfer . One contribution to the diversity detected by RAPD analysis was determined by the presence of large extrachromosomal elements that were amplified during RAPD analysis as shown by Southern hybridization experiments . In contrast to the strains of B . cereus, the 10 strains of B . licheniformis were grouped into two clusters which were the same with all the methods employed . The 16S rRNA genes were identical in all 10 strains when examined using single strand conformation polymorphism analysis after digestion with Alul and Rsal . From these data we hypothesize two different evolutionary schemes for the two species.

Biochemistry, 1998 Mar 24, 37(12), 4275 - 9
Engineering of the nonspecific phospholipase C from Bacillus cereus: replacement of glutamic acid-4 by alanine results in loss of interfacial catalysis and enhanced phosphomonoesterase activity; Tan CA et al.; The nonspecific phospholipase C from Bacillus cereus is a zinc metalloenzyme that catalyzes the hydrolysis of phospholipids to yield diacylglycerol and a phosphate monoester . Glu-4 has been proposed as a potential candidate for the general base in the hydrolysis reaction and was shown to interact with the substrate headgroup . Site-specific mutagenesis studies suggest that Glu-4 is important for substrate binding but not for catalysis . This residue is also critical for the enzyme's preference for a phosphodiester substrate . PA, both monomeric and micellar, is shown to be a poor substrate and inhibitor of wild-type PLC . When Glu-4 was mutated to an alanine, a significant increase in PA hydrolysis and a decrease in PC hydrolysis were observed . Unlike the wild type, kinetic studies suggest that the Glu-4-->Ala mutant does not exhibit interfacial activation and processive catalysis . Glu-4 is part of a highly flexible loop flanking the entrance to the active site, suggesting that this loop might constitute an interfacial binding recognition site . This is the first evidence for the presence of an interfacial binding site distinct from the active site in the nonspecific PLC.

Am J Respir Crit Care Med, 1998 Apr, 157(4 Pt 1), 1324 - 7
Bacillus Calmette-Guérin revaccination questionable with low tuberculosis incidence; Tala-Heikkila MM et al.; Bacillus Calmette-Guerin (BCG) revaccination was discontinued in Finland in 1990 . The objective of this study was to assess the impact of BCG revaccination of tuberculin-negative school-children in prevention of tuberculosis . The tuberculosis cases in 1990-1995 were calculated among age cohorts born 1979-1984 and no longer covered by the BCG revaccination program . Corresponding data were collected for comparison from the period of revaccination in 1980-1985 among age cohorts born in 1969-1974 . The National Tuberculosis Register was reviewed in order to observe the tuberculosis trend since 1980 in the age groups of 10-14 and 15-19 yr . Three cases of tuberculosis have been registered among non-BCG-revaccinated children during 6 yr after discontinuation of the program, i.e., 2.23 cases (95% CI 0.72 to 6.90) per million person yr . The control group revealed five cases, 3.78 (95% CI 1.57 to 9.07) per million person yr . The relative risk of tuberculosis in non-BCG-revaccinated children is 0.59 (95% CI 0.14 to 2.47) compared with the control group . The incidence of tuberculosis has continued to decline among adolescents since 1980 . The follow-up data confirm that the cessation of BCG revaccination program had no effect on the continuing overall decline of tuberculosis in Finland . The efficacy of BCG revaccination seems to be low or nonexistent in countries with low tuberculosis incidence.

Virus Genes, 1998, 16(1), 119 - 31
Origin, adaptation and evolutionary pathways of fungal viruses; Ghabrial SA; Fungal viruses or mycoviruses are widespread in fungi and are believed to be of ancient origin . They have evolved in concert with their hosts and are usually associated with symptomless infections . Mycoviruses are transmitted intracellularly during cell division, sporogenesis and cell fusion, and they lack an extracellular phase to their life cycles . Their natural host ranges are limited to individuals within the same or closely related vegetative compatibility groups . Typically, fungal viruses are isometric particles 25-50 nm in diameter, and possess dsRNA genomes . The best characterized of these belong to the family Totiviridae whose members have simple undivided dsRNA genomes comprised of a coat protein (CP) gene and an RNA dependent RNA polymerase (RDRP) gene . A recently characterized totivirus infecting a filamentous fungus was found to be more closely related to protozoan totiviruses than to yeast totiviruses suggesting these viruses existed prior to the divergence of fungi and protozoa . Although the dsRNA viruses at large are polyphyletic, based on RDRP sequence comparisons, the totiviruses are monophyletic . The theory of a cellular self-replicating mRNA as the origin of totiviruses is attractive because of their apparent ancient origin, the close relationships among their RDRPs, genome simplicity and the ability to use host proteins efficiently . Mycoviruses with bipartite genomes (partitiviruses), like the totiviruses, have simple genomes, but the CP and RDRP genes are on separate dsRNA segments . Because of RDRP sequence similarity, the partitiviruses are probably derived from a totivirus ancestor . The mycoviruses with unencapsidated dsRNA-like genomes (hypoviruses) and those with bacilliform (+) strand RNA genomes (barnaviruses) have more complex genomes and appear to have common ancestry with plant (+) strand RNA viruses in supergroup 1 with potyvirus and sobemovirus lineages, respectively . The La France isometric virus (LIV), an unclassified virus with multipartite dsRNA genome, is associated with a severe die-back disease of the cultivated mushroom . LIV appears to be of recent origin since it differs from its host in codon usage.

Int J Food Microbiol, 1998 Jan 6, 39(1-2), 93 - 9
The adhesion of Bacillus cereus spores to epithelial cells might be an additional virulence mechanism; Andersson A et al.; Four out of ten Bacillus cereus strains produced spores able to adhere to monolayers of Caco-2 cells (human epithelial cells) . One of these strains has been involved in an outbreak of food poisoning where the symptoms were more severe and persisted for longer than a normal B . cereus food poisoning . The hydrophobicity of the spores is a contributing factor for the adhesion to occur . The spores are able to germinate in an environment similar to that of the small intestine and then the vegetative cells can produce the enterotoxin directly at the target place . A concentrated and active form of the enterotoxin will be taken up by the epithelial cells in the small intestine . Spore adhesion could be an important virulence factor for some B . cereus strains.

Biochem J, 1998 May 1, 331 ( Pt 3), 703 - 11
The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent beta-lactamase; Bounaga S et al.; The plot of kcat/Km against pH for the Bacillus cereus 569/H beta-lactamase class B catalysed hydrolysis of benzylpenicillin and cephalosporin indicates that there are three catalytically important groups, two of pKa 5.6+/-0.2 and one of pKa 9.5+/-0.2 . Below pH 5 there is an inverse second-order dependence of reactivity upon hydrogen ion concentration, indicative of the requirement of two basic residues for catalysis . These are assigned to zinc(II)-bound water and Asp-90, both with a pKa of 5.6+/-0.2 . A thiol, N-(2'-mercaptoethyl)-2-phenylacetamide, is an inhibitor of the class B enzyme with a Ki of 70 microM . The pH-dependence of Ki shows similar pH inflections to those observed in the catalysed hydrolysis of substrates . The pH-independence of Ki between pH 6 and 9 indicates that the pKa of zinc(II)-bound water must be 5.6 and not the higher pKa of 9.5 . The kinetic solvent isotope effect on kcat/Km is 1.3+/-0.5 and that on kcat is 1.5 . There is no effect on reactivity by either added zinc(II) or methanol . The possible mechanisms of action for the class B beta-lactamase are discussed, and it is concluded that zinc(II) acts as a Lewis acid to stabilize the dianionic form of the tetrahedral intermediate and to provide a hydroxide-ion bound nucleophile, whereas the carboxylate anion of Asp-90 acts as a general base to form the dianion and also, presumably, as a general acid catalyst facilitating C-N bond fission.

Eur Urol, 1998, 33(3), 278 - 84
Comparative analysis of MiB1 and p53 expression in human bladder tumors and their correlation with cancer progression; Pfister C et al.; Expression of p53 and MiB1, markers of tumor proliferation, was evaluated in human bladder tumors, and correlated with ploidy and cancer progression in 83 consecutive patients . Transurethral resection of a newly diagnosed bladder tumor was performed in 73 cases, and systematic bladder biopsies were performed in 10 cases after bacillus Calmette-Guerin (BCG) treatment . p53 and MiB1 expression were performed by an immunohistochemical technique and the ploidy was determined on a frozen fragment of the tumor . p53 expression was correlated in relation to grade, stage and combination of grade and stage . MiB1 expression was correlated with cytological grade, and a significant difference was demonstrated between pT0 and pTa, pTa, and pT1, pTa and pT2 tumors but not between pT1 and > or = pT2 tumors . A discordance was observed for the comparison of p53 and MiB1 values, stage by stage, suggesting that these two techniques are independent of each other . A larger proportion of aneuploid tumors were positive for p53 and MiB1 (64.8 vs . 86.5%, respectively), but p53 and MiB1 immunostaining were not better indicators than ploidy alone to predict cancer progression.

Curr Microbiol, 1998 May, 36(5), 278 - 82
Spore coat protein synergizes bacillus thuringiensis crystal toxicity for the indianmeal moth
Johnson DE, Oppert B, McGaughey WH.
Spores from Bacillus thuringiensis serovars kurstaki and entomocidus synergized crystal protein toxicity for larvae of the Indianmeal moth (Plodia interpunctella) . Preparations of spore-crystal mixtures of either serovar were more toxic for the larvae than either purified spores or crystals alone (based on dry weight) . Spores lost 53% of their toxicity for the Indianmeal moth after 2 h of UV-irradiation, but remained partially toxic (28%) even after 4 h of irradiation . Spore coat protein was toxic for the Indianmeal moth and was synergistic with B . thuringiensis serovar kurstaki HD-1 crystal protein . Enhanced toxicity of the combined spore-crystal preparation was attributed to a combination of crystal and spore coat protein, and included the effects of spore germination and resulting septicemia in the larval hemolymph . Ultraviolet irradiation of spores reduced the toxicity from septicemia but not the synergism caused by spore coat protein . The potencies of spore-crystal preparations must be carefully evaluated on the basis of contributions from all three factors.

