Microbiology Reader
Equipment to run microbiology work automatically

Growth Curves of any strain.
Microbiological calculations.

Microbiology Home
Microbioloy Reader
Growth Curves
Photo Album
Microorganisms
Software
Download
Purchasing
Contact Us


FEMS Microbiol Lett, 2000 Oct 15, 191(2), 221 - 5
Evidence for intermolecular interaction as a necessary step for pore-formation activity and toxicity of Bacillus thuringiensis Cry1Ab toxin; Soberon M et al.; Based on the observation of large conductance states formed by Bacillus thuringiensis Cry toxins in synthetic planar lipid bilayers and the estimation of a pore size of 10-20 A, it has been proposed that the pore could be formed by an oligomer containing four to six Cry toxin monomers . However, there is a lack of information regarding the insertion of Cry toxins into the membrane and oligomer formation . Here we provide direct evidence showing that the intermolecular interaction between Cry1Ab toxin monomers is a necessary step for pore formation and toxicity . Two Cry1Ab mutant proteins affected in different steps of their mode of action (F371A in receptor binding and H168F in pore formation) were affected in toxicity against Manduca sexta larvae . Binding analysis showed that F371A protein bound more efficiently to M . sexta brush border membrane vesicles when mixed with H168F in a one to one ratio . These mutant proteins also recovered pore-formation activity, measured with a fluorescent dye with isolated brush border membrane vesicles, and toxicity against M . sexta larvae when mixed, showing that monomers affected in different steps of their mode of action can form functional hetero-oligomers.

J Mol Biol, 2000 Oct 20, 303(2), 299 - 310
Stabilization of the transition state for the transfer of tyrosine to tRNA(Tyr) by tyrosyl-tRNA synthetase; Xin Y et al.; Aminoacylation of tRNA(Tyr) involves two steps: (1) tyrosine activation to form the tyrosyl-adenylate intermediate; and (2) transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA(Tyr) . In Bacillus stearothermophilus tyrosyl-tRNA synthetase, Asp78, Tyr169, and Gln173 have been shown to form hydrogen bonds with the alpha-ammonium group of the tyrosine substrate during the first step of the aminoacylation reaction . Asp194 and Gln195 stabilize the transition state complex for the first step of the reaction by hydrogen bonding with the 2'-hydroxyl group of AMP and the carboxylate oxygen atom of tyrosine, respectively . Here, the roles that Asp78, Tyr169, Gln173, Asp194, and Gln195 play in catalysis of the second step of the reaction are investigated . Pre-steady-state kinetic analyses of alanine variants at each of these positions shows that while the replacement of Gln173 by alanine does not affect the initial binding of the tRNA(Tyr) substrate, it destabilizes the transition state complex for the second step of the reaction by 2.3 kcal/mol . None of the other alanine substitutions affects either the initial binding of the tRNA(Tyr) substrate or the stability of the transition state for the second step of the aminoacylation reaction . Taken together, the results presented here and the accompanying paper are consistent with a concerted reaction mechanism for the transfer of tyrosine to tRNA(Tyr), and suggest that catalysis of the second step of tRNA(Tyr) aminoacylation involves stabilization of a transition state in which the scissile acylphosphate bond of the tyrosyl-adenylate species is strained . Cleavage of the scissile bond on the breakdown of the transition state alleviates this strain .

J Mol Biol, 2000 Oct 20, 303(2), 287 - 98
Correlating amino acid conservation with function in tyrosyl-tRNA synthetase; Xin Y et al.; Sequence comparisons have been combined with mutational and kinetic analyses to elucidate how the catalytic mechanism of Bacillus stearothermophilus tyrosyl-tRNA synthetase evolved . Catalysis of tRNA(Tyr) aminoacylation by tyrosyl-tRNA synthetase involves two steps: activation of the tyrosine substrate by ATP to form an enzyme-bound tyrosyl-adenylate intermediate, and transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA(Tyr) . Previous investigations indicate that the class I conserved KMSKS motif is involved in only the first step of the reaction (i.e . tyrosine activation) . Here, we demonstrate that the class I conserved HIGH motif also is involved only in the tyrosine activation step . In contrast, one amino acid that is conserved in a subset of the class I aminoacyl-tRNA synthetases, Thr40, and two amino acids that are present only in tyrosyl-tRNA synthetases, Lys82 and Arg86, stabilize the transition states for both steps of the tRNA aminoacylation reaction . These results imply that stabilization of the transition state for the first step of the reaction by the class I aminoacyl-tRNA synthetases preceded stabilization of the transition state for the second step of the reaction . This is consistent with the hypothesis that the ability of aminoacyl-tRNA synthetases to catalyze the activation of amino acids with ATP preceded their ability to catalyze attachment of the amino acid to the 3' end of tRNA . We propose that the primordial aminoacyl-tRNA synthetases replaced a ribozyme whose function was to promote the reaction of amino acids and other small molecules with ATP .

J Invertebr Pathol, 2000 Oct, 76(3), 191 - 7
Composition and ecological distribution of cry proteins and their genotypes of Bacillus thuringiensis isolates from warehouses in China; Hongyu Z et al.; The composition and distribution of insecticidal crystal proteins (Cry proteins) and their genotypes of Bacillus thuringiensis isolates from warehouses were evaluated through SDS-PAGE and PCR techniques . The results showed that the electrophoretic patterns of delta-endotoxin crystal preparations were divided into five types . The isolates containing approximately 135 kDa with a 65-kDa protein or only a approximately 135-kDa protein, which amounted to 55.74 and 35.25% of all isolates respectively, were the two major profiles of Cry protein isolated . The distribution of cry genes of B . thuringiensis from warehouses was highly variable . Cry protein genotypes detected in B . thuringiensis isolates included cry1Aa5, cry1Ab9, cry1Ac5, cry1Ba, cry1Ca1, cry1Da1, cry1Ea3, cry2, and cry3 genes, but not cry1Fa2 . Among them, cry2, cry1Ac5, and cry1Ab9 genes were the most common in our B . thuringiensis isolates . Most B . thuringiensis isolates contained several cry genes in a total of 18 profiles . Among them, cry1Ac5 with cry1Ea3; cry1Aa5, cry1Ab9, cry1Ac5 with cry1Ea3; and cry1Aa5, cry1Ab9 with cry1Ac5 were the three principal profiles . The distribution of the Cry proteins and cry genes in isolates depended on geography and type of warehouses . Gene profiles may be used as markers for insecticidal activity of B . thuringiensis strains, but they did not directly reflect the toxic level of B . thuringiensis strains . The serotype of B . thuringiensis strains did not directly reflect the specific cry gene profiles in the strains, but certain relationships can be established between the serotype and cry genotype .

J Invertebr Pathol, 2000 Aug, 76(2), 131 - 9
Characterization of a Bacillus thuringiensis delta-endotoxin which is toxic to insects in three orders; Zhong C et al.; We report here the first Bacillus thuringiensis (Bt) toxin which is toxic to insects from three insect orders (Diptera, Coleoptera, and Lepidoptera) . An oligonucleotide probe based on the delta-endotoxin N-terminal sequence was used to detect the gene . A 23-kb BamHI fragment containing the intact gene was identified and cloned from Bt strain YBT-226 plasmid DNA into the vector pBluescript II . Through a series of DNA manipulations the size of this fragment was reduced and the gene sequenced . The deduced amino acid sequence gave a predicted molecular mass of 137 kDa and was identical to a cry1Ba protein from Bt subsp . thuringiensis HD-2, which is now designated as Cry1Ba1 under a new classification scheme . This protein also showed 81.6% similarity with the Cry1B protein (Cry1Bb1) from Bt strain EG 5847 . When the YBT-226 cry1Ba1 gene was expressed in an acrystalliferous Bt subsp . israelensis strain it produced irregular bipyramidal crystals during sporulation, which reacted specifically with anti-Cry1Ba antiserum . Bioassays using these crystals after purification resulted in significant mortality at low to moderate concentrations to larvae of the house fly (Musca domestica, Diptera), cottonwood leaf beetle (Chrysomela scripta, Coleoptera), and tobacco hornworm (Manduca sexta, Lepidoptera) . This broad-spectrum toxicity was not dependent on presolubilization . In assays with insect cell lines not derived from midgut cells, the soluble toxin killed CH1t (Manduca sexta cells) but was inactive against CF1 (Choristoneura fumiferana cells), Aa(s) (Aedes aegypti), and C2 (Culex quinquefasciatus) mosquito cells .

Ecotoxicol Environ Saf, 2000 Oct, 47(2), 112 - 6
Influence of cellular density on determination of EC(50) in microalgal growth inhibition tests; Moreno-Garrido I et al.; Growth inhibition tests for copper were carried out on four marine microalgal species: Chlorella autotrophyca, Nannochloris atomus (Chlorophyceae), Phaeodactylum tricornutum (Bacillariophyceae), and Isochrysis aff . galbana (Primnesiophyceae) . The test initial cellular densities were reduced to 50 and 10% from the recommended initial cellular density in most of standardized assays . OECD test protocol (originally described for freshwater) was adapted for seawater . The EC(50) values were reduced when initial cellular density decreased . The green algae used in this study exhibited lower sensitivity than P . tricornutum and quite lower than I . aff . galbana . The latter species was found to be very sensitive to copper . The concept of cellular toxic quote (amount of toxic per cell) is defined in order to improve the results of toxicity tests .

Enzyme Microb Technol, 2000 Nov 1, 27(8), 545 - 548
High-Producers of Polygalacturonase Selected From Mutants Resistant to Rifampin in Alkalophilic Bacillus sp . NTT33; Cao J et al.; Synthesis of polygalacturonases in alkalophilic Bacillus sp . which is sensitive to rifampin, was clearly repressed by glucose . Mutants resistant to glucose catabolic repression were selected . From spontaneous mutants, NTT33-cs52 and NTT33-cs301 produced polygalacturonase activities 57.2 to 82.4% higher than the wild type.

J Biomol Struct Dyn, 2000 Aug, 18(1), 137 - 44
Homology model of a novel xylanase: molecular basis for high-thermostability and alkaline stability; Mande SS et al.; Xylanases form enzymes of considerable interest to a variety of biotechnological industries . Their industrial usage is especially attractive since they can replace some of the environmental pollutants, and are economically viable . Those with higher thermostability and optimal activity at alkaline pH are of particular importance to the paper and pulp industry due to the demands of conditions under which the enzymatic reactions are carried out . We have earlier isolated a xylanase from Bacillus sp . NG-27, which is active both at high temperature as well as at alkaline pH . In order to find out factors responsible for the adaptation of this enzyme to the extreme conditions, three dimensional structure of NG-27 xylanase has now been obtained by homology modelling . The tertiary structure shows TIM barrel fold consisting of 8 parallel beta-strands surrounded by alpha-helices . The active site is located at the carboxy terminal end of the TIM barrel . Factors which contribute to the thermostability of the enzyme are increased number of salt bridges . The salt bridges occur remarkably on one face of alpha-helices, with oppositely charged residues occupying i, i+4, i+7 positions . A solvent shielded salt bridge interaction is also observed, which is absent in the mesophilic homologous xylanases . Solvent shielding may enhance electrostatic interaction through lowering of the dielectric, and contribute to increased stability of the enzyme.

Biochim Biophys Acta, 2000 Sep 27, 1487(2-3), 286 - 95
Lipid composition and dynamics of cell membranes of Bacillus stearothermophilus adapted to amiodarone; Rosa SM et al.; Bacillus stearothermophilus, a useful model to evaluate membrane interactions of lipophilic drugs, adapts to the presence of amiodarone in the growth medium . Drug concentrations in the range of 1-2 microM depress growth and 3 microM completely suppresses growth . Adaptation to the presence of amiodarone is reflected in lipid composition changes either in the phospholipid classes or in the acyl chain moieties . Significant changes are observed at 2 microM and expressed by a decrease of phosphatidylethanolamine (relative decrease of 23.3%) and phosphatidylglycerol (17.9%) and by the increase of phosphoglycolipid (162%) . The changes in phospholipid acyl chains are expressed by a decrease of straight-chain saturated fatty acids (relative decrease of 12.2%) and anteiso-acids (22%) with a parallel increase of the iso-acids (9.8%) . Consequently, the ratio straight-chain/branched iso-chain fatty acids decreases from 0 . 38 (control cultures) to 0.30 (cultures adapted to 2 microM amiodarone) . The physical consequences of the lipid composition changes induced by the drug were studied by fluorescence polarization of diphenylhexatriene and diphenylhexatriene-propionic acid, and by differential scanning calorimetry . The thermotropic profiles of polar lipid dispersions of amiodarone-adapted cells are more similar to control cultures (without amiodarone) than those resulting from a direct interaction of the drug with lipids, i.e., when amiodarone was added directly to liposome suspensions . It is suggested that lipid composition changes promoted by amiodarone occur as adaptations to drug tolerance, providing the membrane with physico-chemical properties compatible with membrane function, counteracting the effects of the drug.

Annu Rev Microbiol, 2000, 54, 849 - 80
Regulation of carbon catabolism in Bacillus species; Stulke J et al.; The gram-positive bacterium Bacillus subtilisis capable of using numerous carbohydrates as single sources of carbon and energy . In this review, we discuss the mechanisms of carbon catabolism and its regulation . Like many other bacteria, B . subtilis uses glucose as the most preferred source of carbon and energy . Expression of genes involved in catabolism of many other substrates depends on their presence (induction) and the absence of carbon sources that can be well metabolized (catabolite repression) . Induction is achieved by different mechanisms, with antitermination apparently more common in B . subtilis than in other bacteria . Catabolite repression is regulated in a completely different way than in enteric bacteria . The components mediating carbon catabolite repression in B . subtilis are also found in many other gram-positive bacteria of low GC content.

Nat Biotechnol, 2000 Oct, 18(10), 1101 - 4
Field performance of transgenic elite commercial hybrid rice expressing bacillus thuringiensis delta-endotoxin; Tu J et al.; Here we describe development of transgenic elite rice lines expressing a Bt fusion gene derived from cryIA(b) and cryIA(c) under the control of rice actinI promoter . The lines used in the study were indica CMS restorer line of Minghui 63 and its derived hybrid rice Shanyou 63 . The level of Bt fusion protein CryIA(b)/CryIA(c) detected in Minghui 63 (T51-1) plants was 20 ng/mg soluble protein . The Bt Shanyou 63 was field-tested in natural and repeated heavy manual infestation of two lepidopteran insects, leaffolder and yellow stem borer . The transgenic hybrid plants showed high protection against both insect pests without reduced yield.

Mol Gen Genet, 2000 Sep, 264(1-2), 82 - 8
Premature polyadenylation contributes to the poor expression of the Bacillus thuringiensis cry3Ca1 gene in transgenic potato plants; Haffani YZ et al.; The cry genes that code for the insecticidal crystal proteins of Bacillus thuringiensis (B.t.) have been widely used to develop insect-resistant transgenic plants . The cry3Ca1 gene has been reported to code for a crystal protein which is particularly potent against the Colorado potato beetle (CPB) . To explore the biotechnological potential of cry3Ca1, we introduced this gene into transgenic potato plants under the control of the CaMV 35S promoter . In the resulting transformants, the cry3-Ca1 gene was very poorly expressed . In fact, no full-length transcript (2300 nt) could be detected . Instead, only short transcripts of approximately 1100 nt were observed . Analysis of these short transcripts by Northern hybridization, RT-PCR as well as by cloning and sequencing showed that they resulted from premature polyadenylation . These processing events occurred at four sites within the cry3Ca1 coding region (at positions 652, 669, 914 and 981 relative to the translation start site) . The sites at which premature polyadenylation took place were not those that showed the highest degree of identity to the canonical AAUAAA motif . Together with other recent data, our findings suggest that premature polyadenylation is an important mechanism which can contribute to the poor expression of transgenes in a foreign host.

Antonie Van Leeuwenhoek, 2000 Jul, 78(1), 13 - 21
Novel bacterial diversity recovered from the rhizosphere of oilseed rape (Brassica napus) determined by the analysis of 16S ribosomal DNA; Macrae A et al.; Soil was sampled to a distance of 2.5 mm beneath a root mat of oilseed rape (Brassica napus) in a model rhizosphere system . DNA was extracted and the 16S rDNA amplified, cloned and sequenced . Phylogenetic analysis of these sequences with those held on-line, revealed that 37% of the clones fell within the Holophaga /Acidobacterium phylum, 17% were within the proteobacteria, 14% of the clones were close relatives of Bacillus megaterium and 5% were related to Verrucomicrobium spinosum . An additional eleven clones (21%) could not be assigned to any known phylum and may represent novel bacterial lineages . This study highlights the diverse nature of rhizosphere soils and reinforces the role that molecular approaches play in unravelling such diversity.

J Indian Med Assoc, 2000 Mar, 98(3), 115 - 8
Diagnosis of tuberculosis; Ghoshal AG et al.; Presence of tuberculous infection in the body does not necessarily mean disease . Any diagnostic work up of the disease starts from a high index of clinical suspicion . However, diagnostic modalities include: (a) Isolation of the bacillus; (b) Immunologic tests; (c) Chemical markers; (d) FNAC, bronchoscopy and bronchoalveolar lavage; (e) Amplification systems . There exists controversies and limitations about the disease process even then.

J Indian Med Assoc, 2000 Mar, 98(3), 112 - 4
Tuberculosis--historical landmarks; Das RK; Tuberculosis (TB) since time immemorial has inflicted most miseries in mankind . In ancient times TB was called by many names but the modern one comes from the word 'tubercle' . TB as Pott's disease was widely prevalent among Egyptians in 3700-1000 BC . Hippocrates (460-377 BC) recognised symptomatology of TB . The name tuberculosis was first used by Lanneac and Bayle in early 19th century . Robert Koch in 1882 AD discovered tuberculosis bacillus . Calmette and Guerin laid the foundation BCG vaccination . In 1943, chemotherapy began with the advent of streptomycin (SM) followed by PAS in 1946 and then INH in 1951 . Short course chemotherapy results were published in mid 1970s.