Plant Physiol, 1998 Apr, 116(4), 1431 - 41
Extracellular matrix assembly in diatoms (Bacillariophyceae) . Iii . Organization Of fucoglucuronogalactans within the adhesive stalks of achnanthes longipes
Wustman BA, Lind J, Wetherbee R, Gretz MR.
Achnanthes longipes is a marine, biofouling diatom that adheres to surfaces via adhesive polymers extruded during motility or organized into structures called stalks that contain three distinct regions: the pad, shaft, and collar . Four monoclonal antibodies (AL.C1-AL.C4) and antibodies from two uncloned hybridomas (AL.E1 and AL.E2) were raised against the extracellular adhesives of A . longipes . Antibodies were screened against a hot-water-insoluble/hot-bicarbonate-soluble-fraction . The hot-water-insoluble/hot-bicarbonate-soluble fraction was fractionated to yield polymers in three size ranges: F1, >/= 20,000, 000 Mr; F2, congruent with100,000 Mr; and F3, <10,000 Mr relative to dextran standards . The congruent with100,000-Mr fraction consisted of highly sulfated (approximately 11%) fucoglucuronogalactans (FGGs) and low-sulfate (approximately 2%) FGGs, whereas F1 was composed of O-linked FGG (F2)-polypeptide (F3) complexes . AL.C1, AL.C2, AL.C4, AL.E1, and AL.E2 recognized carbohydrate complementary regions on FGGs, with antigenicity dependent on fucosyl-containing side chains . AL.C3 was unique in that it had a lower affinity for FGGs and did not label any portion of the shaft . Enzyme-linked immunosorbent assay and immunocytochemistry indicated that low-sulfate FGGs are expelled from pores surrounding the raphe terminus, creating the cylindrical outer layers of the shaft, and that highly sulfated FGGs are extruded from the raphe, forming the central core . Antibody-labeling patterns and other evidence indicated that the shaft central-core region is related to material exuded from the raphe during cell motility.

Int J Urol, 1998 Mar, 5(2), 185 - 7
Nephrogenic adenoma in a patient with transitional cell carcinoma of the bladder receiving intravesical bacillus Calmette-Guérin; Oyama N et al.; A 76-year-old-man was admitted to our hospital for a recurrent bladder tumor . He had received intravesical bacillus Calmette-Guerin (BCG) treatment for a transitional cell carcinoma of the bladder . A follow-up cystoscopy revealed a solitary papillary tumor in the left bladder wall . A transurethral cold cup biopsy revealed a nephrogenic adenoma without any evidence of malignant cells . We discuss the pathogenesis of nephrogenic adenoma and suggest that prolonged cystitis caused by intravesical BCG may play an etiological role.

Int J Tuberc Lung Dis, 1998 Apr, 2(4), 288 - 95
Nationwide surveillance of drug-resistant tuberculosis in The Netherlands: rates, risk factors and treatment outcome; Lambregts-van Weezenbeek CS et al.; SETTING: The Netherlands, 1993 and 1994 . OBJECTIVE: To determine 1) rates of drug resistance in relation to nationality and country of birth, 2) risk factors for drug resistance, 3) treatment outcome of drug-resistant cases, and 4) rates of primary and acquired drug resistance . DESIGN: Retrospective study of all cases notified with bacillary tuberculosis in The Netherlands in 1993 and 1994 . RESULTS: Drug resistance to one or more drugs was reported in 268 (14.6%) of all 1836 cases, of whom 203 (76%) were foreign born . In Dutch patients rates of isoniazid (H) (2.9%) and streptomycin resistance (3.6%) were lower than in foreign patients (8.6% and 10.6% respectively, P < 0.001) . Multidrug (H and rifampicin {R}) resistance was reported in 0.5% of Dutch-born and 1.4% of foreign cases (P = 0.055) . Rates of acquired resistance to H (11.4%) and HR (5.7%) were higher than rates of primary resistance to these drugs (5.2% and 0.7% respectively, P < 0.05), but the number of retreatment cases was low (6.8% of all cases) . Drug resistance was associated with immigration but not with drug use, homelessness or human immunodeficiency virus (HIV) co-infection . One fifth (20%) of drug-resistant cases was diagnosed by active case finding . Treatment outcome in sensitive and resistant cases was compared . CONCLUSION: These findings suggest that drug resistance is imported, but it is unclear to what extent drug resistance among foreigners has been transmitted or created in The Netherlands . Drug resistance data should be monitored in Dutch and foreign patients separately.

Glycoconj J, 1998 Feb, 15(2), 155 - 60
Purification and properties of recombinant beta-galactosidase from Bacillus circulans; Fujimoto H et al.; A gene encoding beta-galactosidase from Bacillus circulans which had hydrolysis specificity for the beta1-3 linkage was expressed in Escherichia coli . The beta-galactosidase was purified from crude cell lysates of E . coli by column chromatographies on Resource Q and Sephacryl S-200 HR . The enzyme released galactose with high selectivity from oligosaccharides which had terminal beta1-3 linked galactose residues . However it did not hydrolyse beta1-4 linked galactooligosaccharides . Moreover, Galbeta1-3GlcNAc, Galbeta1-3GalNAc, and their p-nitrophenyl glycosides were regioselectively synthesized in 10-46% yield by the transglycosylation reaction using this enzyme.

Microbios, 1997, 91(368-369), 203 - 14
Plasmid patterns of Bacillus thuringiensis strains and isolates; Aptosoglou SG et al.; Bacillus thuringiensis produces crystal proteins which are toxic to several orders of economically important insects and other invertebrates . The genes encoding these toxins reside mainly on plasmids . This report consists of a comparative analysis of the plasmid content of a number of B . thuringiensis strains and isolates which may facilitate the search for novel toxin genes and other important products of this organism.

Arch Intern Med, 1998 Apr 13, 158(7), 801 - 3
Diagnostic utility of the polymerase chain reaction in 2 cases of suspected Whipple disease; Tasken K et al.; We describe 2 patients with a diagnosis of Whipple disease in whom the usual antibiotic therapy failed . A polymerase chain reaction-based test was used to identify the recently described Whipple bacillus, Tropheryma whippelii . In one case, the diagnosis was confirmed, whereas in the second case, which had been histologically diagnosed as Whipple disease of the brain, the process was identified as a monocyte-derived histiocytosis . In conclusion, Whipple disease can be distinguished from other diseases with similar histological features with the use of a polymerase chain reaction-based test.

J Urol, 1998 May, 159(5), 1488 - 92
Activation of human dendritic cells by bacillus Calmette-Guerin; Ramoner R et al.; PURPOSE: Dendritic cells are the most potent antigen presenting cells capable of initiating antitumor immune responses . We previously showed that bacillus Calmette-Guerin (BCG) stimulates cultured human dendritic cells . We extended these studies and tested the ability of cultured human dendritic cells to express interleukin IL-8 in response to BCG . We also investigated the T cell stimulatory potential of BCG treated dendritic cells in mixed leukocyte reactions . MATERIALS AND METHODS: Dendritic cells were obtained by culturing plastic adherent mononuclear cells from peripheral blood for 6 days in the presence of granulocyte-macrophage colony-stimulating factor and IL-4 . Spontaneous and BCG stimulated IL-8 protein release into culture supernatants was measured by a quantitative immunoassay . IL-8 gene transcription was assessed by reverse transcription-polymerase chain reaction . Untreated and BCG exposed dendritic cells were compared as stimulators of allogeneic T cell proliferation, measured as {3H}thymidine incorporation . RESULTS: BCG stimulated IL-8 messenger ribonucleic acid expression and IL-8 protein release . IL-8 secretion occurred in a dose and time dependent fashion . BCG induced IL-8 release was further enhanced in the presence of indomethacin . BCG treated dendritic cells were much more potent T cell stimulators than untreated dendritic cells . CONCLUSIONS: These data demonstrate that BCG enhances the production of IL-8, a potent chemokine of T cells and granulocytes, as well as the T cell stimulatory potential of human dendritic cells.

Immunol Cell Biol, 1998 Feb, 76(1), 41 - 6
IL-4, IL-5 and IL-10 are not required for the control of M . bovis-BCG infection in mice; Erb KJ et al.; Mycobacterial infections in mice are normally characterized by a profound Th1 cell-mediated immune response, in which T cells secrete large amounts of IFN-gamma . Recent evidence suggests that this response also includes a Th2 component . In order to investigate whether production of IL-4, IL-5, or IL-10 influenced the outcome of a Mycobacterium bovis-bacille Calmette-Guerin (BCG) infection, we intranasally infected IL-4, IL-5, and IL-10 gene-deficient and control mice and monitored the resulting immune response and bacterial clearance . IL-4, IL-5, and IL-10 deficient mice cleared the mycobacteria with the same kinetics as control mice . Furthermore, T cells of cytokine deficient and control mice produced similar levels of IFN-gamma following in vitro stimulation with purified protein derivative (PPD) from M . bovis . We conclude that the cytokines IL-4, IL-5 and IL-10 are not essential for and do not negatively influence the protective immune response against M . bovis-BCG in the lung of mice.

Infect Control Hosp Epidemiol, 1998 Mar, 19(3), 191 - 3
In support of bacillus of Calmette and Guérin for healthcare workers; Jenney AW et al.; Although widely used outside the United States, bacillus of Calmette and Guerin (BCG) immunization generally is given scant consideration in the US literature . We believe that the recent resurgence of tuberculosis, including multidrug-resistant tuberculosis, is a compelling argument for the use of BCG in healthcare workers and that BCG given to those at risk of exposure could be more effective than routine tuberculin skin testing and isoniazid prophylaxis

Infect Control Hosp Epidemiol, 1998 Mar, 19(3), 168 - 74
Low risk for tuberculosis in a regional pediatric hospital: nine-year study of community rates and the mandatory employee tuberculin skin-test program; Christie CD et al.; OBJECTIVE: To assess the risk of Mycobacterium tuberculosis infection and disease among patients and workers in a regional pediatric hospital . DESIGN: Descriptive epidemiological study of the mandatory tuberculin skin testing program of hospital employees at hire and during annual reevaluation, pediatric patients with tuberculosis (TB), efficacy of hospital infection control measures, and community rates of TB . SETTING: 361-bed, university, pediatric hospital serving Cincinnati (1.7 million population) . RESULTS: During 1986 through 1994, 2,275 to 4,356 employees were compliant with Mantoux skin testing and screening each year . This represented >97% of the population who were eligible for screening . The cumulative rate of M tuberculosis infection from a previous positive tuberculin skin test was 10% to 12% per year during 1986 through 1994 . Among new Mantoux skin-test converters in employees at annual reevaluation, the risk of TB infection was 0.3% in 1993 and 1994 . There were no active cases of TB identified during new employee screening or annual reevaluation . Of 62 new Mantoux skin-test converters in 9 years, 23% were foreign-born, 13% were Asian, 23% were African American, 11% received the bacillus of Calmette-Guerin vaccine, and 60% had direct patient care or indirect patient contact . A cluster of five converters occurred in a department with no patient care or contact . Mantoux conversion rates were 1.9 per 1,000 employee patient-care or contact-years and 2.2 per 1,000 employee non-patient-contact years . Twenty pediatric patients with active TB were identified during 1991 to 1994, with < or =6 cases per year, placing this hospital in the low-risk category for M tuberculosis disease . Three children with pulmonary TB were admitted without immediate respiratory isolation, possibly exposing 9 patients and 42 employees; none converted their Mantoux skin tests on retesting . Rates of active TB in Cincinnati were stable during the period (eg, 8/100,000 population in 1994) . CONCLUSIONS: Despite intense active surveillance among thousands of hospital employees with >97% annual compliance, tuberculin conversion rates were low, and no cases of active TB were identified during 9 years of follow-up . There was no evidence of transmission of M tuberculosis from infected patients to employees during uncontrolled exposures . Rates of TB in the community were low . These data suggest that rigorous application of the Centers for Disease Control and Prevention guidelines and Occupation Safety and Health Administration regulations for preventing nosocomial TB in pediatric hospitals may be excessive and costly . Special provisions should be made for pediatric hospitals with a proven low risk of transmission of M tuberculosis.

J Immunol, 1998 Jan 1, 160(1), 494 - 501
Distinct classes of chaperoned IL-6 in human blood: differential immunological and biological availability; Ndubuisi MI et al.; Transport of IL-6 in blood is fundamental to the biology of this cytokine . In the present study, IL-6 transport, immunological reactivity, and biological availability were investigated in blood from melanoma patients subjected to different active specific immunization regimens (an anti-idiotypic mAb immunization protocol (mAb-keyhole limpet hemocyanin (KLH)-Calmette-Guerin bacillus (BCG), an autologous anti-cancer vaccine protocol (AAAP), or both) . Sera were subjected to Sephadex G-200 gel filtration chromatography, and the structure and biological activity of IL-6 complexes in the eluate fractions were probed using five IL-6 ELISAs and two bioassays . Sera from patients administered mAb-KLH+BCG followed by AAAP contained three distinct classes of IL-6 eluting at 30, 200, and 450 kDa, each with its characteristic ELISA reactivity and bioactivity: the 30- and 450-kDa complexes were bioactive in the B9 and Hep3B assays, but the 200-kDa complex was not . The 30- and 450-kDa IL-6 complexes were preferentially reactive in the 7IL6/5IL6 ELISA, the 200-kDa IL-6 complexes were preferentially reactive in the 4IL6/5IL6 ELISA, while the three commercial ELISAs (R&D, Endogen, and Genzyme) detected essentially only the 30-kDa IL-6 . In contrast, 1) sera from AAAP patients contained biologically active 30- and 450-kDa IL-6 complexes, while 2) sera from mAb-KLH+BCG patients contained 200-kDa IL-6 complexes inactive in ex vivo bioassays . Both the 450- and 200-kDa complexes included soluble IL-6R, with the 200-kDa complexes additionally containing ligand-occupied anti-IL-6 and anti-soluble IL-6R IgG . The data indicate the existence of specific mechanisms that regulate the transport and function of IL-6 in vivo.

Structure, 1998 Mar 15, 6(3), 281 - 92
Activation of Bacillus licheniformis alpha-amylase through a disorder-->order transition of the substrate-binding site mediated by a calcium-sodium-calcium metal triad; Machius M et al.; BACKGROUND: The structural basis as to how metals regulate the functional state of a protein by altering or stabilizing its conformation has been characterized in relatively few cases because the metal-free form of the protein is often partially disordered and unsuitable for crystallographic analysis . This is not the case, however, for Bacillus licheniformis alpha-amylase (BLA) for which the structure of the metal-free form is available . BLA is a hyperthermostable enzyme which is widely used in biotechnology, for example in the breakdown of starch or as a component of detergents . The determination of the structure of BLA in the metal-containing form, together with comparisons to the apo enzyme, will help us to understand the way in which metal ions can regulate enzyme activity . RESULTS: We report here the crystal structure of native, metal-containing BLA . The structure shows that the calcium-binding site which is conserved in all alpha-amylases forms part of an unprecedented linear triadic metal array, with two calcium ions flanking a central sodium ion . A region around the metal triad comprising 21 residues exhibits a conformational change involving a helix unwinding and a disorder-->order transition compared to the structure of metal-free BLA . Another calcium ion, not previously observed in alpha-amylases, is located at the interface between domains A and C . CONCLUSIONS: We present a structural description of a major conformational rearrangement mediated by metal ions . The metal induced disorder-->order transition observed in BLA leads to the formation of the extended substrate-binding site and explains on a structural level the calcium dependency of alpha-amylases . Sequence comparisons indicate that the unique Ca-Na-Ca metal triad and the additional calcium ion located between domains A and C might be found exclusively in bacterial alpha-amylases which show increased thermostability . The information presented here may help in the rational design of mutants with enhanced performance in biotechnological applications.

Farmaco, 1997 Nov, 52(11), 707 - 10
Synthesis, lipophilicity and biological properties of some novel 1H-1,2,4 triazole derivatives; Papakonstantinou-Garoufalias S et al.; A series of new 1H-1,2,4-triazole derivatives was synthesized and evaluated as potential antiviral (i.e . anti-influenza virus), antibacterial and antifungal agents . The lipophilicity of the compounds was also investigated using calculation procedures . Among the test compounds none showed specific activity against influenza virus, although compound 3a, the most hydrophilic member of the series, showed weak activity against Bacillus subtillis.

PDA J Pharm Sci Technol, 1998 Jan-Feb, 52(1), 5 - 12
Evaluation of high-temperature and short-time sterilization of injection ampules by microwave heating; Sasaki K et al.; The high-temperature and short-time sterilization by microwave heating with a continuous microwave sterilizer (MWS) was evaluated . The evaluation were performed with respect to: {1} lethal effect against microorganisms corresponding to F-value, and {2} reliability of MWS sterilization process . Bacillus stearothermophilus ATCC 7953 spores were used as the biological indicator and the heat-resistance of spores was evaluated with conventional heating method (121-129 degrees C) . In MWS sterilization (125-135 degrees C), the actual lethal effect against B . stearothermophilus spores was almost in agreement with the F-value and the survival curve against the F-value was quite consistent with that for the autoclave . These results suggest that the actual lethal effect could be estimated by the F-value with heat-resistance parameters of spores from lower than actual temperatures and that there was no nonthermal effect of the microwave on B . stearothermophilus spores . The reliability of sterilization with the MWS was confirmed using more than 25,000 test ampules containing biological indicators . All biological indicators were killed, thus the present study shows that the MWS was completely reliable for all ampules.

Biochemistry, 1998 Mar 31, 37(13), 4568 - 80
Mechanism of phosphatidylinositol-specific phospholipase C: a unified view of the mechanism of catalysis; Hondal RJ et al.; The mechanism of phosphatidylinositol-specific phospholipase C (PI-PLC) has been suggested to resemble that of ribonuclease A . The goal of this work is to rigorously evaluate the mechanism of PI-PLC from Bacillus thuringiensis by examining the functional and structural roles of His-32 and His-82, along with the two nearby residues Asp-274 and Asp-33 (which form a hydrogen bond with His-32 and His-82, respectively), using site-directed mutagenesis . In all, twelve mutants were constructed, which, except D274E, showed little structural perturbation on the basis of 1D NMR and 2D NOESY analyses . The H32A, H32N, H32Q, H82A, H82N, H82Q, H82D, and D274A mutants showed a 10(4)-10(5)-fold decrease in specific activity toward phosphatidylinositol; the D274N, D33A, and D33N mutants retained 0 . 1-1% activity, whereas the D274E mutant retained 13% activity . Steady-state kinetic analysis of mutants using (2R)-1, 2-dipalmitoyloxypropane-3-(thiophospho-1d-myo-inositol) (DPsPI) as a substrate generally agreed well with the specific activity toward phosphatidylinositol . The results suggest a mechanism in which His-32 functions as a general base to abstract the proton from 2-OH and facilitates the attack of the deprotonated 2-oxygen on the phosphorus atom . This general base function is augmented by the carboxylate group of Asp-274 which forms a diad with His-32 . The H82A and D33A mutants showed an unusually high activity with substrates featuring low pKa leaving groups, such as DPsPI and p-nitrophenyl inositol phosphate (NPIPs) . These results suggest that His-82 functions as the general acid with assistance from Asp-33, facilitating the departure of the leaving group by protonation of the glycerol O3 oxygen . The Bronsted coefficients obtained for the WT and the D33N mutant indicate a high degree of proton transfer to the leaving group and further underscore the "helper" function of Asp-33 . The complete mechanism also includes activation of the phosphate group toward nucleophilic attack by a hydrogen bond between Arg-69 and a nonbridging oxygen atom . The overall mechanism can be described as "complex" general acid-general base since three elements are required for efficient catalysis.