Arch Insect Biochem Physiol, 2000 Sep, 45(1), 12 - 23
Apoptosis in cultured midgut cells from heliothis virescens larvae exposed to various conditions; Loeb MJ et al.; We exposed midgut cells from primary cultures of Heliothis virescens larvae to cell-free previously used medium, the Vaughn X and HyQ SFtrade mark media used for serum-free culture of insect cell lines which do not support H . virescens midgut cells, and to toxin from Bacillus thuringiensis . A statistically significant increase in the percent of dying cells was counted in cell populations in Vaughn X medium . Use of the TUNEL method to detect apoptosis indicated a low rate (7.2%) of apoptosis in control cultures grown in Heliothis medium, an increase to approximately 20% in previously used and HyQ SFtrade mark media, and to approximately 45% of cells remaining after exposure to and initial destruction by B . thuringiensis toxin . Apoptotic nuclei were predominant (approximately 6%) in mature columnar cells in control cultures . Approximately 1% of goblet, stem, and differentiating cells were apoptotic . However, apoptosis rose to 12% in stem and differentiating cells exposed to used and unsuitable medium . B . thuringiensis exposure to toxin for 2-3 days resulted in visible membrane damage and necrosis, causing the death of 84% of the cells as measured by both the TUNEL and Annexin methods . Some of the columnar cells and stem and differentiating cells that remained also contained apoptotic nuclei . Stem and differentiating cells normally replace dying mature cells in the midgut . Thus, exposure of cultures of H . virescens midgut cells to adverse environments such as unsuitable or poisonous media appeared to induce down-regulation of the cell populations by apoptosis.

Curr Microbiol, 2000 Nov, 41(5), 352 - 6
Analysis of expression of the binary toxin genes from Bacillus sphaericus in Anabaena and the potential in mosquito control; Xu X et al.; Anabaena strains expressing the binary toxin genes of Bacillus sphaericus produce high larvicidal activity with living cells . Western blot analysis showed that the 51-kDa and 42-kDa toxin proteins were stable in Anabaena . When a DNA fragment upstream of the 51-kDa protein gene was deleted, the toxicity was reduced by over a hundred-fold, whereas deletions at the coding regions showed that the cooperation of the two proteins expressed in Anabaena is essential for the larvicidal activity . Outdoor tests showed that the genetically altered Anabaena could keep containers with natural water from being inhabited by Culex larvae for over 2 months.

EMBO J, 2000 Oct 2, 19(19), 5233 - 40
Mapping the fMet-tRNA(f)(Met) binding site of initiation factor IF2; Guenneugues M et al.; The interaction between fMet-tRNA(f)(Met) and Bacillus stearothermophilus translation initiation factor IF2 has been characterized . We demonstrate that essentially all thermodynamic determinants governing the stability and the specificity of this interaction are localized within the acceptor hexanucleotide fMet-3'ACCAAC of the initiator tRNA and a fairly small area at the surface of the beta-barrel structure of the 90-amino acid C-terminal domain of IF2 (IF2 C-2) . A weak but specific interaction between IF2 C-2 and formyl-methionyl was also demonstrated . The surface of IF2 C-2 interacting with fMet-tRNA(f)(Met) has been mapped using two independent approaches, site- directed mutagenesis and NMR spectroscopy, which yielded consistent results . The binding site comprises C668 and G715 located in a groove accommodating the methionyl side-chain, R700, in the vicinity of the formyl group, Y701 and K702 close to the acyl bond between fMet and tRNA(f)(Met), and the surface lined with residues K702-S660, along which the acceptor arm of the initiator tRNA spans in the direction 3' to 5'.

Appl Environ Microbiol, 2000 Oct, 66(10), 4582 - 4
Cross-resistance of pink bollworm (Pectinophora gossypiella) to Bacillus thuringiensis toxins; Tabashnik BE et al.; Two strains of pink bollworm (Pectinophora gossypiella) selected in the laboratory for resistance to Bacillus thuringiensis toxin Cry1Ac had substantial cross-resistance to Cry1Aa and Cry1Ab but not to Cry1Bb, Cry1Ca, Cry1Da, Cry1Ea, Cry1Ja, Cry2Aa, Cry9Ca, H04, or H205 . The narrow spectrum of resistance and the cross-resistance to activated toxin Cry1Ab suggest that reduced binding of toxin to midgut target sites could be an important mechanism of resistance.

Appl Environ Microbiol, 2000 Oct, 66(10), 4568 - 70
Incorporation of protease K into larval insect membrane vesicles does not result in disruption of integrity or function of the pore-forming Bacillus thuringiensis delta-endotoxin; Aronson A; Bacillus thuringiensis delta-endotoxins insert into the brush border membranes of insect larval cells to form ion channels . A possible interaction of these toxins with a cytoplasmic component was examined by preloading vesicles from insect larval cells with protease K followed by incubation with toxin . There was no evidence for toxin antigens smaller than the intact toxin in extracts of solubilized vesicles, nor was there an effect of the inclusion of protease K on either of two functional properties, the formation of toxin aggregates or of ion pores . These toxins, physically and functionally, appear to be confined to the membrane.

Appl Environ Microbiol, 2000 Oct, 66(10), 4449 - 55
Molecular genetic manipulation of truncated Cry1C protein synthesis in Bacillus thuringiensis to improve stability and yield; Park HW et al.; Cry1 protoxins of Bacillus thuringiensis are insecticidal 135-kDa proteins synthesized and assembled into parasporal crystals during sporulation . After ingestion, these crystals dissolve in the midgut and active toxins with molecular masses of about 65-kDa are released from the N-terminal half of the molecule by midgut proteases . Direct synthesis of the toxin-containing N-terminal half of Cry1 molecules using recombinant DNA techniques results in a low level of unstable truncated proteins that do not crystallize . In the present study, inclusions of truncated Cry1C (Cry1C-t) were obtained by combining genetic elements from other endotoxin genes and operons that enhance Cry protein synthesis and crystallization . Increased levels of Cry1C-t synthesis were achieved by using cyt1A promoters to drive expression of the 5' half of cry1C that included in the construct the 5' cry3A STAB-SD mRNA stabilizing sequence and the 3' stem-loop transcription terminator . RNA dot blot analysis showed that the STAB-SD and 3' transcriptional termination sequences were important for stabilization of truncated cry1C (cry1C-t) mRNA . A low level of cry1C-t mRNA was present when only the cyt1A promoters were used to express cry1C-t, but no accumulation of Cry1C-t was detected in Western blots . The orientation of the transcription terminator was important to enhancing Cry1C-t synthesis . Inclusion of the 20- and 29-kDa helper protein genes in cry1C-t constructs further enhanced synthesis . The Cry1C-t protein was toxic to Spodoptera exigua larvae, though the toxicity (50% lethal concentration {LC(50)} = 13.2 microg/ml) was lower than that of full-length Cry1C (LC(50) = 1.8 microg/ml) . However, transformation of the HD1 isolate of B . thuringiensis subsp . kurstaki with the cry1C-t construct enhanced its toxicity to S . exigua as much as fourfold.

Clin Infect Dis, 2000 Sep, 31 Suppl 3, S106 - 8
Role of animal models in understanding intravesical therapy with bacille Calmette-Guérin; Ratliff TL; Animal models provide a vehicle for understanding basic biological questions . Through the use of animal models, adequate control of experimental design is possible so that rigorous experiments can be performed to test a hypothesis . In the case of testing therapeutic mechanisms, it is important to select a model that is most analogous to the clinical setting so that observations can be readily transferred to clinical studies for validation . In mechanistic studies of Mycobacterium bovis strain (bacille Calmette-Guerin) therapy for bladder cancer, the orthotopic animal model most closely mimics the clinical treatment of bladder cancer.

Clin Infect Dis, 2000 Sep, 31 Suppl 3, S101 - 5
Reduction of side effects of intravesical therapy with bacille Calmette-Guérin by pentoxifylline?--an in vitro approach; Bohle A et al.; Immunotherapy with intravesical bacille Calmette-Guerin (BCG) is the treatment of choice against superficial bladder cancer recurrences . However, this therapy is associated with side effects that are considered to be the result of inflammatory cytokines . Since pentoxifylline is known to interfere with the production of cytokines, this drug was tested in vitro with regard to a later clinical application in BCG-treated patients . The cytokine release and the cytotoxicity of interleukin-2 or BCG-stimulated mononuclear cells were analyzed, and the growth of BCG under the influence of pentoxifylline was assayed . The results showed an inhibition of cytokine release of stimulated mononuclear cells . The cytotoxicity of BCG-stimulated mononuclear cells but not of lymphokine-stimulated mononuclear cells against bladder carcinoma cells was significantly inhibited . Restimulation with fresh BCG restored cytotoxicity . Direct coincubation of BCG and pentoxifylline resulted in a reduction of mycobacterial metabolism . From these data, we conclude that the use of pentoxifylline to reduce BCG-related side effects should be tested further in a clinical study.

Clin Infect Dis, 2000 Sep, 31 Suppl 3, S94 - S100
In vitro generation of bacillus Calmette-Guérin-activated killer cells; Brandau S et al.; Tumor regression induced in cancer patients by local instillation of bacillus Calmette-Guerin (BCG) into the bladder is considered to be mediated by cellular immune and inflammatory reactions . In an attempt to elucidate which of these effects are relevant to tumoricidal activity, an in vitro system was employed in which the immunostimulatory effects of BCG could be studied . This report describes the induction of BCG-activated killer (BAK) cells, which effectively lyse bladder tumor cells . Human peripheral blood mononuclear cells (PBMC) were stimulated with viable and sonicated BCG (v-BCG and s-BCG, respectively) to generate BAK cells . Cytotoxicity of BAK cells was comparable with the cytotoxicity exerted by lymphokine-activated killer (LAK) cells generated by interferon (IFN)-gamma but did not reach the level of interleukin-2 (IL-2)-generated LAK cells . Induction of BAK cells was possible only with v-BCG and not with s-BCG . By depletion and enrichment of defined cell populations, the cytotoxic potential of BAK cells could be attributed to a population of CD8(+) and CD56(+) double-positive lymphocytes . Macrophages and CD4(+) cells were required for the induction of killing activity but had no such activity by themselves . Furthermore, the presence of IFN-gamma and IL-2 in the supernatants harvested during the generation of BAK cells was demonstrated . Monoclonal antibodies neutralizing these cytokines abolished BCG-mediated cytotoxicity . From these results, it is concluded that the known beneficial effect of local instillation of BCG on maintenance of the relapse-free state in superficial bladder cancer may be due to local generation of BAK cells.

Clin Infect Dis, 2000 Sep, 31 Suppl 3, S91 - 3
Mechanisms of action of intravesical bacille Calmette-Guérin: local immune mechanisms; Prescott S et al.; The local immune response to mycobacteria is complex, but mycobacterial antigen presentation by phagocytes to T helper cells is the pivotal interaction . Bacille Calmette-Guerin (BCG) vaccination is associated with the development of antituberculosis immunity but not necessarily with antitumor immunity . Animal studies have shown that an intact host immune system is required for the antitumor activity of BCG . Immunosuppressed and, particularly, T cell-depleted individuals fail to respond to BCG immunotherapy . Clinical and laboratory evidence suggest that the antitumor activity is concentrated at the site of BCG administration, which reinforces the view that local immune mechanisms are responsible for this phenomenon.

Clin Infect Dis, 2000 Sep, 31 Suppl 3, S86 - 90
Efficacy and safety of bacille Calmette-Guérin immunotherapy in superficial bladder cancer; Lamm DL; In the United States, bladder cancer is the fourth most common human malignancy . In the past decade, the incidence of bladder cancer has increased by 36% . However, mortality has declined by 8% . Intravesical chemotherapy was considered to be partially responsible for this improvement in survival, but a recent review of clinical studies shows no reduction in disease progression with intravesical chemotherapy . Fortunately, the results of immunotherapy with bacille Calmette-Guerin (BCG) are quite different, and it is expected that patients treated with optimal BCG treatment regimens will have a long-term reduction in tumor recurrence, tumor progression, and cancer mortality.

Clin Infect Dis, 2000 Sep, 31 Suppl 3, S77 - 80
An analysis of some hypotheses related to the Chingelput bacille Calmette-Guérin trial; Smith D et al.; Investigation of several hypotheses related to the outcome of the Chingelput Bacille Calmette-Guerin (BCG) Trial suggests at least 2 factors that might explain the major scientific puzzle of a protective effect expected of 80% and a protective effect observed of 0% (i.e., equivalent protection for BCG and placebo) . One factor that explains some of the low efficacy observed for BCG in this trial is the virtual saturation level of exposure to environmental mycobacteria (EM) . Studies in animal models demonstrated that the protection afforded by infection with EM was equivalent to the protection that resulted from BCG vaccination . The second factor, pathogenetic pathway, explains why there was still a high case rate for tuberculosis, even though the population was fully vaccinated by EM . This hypothesis states that tuberculosis in India, as well as in most developing countries, results primarily from exogenous reinfection, a pathway against which BCG (or EM) exerts no protective effect beyond that induced by the first episode of infection with Mycobacterium tuberculosis.

Clin Infect Dis, 2000 Sep, 31 Suppl 3, S75 - 6
Management of adverse reactions to bacille Calmette-Guérin vaccine; FitzGerald JM; Published reports appear to underestimate the true rate of adverse reactions to bacille Calmette-Guerin (BCG) vaccine . At a recent national conference on tuberculosis control among aboriginal populations, lack of awareness of what constitutes an adverse reaction was considered a possible contributing factor to underreporting . The following review defines a normal BCG response and discusses the management of complications when they occur.

Clin Infect Dis, 2000 Sep, 31 Suppl 3, S71 - 4
What does tuberculin reactivity after bacille Calmette-Guérin vaccination tell us?
Menzies D.
The effect of bacille Calmette-Guerin (BCG) vaccination on tuberculin reactivity is briefly reviewed . BCG vaccination will almost invariably result in tuberculin conversion with a positive tuberculin skin test developing 4-8 weeks after vaccination . However, these tuberculin reactions will wane-rapidly in all individuals who receive the vaccine in the neonatal period and more slowly in those who are vaccinated at an older age such as during the primary-school years . Of BCG vaccine recipients whose initial tuberculin skin test is negative, 10%-25% will have a positive tuberculin skin test if they are retested within 1-4 weeks-the so-called "booster phenomenon . " There is no relationship between tuberculin reactivity after BCG vaccination and the protective efficacy of the vaccine against development of active tuberculosis . Therefore, the ideal BCG vaccine would produce a scar at the site of injection to identify individuals who have been vaccinated but would have no effect on tuberculin reactivity.

Clin Infect Dis, 2000 Sep, 31 Suppl 3, S64 - 7
Preventing tuberculosis with bacillus Calmette-Guérin vaccine: a meta-analysis of the literature; Brewer TF; This article reviews a previously published meta-analysis of 1264 titles or abstracts and 70 selected studies for evaluation of the efficacy of bacillus Calmette-Guerin (BCG) vaccine in preventing tuberculosis . Following review, data from 26 studies were included in the analysis . These 26 studies reveal that vaccination with BCG significantly reduces the risk of tuberculosis by an average of 50% . This level of protection persists across a number of subgroups defined by age at vaccination and study design . Vaccination with BCG was significantly associated with a reduction in the incidence of pulmonary tuberculosis and extrapulmonary disease . In general, the results of this meta-analysis lend weight and confidence to arguments favoring the use of BCG vaccine.

Microbes Infect, 2000 Aug, 2(10), 1193 - 205
Bartonella henselae, B . quintana, and B . bacilliformis: historical pathogens of emerging significance; Karem KL et al.; Bartonella species were virtually unrecognized as modern pathogens of humans until the last decade . However, identification of Bartonella species as the agents of cat-scratch disease, bacillary angiomatosis, urban trench fever, and possible novel presentations of Carrion's disease has left little doubt of the emerging medical importance of this genus of organisms . The three primary human pathogenic bartonellae, Bartonella bacilliformis (Carrion's disease), B . henselae (cat-scratch disease), and B . quintana (trench fever), present noteworthy comparisons in the epidemiology, natural history, pathology, and host-microbe interaction that this review will briefly explore.

Comp Biochem Physiol B Biochem Mol Biol, 2000 Jul, 126(3), 445 - 53
Phosphatidylcholine-specific phospholipase C-mediated induction of phospholipase D activity in Fas-expressing murine cells; Shin I et al.; We have previously reported that Fas cross-linking resulted in the activation of phosphatidylcholine-specific phospholipase C (PC-PLC) and the subsequent activation of protein kinase C (PKC) and phospholipase D (PLD) in A20 cells . In an attempt to correlate the existence of PC-PLC activity and activation of PLD by Fas activation among various Fas-expressing murine cell lines, we have investigated the effect of anti-Fas monoclonal antibody on PC-PLC and PLD activities in A20, P388D1 and YAC-1 cell lines . Upon treatment of anti-Fas monoclonal antibody to these three cell lines, the activation of PLD was only observed in A20 cells . When the effect of anti-Fas monoclonal antibody on PKC and PC-PLC activities in Fas-expressing clones were investigated, the activation of PKC and PC-PLC was detected only in A20 clones . Results presented here also show that exogenous addition of Bacillus cereus PC-PLC activates PC hydrolysis, PKC and PLD in all three murine cell lines . These findings suggest that the activation of PC-PLC is a necessary requirement for the activation of PLD by Fas cross-linking and cell lines devoid of functional PC-PLC activity could exhibit enhanced PLD activity by exogenous addition of PC-PLC.

Can J Microbiol, 2000 Sep, 46(9), 826 - 31
Cholesterol is accumulated by mycobacteria but its degradation is limited to non-pathogenic fast-growing mycobacteria; Av-Gay Y et al.; In this report we show that fast-growing non-pathogenic mycobacteria degrade cholesterol from liquid media, and are able to grow on cholesterol as a sole carbon source . In contrast, slow-growing mycobacteria, including pathogenic Mycobacterium tuberculosis and bacillus Calmette-Guerin (BCG), do not degrade and use cholesterol as a carbon source . Nevertheless, pathogenic mycobacteria are able to uptake, modify, and accumulate cholesterol from liquid growth media, and form a zone of clearance around a colony when plated on solid media containing cholesterol . These data suggest that cholesterol may have a role in mycobacterial infection other than its use as carbon source.