Curr Microbiol, 1998 Mar, 36(3), 152 - 7
A sandwich enzyme-linked immunosorbent assay for the Bacillus sphaericus binary toxin; Jiaviriyaboonya S et al.; Bacillus sphaericus (Bs) binary toxin was purified from recombinant E . coli DH5alpha harboring the recombinant plasmid pAR5, which carries a 3.6-kb DNA fragment of Bs 1593M encoding mosquito larvicidal activity . The binary toxin preparation, designated BsEcAg, contained mainly 51- and 42-kDa toxin proteins and was toxic to 50% of Culex quinquefasciatus larvae at a concentration of 9.22 ng toxin protein/ml . This preparation was used to raise antibodies in sheep and mice . The sandwich ELISA used sheep antitoxin antibody as primary antibody (coating antibody), mouse antitoxin antibody as second antibody, and goat antimouse antibody as an alkaline phosphatase-conjugated detecting antibody . The assay sensitivity was 200 ng/ml for both BsEcAg and binary toxin antigen (BsAg) from Bs 2362 cells . There is a significant correlation between toxin level determined by ELISA and bioassay . This procedure has also been used to monitor toxin levels in batch fermentations of Bs 2362.

Curr Microbiol, 1998 Mar, 36(3), 148 - 51
Psychrotrophic bacteria isolated from a constantly warm tropical environment; Astwood AC et al.; Psychrotrophic bacteria are known to occur in temperate, constantly cold, and artificially cooled environments . This is the first report of their occurrence in a constantly warm (ca . 24 degrees-35 degrees C) tropical environment . Soil samples taken from two sites along the southeastern coastal zone of Jamaica yielded growth of psychotrophic bacteria after 3-4 weeks of enrichment culture in 1/30 strength tryptic soy broth, 20 mg L-1 cycloheximide at 2 degrees C . Growth of individual isolates at 2 degrees C was confirmed . Isolates include aerobic and fermentative Gram-negative rods and sporeforming (Bacillus sp.) and non-sporeforming (Aureobacterium sp.) Gram-positive rods . We determined the effect of temperature on growth rate in four isolates . Strain Y1 has an unusually wide temperature range for growth, 2 degrees-44 degrees C, resembling that of Listeria monocytogenes . In strain R1 the optimum temperature for growth occurred unusually near the maximum temperature for growth . Strains R2 and Y2 displayed cardinal temperatures typical of known psychotrophs but appear to have evolved enhanced growth potential near the optimum temperature in response to a constantly warm environment.

Eur J Biochem, 1998 Mar 15, 252(3), 447 - 57
Comparative study of the catalytic domain of phosphorylating glyceraldehyde-3-phosphate dehydrogenases from bacteria and archaea via essential cysteine probes and site-directed mutagenesis; Talfournier F et al.; Phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GraP-DH) catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate to form 1.3-diphosphoglycerate . The currently accepted mechanism involves an oxidoreduction step followed by a phosphorylation . Two essential aminoacids, Cys149 and His176 are involved in the chemical mechanism of bacterial and eukaryotic GraP-DHs . Roles have been assigned to the His176 as (a) a chemical activator for enhancing the reactivity of Cys149, (b) a stabilizator of the tetrahedral transition states, and (c) a base catalyst facilitating hydride transfer towards NAD . In a previous study carried out on Escherichia coli GraP-DH {Soukri, A., Mougin, A., Corbier, C., Wonacott, A . J., Branlant, C . & Branlant, G . (1989) Biochemistry, 28, 2586-2592}, the role of His176 as an activator of the reactivity of Cys149 was studied . Here, we further investigated the role of the His residue in the chemical mechanism of phosphorylating GraP-DH from E . coli and Bacillus stearothermophilus . The chemical reactivity of Cys149 in the His176Asn mutant was reinvestigated . At neutral pH, its reactivity was shown to be at least as high as that observed in the Cys-/His+ ion pair present in the wild type . No pre-steady state burst of NADH was found with the His176Asn mutant in contrast to what is observed for the wild type, and a primary isotope effect was observed when D-{1-2H}glyceraldehyde-3-phosphate was used as the substrate . Therefore, the major role of the His176 in the catalytic mechanism under physiological conditions is not to activate the nucleophilicity of Cys149 but first to facilitate the hydride transfer . These results hypothesized that a phosphorylating GraP-DH possessing a different protein environment competent to increase the nucleophilic character of the essential Cys residue and to favor the hydride transfer in place of His, could be enzymically efficient . This is most likely the case for archaeal Methanothermus fervidus GraP-DH which shares less than 15% amino-acid identity with the bacterial or eukaryotic counterparts . No Cys-/His+ ion pair was detectable . Only one thiolate entity was observed with an apparent pKa of 6.2 . This result was confirmed by the fact that none of the mutations of the five invariant His changed the catalytic efficiency.

Appl Environ Microbiol, 1998 Apr, 64(4), 1338 - 43
A novel sensitive bioassay for detection of Bacillus cereus emetic toxin and related depsipeptide ionophores; Andersson MA et al.; Of the toxins produced by Bacillus cereus, the emetic toxin is likely the most dangerous but, due to the lack of a suitable assay, the least well known . In this paper, a new, sensitive, inexpensive, and rapid bioassay for detection of the emetic toxin of B . cereus is described . The assay is based on the loss of motility of boar spermatozoa upon 24 h of exposure to extracts of emetic B . cereus strains or contaminated food . The paralyzed spermatozoa exhibited swollen mitochondria, but no depletion of cellular ATP or damage to plasma membrane integrity was observed . Analysis of the purified toxin by electrospray tandem mass spectrometry showed that it was a dodecadepsipeptide with a mass fragmentation pattern similar to that described for cereulide . The 50% effective concentration of the purified toxin to boar spermatozoa was 0.5 ng of purified toxin ml of extended boar semen-1 . This amount corresponds to 10(4) to 10(5) CFU of B . cereus cells . No toxicity was detected for 27 other B . cereus strains up to 10(8) CFU ml-1 . The detection limit for food was 3 g of rice containing 10(6) to 10(7) CFU of emetic B . cereus per gram . Effects similar to those provoked by emetic B . cereus toxin were also induced in boar spermatozoa by valinomycin and gramicidin at 2 and 3 ng ml of extended boar semen-1, respectively . The symptoms provoked by the toxin in spermatozoa indicated that B . cereus emetic toxin was acting as a membrane channel-forming ionophore, damaging mitochondria and blocking the oxidative phosphorylation required for the motility of boar spermatozoa.

Appl Environ Microbiol, 1998 Apr, 64(4), 1328 - 32
Bacterial oxidation of mercury metal vapor, Hg(0); Smith T et al.; We used metalloregulated luciferase reporter fusions and spectroscopic quantification of soluble Hg(II) to determine that the hydroperoxidase-catalase, KatG, of Escherichia coli can oxidize monatomic elemental mercury vapor, Hg(0), to the water-soluble, ionic form, Hg(II) . A strain with a mutation in katG and a strain overproducing KatG were used to demonstrate that the amount of Hg(II) formed is proportional to the catalase activity . Hg(0) oxidation was much decreased in stationary-phase cells of a strain lacking KatG, suggesting that the monofunctional hydroperoxidase KatE is less effective at this reaction . Unexpectedly, Hg(0) oxidation also occurred in a strain lacking both KatE and KatG, suggesting that activities other than hydroperoxidases may carry out this reaction . Two typical soil bacteria, Bacillus and Streptomyces, also oxidize Hg(0) to Hg(II) . These observations establish for the first time that bacteria can contribute, as do mammals and plants, to the oxidative phase of the global Hg cycle.

Blood, 1998 Apr 1, 91(7), 2525 - 35
Granulocyte colony-stimulating factor worsens the outcome of experimental Klebsiella pneumoniae pneumonia through direct interaction with the bacteria; Held TK et al.; Besides its well-established effects on granulocytopoiesis, granulocyte colony-stimulating factor (G-CSF) has been shown to have direct effects on the recruitment and bactericidal ability of neutrophils, resulting in improved survival of experimentally infected animals . We studied the effect of G-CSF on the course of experimental pneumonia induced by Klebsiella pneumoniae, an important gram-negative bacillary pulmonary pathogen . Using a highly reproducible murine model, we here show the paradoxical finding that mortality from infection was significantly increased when animals received G-CSF before induction of pneumonia . Administration of G-CSF promoted replication of bacteria in the liver and spleen, thus indicating an impairment rather than an enhancement of antibacterial mechanisms . By contrast, a monoclonal antibody against Klebsiella K2 capsule significantly reduced bacterial multiplication in the lung, liver, and spleen, and abrogated the increased mortality caused by G-CSF . In vitro studies showed a direct effect of G-CSF on K pneumoniae resulting in increased capsular polysaccharide (CPS) production . When bacteria were coincubated with therapeutically achievable concentrations of G-CSF, phagocytic uptake and killing by neutrophils was impaired . Western blot analysis showed three binding sites of G-CSF to K pneumoniae . Binding of 125I-G-CSF to K pneumoniae was displaced by an excess of unlabeled G-CSF, whereas an unrelated cytokine, interleukin-1alpha, did not compete with G-CSF binding to the bacteria . Thus, in this model, the direct effect of G-CSF on a bacterial virulence factor, CPS production, outweighed any beneficial effect of G-CSF on recruitment and stimulation of leukocytes.

J Invertebr Pathol, 1998 Mar, 71(2), 121 - 7
Toxicity and binding properties of the Bacillus thuringiensis delta-endotoxin Cry1C to cultured insect cells; Kwa MS et al.; A better understanding of the mode of action of Bacillus thuringiensis delta-endotoxins is needed to develop strategies which may prevent or slow down selection for resistance . We studied the effect of Cry1C on several different cultured insect cell lines by means of toxicity assays, ligand blotting, and toxin binding studies . A clear difference in sensitivity toward Cry1C between the insect cell lines was observed . Spodoptera frugiperda cell line Sf9 was most sensitive, whereas Spodoptera exigua cell lines SeUCR and SelZD2109 showed intermediate sensitivity . Mamestra brassicae (Mb0503) and Drosophila melanogaster (Dm1) cells were the least sensitive as compared to Sf9 cells . Ligand blot analysis of SDS-PAGE size-separated proteins showed that Cry1C specifically binds to a 40-kDa protein in Sf9, SeUCR, and SelZD2109 cells . Cry1Ab does not bind to this protein . The Cry1C-binding protein was not observed in Mb0503 and Dm1 cells, suggesting that the presence of the 40-kDa Cry1C-binding protein is correlated with sensitivity toward Cry1C .

FEMS Immunol Med Microbiol, 1998 Feb, 20(2), 99 - 102
Effect of trifluoperazine on in vitro ATP synthesis by Mycobacterium leprae; Katoch VM et al.; The effect of trifluoperazine (TFP), a calmodulin antagonist, was investigated on in vitro ATP levels of human derived Mycobacterium leprae . M . leprae were obtained from biopsies from multi-bacillary forms of leprosy and were incubated in a modified Dubos medium system which supports limited in vitro synthesis of M . leprae . This incubation was carried out in the absence and presence of different concentrations of trifluoperazine . Samples for estimation of bacillary ATP levels were taken at day 0 and at 14 days of incubation . TFP inhibited ATP levels in M . leprae and this inhibitory effect was marginal at 2.5 microg ml(-1) (35% inhibition), highly significant at 5 microg ml(-1) (87% inhibition) and almost total at 10 microg ml(-1) (98.5% inhibition) . This compound appears to have potential as an anti-leprotic drug and also as a broad spectrum anti-mycobacterial agent in view of its anti-tubercular activity reported earlier.

Can J Microbiol, 1998 Feb, 44(2), 175 - 80
Comparative genome analysis of Bacillus sphaericus by ribotyping, M13 hybridization, and M13 polymerase chain reaction fingerprinting; Miteva V et al.; A comparative genome analysis of 15 strains representing the five homology groups of the highly heterogeneous species Bacillus sphaericus was performed by M13 hybridization fingerprinting, M13 polymerase chain reaction fingerprinting, and ribotyping with the whole rrn operon . The computer cluster analyses of the polymorphic patterns, presented in dendrograms, showed that these methods allow the differentiation of the individual strains and some homology groups . Our results confirm the close genetic relatedness of the mosquito pathogenic strains of group IIA and support the idea for differentiation of a separate species . At the same time, we present additional proof of the significant genetic heterogeneity of B . sphaericus and the necessity of reconsideration of its present classification.

Clin Oncol (R Coll Radiol), 1998, 10(1), 59 - 61
Tuberculosis of the nasopharynx following radiotherapy; Chua BL et al.; We report the case history of a patient who was treated with radiotherapy for nasopharyngeal carcinoma . During follow-up, she showed signs, symptoms and radiological evidence of disease recurrence and progression . However, repeated biopsies of the posterior nasal space (PNS) failed to demonstrate malignancy . A diagnosis of nasopharyngeal tuberculosis was finally made when tissue from a PNS biopsy stained positive for acid-fast bacillus . The patient responded to antituberculous chemotherapy.

Biochem J, 1998 Feb 15, 330 ( Pt 1), 295 - 302
Identification of the structural similarity in the functionally related amidohydrolases acting on the cyclic amide ring; Kim GJ et al.; The functionally related amidohydrolases, including D-hydantoinases, dihydropyrimidinases, allantoinases and dihydro-orotases, share a similar catalytic function of acting on the cyclic amide ring . We aligned 16 amidohydrolases by taking account of the conservative substitution and found a number of highly conserved regions and invariant amino acid residues . Analyses of the secondary structure and hydropathy profile of the enzymes revealed a significant degree of similarity in the conserved regions . Among the regions, the long stretched region I is of particular interest, because it is mainly composed of invariant amino acid residues, showing a similarity of 69% for the enzymes . A search of the protein data bank using the sequence of the conserved region I identified a number of proteins possessing a similar catalytic property, providing a clue that this region might be linked with the catalytic function . As a particular sequence, one aspartic acid and four histidine residues are found to be rigidly conserved in the functionally related amidohydrolases . In order to investigate the significance of the conserved residues, site-directed mutagenesis was carried out typically for the D-hydantoinase gene cloned from Bacillus stearothermophilus SD1 . These residues were found to be essential for metal binding as well as catalysis, strongly implying that these invariant residues play a critical role in other enzymes as well as in D-hydantoinase . On the basis of the similar catalytic function and existence of the rigidly conserved sequence, we propose a close evolutionary relationship among the functionally related amido hydrolases, including D-hydantoinase, dihydropyrimidinase, allantoinase and dihydroorotase.

Biochem Biophys Res Commun, 1998 Mar 27, 244(3), 868 - 72
Induction of CYP102 (cytochrome P450BM-3) in Bacillus megaterium by 17 beta-estradiol and 4-sec-butylphenol; Hopkins NE et al.; CYP102 (Cytochrome P450BM-3) is induced in Bacillus megaterium by barbiturates, peroxisome proliferators, and nonsteroidal anti-inflammatory drugs . We now describe the induction of CYP102 in B . megaterium by 17 beta-estradiol and by 4-sec-butylphenol . These estrogens interact with the repressor protein Bm3R1, causing it to dissociate with the operator of the CYP102 gene and allowing transcription to occur . We have developed a stable transfection of a construct into B . megaterium of a truncated CYP102 gene coupled with the luciferase gene in a promoterless plasmid and have used this construct to test the induction of CYP102 by these estrogens . Estradiol demonstrated a dose-dependent induction of CYP102 which saturated at a 2-fold increase at 150 microM 4 hr post-addition . 4-sec-Butylphenol produced a dose-dependent and time-dependent induction up to 300 microM and 6 hr post-induction.

Appl Microbiol Biotechnol, 1998 Feb, 49(2), 164 - 7
Increased toxicity of modified mosquitocidal binary toxins of Bacillus sphaericus expressed in Escherichia coli; Ahmad S et al.; The binary mosquitocidal genes of 51-kDa and 42-kDa proteins isolated from Bacillus sphaericus 1593 have been expressed at moderate levels in Escherichia coli employing the pQE expression system . The expressed proteins are readily visible in Coomassie blue-stained protein gels . The recombinant E . coli cells expressing toxic proteins were toxic towards Culex larvae . During the assembly of crystals in B . sphaericus, the 42-kDa toxin is first cleaved at the N-terminal end by a specific B . sphaericus protease . To express the toxins in E . coli the B . sphaericus specific protease-recognition site was deleted at the N-terminal end of the 42-kDa toxin, thereby mimicking the structure of the toxin as present in the crystal . This modification resulted in a twofold increase in the toxicity of the E . coli cells expressing the modified 42-kDa toxin as a constituent of the binary toxin . Our results demonstrate the utility of this modification for heterologous expression of the binary toxin genes from B . sphaericus.

Microbiology, 1998 Mar, 144 ( Pt 3), 609 - 20
Horizontal spread of mer operons among gram-positive bacteria in natural environments; Bogdanova ES et al.; Horizontal dissemination of the genes responsible for resistance to toxic pollutants may play a key role in the adaptation of bacterial populations to environmental contaminants . However, the frequency and extent of gene dissemination in natural environments is not known . A natural horizontal spread of two distinct mercury resistance (mer) operon variants, which occurred amongst diverse Bacillus and related species over wide geographical areas, is reported . One mer variant encodes a mercuric reductase with a single N-terminal domain, whilst the other encodes a reductase with a duplicated N-terminal domain . The strains containing the former mer operon types are sensitive to organomercurials, and are most common in the terrestrial mercury-resistant Bacillus populations studied in this work . The strains containing the latter operon types are resistant to organomercurials, and dominate in a Minamata Bay mercury-resistant Bacillus population, previously described in the literature . At least three distinct transposons (related to a class II vancomycin-resistance transposon, Tn1546, from a clinical Enterococcus strain) and conjugative plasmids are implicated as mediators of the spread of these mer operons.

J Biol Chem, 1998 Mar 6, 273(10), 5771 - 9
Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1; Wind RD et al.; The product specificity and pH optimum of the thermostable cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes EM1 was engineered using a combination of x-ray crystallography and site-directed mutagenesis . Previously, a crystal soaking experiment with the Bacillus circulans strain 251 beta-CGTase had revealed a maltononaose inhibitor bound to the enzyme in an extended conformation . An identical experiment with the CGTase from T . thermosulfurigenes EM1 resulted in a 2.6-A resolution x-ray structure of a complex with a maltohexaose inhibitor, bound in a different conformation . We hypothesize that the new maltohexaose conformation is related to the enhanced alpha-cyclodextrin production of the CGTase . The detailed structural information subsequently allowed engineering of the cyclodextrin product specificity of the CGTase from T . thermosulfurigenes EM1 by site-directed mutagenesis . Mutation D371R was aimed at hindering the maltohexaose conformation and resulted in enhanced production of larger size cyclodextrins (beta- and gamma-CD) . Mutation D197H was aimed at stabilization of the new maltohexaose conformation and resulted in increased production of alpha-CD . Glu258 is involved in catalysis in CGTases as well as alpha-amylases, and is the proton donor in the first step of the cyclization reaction . Amino acids close to Glu258 in the CGTase from T . thermosulfurigenes EM1 were changed . Phe284 was replaced by Lys and Asn327 by Asp . The mutants showed changes in both the high and low pH slopes of the optimum curve for cyclization and hydrolysis when compared with the wild-type enzyme . This suggests that the pH optimum curve of CGTase is determined only by residue Glu258.