Can J Microbiol, 2000 Sep, 46(9), 784 - 9
Stoichiometry of diauxic growth of a xylanase-producing Bacillus strain; Schneider G et al.; In this work, the establishment of material balances and stoichiometry of the growth of Bacillus sp . was undertaken . This strain produces high quantities of a xylanase suitable for use as bleach boost agent in chlorine-free bleaching sequences of paper pulp . As carbon dioxide plays an important role as a growth factor, bacterial growth in two fermentations, one fed with air and another fed with carbon-dioxide-enriched air, were compared . For this purpose, a method permitting the determination of the consumption of the two carbon sources, xylan and peptone, was proposed . The material balances revealed that in both cases, the bacteria first use peptone as their carbon source, and then xylan in the second part of the growth phase . The aerated culture showed diauxic growth on these two substrates, whereas carbon-dioxide-enriched air caused disappearance of the metabolic adaptation phase, and rendered biomass production more economic . The fermentation fed with air needed 30% more xylan than the fermentation fed with carbon-dioxide-enriched air for the same quantity of biomass produced.

Lab Invest, 2000 Sep, 80(9), 1385 - 97
Lethal Mycobacterium bovis Bacillus Calmette Guérin infection in nitric oxide synthase 2-deficient mice: cell-mediated immunity requires nitric oxide synthase 2; Garcia I et al.; The role of nitric oxide (NO) in Mycobacterium bovis Bacillus Calmette Guerin (BCG) infection was investigated using nitric oxide synthase 2 (nos2)-deficient mice, because NO plays a pivotal protective role in M . tuberculosis infection . We demonstrate that nos2-deficient mice were unable to eliminate BCG and succumbed within 8 to 12 weeks to BCG infection (10(6) CFU) with cachexia and pneumonia, whereas all infected wild-type mice survived . The greatest mycobacterial loads were observed in lung and spleen . Nos2-deficient mice developed large granulomas consisting of macrophages and activated T cells and caseous necrotic lesions in spleen . The macrophages in granulomas from nos2-deficient mice had reduced acid phosphatase activities, suggesting that NO is required for macrophage activation . The absence of NOS2 affected the cytokine production of the Th1 type of immune response, except IL-18 . Serum amounts of IL-12p40 were increased and IFN-gamma was decreased compared with wild-type mice . The lack of NOS2 resulted in an overproduction of TNF, observed throughout the infection period . Additionally, TNFR1 and TNFR2 shedding was altered compared with wild-type mice . Up-regulation of TNF may be compensatory for the lack of NOS2 . The late neutralization of TNF by soluble TNF receptors resulted in heightened disease severity and accelerated death in nos2-deficient mice but had no effect in wild-type mice . In conclusion, the inability of nos2-deficient mice to kill M . bovis BCG resulted in an accumulation of mycobacteria with a dramatic activation of the immune system and overproduction of pro-inflammatory cytokines, which resulted in death.

J Bacteriol, 2000 Oct, 182(20), 5765 - 70
A truncated soluble Bacillus signal peptidase produced in Escherichia coli is subject to self-cleavage at its active site; van Roosmalen ML et al.; Soluble forms of Bacillus signal peptidases which lack their unique amino-terminal membrane anchor are prone to degradation, which precludes their high-level production in the cytoplasm of Escherichia coli . Here, we show that the degradation of soluble forms of the Bacillus signal peptidase SipS is largely due to self-cleavage . First, catalytically inactive soluble forms of this signal peptidase were not prone to degradation; in fact, these mutant proteins were produced at very high levels in E . coli . Second, the purified active soluble form of SipS displayed self-cleavage in vitro . Third, as determined by N-terminal sequencing, at least one of the sites of self-cleavage (between Ser15 and Met16 of the truncated enzyme) strongly resembles a typical signal peptidase cleavage site . Self-cleavage at the latter position results in complete inactivation of the enzyme, as Ser15 forms a catalytic dyad with Lys55 . Ironically, self-cleavage between Ser15 and Met16 cannot be prevented by mutagenesis of Gly13 and Ser15, which conform to the -1, -3 rule for signal peptidase recognition, because these residues are critical for signal peptidase activity.

An Esp Pediatr, 2000 Mar, 52(3), 232 - 7
{Tuberculous meningitis: a disease in regression in our country?}; Parrilla Parrilla JS et al.; OBJECTIVE: Our aim was to analyse clinical, diagnostic, therapeutical and evolutionary features in a pediatric population with tuberculous meningitis . PATIENTS AND METHODS: The medical records of thirteen children with this diagnosis admitted to Hospital Infantil Virgen del Rocio from Seville (Spain) between 1984 and 1999 were reviewed . RESULTS: The mean age was 2,35 +/- 2,3 years . The symptoms upon admission were: fever in 11 children, anorexia and vomiting in 8, disturbance of the consciousness in 7 . Meningeal signs in 6, all of them older than 20 months, the remaining seven showed irritability and four of these ones hypertense fontanelles . Three patients were in the first stage of the disease, 9 in the second and 1 in the third, according to the Medical Research Council . CSF findings were indicative in all the cases . Five children had bacilloscopy positive and Mycobacterium tuberculosis was isolated in 6 patients, sometimes in CSF others in gastric juice . Mantoux skin test was positive in 11 . Radiographic studies demonstrated abnormal chest findings in 8 patients (hiliar adenopathy, 1; miliary pattern, 2; and infiltrates, 5) . Pathology cranial computed tomography showed in all the cases and the electroencephalogram was slowed down in the initial phases in 11 . Two children died and the neurological complications were the most frequent, appearing in 9 patients . Without consequences cured 4 patients, the rest presented cognitive, visual and motor deficits, sensibility skin disturbance and late seizures . No case has been observed during the last 5 years . CONCLUSIONS: Fast diagnosis tests used for M . tuberculosis identification were useful to begin an antituberculous treatment in a high suspicion of meningeal affectation by this German patient . The early treatment will decrease complications and consequences by this disease . A decrease in the incidence looks to be in spite of the VIH infection increase nowadays.

Bioorg Med Chem, 2000 Aug, 8(8), 1957 - 68
Covalent modification of subtilisin Bacillus lentus cysteine mutants with enantiomerically pure chiral auxiliaries causes remarkable changes in activity; Dickman M et al.; Methanethiosulfonate reagents may be used to introduce virtually unlimited structural modifications in enzymes via reaction with the thiol group of cysteine . The covalent coupling of enantiomerically pure (R) and (S) chiral auxiliary methanethiosulfonate ligands to cysteine mutants of subtilisin Bacillus lentus induces spectacular changes in catalytic activity between diastereomeric enzymes . Amidase and esterase kinetic assays using a low substrate approximation were used to establish kcat/KM values for the chemically modified mutants, and up to 3-fold differences in activity were found between diastereomeric enzymes . Changing the length of the carbon chain linking the phenyl or benzyl oxazolidinone ligand to the mutant N62C by a methylene unit reverses which diastereomeric enzyme is more active . Similarly, changing from a phenyl to benzyl oxazolidinone ligand at S166C reverses which diastereomeric enzyme is more active . Chiral modifications at S166C and L217C give CMMs having both high esterase kcat/KM's and high esterase to amidase ratios with large differences between diastereomeric enzymes.

Bull Soc Pathol Exot . 1999 Dec;92(5 Pt 2):418.
{The plague reviewed by way of molecular biology}; Carniel E; The aetiology of plague was first discovered during the third pandemic of the disease occurring in Hong Kong in 1894 . After Alexandre Yersin had identified the causal agent (Y . Pestis), Paul-Louis Simond proved the flea's role as vector . These discoveries were of prime importance for the subsequent development of efficient means for fighting plague as well as for preventive and curative treatments and vaccines . Vaccination brought about a sharp decrease in plague mortality and morbidity . However, the disease has never been eradicated . It is still prevalent in various Asian, African and American countries and is among the re-emerging diseases at the present time . The genetic basis of transmission mechanisms and pathogenicity of the bacillus are only beginning to be understood . We now know that the attenuation of the EV76 strain used by Girard and Robic as an anti-plague vaccine in Madagascar is due to the spontaneous excision of a large chromosomal DNA fragment of 102 kb, a part of which contains a group of genes implicated in the pathogenicity and appropriately called high pathogenicity island . These mechanisms of flea bacillus transmission are also beginning to be known . Two bacterial loci participating in the blocking of the ectoparasite's proventriculus have been identified . One is situated next to the high pathogenicity island on the unstable 102 kb chromosomal fragment, the other--on the large 95 kb plasmid specific to Y . pestis . The molecular basis of the bacillus' acquisition of multi-resistance to antibiotics have likewise recently been characterised . However, although Y . pestis is one of the most pathogenic micro-organisms of the bacterial world, the mechanisms responsible for this high level of pathogenecity have still not been identified . This is well worth noting, since a certain number of genes acting as pathogenicity factors in other species are present but altered in Y . pestis . Plague still withholds many secrets.

Bull Soc Pathol Exot, 1999 Dec, 92(5 Pt 2), 383 - 7
{The discovery by Paul-Louis Simond of the role of the flea in the transmission of the plague}; Mollaret HH; After Yersin's two fundamental discoveries of the plague bacillus and of the rat's role in its propagation, no one had sought to solve the riddle of how the bacillus itself spread and how it contaminated man . P . L . Simond was the first to realise that manipulating a rat that had recently died could be extremely dangerous whereas after a time lapse of several hours the same dead rat presented no risks to man . He was also the first to detect an insect bite as being responsible for the lesions he had observed at the beginning of bubonic plague outbreaks and called "precocious plyctenas" . After having verified the presence of the bacillus in fleas of dying rats, on 2 June 1898 Simond carried out his princeps experiment on the transmission of plague to rats by fleas . From then onwards, disinsectisation was added to deratisation in plague prophylaxis.

Clin Cancer Res, 2000 Sep, 6(9), 3729 - 38
Perforin-mediated lysis of tumor cells by Mycobacterium bovis Bacillus Calmette-Guérin-activated killer cells; Brandau S et al.; Immunotherapy with Bacillus Calmette-Guerin (BCG) is clinically established in the treatment of superficial bladder cancer . In our attempt to clarify the underlying immunological mechanism, we could previously show that stimulation of PBMC with BCG leads to the generation of cytotoxic BCG-activated killer (BAK) cells . Among others, these BAK cells as well as lymphokine-activated killer (LAK) cells have been suggested as possible effector cells during BCG therapy . To understand BCG-induced activation of effector lymphocytes more precisely, we investigated the lytic pathways of human BAK cells and compared BAK cell cytotoxicity with LAK cell cytotoxicity . Perforin and Fas ligand (FasL) are the major cytolytic molecules of cytotoxic lymphocytes . Our results demonstrate that BAK and LAK cells showed an increased expression of perforin and FasL as compared with unstimulated controls . Killing of T-24 bladder tumor as well as Jurkat cells by BAK and LAK cells was predominantly mediated via perforin as demonstrated by a drastically reduced lysis in the presence of concanamycin A and EGTA/MgCl2, respectively . In contrast, lysis (radioactive release assay) and membrane disintegration (Annexin V binding) of both targets by BAK and LAK cells could not be blocked with an inhibitory anti-FasL monoclonal antibody (NOK-1) . Nevertheless, T-24 and Jurkat were susceptible to killing by recombinant soluble FasL and by Chinese hamster ovary cells expressing membrane-bound FasL . We conclude that cellular mediators of BCG effector mechanisms, such as BAK and LAK cells, kill their targets via perforin and independent of the FasL pathway . Because we also found increased numbers of perforin-expressing lymphocytes in patients after BCG therapy, our findings have potential clinical relevance because BCG therapy would not be impaired by FasL resistance of target cells, which recently has been described for some tumors.

Cancer Immunol Immunother, 2000 Sep, 49(7), 369 - 76
Killing of Fas ligand-resistant renal carcioma cells by interleukin-2- and BCG-activated effector cells; Brandau S et al.; Activated cytolytic effector cells like lymphokine-activated killer (LAK) and the recently described bacillus-Calmette-Guerin-activated killer (BAK) cells are thought to mediate antitumor effects against metastatic renal cell carcinoma (RCC) and superficial bladder cancer respectively . Perforin and Fas ligand (FasL) have been described as the major lytic principles in cellular cytotoxicity . Using a radioactive-release assay and specific inhibitors, we investigated the molecular mechanisms used by LAK and BAK cells in the lysis of renal carcinoma cells . In addition, we evaluated the susceptibility of RCC cells to FasL-mediated cytotoxicity . LAK and BAK cells effectively lysed the renal cancer cell line SK-RC-35 upon cell-cell contact . Both effector cell populations were shown to produce perforin and FasL as determined by reverse transcriptase/polymerase chain reaction (RT-PCR) . Using fluorescence-activated cell sorting analyses and RT-PCR, we detected a marked Fas receptor (Fas, CD95) expression on RCC cells . However, RCC cells were shown to be resistant to killing by recombinant FasL and lysis by BAK and LAK cells was not inhibited in the presence of anti-FasL antibody . In contrast, the cytotoxicity exerted by LAK and BAK cells was drastically reduced in the presence of the Ca2+-chelating agent EGTA as well as concanamycin A, a specific inhibitor of perforin-mediated lysis . These results demonstrate that cytolysis of FasL-resistant RCC cells by activated immune cells is mediated via perforin . Our findings give further insights into the molecular mechanisms involved in the elimination of RCC by cytotoxic lymphocytes activated with biological response modifiers.

Mem Inst Oswaldo Cruz, 2000 Sep-Oct, 95(5), 693 - 700
Proteolytic processing of the Cyt1Ab1 toxin produced by Bacillus thuringiensis subsp . medellin; Escobar E et al.; Bacillus thuringiensis produces d-endotoxins that require proteolytic processing to become active . The activation of the B . thuringiensis subsp . medellin 28 kDa (Cyt1Ab1) cytolytic toxin by trypsin, chymotrypsin and gut extract from Culex quinquefasciatus larvae was analyzed . The Cyt1Ab1 toxin of B . thuringiensis subsp . medellin was processed by all proteases tested to fragments between 23 and 25 kDa, while processing of the Cyt1Aa1 toxin produce fragments between 22.5 and 24.5 kDa . The Cyt1Ab1 toxin was preferentially processed at the alkaline pH of 12 . The in vitro proteolytic processing of the Cyt1Ab1 toxin by C . quinquefasciatus larvae midgut extract showed a 25 kDa fragment; a similar result was observed when the activation was performed in the in vivo experiments . The solubilized Cyt1Ab1 toxin and the protease resistant cores generated by in vitro processing showed hemolytic activity but not mosquitocidal activity . Amino terminal sequence of the C . quinquefasciatus gut extract resistant fragment indicated that the cutting site was located between Lys31 and Asp32, with a sequence DDPNEKNNHNS; while for the trypsin-resistant fragment the cutting site was determined between Leu29 and Arg30, and for the chymotrypsin-resistant fragment between Arg30 and Lys31.

Mol Med, 2000 Jul, 6(7), 581 - 90
T cells recognize an immunodominant epitope of heat shock protein 65 in Kawasaki disease; Sireci G et al.; BACKGROUND: Kawasaki disease (KD) is an acute systemic vasculitis of infancy and early childhood that is characterized by endothelial cell damage associated with T-cell activation . Lymphocytes infiltrating damaged tissues might be responsible for the disease through secretion of cytokines, such as tumor necrosis factor (TNF)-alpha, that could cause fever, as well as endothelial tissue damage . Debate is growing about the nature of antigen responsible for T-cell activation in KD . Bacillus Calmette Guerin (BCG) and purified protein derivative (PPD) hyperresponsiveness was observed in KD patients and this phenomenon was hypothetically ascribed to cross-reactivity between mycobacterial Heat Shock Protein (HSP) 65 and human homologue HSP63 . MATERIALS AND METHODS: CD4+ and CD8+ T-cell clones were obtained from peripheral blood of KD patients in acute phase, or control subjects . The clones were tested for reactivity toward HSP65 and derived peptides . Both proliferation and cytokine production were analyzed . RESULTS: A significant fraction of CD4 and CD8 T-cell clones from KD patients recognized an epitope from HSP65, spanning amino acids 65-85 . T-cell clones cross-reacted with the corresponding 90-110 peptide sequence of human HSP-63 . CONCLUSIONS: Cross-reactivity between specific epitopes of mycobacterial and human HSP could play a role in the development of the tissue-damage characteristic of KD.

Cancer Lett, 2000 Oct 31, 159(2), 127 - 34
Effects of tumor necrosis factor-alpha and interferon-gamma on expressions of matrix metalloproteinase-2 and -9 in human bladder cancer cells; Shin KY et al.; We have investigated the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon (INF-gamma), the potent Bacillus Calmette-Guerin (BCG)-induced cytokines on the production of MMP-2, MMP-9, TIMP-1, TIMP-2 and MT1-MMP in high grade human bladder cancer cell lines, T-24, J-82 and HT-1376 cell lines . MMP-2 expression and activity were decreased in T-24 cells treated with both cytokines in a dose dependent manner . However, J-82 cells treated with TNF-alpha and INF-gamma revealed dose dependent increases of MMP-9 expression and activity with similar baseline expression and activity of MMP-2 . HT-1376 cells after exposure to TNF-alpha only enhanced the expression and activity of MMP-9 . These results indicate that TNF-alpha and INF-gamma could regulate the production of MMP-2 or MMP-9 on bladder cancer cells and their patterns of regulation are cell specific . Furthermore, this diverse response of bladder cancer cells to TNF-alpha and INF-gamma suggests that BCG immunotherapy may enhance the invasiveness of bladder cancer in certain conditions with induction of MMPs.