J Biol Chem, 1998 Mar 6, 273(10), 5697 - 701
Intramolecular processing of prothermolysin; Marie-Claire C et al.; Thermolysin, an extracellular zinc endopeptidase from Bacillus thermoproteolyticus, is synthesized as a pre-proenzyme and the prosequence has been shown to assist the refolding of the denatured enzyme in vitro and to inhibit enzyme activity (O'Donohue, M . J., and Beaumont, A . (1996) J . Biol . Chem . 271, 26477-26481) . To determine whether prosequence cleavage from the mature enzyme is autocatalytic and if so, whether it is an intermolecular or intramolecular process, N-terminal histidine-tagged prothermolysin was expressed in Escherichia coli . Although partial processing to mature enzyme occurred, most of the proenzyme was recovered intact from inclusion bodies . This was then solubilized in guanidinium hydrochloride, immobilized on a cobalt-containing resin, and after dialysis against renaturation buffer, was quantitatively transformed to mature enzyme . However, when a mutation was introduced into the mature sequence to inactivate thermolysin, the proenzyme was not processed either in vivo or in vitro . In addition, mutated prothermolysin was not processed by exogenous thermolysin under a variety of experimental conditions . The results demonstrate that thermolysin maturation can proceed via an autocatalytic intramolecular pathway.

Proc Natl Acad Sci U S A, 1998 Mar 3, 95(5), 2056 - 60
Engineering an enzyme to resist boiling; Van den Burg B et al.; In recent years, many efforts have been made to isolate enzymes from extremophilic organisms in the hope to unravel the structural basis for hyperstability and to obtain hyperstable biocatalysts . Here we show how a moderately stable enzyme (a thermolysin-like protease from Bacillus stearothermophilus, TLP-ste) can be made hyperstable by a limited number of mutations . The mutational strategy included replacing residues in TLP-ste by residues found at equivalent positions in naturally occurring, more thermostable variants, as well as rationally designed mutations . Thus, an extremely stable 8-fold mutant enzyme was obtained that was able to function at 100 degrees C and in the presence of denaturing agents . This 8-fold mutant contained a relatively large number of mutations whose stabilizing effect is generally considered to result from a reduction of the entropy of the unfolded state ("rigidifying" mutations such as Gly --> Ala, Ala --> Pro, and the introduction of a disulfide bridge) . Remarkably, whereas hyperstable enzymes isolated from natural sources often have reduced activity at low temperatures, the 8-fold mutant displayed wild-type-like activity at 37 degrees C.

Proteins, 1998 Mar 1, 30(4), 372 - 80
A general method of domain closure is applied to phosphoglycerate kinase and the result compared with the crystal structure of a closed conformation of the enzyme; Chandra NR et al.; The occurrence of large domain motions associated with the mechanism of action of many proteins is well established . We present a general method of predicting domain closure applicable to proteins containing domains separated by an apparent hinge . The method attempts to allow for natural directional bias within the closing protein by repeatedly applying a weak pulling force over a short distance between pairs of atoms chosen at random in the two domains in question . Appropriate parameters governing the pulling function were determined empirically . The method was applied to the bi-lobal protein PGK and a closed-form activated ternary complex generated for Bacillus stearothermophilus PGK . This model was compared with the recently determined crystal structure of closed-form Trypanosoma brucei PGK . The model predicts the correct hinge regions, although the magnitude of movement at one hinge point was overestimated, and provides a reasonable representation of the closed-form ternary complex.

Biosci Biotechnol Biochem, 1998 Feb, 62(2), 393 - 5
Extracellular dextran-induced p-nitrophenyl-alpha-D-glucoside-hydrolyzing enzyme of Bacillus circulans KA-304: a producer of Schizophyllum commune-lytic enzyme; Mizuno K et al.; p-NP-alpha-D-Glucoside-hydrolyzing activity in the culture filtrate of Bacillus circulans KA-304, a producer of Schizophyllum commune cell-wall lytic enzyme, increased remarkably when the bacterium was grown on dextran as a carbon source . It was suggested that the increase of the activity was caused by increases of two major species, alpha-D-glucosidase I and alpha-D-glucosidase II . alpha-D-Glucosidase I, which showed a certain reactivity toward dextran, was isolated from the filtrate (MW 70 kDa, 35-fold, 10% recovery) . The enzyme was stable around pH 6.5-7.5 and showed its highest activity at pH 6.5 . The enzyme preparation inactivated with p-chloromerucuribenzoic acid recovered its activity by incubating with ditiothereitol . Its substrate specificity suggested that the enzyme was an exo-type enzyme with certain affinity toward alpha-1,6-glucosidic linkage.

Biosci Biotechnol Biochem, 1998 Feb, 62(2), 268 - 74
A cysteine-dependent serine protease associated with the dormant spores of Bacillus cereus: purification of the protein and cloning of the corresponding gene; Moriyama R et al.; Subtilisin-like serine protease, which is associated with the dormant spores of Bacillus cereus, was solubilized by washing the spores with 2 M KCl and purified to homogeneity by carbobenzoxy-D-phenylalanine-liganded affinity column chromatography and hydrophobic interaction column chromatography . Enzyme activity was completely inhibited by reagents for sulfhydryl groups such as HgCl2 as well as by conventional subtilisin inhibitors, suggesting the enzyme to be cysteine-dependent . The enzyme retained activity in 5 M urea at 4 degrees C for at least 2 months, and the specific activity was 50 times that of subtilisin BPN when measured for a common chromogenic substrate, carbobenzoxy-glycyl-glycyl-L-leucine p-nitroanilide . The gene encoding this protease was cloned in Escherichia coli, and its nucleotide sequence was analyzed . The deduced amino acid sequence suggested that the protease is produced as a precursor comprising three portions; a signal sequence (28 amino acid residues), a prosequence (80 amino acid residues) and a mature enzyme (289 amino acid residues) . The mature region of the enzyme had high similarity with a thermitase from Thermoactinomyces vulgaris (72% identity) and a thermostable alkaline protease from Thermoactinomyces sp . E79 (66% identity), which have the N-terminal sequence showing scarcely noticeable similarity with corresponding stretches of subtilisins and mercuric ion-sensitive free cysteine in the equivalent position of the primary structure.

Biosci Biotechnol Biochem, 1998 Feb, 62(2), 221 - 4
Enzymatic synthesis of a new derivative of thiamin, O-alpha-glucosylthiamin; Uchida K et al.; A new transglucosylated derivative of thiamin could be synthesized by the actions of cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus and glucoamylase from Rhizopus sp., in this order, on a mixture of dextrin and thiamin . The derivative was isolated in crystalline form and identified as 5'-O-(alpha-D-glucopyranosyl)thiamin by spectroscopy (FAB-MS, UV, 1H-NMR, and 13C-NMR), thiochrome formation with K3 {Fe(CN)6}-NaOH reagent, and the hydrolysis products by alpha- and beta-glucosidases . O-alpha-Glucosylthiamin was odorless and mildly sweet with no tongue-pricking taste, and was more stable than thiamin hydrochloride in aqueous solutions at pHs 7.0 and 9.3.

Anal Chem, 1998 Mar 15, 70(6), 1203 - 7
Heterogeneity in Bacillus cereus PCR products detected by ESI-FTICR mass spectrometry; Wunschel DS et al.; PCR amplification of a segment of the 16/23S rDNA interspace region (ISR) from Bacillus cereus 6464 produced a mixture of products . An 89-bp product was predicted on the basis of the reported sequence . The ESI-FTICR analysis revealed three double-stranded products, differing in size by a single nucleotide corresponding to two homoduplexes of 89 and 88 base pairs and a heteroduplex of 89 and 88 nucleotide strands . These were produced from a single preparation of genomic DNA and a single primer pair . ESI-FTICR analysis of the single strands identified a deletion of a T in the coding strand and a corresponding loss of an A in the noncoding strand of this product . The ESI-FTICR analysis indicated the presence of an unreported sequence variation between rRNA operons in this organism . This report illustrates that PCR products amplified from templates differing by a single nucleotide can be resolved and identified using ESI-FTICR at the 89-bp level . Furthermore, the ESI-FTICR mass measurements provided the identity of the deletion, which is indicative of interoperon variability.

Biochim Biophys Acta, 1998 Feb 2, 1369(1), 51 - 60
Voltage clamp studies on S-layer-supported tetraether lipid membranes; Schuster B et al.; Isolated subunits from the cell surface proteins (S-layer) of Bacillus coagulans E38-66 have been recrystallized on a glycerol dialkyl nonitol tetraether lipid (GDNT)-monolayer and the electrophysical features of this biomimetic membrane have been investigated in comparison to unsupported GDNT-monolayers . The GDNT-monolayer, spread on a Langmuir-Blodgett trough, was clamped with the tip of a glass patch pipette . In order to investigate the barrier function and potential to incorporate functional molecules, voltage-clamp examinations on plain and S-layer-supported GDNT-monolayers were per-formed . Our results indicate the formation of a tight GDNT-monolayer sealing the tip of the glass pipette, and a decrease in conductance of the GDNT-monolayer upon recrystallization of the S-layer protein . Thus, the S-layer protein, apparently, did not penetrate or rupture the lipid monolayer . The valinomycin-mediated increase in conductance was less pronounced for the S-layer-supported than for the plain GDNT-monolayer, indicating differences in the accessibility and/or in the fluidity of the lipid membranes . Furthermore . in contrast to plain GDNT-monolayers . S-layer supported GDNT-monolayers with high valinomycin-mediated conductance persisted over long, periods of time, indicating enhanced stability . These composite S-layer/lipid films may constitute a new tool for electrophysical and electrophysiological studies on membrane-associated and membrane-integrated biomolecules.