J Biol Chem, 2000 Dec 15, 275(50), 39734 - 40
Fusarium oxysporum fatty-acid subterminal hydroxylase (CYP505) is a membrane-bound eukaryotic counterpart of Bacillus megaterium cytochrome P450BM3; Kitazume T et al.; The gene of a fatty-acid hydroxylase of the fungus Fusarium oxysporum (P450foxy) was cloned and expressed in yeast . The putative primary structure revealed the close relationship of P450foxy to the bacterial cytochrome P450BM3, a fused protein of cytochrome P450 and its reductase from Bacillus megaterium . The amino acid sequence identities of the P450 and P450 reductase domains of P450foxy were highest (40.6 and 35.3%, respectively) to the corresponding domains of P450BM3 . Recombinant P450foxy expressed in yeast was catalytically and spectrally indistinguishable from the native protein, except most of the recombinant P450foxy was recovered in the soluble fraction of the yeast cells, in marked contrast to native P450foxy, which was exclusively recovered in the membrane fraction of the fungal cells . This difference implies that a post (or co)-translational mechanism functions in the fungal cells to target and bind the protein to the membrane . These results provide conclusive evidence that P450foxy is the eukaryotic counterpart of bacterial P450BM3, which evokes interest in the evolutionary aspects concerning the P450 superfamily along with its reducing systems . P450foxy was classified in the new family, CYP505.

Prikl Biokhim Mikrobiol, 2000 Jul-Aug, 36(4), 395 - 401
{New cyclomaltodextrin-glucanotransferases, produced by Bacillus macerans}; Abelian VA et al.; For the first time, microorganisms producing cyclomaltodextrin glucan transferases (CGT, EC 2.4.1.19) were isolated from soil samples of various ecogeographical regions . These microorganisms were identified as Bacillus macerans . The enzymes were purified by affinity chromatography on a alpha-cyclodextrin polymer and gel filtration on Biogel P-450 and proved to be electrophoretically homogeneous . Some of their physicochemical and biochemical properties are reported.

J Gen Virol, 2000 Oct, 81(Pt 10), 2525 - 9
Three functionally diverged major structural proteins of white spot syndrome virus evolved by gene duplication; van Hulten MC et al.; White spot syndrome virus (WSSV) is an invertebrate virus causing considerable mortality in penaeid shrimp . The oval-to-bacilliform shaped virions, isolated from infected Penaeus monodon, contain four major proteins: VP28, VP26, VP24 and VP19 (28, 26, 24 and 19 kDa, respectively) . VP26 and VP24 are associated with the nucleocapsid and the remaining two with the envelope . Forty-one N-terminal amino acids of VP24 were determined biochemically allowing the identification of its gene (vp24) in the WSSV genome . Computer-assisted analysis revealed a striking similarity between WSSV VP24, VP26 and VP28 at the amino acid and nucleotide sequence level . This strongly suggests that these structural protein genes may have evolved by gene duplication and subsequently diverged into proteins with different functions in the WSSV virion, i.e . envelope and nucleocapsid . None of these three structural WSSV proteins showed homology to proteins of other viruses including baculoviruses, underscoring the distinct taxonomic position of WSSV among invertebrate viruses.

Microb Pathog, 2000 Oct, 29(4), 213 - 22
Identification and characterization of the ribosome-associated protein, HrpA, of Bacillus Calmette-Guérin; Tabira Y et al.; HrpA was found as a ribosome-associated protein which appeared in heat-stressed Mycobacterium bovis Bacillus Calmette-Guerin . Here, we have studied the function of HrpA in vitro . HrpA is a heat shock protein belonging to a small heat shock protein family . The putative molecular mass was 17784.86 kDa . Recombinant HrpA formed large complexes of nonamer or dodecamer . HrpA prevented the aggregation of enzymes under heat shock conditions, and it formed stable complexes with partially denatured enzymes . HrpA was induced temporarily by oxygen repletion after anaerobic condition .

Biosci Biotechnol Biochem, 2000 Aug, 64(8), 1743 - 6
Efficient synthesis of a sialyl T-antigen-linked glycopeptide by the chemoenzymatic method; Ajisaka K et al.; A sialyl T-antigen-linked tetrapeptide was prepared by the combined method of chemical synthesis and enzymatic synthesis . The GalNAc-linked peptide was first obtained by using a commercial peptide synthesizer, and then a galactose residue was attached with beta-(1-->3)-linkage by transglycosylating with a recombinant beta-galactosidase from Bacillus circulans . The sialic acid residue was then combined by alpha-(2-->3)-linkage with sialytransferase from rat liver.

Biosci Biotechnol Biochem, 2000 Aug, 64(8), 1722 - 5
Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp . strain DJ-4 screened from Doen-Jang; Kim SH et al.; Bacillus sp . strain DJ-4, which produces extracellular proteases, was screened from Doen-Jang, a traditional Korean fermented food . A fibrinolytic enzyme (subtilisin DJ-4) was purified using commercial chromatographic techniques . The relative molecular mass of the isolated protein was 29 kDa by SDS-PAGE and fibrin zymography assay . The enzyme was characterized as a serine protease by an inhibitor assay on the fibrin zymography gel and by an amidolytic assay using a chromogenic substrate . The enzyme was inhibited by PMSF, but not by EDTA or leupeptin . The first 14 amino acids of the N-terminal sequence were identical to that of subtilisin BPN', but the activity of subtilisin DJ-4 was 2.2 and 4.3 times higher than those of subtilisin BPN' and subtilisin Carlsberg, respectively.

Biosci Biotechnol Biochem, 2000 Aug, 64(8), 1702 - 6
Hydrolysis of beta-galactosyl ester linkage by beta-galactosidases; Kiso T et al.; p-Hydroxybenzoyl beta-galactose (pHB-Gal) was synthesized chemically to examine the hydrolytic activity of beta-galactosyl ester linkage by beta-galactosidases . The enzyme from Penicillium multicolor hydrolyzed the substrate as fast as p-nitrophenyl beta-galactoside (pNP-Gal), a usual substrate with a beta-galactosidic linkage . The enzymes from Escherichia coli and Aspergillus oryzae hydrolyzed pHB-Gal with almost the same rates as pNP-Gal . The enzymes from Bacillus circulans, Saccharomyces fragilis, and bovine liver showed much lower activities . pH-activity profiles, inhibition analysis, and kinetic properties of the enzymic reaction on pHB-Gal suggested that beta-galactosidase had only one active site for hydrolysis of both galactosyl ester and galactoside . The Penicillium enzyme hydrolyzed pHB-Gal in the presence of H218O to liberate galactose containing 18O . This result suggests the degradation occurs between the anomeric carbon and an adjacent O atom in the ester linkage of pHB-Gal.

Indian J Lepr, 1998, 70 Suppl, 11S - 16S
Incidence of disabilities among multi-bacillary cases after initiation of multidrug therapy and factors associated with the risk of developing disabilities; Selvaraj G et al.; Out of the 1724 new cases registered between 1985 to 1992, 1169 could be contacted . The overall incidence of disabilities was 6.8% . Age above 45 years, bacteriopositivity and thickening of three or more trunk nerves were associated with a higher risk of disabilities . Staff training, patient education and steroid availability in the field were the suggested methods of reducing the occurrence of disabilities in leprosy patients.

Infect Immun, 2000 Oct, 68(10), 5960 - 9
Infection of human endothelial cells with Bartonella bacilliformis is dependent on Rho and results in activation of Rho; Verma A et al.; Bartonella bacilliformis was continuously internalized into human endothelial cells beginning shortly after addition of the bacteria and continuing for at least 24 h after infection in vitro, with a major increase in uptake occurring between 16 and 24 h . Preincubation of endothelial cells with C3 exoenzyme, which inactivated intracellular Rho-GTPase, blocked internalization of the bacteria . Addition of C3 exoenzyme at any time after addition of the bacteria blocked further internalization of bacteria, including the major uptake of bacteria internalized at 16 to 24 h . Rho, a key signaling protein in pathways involving actin organization, was directly shown to be activated in endothelial cells undergoing infection with B . bacilliformis, with maximal activation and translocation to the plasma membrane at 12 to 16 h . At late times of infection, most of the bacteria were found in a perinuclear location . Staining of the Golgi complex with specific markers, anti-human Golgin-97, anti-KDEL receptor, and BODIPY-TR ceramide, showed colocalization of bacteria in the Golgi complex region . Disruption of the Golgi complex with brefeldin A scattered the bacteria from this perinuclear location and resulted in inhibition of internalization of the bacteria in endothelial cells.

Pharmazie, 2000 Aug, 55(8), 595 - 600
{Gyrase inhibitors . 3 . Synthesis and reactions of 1,4-dihydro-4-oxo-(1)benzothieno(3,2-b)pyridin-3-carboxylic acid esters}; Gorlitzer K et al.; The title compound 4a is synthesized from potassium 3-aminobenzo{b}thiophene-2-carboxylate (1) by Gould-Jacobs reaction . Compound 4a reacts with alkyl halides and sodium hydride in DMF to yield the N-alkylpyridones 5a-c as well as the 4-alkoxypyridines 6a-c; with phosphoryl chloride the 4-chloropyridine 7a is obtained . The carboxylic acids 4b, 5d, 5e, 6d and 7b are received by alkaline saponification of the esters 4a, 5a-c, 6a-c and 7a . The carbinoles 8 and 9, formed by boranate reduction of the esters 6a and 7a, are transformed to the aldehydes 10 and 11 by activated manganese dioxide oxidation . The aldoxime 12 from the carbaldehyde 11 is dehydrated to yield the nitrile 13 . The carbaldehyde 11 reacts with methyl beta-aminocrotonate to yield the 1,4-dihydropyridine (DHP) 14, which is dehydrogenated to give the 3,4'-bipyridine 15 . The pyridone 4a reacts with tosylisocyanate to yield the 4-tosylaminopyridine 16a . The antibacterial activity of the carboxylic acids 4b, 5d, 5e and 6d is proved . The growth of Escherichia coli and Bacillus megaterium is inhibited by 5d in the same range as nalidixic acid does.

Insect Biochem Mol Biol, 2000 Nov, 30(11), 1069 - 78
Binding of Bacillus thuringiensis delta-endotoxins Cry1Ac and Cry1Ba to a 120-kDa aminopeptidase-N of Epiphyas postvittana purified from both brush border membrane vesicles and baculovirus-infected Sf9 cells; Simpson RM et al.; A 120-kDa protein was purified from brush border membrane vesicles of the tortricid moth Epiphyas postvittana (Walker) based both on its activity as an aminopeptidase and the ability to bind the Bacillus thuringiensis delta-endotoxin Cry1Ac . The purified enzyme had a pI of 5.6 and was a leucine aminopeptidase, with some isoleucine, phenylalanine and tryptophan aminopeptidase activity . Further characterisation showed that the protein was also able to bind Cry1Ba . During purification, the molecular weight of the protein decreased from 120 to 115 kDa due to the loss of a glycophosphatidinyl anchor . The protein was N-terminally sequenced and, using this information and conserved regions within other insect aminopeptidase-N (APN) sequences, redundant primers were designed to amplify the aminopeptidase coding sequence from E . postvittana midgut cDNA . The predicted protein sequence from the full-length cDNA was most closely related to the APN protein sequence from Heliothis virescens (61% identity) and shared other features of insect APNs including a Zn(2+) binding site motif and four conserved cysteines . The E . postvittana was expressed in Sf9 cells using baculovirus, yielding a protein of molecular weight 130 kDa, but with unchanged N-terminal sequence . Purified recombinant protein bound both Cry1Ac and Cry1Ba by ligand blot assays . However, despite the protein being expressed on the external surface of the Sf9 cells, it bound neither Cry1Ac nor Cry1Ba in vivo.

Insect Biochem Mol Biol, 2000 Nov, 30(11), 1027 - 35
Trypsinogen-like cDNAs and quantitative analysis of mRNA levels from the Indianmeal moth, Plodia interpunctella; Zhu YC et al.; Two cDNA fragments encoding full-length trypsinogen-like proteins were cloned from larvae of two strains (RC688s and HD198r) of the Indianmeal moth, Plodia interpunctella (Hubner), which differed in their sensitivity to Bacillus thuringiensis protoxins . One cDNA fragment contained 874 nucleotides, including a 780-nucleotide open reading frame that encoded a trypsinogen-like protein (PiT2b) . Another cDNA fragment amplified from both P . interpunctella strains contained 864 nucleotides including a 780 bp open reading frame encoding a second trypsinogen-like protein (PiT2c) . The cDNA sequence of PiT2b shared 89% sequence identity with PiT2a, a trypsinogen-like protein cloned previously from this species . The cDNA sequences of PiT2a and PiT2c shared 83% identity . The cDNA sequence identity between PiT2b and PiT2c was 80% . The cDNA for PiT2b from strain RC688s was different at six nucleotide positions from that of PiT2b from strain HD198r . Five nucleotide replacements occurred in the open reading frame leading to amino acid changes at all five positions . There were five nucleotide differences in the cDNAs for PiT2c trypsinogen-like proteins from the two strains . Two nucleotide substitutions in the open reading frame resulted in replacements of two amino acid residues in the deduced protein sequences . Amino acid sequences for PiT2a and PiT2b shared 84% identity, but only 50% identity was observed between PiT2c and the other two trypsinogen-like proteins . The deduced amino acid sequences for PiT2b and PiT2c included both signal and zymogen activation peptides and amino acid sequence motifs which are conserved in seven homologous trypsinogen-like proteins from other insects . Typical features of the putative trypsinogen-like proteins from P . interpunctella included the serine proteinase active site triad (His(81), Asp(133), and Ser(233)), three pairs of cysteine residues for disulfide bridges, and three residues, Asp(227), Gly(250), and Gly(260), that help to confer trypsin-like specificity to the enzymes . Quantitative RT-PCR analyses showed that, in fourth instar larvae, RC688s had 1.6-fold higher PiT2a trypsinogen-like mRNA than did HD198r . Expression of PiT2b mRNA was 3.4-fold higher in HD198r than in RC688s . Expression of PiT2c mRNA was 2.8-fold higher in RC688s than in HD198r . Mean accumulation levels of mRNAs for all three trypsinogen-like proteins were slightly higher in RC688s than in HD198r based on total RNA, and 1.3-fold higher in RC688s than in HD198r based on wet weight of larval body tissues.

J Biotechnol, 2000 Aug 25, 81(2-3), 199 - 204
Purification and characterization of a new xylanase from Acrophialophora nainiana; Salles BC et al.; A new xylanase activity (XynII) was isolated from liquid state cultures of Acrophialophora nainiana containing birchwood xylan as carbon source . XynII was purified to apparent homogeneity by gel filtration and ion exchange chromatographies . The enzyme was optimally active at 55 degrees C and pH 7.0 . XynII had molecular mass of 22630+/-3.0 and 22165 Da, as determined by mass spectrometry and SDS-PAGE, respectively . The purified enzyme was able to act only on xylan as substrate . The apparent K(m) values on soluble and insoluble birchwood xylans were 40.9 and 16.1 mg ml(-1), respectively . The enzyme showed good thermal stability with half lives of 44 h at 55 degrees C and ca . 1 h at 60 degrees C The N-terminal sequence of XynII showed homology with a xylanase grouped in family G/11 . The enzyme did not show amino acid composition similarity with xylanases from some fungi and Bacillus amyloliquefaciens.

J Biochem Biophys Methods, 2000 Sep 11, 45(2), 211 - 9
Influence of a poly-ethylene glycol spacer on antigen capture by immobilized antibodies; Weimer BC et al.; The use of spacers to distance an immobilized antibody from the surface of a support matrix introduces flexibility, which can reduce steric interferences between antibodies leading to a higher antigen capture efficiency . In this paper we investigated the use of a spacer molecule, poly-ethylene glycol (PEG), between the matrix surface and antibodies for the capture of Bacillus globigii, E . coli O157:H7, and ovalbumin . The antigen capture efficiency was determined using a surface ELISA method . Antibodies against the antigens were covalently immobilized either directly or via PEG to glass surfaces using a one-step EDC reaction . The amount of antibody immobilized was determined before blocking the nonspecific binding sites with bovine serum albumin . Antibodies immobilized via a PEG spacer showed a higher capture efficiency compared to direct immobilization, which was more pronounced with large antigens . Antibodies immobilized on glass supports were stable at 65 degrees C for at least 80 min, and the capture efficiency increased with heating at 65 degrees C for 20 min.

Acta Leprol, 1999, 11(4), 179 - 82
Effect of treatment on PCR positivity in multibacillary leprosy patients treated with conventional and newer drugs ofloxacin and minocycline; Singh HB et al.; In order to develop objective criteria to monitor trends of therapeutic responses positivity of PCR signals and ATP assay methods has been compared in multibacillary (MB) leprosy patients . Biopsies from lesions of 95 BL/LL patients before and after one year of treatment with a new drug regimen comprising of conventional and newer drugs ofloxacin and minocycline have been studied . These biopsies were processed for bacillary ATP assay and PCR positivity for a 36 kDa gene target by earlier published methods . In the untreated patients bacillary ATP levels were detectable in all specimens and ranged from 0.02 to more than 36 pg/millions organisms . After one year of treatment ATP levels were not detectable in any of the 57 biopsies specimens available for analysis . However, PCR signals were detectable in 3 out of 57 biopsies . In two specimens signals were very weak detectable only by hybridization . It may be concluded that DNA based PCR assay may be useful in monitoring the trends of therapeutic responses in MB patients under treatment.

J Basic Microbiol, 2000, 40(4), 223 - 32
Studies on the extracellular tannase from newly isolated Bacillus licheniformis KBR 6; Mondal KC et al.; A tannase producing bacterial strain KBR 6 has been isolated from lateritic soil and identified as Bacillus licheniformis . It is capable of producing tannase in the medium containing only tannic acid . The rapid degradation of tannic acid and production of extracellular tannase was observed in three different media containing tannic acid (M1), tannic acid + basal salt (M2) and tannic acid + basal salt + glucose (M3) . Maximum enzyme production and growth of the organism was obtained at 18-21 h and 30-36 h, respectively . The increased order of enzyme production in relation to different media is as per the following sequence, M3 > M2 > M1 . The maximum growth and enzyme production was observed at pH 5.0 . The pH and temperature optima of the enzyme activity were found to be at 5.75 and 60 degrees C respectively . Paper chromatographic analysis indicates that gallic acid is the enzymatic degradative product of tannic acid.