Immunity, 1998 Mar, 8(3), 383 - 90
Defective NK cell activity and Th1 response in IL-18-deficient mice; Takeda K et al.; IL-18 is a cytokine that is secreted from activated macrophages and induces IFNgamma production . To investigate the in vivo role of IL-18, we generated IL-18-deficient mice . In Propionibacterium acnes (P . acnes)-primed IL-18-deficient mice, LPS-induced IFNgamma production was markedly reduced, despite normal IL-12 induction . Natural killer cell activity was significantly impaired . Th1 cell response after injection of P . acnes or Mycobacterium bovis (bacillus Calmette-Guerin {BCG}) was significantly reduced . Similar results were observed in IL-12-deficient mice . Interestingly, Th1 response was induced after BCG infection in IL-12-deficient mice . We therefore generated mice lacking both IL-18 and IL-12 . In these mice, NK activity and Th1 response were further impaired . This demonstrates the important role of both IL-18 and IL-12 in NK activity, as well as in in vivo Th1 response.

Biochim Biophys Acta, 1998 Feb 2, 1369(1), 71 - 84
Lipid composition changes induced by tamoxifen in a bacterial model system; Luxo C et al.; A putative relationship between growth impairment of Bacillus stearothermophilus by tamoxifen (TAM) and TAM-induced perturbation of the physical properties of bacterial membrane lipids has been observed . The supplementation of the growth medium with Ca2+ (a membrane stabilizer) partially relieves growth inhibition by TAM, allowing growth at TAM concentrations that fully impair growth in the basal medium . B . stearothermophilus modifies the membrane lipid composition in response to the addition of TAM to the growth medium and the response is sensitive to Ca2+ . Changes in lipid composition are observed in the acyl chains and in the polar head groups of phospholipids . The physical effects of alteration in these lipids was studied by fluorescence polarization of DPH and DPH-PA . Polar lipid dispersions from TAM-adapted cells grown in a Ca2+ medium show a shift of Tm to higher temperatures and a significant increase of the structural order as compared to lipids from control cells, suggesting that TAM-induced lipid composition changes compensate for the destabilizing effects of the cytostatic on membrane organization . The polar lipids from cells grown in the basal medium containing tamoxifen are also altered, but these alterations do not promote order increase of the bilayer in spite of a deviation of Tm to higher temperatures as detected by DPH . Data indicate that B . stearothermophilus controls the membrane lipid composition in response to tamoxifen, to compensate for TAM-promoted disordering in membranes and to provide an appropriate packing of phospholipid molecules in a stable bilayer, putatively disturbed by TAM incorporation.

Lakartidningen, 1998 Mar 4, 95(10), 1010 - 2, 1015-6
{Policy program to minimize spread of infection . Prolonged cough may be a sign of tuberculosis}; Fredlund H et al.; In a worldwide epidemiological perspective, Sweden is well favoured with an annual tuberculosis incidence of approximately six cases per 100,000 of the population . Neither the impact of the HIV pandemic nor the occurrence of multiresistant strains of Mycobacterium tuberculosis has yet become a major problem in the care of tuberculosis patients in Sweden . Only a few per cent of HIV patients have developed tuberculosis, and during the period, 1991-94, only one per cent of M . tuberculosis isolates in Sweden were resistant to such antimycobacterials as isoniazid and rifampicin . However, the epidemiological situation in the neighbouring Baltic states is a matter for concern . Bovine tuberculosis has been eradicated in Sweden, the last case having been diagnosed in 1978 . Although the reported efficacy of BCG (bacillus Calmette-Guerin) tuberculosis vaccine varies according to the population studied, protective rates of 70-85 per cent have been reported for Sweden and other west European countries . Re-vaccination of tuberculin-negative individuals has not been shown to yield added protection . The aim of a national programme for protection against tuberculosis is to preserve our favourable epidemiological situation by early detection of new cases, effective contact tracing, and BCG vaccination of children in population groups at risk . The primary means of achieving this is the education of health care personnel to retain tuberculosis as a differential diagnosis . Moreover, national guidelines for contact tracing must be duly observed, and immigrants from high prevalence areas need to be screened for tuberculosis . Registration of all cases of tuberculosis should be maintained at regional and national levels, and follow-up must be meticulous until a successful outcome of treatment is accomplished . Recommendations for dealing with tuberculosis should be made available and duly implemented at all hospitals caring for tuberculosis patients, in order to avoid nosocomial transmission . Although BCG vaccination at birth was formerly general in Sweden, since 1975 only children considered to be at risk have been vaccinated . Thus, non-vaccinated young adults are now entering the health care sector as students or employees, and should be offered BCG vaccination . Moreover, the epidemiological situation both in Sweden and in neighbouring countries needs to be monitored carefully in order that recommendations concerning BCG vaccination and other preventive measures can be modified if necessary.

J Neuroimmunol, 1998 Feb, 82(1), 73 - 80
Bacillus Calmette-Guérin sequestered in the brain parenchyma escapes immune recognition; Matyszak MK et al.; We have previously shown that heat-killed bacillus Calmette-Guerin (BCG) injected into the brain parenchyma becomes sequestered behind the blood-brain barrier for months, apparently unrecognised by the immune system (Matyszak and Perry, 1995, 1996a,b) . In this paper we have studied T-cell and antibody responses to purified protein derivative (PPD) at different times after intracranial injection of BCG or after the same dose of BCG was injected intradermally . We detected no antibody to PPD in the sera of animals which received intracranial injection, although there was a clear antibody response in the sera of animals injected intradermally, as shown using immunoblot analysis . The skin contact sensitivity to PPD was robust in animals which had received a previous intradermal injection of BCG . 72 h after a PPD injection, the injected site showed many MHC class II + macrophages and T-cells . However, the response in skin following PPD challenge, in animals injected intracranially (i.c.), was comparable with that of naive animals which had received no previous BCG challenge . The skin lesions in animals injected i.c . and in naive animals, were characterised by a small number of MHC class II + cells and rare T-cells . T-cell responses were also studied in an in vitro proliferation assay . The proliferative response was measured for cells isolated from the cervical lymph nodes and the spleen . Cells purified from the spleen and the cervical lymph nodes of animals injected with BCG i.c . showed no specific proliferative response to PPD . The response was comparable to that found in naive, uninjected animals . However, spleen and cervical lymph node cells from animals injected intradermally with BCG showed a significant proliferative response to PPD . These results show that a dose of bacteria injected into the brain parenchyma fails to prime the immune system even though the same dose injected subcutaneously will do so . This response to bacteria in the CNS differs from that previously reported for soluble proteins.

Acta Leprol, 1997, 10(4), 195 - 8
{Atypical presentations of leprosy: apropos of 2 cases}; Benzekri L et al.; This report describes two atypical cases of leprosy . A 48 year old male patient presented laryngeal dyspnea with adhesions of the oropharynx of which the biopsies were inconclusive . The patient was cachectic with hyperesthesia of the extremities and two subcutaneous nodules . The biopsy of one nodule evoked thesaurismosis or dyslipoidosis while the bacilloscopy was positive in nasal smears . A 14 year old female patient suffered from bullae which appeared spontaneously on erythematous skin on the legs and upper arms . Upon examination those areas were found to be hypoaesthetic, as was a very large hamartoma on the left half of the body . A biopsy of healthy skin evoked the diagnosis of leprosy . The patient then developed BT leprosy and episodes of hysteria . The first observation led to several diagnoses: while laryngeal dyspnea is unusual in LL and while cutaneous histology of regressive LL contrasted with the abundance of the bacilloscopy . The diagnosis of the second case is that of indeterminate leprosy with premonitory neurological signs associated with pathomania and evolution to a multibacillary form.

Int J Tuberc Lung Dis, 1998 Mar, 2(3), 200 - 7
Does the efficacy of BCG decline with time since vaccination?
Sterne JA, Rodrigues LC, Guedes IN.
OBJECTIVE: To investigate whether the protective efficacy of bacille Calmette-Guerin (BCG) against tuberculosis decreases with time since vaccination . DESIGN: A quantitative review of all 10 randomized trials of BCG against tuberculosis in purified protein derivative (PPD)-negative individuals, that presented data for discrete periods . For each trial, we derived log rate ratios for the annual change in the efficacy of BCG . We also compared efficacy in the first two years, and the first 10 years, to that in the rest of the trial . RESULTS: There was considerable heterogeneity between trials in the annual change in the efficacy of BCG . In seven efficacy decreased overtime, while in three it increased . Average annual change in efficacy was not related to overall efficacy . Efficacy also varied between trials in the first two years after vaccination, at more than two years after vaccination and in the first ten years after vaccination . However the variation in efficacy between trials more than 10 years after vaccination was not statistically significant (P = 0.26) . We therefore calculated that the average efficacy more than 10 years after vaccination was 14% (95% confidence interval -9% to 32%) . CONCLUSION: BCG protection can wane with time since vaccination . There is no good evidence that BCG provides protection more than 10 years after vaccination.

Nucleic Acids Res, 1998 Mar 1, 26(5), 1288 - 93
Bacillus popilliae cry18Aa operon is transcribed by sigmaE and sigmaK forms of RNA polymerase from a single initiation site; Zhang J et al.; Bacillus popilliae is an obligate pathogen for larvae of the insect family Scarabaeidae (Coleoptera) . It forms parasporal crystals upon sporulation . The gene cry18Aa coding for the parasporal crystal protein and an upstream open reading frame, orf1, were previously isolated from B.popilliae . Here we report an analysis of cry18Aa transcription in Bacillus thuringiensis . The only transcriptional start site of cry18Aa was found 29 bp upstream of the open reading frame orf1, suggesting that orf1 and cry18Aa are transcribed as an operon . lacZ fusion to the cry18Aa promoter was used to follow the time-course of cry18Aa transcription in wild type B.thuringiensis and in various B.thuringiensis sporulation-deficient mutants (spo0A, sigE or sigK) . In wild type B.thuringiensis, the cry18Aa promoter was activated 2 h after the end of exponential growth and the expression lasted to the late sporulation phase . The results of promoter activity in Spo+or Spo-backgrounds together with the results of primer extension experiments suggest that the transcription from this promoter can be driven by both sigmaE and sigmaK types of RNA polymerase at a single start site . The promoter region of cry18Aa operon fits the consensus sequences of both sigmaE and sigmaK dependent promoters of Bacillus.