J Basic Microbiol, 2000, 40(4), 215 - 21
Effects of UV-light on Bacillus sphaericus and its protection by chemicals; Cokmus C et al.; Although when UV-irradiated seven most toxic strains of Bacillus sphaericus lost their viabilities between 2.5-4.5 min, their larvicidal activity was protected for longer periods . Benzaldehyde, cinnamaldehyde, salicylaldehyde, methylene blue and yeast extract showed good protective effect for spore viability and larvicidal activity from UV inactivation in B . sphaericus . This protective effect has also been confirmed by SDS-PAGE analyses whereby the 42 kDa and 51 kDa toxic proteins bands did not disappear following UV treatment.

Dis Aquat Organ, 2000 Aug 10, 42(1), 1 - 9
Isolation of a lethal rhabdovirus from the cultured Chinese sucker Myxocyprinus asiaticus; Zhang QY et al.; A rhabdovirus was found to be associated with a lethal hemorrhagic disease in the cultured Chinese sucker Myxocyprinus asiaticus Bleeker . The rhabdovirus was amplified and isolated from the infected GCO (grass carp ovary) cells . In ultrathin sections of liver cells from the diseased fish, the virus particles exhibited the characteristic bacilliform morphology, and budded through vesicle membranes of the infected cells . The isolated rhabdovirus particles were found to have a bacilliform morphology with 2 rounded ends rather than a typical flat base . The virus particles were measured and ranged in size from 150 to 200 nm in length and 50 to 60 nm in diameter . Most other characteristics, including their size, extensive virus infectivity to fish cell lines, strong cytopathogenic effects, stability at high temperatures, vesicle formation in infected cells, structure protein electrophoretic patterns and the presence of an RNA genome, very closely resembled those of other fish rhabdoviruses . At present it is not known if this is a novel virus species or if it is an isolate of a known fish rhabdovirus . Until a confirmed identification can be made, we will temporarily refer to this virus as Chinese sucker rhabdovirus (CSRV).

Vet J, 2000 Sep, 160(2), 92 - 106
Cattle-to-cattle transmission of bovine tuberculosis; Menzies FD et al.; In developed countries, Mycobacterium bovis infection in cattle is now mostly confined to the respiratory system, which reflects transmission and establishment of infection mainly by this route . A single bacillus transported within a droplet nucleus is probably sufficient to establish infection within the bovine lung . Infected cattle should always be considered as potential sources of infection, since studies have demonstrated that a significant proportion of tuberculous cattle excrete M . bovis.In general, the dynamics of M . bovis transmission are poorly understood and the conditions under which a tuberculous animal becomes an effective disseminator of infection are currently not defined although environmental contamination appears to be a less effective method of disease transmission . Field studies indicate a wide spectrum of transmission rates but generally the spread of M . bovis infection is still considered to be a relatively slow process . Slaughter of diseased cattle detected by tuberculin testing and at meat plant inspection has been shown to be an effective policy for tuberculosis eradication, provided there are no other reservoirs of infection and all involved in the cattle industry are committed to a policy of eradication . Epidemiological approaches, particularly case-control studies, seem to provide the best method for quantifying the relative importance of the various sources of M . bovis transmission to cattle and modelling techniques can be used to assist in the design of cost-effective control measures that may lead to tuberculosis eradication .

J Econ Entomol, 2000 Aug, 93(4), 1300 - 7
Feeding behavior of bollworm and tobacco budworm (Lepidoptera: Noctuidae) larvae in mixed stands of nontransgenic and transgenic cotton expressing an insecticidal protein; Halcomb JL et al.; Feeding behavior of third-instar bollworm, Helicoverpa zea (Boddie), and tobacco budworm, Heliothis virescens (F.), was observed in pure and mixed stands of nontransgenic and transgenic cotton (BTK), Gossypium hirsutum L., expressing an insecticidal protein CryIA(c) from a bacterium, Bacillus thuringiensis Berliner subsp . kurstaki . Five plant stands composed of BTK and non-BTK plants were evaluated; two pure stands and three mixed stands . Percentage ratios of BTK to non-BTK plants in the stands were 100:0, 75:25, 50:50, 25:75 and 0:100, respectively . In all stands with BTK plants, fewer bollworm and tobacco budworm larvae were found on BTK plants than non-BTK plants 24 h after infestation with third instars . At 48 h, significantly fewer tobacco budworm larvae, but not fewer bollworm larvae, were found on BTK plants . However, the number of larvae of either insect did not increase on non-BTK plants compared with the initial infestation density of three larvae per plant . The number of obacco budworm injured flower buds, and capsules was lower in all plant stands containing BTK plants compared with the pure stand of non-BTK at 48 h after infestation . Higher numbers of larvae on non-BTK plants were possibly the result of larval intoxication, reduced feeding, and increased plant abandonment and death on BTK plants rather than a classical feeding preference . Unexpectedly, the number of flower buds and capsules injured by bollworm and tobacco budworm when averaged per plant for all plants in a stand, differed little among the 75:25, 50:50 and 25:75 plant mixtures . These data suggest that larvae of both species frequently moved among plants, feeding indiscriminately on BTK and non-BTK plants.

J Econ Entomol, 2000 Aug, 93(4), 1293 - 9
Plant-toxin interactions in transgenic Bt cotton and their effect on mortality of Helicoverpa armigera (Lepidoptera: Noctuidae); Olsen KM et al.; In Australia, transgenic cotton plants expressing the cry1Ac gene from Bacillus thuringiensis Berliner variety kurstaki are less toxic to first-instar Helicoverpa armigera (Hubner) after the plant is producing fruit . We developed two bioassay methods (leaf mush, leaf disk) to test if the physiological state of the plants explained changes in toxicity and a third method (diet incorporation) was developed to quantify the toxicity of Bt leaves when mixed in chickpea diet . Cry1Ac protein was less toxic to H . armigera larvae when the protein was mixed with leaves from fruiting versus presquare conventional cotton . Differences in LC50 varied from 2.4- to 726-fold, depending on the source of toxin and conventional plant material . These results suggest that plant-toxin interactions in fruiting cotton are reducing the toxicity of the Cry1Ac protein . The possible role of tannins in these changes is discussed.

J Econ Entomol, 2000 Aug, 93(4), 1276 - 85
Late-instar European corn borer (Lepidoptera: Crambidae) tunneling and survival in transgenic corn hybrids; Walker KA et al.; Field studies were conducted in 1996 and 1997 to determine injury by and survival of late-instar European corn borer, Ostrinia nubilalis (Hubner), on genetically altered Bacillus thuringiensis Berliner corn, Zea mays L . Cry1Ab events 176, Bt11, MON810, and MON802; Cry1Ac event DBT418; and Cry9C event CBH351 were evaluated . Plants of each corn hybrid were manually infested with two third-, fourth-, or fifth-instar O . nubilalis . Larvae were held in proximity to the internode of the plant above the ear with a mesh sleeve . Larvae were put on the plants during corn developmental stages V8, V16, R1, R3, R4, R5, and R6 . This study shows that not all B . thuringiensis hybrids provide the same protection against O . nubilalis injury . Hybrids with B . thuringiensis events Bt11, MON810, MON802, and CHB351 effectively protected the corn against tunneling by late-instar O . nubilalis . Event 176 was effective in controlling late-instar O . nubilalis during V12 and V16 corn developmental stages; however, significant tunneling occurred by fourth instars during R3 and R5 . Event DBT418 was not effective in controlling late-instar O . nubilalis during corn vegetative or reproductive stages of development . Whether the B . thuringiensis hybrids satisfied high- and ultra-high-dose requirements is discussed.

J Econ Entomol, 2000 Aug, 93(4), 1265 - 8
Baseline susceptibility of the corn earworm (Lepidoptera: Noctuidae) to the Cry1Ab toxin from Bacillus thuringiensis; Siegfried BD et al.; Susceptibility to Cry1Ab toxin from Bacillus thuringiensis (Bt) was determined for 12 field populations of neonate corn earworm, Helicoverpa zea (Boddie), from the United States . Earworm larvae were exposed to artificial diet treated with increasing Bt concentrations, and mortality and growth inhibition were evaluated after 7 d . The range of variation in Bt susceptibility indicated by growth inhibition was very similar to that indicated by mortality . Although interpopulation variation in susceptibility to both proteins was observed, the magnitude of the differences was small (less than or equal to fivefold) . These results suggest that the observed susceptibility differences reflect natural variation in Bt susceptibility among corn earworm populations rather than variation caused by prior exposure to selection pressures . Therefore, corn earworms apparently are susceptible to Bt toxins across most of their geographic range.

J Econ Entomol, 2000 Aug, 93(4), 1096 - 104
Comparative insecticidal properties of two nucleopolyhedrovirus vectors encoding a similar toxin gene chimer; Treacy MF et al.; Laboratory, greenhouse and field studies were conducted to characterize the insecticidal properties of genetically altered forms of Autographa californica (Speyer) nucleopolyhedrovirus (AcNPV) and Helicoverpa zea (Boddie) NPV (HzNPV) against selected heliothine species . The altered viruses each contained a chimeric 0.8-kb fragment encoding the insect-specific, sodium channel neurotoxin from the Algerian scorpion Androctonus australis Hector (AaIT, hence recombinant viruses designated Ac-AaIT and Hz-AaIT) . Based on LD50 values, results from diet-overlay bioassays showed Ac-AaIT and Hz-AaIT to be equally virulent against larval tobacco budworm, Heliothis virescens (F.), but Hz-AaIT averaged 1,335-fold greater bioactivity than Ac-AaIT against larval cotton bollworm, Helicoverpa zea (Boddie) . Hz-AaIT killed larvae of both heliothine species at rates significantly faster than those imparted by HzNPV (viral LT50 values averaged 2.5 and 5.6 d, respectively) . In greenhouse studies, foliar sprays of Ac-AaIT and Hz-AaIT were equally effective in controlling H . virescens on cotton; however, Hz-AaIT provided control of H . zea on cotton at a level superior to that of Ac-AaIT . For example, after three weekly sessions of foliar application and H . zea artificial infestation, cotton treated with Ac-AaIT or Hz-AaIT at 10 x 10(11) occulsion bodies (OB)/ha averaged 2.5 and 16.2 nondamaged flower buds per plant, respectively . Another greenhouse study conducted against heliothine species on cotton showed that the quicker killing speed exhibited by Hz-AaIT led to improved plant protection versus HzNPV . Finally, results from three field trials demonstrated that Hz-AaIT at 5-12 x 10(11) OB/ha provided control of the heliothine complex in cotton at levels slightly better than Bacillus thuringiensis, equal to the macrolide, spinosad, and only slightly less than that of selected pyrethroid and carbamate insecticides . Overall, results from these studies indicate that, because of host range differences between the two wild-type viruses, HzNPV is the better vectoring agent (versus AcNPV) for designing recombinant clones as insecticides targeted at the multi-species heliothine complex . Further, these studies suggest that if appropriately tailored for the pest complex, recombinant NPVs may be very effective, insect-specific approaches to managing pests in many cropping scenarios . Possible Hz-AaIT deployment strategies for control of heliothine species on conventional and transgenic cotton varieties are discussed.

J Econ Entomol, 2000 Aug, 93(4), 1076 - 9
Lack of toxicity to adults of the Mexican fruit fly (Diptera: Tephritidae) of beta-exotoxin in Bacillus thuringiensis endotoxin preparations; Robacker DC et al.; beta-Exotoxin (thuringiensin) was found in high titers in centrifugation supernatants and acetone/lactose powders produced from centrifugation pellets of strains Guat 1 and HD 2 of Bacillus thuringiensis (Berliner) . Diets containing powders of either strain were toxic, diets containing Guat 1 supernatant were not toxic, diets containing HD 2 supernatant were slightly toxic, and diets containing powders or supernatants from uninoculated culturing medium spiked with beta-exotoxin were not toxic . Most mortality occurred within 3 d when flies fed on powders but not until 6-7 d when flies fed on HD 2 supernatant . These results indicated that the primary toxic principals of the powders were endotoxins/spores and that beta-exotoxin alone was not toxic to adult flies at the concentrations found in the supernatants or powders.

J Econ Entomol, 2000 Aug, 93(4), 1055 - 64
An in-field screen for early detection and monitoring of insect resistance to Bacillus thuringiensis in transgenic crops; Venette RC et al.; We present a field-based approach to detect and monitor insects with resistance to insecticidal toxins produced by transgenic plants . Our objective is to estimate the phenotypic frequency of resistance in a population by relating the densities of insects on genetically transformed plants to densities on nontransformed plants . We focus on European corn borer, Ostrinia nubilalis (Hubner), in sweet corn, Zea mays L., expressing Cry1Ab from Bacillus thuringiensis subsp . kurstaki Berliner to illustrate principles underlying the method . The probability of detecting one or more rare, resistant larvae depends on sample size, the density of larvae on nontransformed plants, and an assumed frequency of resistant phenotypes in a given population . Probability of detection increases with increases in sample size, background density, or the frequency of resistant individuals . Following binomial probability theory, if a frequency of 10(-4) is expected, 10(3)-10(4) samples must be collected from a B . thuringiensis (Bt) crop to have at least a 95% probability of locating one or more resistant larvae . In-field screens using transgenic crops have several advantages over traditional laboratory-based methods, including exposure to a large number of feral insects, discrimination of resistant individuals based on Bt dosages expressed in the field, incorporation of natural and Bt-induced mortality factors, simultaneous monitoring for more than one insect species, and ease of use . The approach is amenable to field survey crews working in research, extension, and within the seed corn industry . Estimates of the phenotypic frequency of resistance from the in-field screen can be useful for estimating initial frequency of resistant alleles . Bayesian statistical methods are outlined to estimate phenotype frequencies, allele frequencies, and associated confidence intervals from field data . Results of the approach are discussed relative to existing complementary methods currently available for O . nubilalis and corn earworm, Helicoverpa zea (Boddie).

Folia Microbiol (Praha), 1999, 44(4), 367 - 71
Characterization and some reaction-engineering aspects of thermostable extracellular beta-galactosidase from a new Bacillus species; Sani RK et al.; A new strain of Bacillus sp . was isolated from a hot water spring in India . This strain generated a high activity of extracellular beta-galactosidase at 37 degrees C in shake flasks . The beta-galactosidase activity was found to increase continuously but the production rate was slower than with some other organisms reported in the literature . There were noteworthy differences in the time-domain profiles of bacterial concentration and beta-galactosidase activity when the starting concentration of substrate (glucose) was tripled from 10 g/L . These differences may be explained in terms of the relative rates of enzyme synthesis and its diffusion across the cell wall . The enzyme produced by this organism is more stable than other beta-galactosidases; its half-life is 408 h at 50 degrees C and 94 h at 55 degrees C, while the reported enzymes showed perceptible loss of activity within 2 h.

Appl Biochem Biotechnol, 2000 Jun, 87(3), 219 - 32
Toxicity assessment of tamoxifen by means of a bacterial model; Luxo C et al.; A strain of Bacillus stearothermophilus was used as a model to study physical perturbations induced in the membrane by the cytostatic tamoxifen (TAM) . This study was carried out using two lines of criteria: (1) bacterial growth, and temperature growth range, with determination of growth parameters as a function of TAM concentration; and (2) biophysical studies by differential scanning calorimetry (DSC) and by means of two fluorescent probes to evaluate perturbations promoted by the drug on the structural order of bacterial lipid membranes . The inhibition of growth induced by TAM, the structural bilayer disordering, and the shift in the phase transition temperature to a lower range were also determined in the presence of Ca2+, i.e., a natural membrane stabilizer, to elucidate further perturbing effects of TAM on membranes with putative implications in cell toxicity . Growth inhibition promoted by TAM is potentiated by an increase in growth temperature above the optimal range, but attenuated or relieved by the addition of 2.5 mM Ca2+ to the culture medium . Consistently, fluorescence polarization and DSC studies showed that Ca2+ ions (2.5 mM) effectively compensated for the destabilizing effects promoted by TAM in bacterial lipid membranes.

Neurosurgery, 2000 Sep, 47(3), 773 - 7
Hydrocephalus in coccidioidal meningitis: case report and review of the literature; Romeo JH et al.; OBJECTIVE AND IMPORTANCE: Coccidioidomycosis was once confined to the southwest United States and northern Mexico . It has become a larger concern because of the concentration of military bases in these areas, the increasing mobility of populations, and the rising population of immunocompromised persons . Outside endemic areas, the diagnosis is rarely considered . Patients with coccidioidomycosis may develop occult basilar meningitis progressing to communicating hydrocephalus and death . CLINICAL PRESENTATION: A 60-year-old white man presented with a 1-month history of vertigo, falls, and vomiting . Computed tomography of the head revealed mild hydrocephalus . Lumbar puncture results were remarkable for 1065 mg/dl protein; acid-fast bacillus stain, Gram's stain, and culture results were negative . Postgadolinium magnetic resonance imaging demonstrated enhancement of basilar and cervical meninges, suggesting inflammation, and communicating hydrocephalus . For 48 hours, the patient's level of consciousness decreased progressively . INTERVENTION: A ventriculoperitoneal shunt was placed, and antifungal agents were initiated on an emergent basis . CONCLUSION: Coccidioidomycosis should be considered in the differential diagnosis of occult basilar meningitis . The diagnosis is established by the discovery of a high (>1:2) titer of complement-fixing antibody in the cerebrospinal fluid . Communicating hydrocephalus is a common complication of untreated coccidioidal meningitis, and it may develop during appropriate treatment (oral fluconazole, 200-400 mg/d, continued indefinitely) . Patients with hydrocephalus and evidence of increased intracranial pressure require a shunt.

FEMS Microbiol Lett, 2000 Sep 1, 190(1), 151 - 5
Common occurrence of enterotoxin genes and enterotoxicity in Bacillus thuringiensis; Gaviria Rivera AM et al.; Seventy-four strains of Bacillus thuringiensis thuringiensis representing 24 serovars were examined for the presence of three enterotoxin genes/operons; the non-haemolytic enterotoxin Nhe, the haemolytic enterotoxin hbl and the Bacillus cereus toxin bceT using polymerase chain reaction . The nheBC genes were found in all strains examined, the hblCD genes in 65 of the 74 strains and bceT in 63 strains . There was little consistency of the distribution of enterotoxin loci among strains of the same serovar in serovars that were well represented in our collection . Culture supernatants from all but one strain inhibited protein synthesis in Vero cells, generally with a toxicity equivalent to that seen in strains of B . cereus isolated from incidents of food poisoning . Microbiological Societies.

Harefuah, 2000 Jun 15, 138(12), 1034 - 6, 1086
{Familial parinaud oculo-glandular syndrome in cat-scratch disease}; Shoham N et al.; Cat-scratch disease is manifested by subacute, regional lymphadenitis and occurs mainly in children . The causative agent is a pleomorphic, gram-negative bacillus, Bartonella henselae carried by asymptomatic cats . Parinaud oculoglandular syndrome is the most common ocular manifestation of this disease . It is characterized by unilateral conjunctivitis with polypoid granuloma, usually of the palpebral conjunctiva, and preauricular lymphadenopathy . The diagnosis is supported by a history of exposure to cats and is confirmed by positive serologic tests or positive PCR assay . The occurrence of more than 1 case of Parinaud syndrome in a family is rare . We describe 2 sisters with Parinaud oculoglandular syndrome, proven by serologic tests . They reported that they used to cuddle with their cats, among them a kitten . Because of the refractory conjunctivitis and signs of imminent periorbital cellulitis, they were treated with oral tetracycline with apparently good responses . We recommend asking about contacts with cats in any atypical conjunctivitis accompanied by regional lymphadenopathy, especially in young patients . Systemic antibiotics should be given when there is any suspicion of significant ocular involvement, if the patient is immunosuppressed, or if there are systemic manifestations of cat-scratch disease.

J Nat Prod, 2000 Aug, 63(8), 1131 - 5
Biological characterization of fusapyrone and deoxyfusapyrone, two bioactive secondary metabolites of Fusarium semitectum; Altomare C et al.; Fusapyrone (1) and deoxyfusapyrone (2), two alpha-pyrones originally isolated from rice cultures of Fusarium semitectum, were tested in several biological assays . Compounds 1 and 2 showed considerable antifungal activity against several plant pathogenic and/or mycotoxigenic filamentous fungi, although they were inactive toward yeasts isolated from plants and the Gram-positive bacterium Bacillus megaterium in disk diffusion assays . Compound 1 was consistently more active than 2 . Among the tested fungi, Fusarium species were the least sensitive to the two pyrones, while Alternaria alternata, Ascochyta rabiei, Aspergillusflavus, Botrytis cinerea, Cladosporium cucumerinum, Phoma tracheiphila, and Penicillium verrucosum were the most sensitive . Compounds 1 and 2 also showed good inhibitory activity toward agents of human mycoses . Aspergilli were the most sensitive, while some species-specific variability was found among the Candida spp . In an Artemia salina larvae bioassay, 1 was not toxic at the highest concentration tested (500 microM), whereas the LC(50) of 2 was 37.1 microM (21.8 microg/mL) . Neither 1 nor 2 was phytotoxic in a panel of assays that monitored plant-cell toxicity, as well as wilt-, chlorosis-, and necrosis-inducing activity . Moreover, 2 stimulated the root elongation of tomato seedlings at doses of 10 and 100 microM . In consideration of the biological activities evidenced in this study, 1 and 2 appear to be potential candidates for biotechnological applications, as well as good models for studies on mechanism(s) of action and structure-activity relationships.

Curr Microbiol, 2000 Oct, 41(4), 276 - 83
Toxicity and receptor binding properties of Bacillus thuringiensis delta-endotoxins to the midgut brush border membrane vesicles of the rice leaf folders, Cnaphalocrocis medinalis and Marasmia patnalis; Karim S et al.; Pesticidal activity and receptor-binding properties of Bacillus thuringiensis toxins to rice leaf folders, Cnaphalocrocis medinalis and Marasmia patnalis, were investigated . Saturation and competition binding experiments were done with iodine ((125)1)-labeled Bt proteins and brush border membrane vesicles prepared from the midgut of C . medinalis and M . patnalis . The results show saturable, specific, and high-affinity binding of all toxins except Cry2A toxin . Cry1Aa and Cry2A toxins were bound with low affinity but with high binding site concentration . Heterologous competition experiments showed that Cry1Aa, Cry1Ab, and Cry1Ac recognized or shared the same binding site that is different from the binding site for Cry2A toxin . Iodine ((125)I)-labeled Cry1Ac and Cry1Ab toxins were used in ligand blot experiments to detect specific binding proteins in brush border membrane vesicles of C . medinalis and M . patnalis . Cry1Ab toxin protein binds to 205-kDa and 200-kDa proteins respectively in case of C . medinalis and M . patnalis . The apparent molecular mass of the protein bound to labeled Cry1Ac toxins was identified as a 120-kDa protein in both C . medinalis and M . patnalis.

Curr Microbiol, 2000 Oct, 41(4), 262 - 6
Correlation of the insecticidal activity of the Bacillus thuringiensis A4 strain against Bactrocera oleae (Diptera) with the 140-kDa crystal polypeptide; Sivropoulou A et al.; The crystals of the soil-isolated Bacillus thuringiensis (Bt) strain A4 consist of two polypeptides with molecular mass of 140 kDa and 32 kDa that exhibit insecticidal activity against adult flies of Bactrocera oleae (Diptera) . Plasmid curing applied to this strain resulted in the isolation of several subclones exhibiting alterations in their crystal polypeptides as well as two acrystalliferous subclones . The crystals of subclone 1.1 lacked the 32-kDa polypeptide and consisted uniquely of a 140-kDa polypeptide antigenically related to the parental 140-kDa crystal polypeptide . Additionally, the crystals of this subclone exhibited insecticidal activity against B . oleae equivalent to that of the parental strain . Therefore, the 32-kDa crystal polypeptide is dispensable for insecticidal activity, which appears to be dependent on the presence of the 140-kDa crystal polypeptide.

Curr Microbiol, 2000 Oct, 41(4), 250 - 6
Comparative study of the frequency, flagellar serotype, crystal shape, toxicity, and cry gene contents of Bacillus thuringiensisfrom three environments; Kim HS; A number of Bacillus thuringiensis isolates from sericultural farm, soil, and granary samples in Korea were found . B . thuringiensis isolates were predominant in granary (40%), followed by sericultural farm (33% and 25% in the spring and fall isolation), and soil (10%) . In toxicity tests for three areas, lepidopteran-active isolates were rich in the spring of sericultural farm and granary, but the fall isolation of sericultural farm displayed that a large number of B . thuringiensis isolates having dual specificity against both lepidopteran and dipteran larvae were found . The soil showed even distribution against lepidoptera and/or diptera . Most of B . thuringiensis isolates showed strong toxicity against tested insects . PCR analysis using cryI, cryII, cryIII, cryIV, and cryV gene-specific primers for determination of the cry gene contents of B . thuringiensis isolates indicated that the frequency of the cryIA, cryIC, cryID, and cryII among cry genes predominated, and the cryIB, cryIE, cryIF, cryIG, and cryIV were not popular . In contrast, no PCR products were detected for the cryIII and cryV templates . Several B . thuringiensis isolates produced unusual PCR products and complicated combinations consisting of multiple cry genes . Seven out of 11 B . thuringiensis isolates undetected by specific primers from sericultural farm, all out of 9 isolates from soil, and 19 out of 25 isolates from granary were toxic to lepidoptera and/or diptera . In addition, five nontoxic isolates of sericultural farm, all of five nontoxic isolates of soil, and 13 nontoxic isolates of granary produced the expected PCR products . PCR results showed varied distribution of cry genes for three areas, respectively . An evaluation of this novel activity demands that several criteria be measured: the frequency, flagellar serotype, crystal morphology, toxicity, and combination of the cry genes.

Curr Microbiol, 2000 Oct, 41(4), 246 - 9
Enzyme activity of lectins from the nitrogen-fixing soil bacterium Bacillus polymyxa; Mel'nikova UY et al.; Lectins LI and LII, localized on the surface of the nitrogen-fixing soil bacterium Bacillus polymyxa 1460, were shown to possess proteolytic activity . A relationship was found between the proteolytic and hemagglutinating activities of the lectins . Blocking of hemagglutinating activity with specific carbohydrate haptens led to significant changes in the enzyme activity of both lectins . When lectin activity was blocked with glucuronic acid and fructose-1, 6-diphosphate, the proteolytic activity of both LI and LII declined, whereas incubation with d-galactosamine and d-glucosamine promoted increases in the proteolytic activity of LII . This study proposes that the molecules of the B . polymyxa lectins may have two centers on their surfaces: one responsible for lectin activity and the other for proteolytic activity.

Bioorg Med Chem, 2000 Jul, 8(7), 1537 - 44
Site-selective glycosylation of subtilisin Bacillus lentus causes dramatic increases in esterase activity; Lloyd RC et al.; Using site directed mutagenesis combined with chemical modification, we have developed a general and versatile method for the glycosylation of proteins which is virtually unlimited in the scope of proteins and glycans that may be conjugated and in which the site of glycosylation and the nature of the introduced glycan can be carefully controlled . We have demonstrated the applicability of this method through the synthesis of a library of 48 glycosylated forms of the serine protease subtilisin Bacillus lentus (SBL) as single, pure species . As part of our ongoing program to tailor the activity of SBL for use in peptide synthesis, we have screened these enzymes for activity against the esterase substrate succinyl-Ala-Ala-Pro-Phe-S-benzyl . Gratifyingly, 22 enzymes displayed greater than wild type (WT) activity . Glycosylation at positions 62, in the S2 pocket, resulted in five glycosylated forms of SBL that were 1.3- to 1.9-fold more active than WT . At position 217, in the S1' pocket, all glycosylations increased kcat/KM up to a remarkable 8.4-fold greater than WT for the glucosylated enzyme L217C-S-beta-Glc(Ac)3 . Furthermore, the ratio of amidase to esterase activity, (kcat/KM)esterase/(kcat/KM)amidase (E/A), is increased relative to wild type for all 48 glycosylated forms of SBL . Again, the most dramatic changes are observed at positions 62 and 217 and L217C-S-beta-Glc(Ac)3 has an E/A that is 17.2-fold greater than WT . The tailored specificity and high activity of this glycoform can be rationalized by molecular modeling analysis, which suggests that the carbohydrate moiety occupies the S1' leaving group pocket and enhances the rate of deacylation of the acyl-enzyme intermediate . These glycosylated enzymes are ideal candidates for use as catalysts in peptide synthesis as they have greatly increased (kcat,KM)esterase and severely reduced (kcat/KM)amidase and will favor the formation of the amide bond over hydrolysis.

Bioorg Med Chem, 2000 Jul, 8(7), 1527 - 35
Controlled site-selective protein glycosylation for precise glycan structure-catalytic activity relationships; Davis BG et al.; Glycoproteins occur naturally as complex mixtures of differently glycosylated forms which are difficult to separate . To explore their individual properties, there is a need for homogeneous sources of carbohydrate-protein conjugates and this has recently prompted us to develop a novel method for the site-selective glycosylation of proteins . The potential of the method was illustrated by site-selective glycosylations of subtilisin Bacillus lentus (SBL) as a model protein . A representative library of mono- and disaccharide MTS reagents were synthesized from their parent carbohydrates and used to modify cysteine mutants of SBL at positions 62 in the S2 site, 156 and 166 in the S1 site and 217 in the S1' site . These were the first examples of preparations of homogeneous neoglycoproteins in which both the site of glycosylation and structure of the introduced glycan were predetermined . The scope of this versatile method was expanded further through the combined use of peracetylated MTS reagents and careful pH adjustment to introduce glycans containing different numbers of acetate groups . This method provides a highly controlled and versatile route that is virtually unlimited in the scope of the sites and glycans that may be conjugated, and opens up hitherto inaccessible opportunities for the systematic determination of the properties of glycosylated proteins . This potential has been clearly demonstrated by the determination of detailed glycan structure-hydrolytic activity relationships for SBL . The 48 glycosylated CMMs formed display kcat/KM values that range from 1.1-fold higher than WT to 7-fold lower than WT . The anomeric stereochemistry of the glycans introduced modulates changes in kcat/KM upon acetylation . At positions 62 and 217 acetylation enhances the activity of alpha-glycosylated CMMs but decreases that of beta-glycosylated . This trend is reversed at position 166 where, in contrast, acetylation enhances the kcat/KMs of beta-glycosylated CMMs but decreases those of alpha-glycosylated . Consistent with its surface exposed nature changes at position 156 are more modest, but still allow control of activity, particularly through glycosylation with disaccharide lactose.

J Natl Med Assoc, 2000 Apr, 92(4), 206 - 8
Fatal Bacillus cereus bacteremia in a patient with diabetes; Orrett FA; This report describes a fatal case of Bacillus cereus septicemia in a patient with uncontrolled diabetes and re-emphasizes the potential seriousness of Bacillus infections in patients with compromised immune function.

Acta Cient Venez, 1999, 50(4), 195 - 200
{Chemical disturbance of the phytoplankton structure in an artificial pond (Guaicaipuru, Edo . Miranda, Venezuela)}; Gonzalez F et al.; When an ecosystem is disturbed, it reverts to an early successional state, characterized by changes in community structure and function, that can be anticipated as response to stress . The purpose of this paper was to check the effect of a disturbance caused by application of chemical compound (Copper Sulfate, to 100 micrograms L-1 granular of 92%) on the phytoplankton community in a pond located in Guaicaipuro, Edo . Miranda (Venezuela), and compare the diversity and composition of the community before and after the chemical application . An additional purpose was, to test the Odum's hypothesis about the community response to chemical stress . The chemical compound was apply in different random points to assure homogeneous distribution in the water body . Only one application of the chemical was used 6 hours after the first sample was taken . Dissolved oxygen in surface and depth were measured . In order to estimate the diversity and composition of phytoplankton community, samples were taken in 5 random points of the pond, until a 2 liter sample was completed in an intermediary basis, during 30 days in the beginning of rainy season, between june and july of 1994 . Oxygen showed a maximum value at surface of 9.3 mg O2/L and minimal value at depth (2.3 mg O2/L) . Species richness varied between 4 and 18 . Phytoplankton community varied between 7.1 x 10(4) and 7.8 x 10(6) cel/L, and diversity index between 0.12 and 1.39 . Oxygen, species richness and phytoplankton minimal values were noticed at the fifth day after chemical application . Before chemical application, phytoplankton community was dominated by filamentous microalgae, especially Chlorophyta . After the fifth day, this group decreased in abundance and species number . On the seventh day small-size Cyanobacteria became dominant, particularly Chroococcoid forms . The community reverted to an early stage of ecological succession . Since the ninth day, community showed the following abundance sequence: Chrysophyta, Pyrrophyta, Bacillariophyta, and Euglenophyta, with an increased species richness . At the end of the study period, filamentous Chlorophyta species appeared . These results support Odum's hypothesis.

Microbiology, 2000 Sep, 146 ( Pt 9), 2175 - 83
ISBst12, a novel type of insertion-sequence element causing loss of S-layer-gene expression in Bacillus stearothermophilus ATCC 12980; Egelseer EM et al.; The cell surface of the surface layer (S-layer)-carrying strain of Bacillus stearothermophilus ATCC 12980 is completely covered with an oblique lattice composed of the S-layer protein SbsC . In the S-layer-deficient strain, theS-layer gene sbsC was still present but was interrupted by a novel type of insertion sequence (IS) element designated ISBst12 . The insertion site was found to be located within the coding region of the sbsC gene, 199 bp downstream from the translation start of SbsC . ISBst12 is 1612 bp long, bounded by 16 bp imperfect inverted repeats and flanked by a directly repeated 8 bp target sequence . ISBst12 contains an ORF of 1446 bp and is predicted to encode a putative transposase of 482 aa with a calculated theoretical molecular mass of 55562 Da and an isoelectric point of 9.13 . The putative transposase does not exhibit a typical DDE motif but displays aHis-Arg-Tyr triad characteristic of the active site of integrases from the bacteriophage lambda Int family . Furthermore, two overlapping leucine-zipper motifs were identified at the N-terminal part of the putative transposase . As revealed by Southern blotting, ISBst12 was present in multiple copies in the S-layer-deficient strain as well as in the S-layer-carrying strain . Northern blotting indicated that S-layer gene expression is already inhibited at the transcriptional level, since no sbsC-specific transcript could be identified in the S-layer-deficient strain . By using PCR, ISBst12 was also detected in B . stearothermophilus PV72/p6, in its oxygen-induced strain variant PV72/p2 and in the S-layer-deficient strain PV72/T5.

Microbiology, 2000 Sep, 146 ( Pt 9), 2161 - 73
Study of the bldG locus suggests that an anti-anti-sigma factor and an anti-sigma factor may be involved in Streptomyces coelicolor antibiotic production and sporulation; Bignell DR et al.; A cloned 2.5 kb DNA fragment that can restore antibiotic production and sporulation to a bldG mutant encodes a 113 aa protein showing similarity to a family of anti-anti-sigma factors from BACILLUS: and STAPHYLOCOCCUS:; and the deduced product of a closely spaced downstream ORF, designated ORF3, shows similarity to cognate anti-sigma factors . The homologues in BACILLUS: regulate the activity of sporulation- and stress-response-specific sigma factors . However, there is no sigma factor gene near bldG and ORF3 . bldG is transcribed both as a monocistronic and a polycistronic mRNA, the latter including the downstream ORF3 gene . The two transcripts were present at all time points during growth and both were upregulated when aerial mycelium and pigmented antibiotics were seen . At all time points, the monocistronic bldG transcript was two- to threefold more abundant than the polycistronic transcript . Mapping of the mRNA 5' ends indicated that bldG transcription is initiated from two transcription start sites located 82 and 123 bp upstream of the bldG translation start . A constructed bldG null mutant had the same phenotype as previously isolated bldG point mutations, some of which were shown to have potentially significant base changes within bldG . When compared to the wild-type strain, the null mutant showed no differences in the levels of transcription from the two bldG promoters . These results suggest that bldG is not involved in autoregulation.