Curr Microbiol, 1998 Mar, 36(3), 175 - 9
Susceptibility of the coffee leaf miner (Perileucoptera spp.) to bacillus thuringiensis delta-endotoxins: A model for transgenic perennial crops resistant to endocarpic insects
Filho OG, Denolf P, Peferoen M, Decazy B, Eskes AB, Frutos R.
Binding of several Bacillus thuringiensis delta-endotoxins was studied on histological midgut sections of larvae of coffee leaf miner Perileucoptera coffeella from Brazil and Perileucoptera sp from Madagascar . CryIA(a), CryIA(b), CryIA(c), CryIB, CryIE, and CryIIA were tested for binding, and only CryIA(c), CryIB, and CryIE yielded a positive response . The toxins bound to the whole midgut, and the result was identical on both insect populations . The same toxins, to the number of which CryIC was added, were tested on larvae of P . coffeella . CryIA(c) and CryIB were toxic with an LC50 of 1.47 &mgr;g/ml and 21.93 &mgr;g/ml, respectively . CryIE was not toxic to P . coffeella . CryIA(c) and CryIB were tested for synergistic activity and were shown to act by cumulative effect when delivered to the insect larvae as a mixture.

J Hosp Infect, 1998 Feb, 38(2), 139 - 46
Growth and enterotoxin production by diarrhoeagenic Bacillus cereus in dietary supplements prepared for hospitalized HIV patients; Rowan NJ et al.; This study was initiated because of an increase in diarrhoeal episodes in a ward caring for patients infected with the human immunodeficiency virus (HIV) . An examination of hospital-prepared dietary supplements (build-up food) found Bacillus cereus to be a potential problem . Due in part to inadequate refrigeration conditions (13 +/- 4 degrees C), the microbial flora in commercially pasteurized semi-skimmed milk (PSSM) reached potentially hazardous levels (> 10(6) cfu/mL) . While refrigerated PSSM did not support enterotoxin production, reconstitution of build-up powder in PSSM followed by storage in the HIV ward (4 h at 28 +/- 3 degrees C) resulted in growth of B . cereus (> 10(7) cfu/mL) and synthesis of diarrhoeal enterotoxin . While insufficient epidemiological data was available to establish conclusively a causal relationship between patients' symptoms and source, the study highlights a potential B . cereus problem with hospital-prepared dietary supplements and recommendations are proposed to prevent this re-occurrence.

Acta Microbiol Pol, 1997, 46(4), 357 - 62
beta-Amylase production by some Bacillus cereus, Bacillus megaterium and Bacillus polymyxa {correction of polymaxa} strains; Niziolek S; The production of extracellular beta-amylase by some Bacillus cereus, Bacillus megaterium and Bacillus polymyxa {corrected} strains was investigated, and the maximal yields of the enzyme were 3.6; 9.3 and 20.4 U/mL of the culture fluid, respectively (U, 1 mumol of maltose equivalent per min at 30 degrees C) . Several cultivation media were used for beta-amylase production . Bacillus cereus and some strains of Bacillus megaterium gave good yields of beta-amylase only in medium with the addition of nutrient broth . However, beta-amylase produced during growth in protein rich medium (nutrient broth) was highly unstable, probably due to inactivation by proteolytic enzymes co-existing in the culture fluid . Bacillus polymyxa {corrected} strains can produce good yields of beta-amylase on a semi-synthetic medium consisting of inorganic salts, potato starch and inexpensive soybean extract instead of costly peptone and meat extract . The most potential beta-amylase producer was the strain Bacillus polymyxa {corrected} NCIB 8524 . The tested Bacillus megaterium and Bacillus polymyxa {corrected} strains were apparently differentiated by temperature cultivation (30 and 37 degrees C) suitable for beta-amylase amylase yield.

J Exp Med, 1998 Feb 16, 187(4), 561 - 9
Infection of mice with Mycobacterium bovis-Bacillus Calmette-Guérin (BCG) suppresses allergen-induced airway eosinophilia; Erb KJ et al.; It has been proposed that the increase in prevalence and severity of atopic disorders inversely correlates with exposure to infectious diseases such as tuberculosis . We have investigated this issue by combining an intranasal Mycobacterium bovis-Bacillus Calmette-Guerin (BCG) infection with a murine model of allergen, (ovalbumin {OVA}) induced airway eosinophilia . BCG infection either 4 or 12 wk before allergen airway challenge resulted in a 90-95 and 60-70% reduction in eosinophilia within the lungs, respectively, compared to uninfected controls . The inhibition of airway eosinophilia correlated with a reduced level of IL-5 production by T cells from the lymph node draining the site of OVA challenge . Interestingly, BCG infection of the lung had no effect on IgG1 and IgE OVA-specific serum immunoglobulin or blood eosinophil levels . Furthermore, BCG-induced inhibition of airway eosinophilia was strongly reduced in interferon (IFN)-gamma receptor-deficient mice and could be partially reversed by intranasal IL-5 application . Intranasal BCG infections could also reduce the degree of lung eosinophilia and IL-5 produced by T cells after Nippostrongylus brasiliensis infection . Taken together, our data suggest that IFN-gamma produced during the T helper cell (Th)1 immune response against BCG suppresses the development of local inflammatory Th2 responses in the lung . Most importantly, this inhibition did not extend to the systemic immunoglobulin response against OVA . Our data support the view that mycobacterial infections have the potential to suppress the development of atopic disorders in humans.

J Exp Biol, 1998 Feb, 201 ( Pt 4), 599 - 608
Are integrins involved in the aggregatory and phagocytic behaviour of fish haemostatic cells?
Hill DJ, Rowley AF.
The involvement of a putative integrin-like fibrinogen receptor in the aggregatory and phagocytic behaviour of thrombocytes (platelet equivalents of fish) from the rainbow trout Oncorhynchus mykiss was studied . Aggregation of trout thrombocytes was induced by the thromboxane mimetic U-46619 in the presence of trout fibrinogen . Thrombocyte aggregation was inhibited by the tetrapeptide RGDS, but not by RGES or fibrinogen binding inhibitor peptide (HHLGGAKQAGDV) . A range of monoclonal antibodies against the human platelet integrin alphaIIbbeta3 (anti-CD41a, anti-beta3 and LK7r) showed no reactivity with trout thrombocytes . Subsequently, a panel of monoclonal antibodies was raised against thrombocyte membrane preparations in an attempt to obtain an antibody against the putative integrin fibrinogen receptor . Of these monoclonal antibodies, four were found to inhibit thrombocyte aggregation, namely 12G2, 30D8, 32F8 and 32H10 . The antibody 32H10 was shown significantly to inhibit the attachment of thrombocytes to immobilised trout fibrinogen, suggesting that it and the other antibodies recognise the putative fibrinogen receptor on trout thrombocytes . FITC-labelled Bacillus cereus were employed as test particles to prove that thrombocytes internalise bacteria via an active process and not simply by passive sequestration into the open canalicular system . Preincubation of bacteria with trout fibrinogen resulted in a significant increase in the number of thrombocytes exhibiting phagocytosis . This enhancement of phagocytosis by preincubation of B . cereus with trout fibrinogen could be inhibited by the tetrapeptide RGDS, but not by RGES, hence implicating the putative fibrinogen receptor in the internalisation of microorganisms . The relevance of these findings to the possible existence of an integrin-like receptor on trout thrombocytes is discussed.

J Bacteriol, 1998 Mar, 180(6), 1582 - 5
Isolation and purification of two novel streptomycete RNase inhibitors, SaI14 and SaI20, and cloning, sequencing, and expression in Escherichia coli of the gene coding for SaI14; Krajcikova D et al.; Two new RNase inhibitors, SaI14 (Mr, approximately 14,000) and SaI20 (Mr, approximately 20,000), were isolated and purified from a Streptomyces aureofaciens strain . The gene sai14, coding for SaI14 protein, was cloned and expressed in Escherichia coli . The alignment of the deduced amino acid sequence of SaI14 with that of barstar, the RNase inhibitor from Bacillus amyloliquefaciens, showed significant similarity between them, especially in the region which contains most of the residues involved in barnase-barstar complex formation.

J Bacteriol, 1998 Mar, 180(6), 1488 - 95
The S-layer proteins of two Bacillus stearothermophilus wild-type strains are bound via their N-terminal region to a secondary cell wall polymer of identical chemical composition; Egelseer EM et al.; Two Bacillus stearothermophilus wild-type strains were investigated regarding a common recognition and binding mechanism between the S-layer protein and the underlying cell envelope layer . The S-layer protein from B . stearothermophilus PV72/p6 has a molecular weight of 130,000 and assembles into a hexagonally ordered lattice . The S-layer from B . stearothermophilus ATCC 12980 shows oblique lattice symmetry and is composed of subunits with a molecular weight of 122,000 . Immunoblotting, peptide mapping, N-terminal sequencing of the whole S-layer protein from B . stearothermophilus ATCC 12980 and of proteolytic cleavage fragments, and comparison with the S-layer protein from B . stearothermophilus PV72/p6 revealed that the two S-layer proteins have identical N-terminal regions but no other extended structurally homologous domains . In contrast to the heterogeneity observed for the S-layer proteins, the secondary cell wall polymer isolated from peptidoglycan-containing sacculi of the different strains showed identical chemical compositions and comparable molecular weights . The S-layer proteins could bind and recrystallize into the appropriate lattice type on native peptidoglycan-containing sacculi from both organisms but not on those extracted with hydrofluoric acid, leading to peptidoglycan of the A1gamma chemotype . Affinity studies showed that only proteolytic cleavage fragments possessing the complete N terminus of the mature S-layer proteins recognized native peptidoglycan-containing sacculi as binding sites or could associate with the isolated secondary cell wall polymer, while proteolytic cleavage fragments missing the N-terminal region remained unbound . From the results obtained in this study, it can be concluded that S-layer proteins from B . stearothermophilus wild-type strains possess an identical N-terminal region which is responsible for anchoring the S-layer subunits to a secondary cell wall polymer of identical chemical composition.

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