Clin Diagn Lab Immunol, 2000 Sep, 7(5), 794 - 802
Subtilisin increases macromolecular efflux from the oral mucosa; Rubinstein I; The purpose of this study was to determine whether subtilisin, a potent serine proteinase derived from Bacillus species contaminating smokeless tobacco, increases macromolecular efflux from the oral mucosa and, if so, whether local elaboration of bradykinin mediates this response . Using intravital microscopy, I found that suffusion of subtilisin elicits significant, concentration-dependent leaky site formation and an increase in the clearance of fluorescein isothiocyanate-labeled dextran (molecular mass, 70 kDa) from the in situ hamster cheek pouch (P<0.05) . Heat-inactivated subtilisin had no significant effects on macromolecular efflux . Subtilisin-induced responses were significantly attenuated by Hoe 140 and NPC 17647, two structurally distinct selective bradykinin B(2) receptor antagonists, but not by des-Arg(9)-{Leu(8)}bradykinin, a selective bradykinin B(1) receptor antagonist, or CP-96,345, a selective neurokinin-1 receptor antagonist . Aprotinin, but not leupeptin, significantly attenuated subtilisin-induced increase in macromolecular efflux . Indomethacin had no significant effects on subtilisin-induced responses . Collectively, these data indicate that subtilisin increases the macromolecular efflux from the in situ hamster cheek pouch in a catalytic-site-dependent fashion through local elaboration of bradykinin . This response does not involve the stimulation of local afferent nerves or the production of prostaglandins.

Biotechnol Bioeng, 2000 Oct 20, 70(2), 208 - 16
Enzyme-catalyzed synthesis of sugar-containing monomers and linear polymers; Park OJ et al.; Commercially available proteases and lipases were screened for their ability to acylate regioselectively sucrose and trehalose with divinyladipic acid ester . Opticlean M375 (subtilisin from Bacillus licheniformis) was observed to form sucrose 1'-O-adipate and trehalose 6-O-adipate in anhydrous pyridine . Novozym-435 (lipase B from Candida antarctica) catalyzed the synthesis of sucrose 6, 6'-O-divinyladipate and trehalose 6, 6'-O-divinyladipate in acetone . These diesters were then employed as monomers in polycondensation reactions with various diols (aliphatic and aromatic) catalyzed by Novozym-435 in organic solvents to yield linear polyesters with M(w)'s up to 22,000 Da . Spectroscopic analysis confirmed that only the vinyl end groups of sugar esters reacted in the enzymatic polymerization with the diol, and not the internal sugar-adipate linkages . The two-step enzymatic strategy to yield sugar-based polyesters, which is the first report of its kind, results in higher molecular weights and faster reaction times than one-step enzymatic polyester synthesis .

Scand J Immunol, 2000 Sep, 52(3), 285 - 91
Dendritic cells induce CD4+ and CD8+ T-cell responses to Mycobacterium bovis and M . avium antigens in Bacille Calmette Guérin vaccinated and nonvaccinated cattle; Hope JC et al.; Few data are available regarding the induction of memory T-lymphocyte responses in cattle following Bacille Calmette Guerin (BCG) vaccination . Studies of the immune response induced by BCG vaccination provide an insight into the basis of antimycobacterial immunity that could be exploited for the development of more effective vaccination strategies . We used autologous dendritic cells (DC) infected with Mycobacterium bovis Bacille Calmette Guerin (BCG) or pulsed with purified protein derivative from M . bovis (PPD-B) or M . avium (PPD-A) to assess responses of CD4+, CD8+ and WC1+ gammadelta TCR+ lymphocytes from BCG vaccinated and nonvaccinated cattle . Mycobacteria-specific CD4+ and CD8+, but not WC1+ gammadelta TCR+, memory T lymphocytes were demonstrated in BCG-vaccinated cattle . CD4+ and CD8+ lymphocytes proliferated and produced interferon (IFN)-gamma in response to BCG-infected or PPD-B-pulsed DC . Proliferative responses were greater for CD4+ than CD8+ lymphocytes, although secretion of IFN-gamma was higher from the CD8+ T cells . Responses to PPD-A-pulsed DC were lower, with no CD8+ response . Lymphocytes from nonvaccinated calves were also stimulated to proliferate by BCG-infected DC, although the magnitude of proliferation was lower . The findings suggest that immunity to M . bovis induced by BCG vaccination in cattle may involve CD8+ memory T cells which produce IFN-gamma, as well as CD4+ memory T cells.

Lett Appl Microbiol, 2000 Sep, 31(3), 187 - 92
Selective amplification of bacterial RNA: use of a DNA primer containing mismatched bases near its 3' terminus to reduce false-positive signals; Koo K et al.; A reverse transcription PCR (RT-PCR) method designed to reduce false-positive results due to the co-amplification of contaminating genomic DNA is reported . Feasibility of the method was evaluated using 16S rRNA sequences specific to Bacillus cereus . A DNA oligonucleotide primer, consisting of 22-bases containing three consecutive mismatched bases near its 3' terminus (primer B16RT), was used for reverse transcription and in subsequent cDNA amplification . Specific rRNA was reverse transcribed at low temperature (40 degrees C or 45 degrees C) in the presence of primer B16RT . As subsequent PCR using primer B16RT at high (62 degrees C) annealing temperatures is not nearly as efficient as amplification using the specific primer, amplification of genomic DNA was hindered relative to the amplification of cDNA . The method was readily adapted to the selective amplification of mRNA of the Listeria monocytogenes listeriolysin O (hly) gene.

Lett Appl Microbiol, 2000 Aug, 31(2), 123 - 8
Improved shuttle vector for expression of chitinase gene in Bacillus thuringiensis; Lertcanawanichakul M et al.; A 6.96-kbp plasmid vector pBCX was constructed from the plasmid pBC16 (4.4 kbp) and a 2.56-kbp fragment of pBluescript II KS . The bifunctional plasmid pBCX conferred ampicillin and tetracycline resistance in Escherichia coli but only tetracycline resistance in Bacillus thuringiensis . It has unique sites for BamHI, SmaI, PstI, HindIII, SalI, XhoI, DraII, ApaI and KpnI derived from pBluescript II KS and was lost at a low rate in B . thuringiensis subsp . israelensis when cultured in Luria-Bertani broth without antibiotic . The chitinase gene from B . circulans number 4.1 (pCHIB1) was subcloned into the HindIII sites of this vector and designated as pBX43 (9.56 kbp) . This plasmid produced three times as much chitinase in B . thuringiensis subsp . israelensis strain c4Q272 as pHYB43, which comprises the commercial shuttle vector pHY300PLK plus the chitinase gene.

Lett Appl Microbiol, 2000 Aug, 31(2), 110 - 4
Pulsed electric field inactivation of diarrhoeagenic Bacillus cereus through irreversible electroporation; Rowan NJ et al.; The physical effects of high-intensity pulsed electric fields (PEF) on the inactivation of diarrhoeagenic Bacillus cereus cells suspended in 0.1% peptone water were examined by transmission electron microscopy (TEM) . The levels of PEF-induced microbial cell death were determined by enumeration on tryptone soy yeast extract agar and Bacillus cereus-selective agar plates . Following exposure to lethal levels of PEF, TEM investigation revealed irreversible cell membrane rupture at a number of locations, with the apparent leakage of intracellular contents . This study provides a clearer understanding of the mechanism of PEF-induced cellular damage, information that is essential for the further optimization of this emerging food-processing technology.

Extremophiles, 2000 Aug, 4(4), 209 - 14
Characterization and comparative study of the rrn operons of alkaliphilic Bacillus halodurans C-125; Nakasone K et al.; The ribosomal RNA operons (rrn) of alkaliphilic Bacillus halodurans C-125 were characterized and compared with those of B . subtilis . We isolated clones containing rrn operons from a lambda phage library of the C-125 chromosome, and the complete nucleotide sequence of each was determined . Eight rrn operons were identified by PFGE analysis of the C-125 chromosome digested with I-CeuI . The transcriptional orientation of the rrn operons mapped on the chromosome by Southern hybridization analysis was the same as the direction of replication of the chromosome . These operons were designated as rrnA-H, starting from the oriC locus in clockwise rotation . Sequence and structural analyses of these operons suggested that six of the rrn operons in the C-125 chromosome, rrnA, rrnB, rrnC-rrnD, rrnE, and rrnH, correspond to rrnO, rrnA, rrnJ-rrnW, rrnI, and rrnD in B . subtilis, whereas the other rrn operons (rrnF and rrnG) were specifically observed in C-125 . The rrn loci were positioned from 0 degrees to 90 degrees on the physical map, with the oriC locus assigned the position zero degrees . Two ORFs annotated as tnpA and ykfC, whose gene products are likely to act as transposases, were found downstream of these six operons . Comparative analysis of the 16S-23S and 23S-5S ITS (internally transcribed sequence) regions of B . halodurans C-125 and those of B . subtilis revealed that the ITS regions in C-125 were much longer than those in B . subtilis . There was no substantial difference in the length of potential promoter sequences in B . halodurans and B . subtilis.

Extremophiles, 2000 Aug, 4(4), 201 - 8
Bacillus sp . WW3-SN6, a novel facultatively alkaliphilic bacterium isolated from the washwaters of edible olives; Ntougias S et al.; A novel Gram-positive facultatively alkaliphilic, sporulating, rod-shaped bacterium, designated as WW3-SN6, has been isolated from the alkaline washwaters derived from the preparation of edible olives . The bacterium is nonmotile, and flagella are not observed . It is oxidase positive and catalase negative . The facultative alkaliphile grows from pH 7.0 to 10.5, with a broad optimum from pH 8.0 to 9.0 . It could grow in up to 15% (w/v) NaCl, and over the temperature range from 4 degrees to 37 degrees C, with an optimum between 27 degrees and 32 degrees C: therefore, it is both halotolerant and psychrotolerant . The bacterium is sensitive to a range of beta-lactam, sulfonamide, and aminoglycoside antibiotics, but resistant to trimethoprim . The range of amino acids, sugars, and polyols utilized as growth substrates indicates that this alkaliphile is a heterotrophic bacterium . D(+)-glucose, D(+)-glucose-6-phosphate, D(+)-cellobiose, starch, or sucrose are the substrates best utilized . The major membrane lipids are phosphatidylglycerol and diphosphatidylglycerol, with smaller amounts of phosphatidylethanolamine and an unknown phospholipid . During growth at high pH, the proportion of phosphatidylglycerol is increased relative to phosphatidylethanolamine . The fatty acyl components in the membrane phospholipids are mainly branched chain, with 13-methyl tetradecanoic and 12-methyl tetradecanoic acids as the predominant components . The G + C content of the genomic DNA is 41.1 +/- 1.0 mol% . The results of 16S ribosomal RNA sequence analysis place this alkaliphilic bacterium in a cluster, together with an unnamed alkaliphilic Bacillus species (98.2% similarity).

Med J Malaysia, 1998 Dec, 53(4), 354 - 7
Exposure to sputum positive cases of tuberculosis in a government hospital; Jeyakumar D; A retrospective study was carried out to ascertain the degree of exposure to the tubercle bacillus within Ipoh Hospital . This study reveals that, over a one year period, 92 sputum positive cases were admitted to the general wards . In 11 of these cases, drug resistance was considered to be possible . The mean time from admission to the commencement of treatment was seven days for the newly diagnosed cases . This study thus documents a significant degree of in-hospital exposure to the tubercle bacillus.

J Appl Microbiol, 2000 Aug, 89(2), 361 - 9
Characterization and mechanism of action of cerein 7, a bacteriocin produced by Bacillus cereus Bc7; Oscariz JC et al.; Cerein 7 is a peptidic antibiotic produced by Bacillus cereus Bc7 (CECT 5148) at the end of exponential growth but before sporulation onset . Cerein 7 has a broad spectrum of antibacterial activity against Gram-positive bacteria, but it is inactive against Gram-negative bacteria . The sequence of its amino-terminal end and its characteristics of hydrophobicity and molecular mass make cerein 7 unique among the bacteriocins produced by the soil bacterium B . cereus . In this paper a further characterization of cerein 7 is presented, it is shown that it can be classified as a Klaenhammer's class II bacteriocin and that its mode of action corresponds to that of a membrane-active compound.

J Appl Microbiol, 2000 Aug, 89(2), 309 - 16
Molecular and insecticidal characterization of a Bacillus thuringiensis strain isolated during a natural epizootic; Porcar M et al.; A new Bacillus thuringiensis strain belonging to the serovar aizawai was isolated from a dead larva of the lepidopteran Mythimna loreyi collected in a corn crop in Spain during a natural epizootic . This strain, which was named Leapi01, was compared with the kurstaki and aizawai strains isolated from Dipel(R) and Xentari(R), by electron microscopy, SDS-PAGE, plasmid pattern, PCR and insecticidal activity . This strain showed similar morphological and biochemical characteristics to the standard strains . The content in cry genes of Leapi01 was analysed with a set of general and specific primers recognizing most of the cry genes reported to date . DNA amplification was obtained with primers corresponding to six genes and, to clearly determine the identity of the genes, the amplified fragments were sequenced and corresponded to cry1Aa, cry1Ab, cry1Ca, cry1Da, cry2Ab and cry1Ia . However, the proteins encoded by two of these genes, Cry2 and Cry1I, were not detected in the SDS-PAGE of the purified parasporal bodies . The insecticidal activity of Leapi01 was determined by bioassays against two Lepidoptera species, Helicoverpa armigera and Spodoptera littoralis, that were found to be very susceptible to Leapi01 purified crystals . Since two of the cry genes identified in Leapi01 appear to be silent, other factors may be involved in the toxicity of the strain . As a result of this study, the potential of Leapi01 as biological control agent is discussed, with special emphasis on the high toxicity and relatively broad spectrum activity compared with two B . thuringiensis strains that are the active ingredients of commercial preparations commonly used as bioinsecticides.

Tuber Lung Dis, 2000, 80(3), 141 - 59
Characterization of a two-component system, devR-devS, of Mycobacterium tuberculosis; Dasgupta N et al.; By subtractive hybridization, we isolated genes, differentially expressed in virulent strain (dev), that are expressed at higher levels in the virulent Mycobacterium tuberculosis H37Rv strain in comparison to its avirulent counterpart, H37Ra, and consequently may be associated with the virulence phenotype of M . tuberculosis . A two-component system, devR-devS, was identified by DNA sequencing of a dev clone . DevR, the predicted gene product of devR, is a response regulator (RR) in the NarL/ UhpA subfamily of two-component systems . The devS gene product displayed homology with histidine protein kinases (HPKs) including UhpB, NarX and NarQ . The devR-devS locus is preceded by gene Rv3134c that encodes a putative alanine-aline- rich protein . This locus was conserved in M . tuberculosis and M . bovis BCG but not in other mycobacteria . A devR -lacZ transcription fusion demonstrated beta-galactosidase activity in M . smegmatis and in M . tuberculosis . The devR and devS genes were cotranscribed and the levels of their transcripts were lower in two isolates of the avirulent H37Ra strain in comparison to the virulent H37Rv strain of M . tuberculosis . The level of DevR protein was also lower in one of the H37Ra strains in comparison to the H37Rv strain . However, in a third isolate of H37Ra, RNA and protein expression was equivalent to that in the H37Rv strain . Electron microscopic immunogold analysis of M . tuberculosis grown in laboratory medium and within human monocytes revealed specific labelling for DevR protein within the bacteria and the phagosomal lumen of infected monocytes . These findings collectively suggest a potential role for devR-devS in the regulation of genetic programmes unique to the tubercle bacillus .

J Pept Sci, 2000 Aug, 6(8), 366 - 71
Enzymatic formation of Glu-Xaa and Asp-Xaa bonds using Glu/Asp-specific endopeptidase from Bacillus licheniformis in frozen aqueous systems; Haensler M et al.; The capability of Glu/Asp-specific endopeptidase from Bacillus licheniformis to form Glu/Asp-Xaa bonds in frozen aqueous systems was investigated . Under frozen state conditions, the enzyme was able to catalyse peptide bond formation more effectively than in liquid reaction mixtures . The acceptance of amino components which were completely inefficient nucleophiles at room temperature indicates a changed specificity of Glu/Asp-specific endopeptidase under frozen state conditions . Protease-catalysed coupling of two acidic amino acids was demonstrated for the first time . The utilization of Glu/Asp-specific endopeptidase from Bacillus licheniformis in frozen aqueous systems offers new possibilities in enzyme-catalysed peptide synthesis.

Diabetes, 2000 Sep, 49(9), 1459 - 67
Prevention of encephalomyocarditis virus-induced diabetes by live recombinant Mycobacterium bovis bacillus Calmette-Guérin in susceptible mice; Choi BK et al.; The D variant of encephalomyocarditis (EMC-D) virus causes diabetes in susceptible mice by direct cytolysis of pancreatic beta-cells . cDNA covering the major outer capsid protein (VP1) of the EMC-D virus was cloned into Mycobacterium bovis bacillus Calmette-Guerin (BCG) . None of the SJL/J mice immunized with live recombinant BCG-VP1 (rBCG-VP1) became diabetic when challenged with the highly diabetogenic EMC-D virus, but the control mice inoculated with normal BCG developed diabetes during the same challenge . VP1-specific antibodies (including neutralizing antibodies) were markedly increased over time and reached the maximum titer at week 10 after a single immunization . The plateau of the titer lasted longer than 4 weeks . Mice and guinea pigs immunized with live rBCG-VP1 showed strong delayed-type hypersensitivity to the VP1 of the EMC-D virus . The preventive immunity still worked effectively 10 months after the primary immunization . At that time, the VP1-specific antibody was almost undetectable in the bloodstream, but a large number of VP1-specific lymphocytes was found in the spleen of the immunized mice . Our results show that live rBCG-VP1 elicits effective humoral and long-lasting cellular immune responses against EMC-D virus infection that results in the prevention of virus-induced diabetes in susceptible mice.

Microb Pathog, 2000 Sep, 29(3), 175 - 85
Aerosol infection of mice with recombinant BCG secreting murine IFN-gamma partially reconstitutes local protective immunity; Moreira AL et al.; To better understand the contribution of interferon-gamma (IFN-gamma) to the immune response during the first 60 days of mycobacterial infection in the lungs, IFN-gamma gene disrupted (IFN-gamma-/-) mice were infected via aerosol with recombinant Mycobacterium bovis Bacillus Calmette-Guerin (BCG) secreting murine IFN-gamma (BCG-IFN-gamma) and compared to mice infected with recombinant BCG containing the vector only (BCG-vector) . When IFN-gamma-/- mice were infected with BCG-vector, increasing bacillary loads and large undifferentiated granulomas that did not express inducible nitric oxide synthase (iNOS) were observed in the lungs . In contrast, infection with BCG-IFN-gamma resulted in reduced bacillary load and better differentiated granulomas containing epithelioid macrophages expressing iNOS as well as reduced levels of interleukin 10 (IL-10) mRNA . However, local production of IFN-gamma by the recombinant BCG did not protect IFN-gamma-/- mice from subsequent challenge with M . tuberculosis . Infection of IFN-gamma-/- peritoneal macrophages in vitro with BCG-IFN-gamma led to induction of iNOS expression and lower IL-10 mRNA levels . Nevertheless, the growth of the intracellular BCG was unaffected . Since IFN-gamma induced-iNOS protein and reduced IL-10 production were insufficient to control mycobacterial growth in vitro, the results suggest that additional mediator(s) present in vivo are required for control of mycobacterial growth .

Microb Pathog, 2000 Sep, 29(3), 165 - 74
Interaction of Bartonella bacilliformis with human erythrocyte membrane proteins; Buckles EL et al.; Intracellular invasion is an important aspect of Carrion's disease caused by Bartonella bacilliformis . Both the hematic and tissue phases of the disease involve the initial attachment of the organism to erythrocytes and endothelial cells, respectively . Using two different approaches, preliminary evidence is provided that B . bacilliformis interacts with multiple surface-exposed proteins on human erythrocytes . Utilizing Western blot analysis, it was demonstrated that the organism binds several biotinylated erythrocyte proteins with approximate molecular masses of 230, 210, 100, 83 and 44 kDa . There was enhanced Bartonella binding to the 44 kDa protein and binding to a 25 kDa protein following exposure of intact red cells to trypsin . Moreover, there was a complete abrogation of binding to these proteins following exposure of erythrocytes to sodium metaperiodate oxidation, indicating the significance of carbohydrate moieties in the interactions of Bartonella with the erythrocyte . In a second approach, similar binding proteins or putative receptors were identified when Bartonella was co-incubated with isolated membrane proteins from red cell ghosts . A comparison of the molecular weights of these putative receptors with known erythrocyte proteins and their immunoreactivity to specific antisera suggested that the 230 and 210 kDa proteins are the alpha and beta subunits of spectrin; the 100 and 83 kDa proteins are band 3 protein and glycophorin A, respectively; and the 44 and 25 kDa proteins are the respective dimeric and monomeric forms of glycophorin B . Consistent with this notion was the binding of Bartonella to purified preparations of alpha and beta spectrin and glycophorin A/B .

Appl Microbiol Biotechnol, 2000 Aug, 54(2), 195 - 200
Coexpression of the Bacillus pumilus beta-xylosidase (xynB) gene with the Trichoderma reesei beta xylanase 2 (xyn2) gene in the yeast Saccharomyces cerevisiae; La Grange DC et al.; The xynB gene encoding the Bacillus pumilus beta-xylosidase was expressed separately and jointly with the Trichoderma reesei beta-xylanase (xyn2) gene in the yeast Saccharomyces cerevisiae . Both genes were placed under the transcriptional control of the glucose-derepressible alcohol dehydrogenase 2 promoter (ADH2p) and terminator (ADH2T) sequences . The xynB gene was fused in frame to the yeast mating factor alpha1 secretion sequence (MFalpha1s) to effect secretion in S . cerevisiae . The fusion protein was designated Xlo1 . Xlo1 produced in S . cerevisiae exhibited low affinity for xylobiose, but eventually hydrolyzed xylobiose and xylotriose to the monomeric constituent, D-xylose . Coproduction of Xyn2 and Xlo1 by S . cerevisiae led to a 25% increase in the amount of reducing sugars released from birchwood xylan compared to S . cerevisiae producing only the Xyn2 beta-xylanase . However, no D-xylose was produced from birchwood xylan, presumably due to very low Xlo1 beta-xylosidase activity and its low affinity for xylobiose.

FEMS Immunol Med Microbiol, 2000 Sep, 29(1), 27 - 33
Calprotectin (MRP8/14 protein complex) release during mycobacterial infection in vitro and in vivo; Pechkovsky DV et al.; The calprotectin (MRP8/14) protein complex belongs to the S100 family of Ca2+ binding proteins and is expressed during myelomonocytic differentiation . MRP8/14 plasma levels were determined by ELISA in 35 patients with active pulmonary tuberculosis (TB) showing mild (n = 12), moderate (n = 11) or severe (n = 12) disease, 13 patients with active pulmonary sarcoidosis (SR) and 21 healthy controls . TB patients had significantly increased plasma levels of MRP8/14 in comparison with SR and controls, which significantly depended on the volume of lung tissue involved in the inflammatory process . In TB patients, there was no correlation between plasma levels of MRP8/14 and total white blood cell (WBC) count, and blood polymorphonuclear neutrophil (PMN) count . In SR patients, MRP8/14 plasma levels were twofold higher in comparison with controls, but were lower compared with mild TB, and correlated with PMN and WBC counts . Human monocytes infected and cultured for 7 days with Mycobacterium bovis bacillus Calmette-Guerin showed fivefold higher MRP8/14 levels in supernatants compared with unstimulated or purified protein derivative-stimulated cells . Human MRP8/14 significantly increased Mycobacterium tuberculosis H37Rv growth in liquid medium in a dose- and time-dependent manner . These findings suggest that MRP8/14 plays an important role in the immunopathogenesis of tuberculosis.

J Mol Biol, 2000 Aug 25, 301(4), 1041 - 57
Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase; Declerck N et al.; Bacillus licheniformis alpha-amylase (BLA) is a starch-degrading enzyme that is highly thermostable although it is produced by a rather mesophilic organism . Over the last decade, the origin of BLA thermal properties has been extensively investigated in both academic and industrial laboratories, yet it is poorly understood . Here, we have used structure-based mutagenesis in order to probe the role of amino acid residues previously proposed as being important for BLA thermostability . Residues involved in salt-bridges, calcium binding or potential deamidation processes have been selected and replaced with various amino acids using a site-directed mutagenesis method, based on informational suppression . A total of 175 amylase variants were created and analysed in vitro . Active amylase variants were tested for thermostability by measuring residual activities after incubation at high temperature . Out of the 15 target residues, seven (Asp121, Asn126, Asp164, Asn192, Asp200, Asp204 and Ala269) were found to be particularly intolerant to any amino acid substitutions, some of which lead to very unstable mutant enzymes . By contrast, three asparagine residues (Asn172, Asn188 and Asn190) could be replaced with amino acid residues that significantly increase the thermostability compared to the wild-type enzyme . The highest stabilization event resulted from the substitution of phenylalanine in place of asparagine at position 190, leading to a sixfold increase of the enzyme's half-life at 80 degrees C (pH 5.6, 0.1 mM CaCl(2)).These results, combined with those of previous mutational analyses, show that the structural determinants contributing to the overall thermostability of BLA concentrate in domain B and at its interface with the central A domain . This region contains a triadic Ca-Na-Ca metal-binding site that appears extremely sensitive to any modification that may alter or reinforce the network of electrostatic interactions entrapping the metal ions . In particular, a loop spanning from residue 178 to 199, which undergoes pronounced conformational changes upon removal of calcium, appears to be the key feature for maintaining the enzyme structural integrity . Outside this region, most salt-bridges that were destroyed by mutations were found to be dispensable, except for an Asp121-Arg127 salt-bridge that contributes to the enhanced thermostability of BLA compared to other homologous bacterial alpha-amylases . Finally, our studies demonstrate that the natural resistance of BLA against high temperature is not optimized and can be enhanced further through various means, including the removal of possibly deamidating residues .

Appl Environ Microbiol, 2000 Sep, 66(9), 3998 - 4003
Response of a soil bacterial community to grassland succession as monitored by 16S rRNA levels of the predominant ribotypes; Felske A et al.; The composition of predominant soil bacteria during grassland succession was investigated in the Dutch Drentse A area . Five meadows, taken out of agricultural production at different time points, and one currently fertilized plot represented different stages of grassland succession . Since fertilization and agricultural production were stopped, the six plots showed a constant decline in the levels of nutrients and vegetation changes . The activity of the predominant bacteria was monitored by direct ribosome isolation from soil and temperature gradient gel electrophoresis of reverse transcription (RT)-PCR products generated from bacterial 16S rRNA . The amounts of 16S rRNA of 20 predominant ribosome types per gram of soil were monitored via multiple competitive RT-PCR in six plots at different succession stages . These ribosome types mainly represented Bacillus and members of the Acidobacterium cluster and the alpha subclass of the class Proteobacteria . The 20 16S rRNA molecules monitored represented approximately half of all bacterial soil rRNA which was estimated by dot blot hybridizations of soil rRNA with the Bacteria probe EUB338 . The grasslands showed highly reproducible and specific shifts of bacterial ribosome type composition . The total bacterial ribosome level increased during the first years after agricultural production and fertilization stopped . This correlated with the collapse of the dominant Lolium perenne population and an increased rate of mineralization of organic matter . The results indicate that there is a true correlation between the total activity of the bacterial community in soil and the amount of bacterial ribosomes.

Appl Environ Microbiol, 2000 Sep, 66(9), 3850 - 5
Biotransformation of the antimelanoma agent betulinic acid by Bacillus megaterium ATCC 13368; Chatterjee P et al.; Microbial transformation of the antimelanoma agent betulinic acid was studied . The main objective of this study was to utilize microorganisms as in vitro models to predict and prepare potential mammalian metabolites of this compound . Preparative-scale biotransformation with resting-cell suspensions of Bacillus megaterium ATCC 13368 resulted in the production of four metabolites, which were identified as 3-oxo-lup-20(29)-en-28-oic acid, 3-oxo-11alpha-hydroxy-lup-20(29)-en-28-oic acid, 1beta-hydroxy-3-oxo-lup-20(29)-en-28-oic acid, and 3beta,7beta, 15alpha-trihydroxy-lup-20(29)-en-28-oic acid based on nuclear magnetic resonance and high-resolution mass spectral analyses . In addition, the antimelanoma activities of these metabolites were evaluated with two human melanoma cell lines, Mel-1 (lymph node) and Mel-2 (pleural fluid).

Appl Environ Microbiol, 2000 Sep, 66(9), 3784 - 9
Development and characterization of diamondback moth resistance to transgenic broccoli expressing high levels of Cry1C; Zhao JZ et al.; A field-collected colony of the diamondback moth, Plutella xylostella, had 31-fold resistance to Cry1C protoxin of Bacillus thuringiensis . After 24 generations of selection with Cry1C protoxin and transgenic broccoli expressing a Cry1C protein, the resistance that developed was high enough that neonates of the resistant strain could complete their entire life cycle on transgenic broccoli expressing high levels of Cry1C . After 26 generations of selection, the resistance ratios of this strain to Cry1C protoxin were 12,400- and 63,100-fold, respectively, for the neonates and second instars by a leaf dip assay . The resistance remained stable until generation 38 (G38) under continuous selection but decreased to 235-fold at G38 when selection ceased at G28 . The Cry1C resistance in this strain was seen to be inherited as an autosomal and incompletely recessive factor or factors when evaluated using a leaf dip assay and recessive when evaluated using Cry1C transgenic broccoli . Saturable binding of (125)I-Cry1C was found with brush border membrane vesicles (BBMV) from both susceptible and Cry1C-resistant strains . Significant differences in Cry1C binding to BBMV from the two strains were detected . BBMV from the resistant strain had about sevenfold-lower affinity for Cry1C and threefold-higher binding site concentration than BBMV from the susceptible strain . The overall Cry1C binding affinity was just 2.5-fold higher for BBMV from the susceptible strain than it was for BBMV from the resistant strain . These results suggest that reduced binding is not the major mechanism of resistance to Cry1C.

Farmaco, 2000 Apr, 55(4), 264 - 9
Therapeutic proteins: a comparison of chemical and biological properties of uricase conjugated to linear or branched poly(ethylene glycol) and poly(N-acryloylmorpholine); Schiavon O et al.; Uricase from Bacillus fastidiosus (UC) was covalently linked to linear PEG (PEG-1) (Mw 5 kDa), branched PEG (PEG-2) (Mw 10 kDa) and to poly(N-acryloylmorpholine) (PAcM) (Mw 6 kDa) . The conjugation of UC with linear PEG and PAcM was accompanied by complete loss of enzymatic activity but, if uric acid as site protecting agent was included in the reaction mixture, the conjugate protein retained enzymatic activity . On the other hand, the modification with PEG-2 gave a conjugate that also maintained enzymatic activity in the absence of any active site protection . This behaviour must be related to hindrance of the branched polymer in reaching the enzyme active site . The UC conjugates exhibited increased resistance to proteolytic digestion while minor variations in the inhibitory constant, optimal pH, heat stability, affinity for substrate, were observed . Pharmacokinetic investigations in mice demonstrated increased residence time in blood for all the conjugates as compared with native uricase . Uricase conjugated with linear PEG was longer lasting in blood UC derivative, followed by branched PEG and the PAcM conjugates . Unconjugated uricase was rapidly removed from circulation . All these data are in favour of the use of the less known amphiphilic polymer PAcM as an alternative to PEGs in modification of enzymes devised for therapeutic applications.

Protist, 2000 Aug, 151(2), 161 - 9
Paramecium GPI proteins: variability of expression and localization; Capdeville Y; In Paramecium primaurelia, the two major classes of cell surface proteins, the surface antigen (SAg) and the surface GPI proteins (SGPs), are linked to the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor . In the present study, we have characterized the expression of the SGPs in several geographical strains of P . primaurelia and P . tetraurelia at different temperatures, 23 degrees C and 32 degrees C . The identification of the expressed SGPs was performed on purified cilia, by establishing the SGP SDS-PAGE profiles under four different conditions: with or without their anchoring lipid, cleaved with a Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), and either in a reduced or in an unreduced state . This screening revealed the existence of specific sets of ciliary SGPs, as a function of temperature and the geographical origin of the strains . The SGPs the most abundant at 23 degrees C and 32 degrees C displayed a rapid turnover . We also looked for the presence of PI-PLC releasable proteins in purified cortices . In addition to the SAg and SGPs, the cortical fraction was shown to contain other PI-PLC releasable proteins, not found in the ciliary fraction, thus localized exclusively in the interciliary region.

J Invertebr Pathol, 2000 Jul, 76(1), 70 - 5
Screening for Bacillus thuringiensis crystal proteins active against the cabbage looper, Trichoplusia ni; Iracheta MM et al.; Toxicity tests were performed to find among Cry1 and Cry2 Bacillus thuringiensis crystal proteins those with high activity against the cabbage looper . Tests were performed with neonate larvae on surface-contaminated artificial diet . The crystal proteins found to be toxic were, from higher to lower toxicity: Cry1Ac, Cry1Ab, Cry1C, Cry2Aa, Cry1J, and Cry1F (LC50 of 1.14.1, 3.4-4.4, 12, 34, 87, and 250 ng/cm2, respectively) . Cry1B, Cry1D, and Cry1E can be considered nontoxic (LC50 higher than 2500 ng/cm2) . Cry1Aa was moderately toxic to nontoxic, depending on the source (LC50 of 420 ng/cm2 from PGS and 8100 ng/cm2 from Ecogen) . In vitro binding assays with trypsin-activated 125I-labeled Cry1Aa, Cry1Ab, and Cry1Ac crystal proteins and brush border membrane vesicles from midgut larvae showed a direct correlation between toxicity and binding affinity . Heterologous competition experiments indicated that Cry1Aa and Cry1F bind, though only at very high concentrations, to the Cry1Ab/Cry1Ac shared high-affinity binding site.

J Invertebr Pathol, 2000 Jul, 76(1), 56 - 62
Activation pattern and toxicity of the Cry11Bb1 toxin of Bacillus thuringiensis subsp . medellin; Segura C et al.; Bacillus thuringiensis protoxins undergo proteolytic processing in the midgut of susceptible insects to become active . The ability to process the Cry11Bb1 protoxin by trypsin and Culex quinquefasciatus larval gut extracts was tested . The protease activity indicated by the appearance of proteolytic products increased with an increment in pH, with the highest activity being observed at pH 10.6 . A time course study showed the proteolysis of the 94-kDa Cry11Bb protein ending with the production of fragments of relative molecular mass of 30 and 35 kDa within 5 min . In vitro, gut proteases extract cleaved the solubilized toxin between Ser59 and Ile60 and between Ala395 and Asn396, generating a 30-kDa N-terminal and a 35-kDa C-terminal fragment, respectively . Similarly, mosquito larvae processed in vivo the parasporal inclusions, generating the same fragments as those observed in vitro . The Cry11Bb1 protoxin activated with trypsin or gut proteases showed larvicidal activity against C . quinquefasciatus first instar larvae . The data suggest that gut proteases participate in the activation of CryllBbl protoxin, generating at least two different fragments on which the activity could reside.

Rapid Commun Mass Spectrom, 2000, 14(18), 1701 - 6
Matrix-assisted laser desorption/ionization time-of-flight analysis of Bacillus spores using a 2.94 microm infrared laser; Ryzhov V et al.; The performance of infrared (2.94 microm) and ultraviolet (337 nm) lasers were compared for analysis of purified spores of B . subtilis, B . cereus and B . globigii on a four-inch end-cap reflectron time-of-flight instrument . Infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectra of these microorganisms displayed a larger number of biomarker peaks above m/z 4000, compared with UV-MALDI . Biomarker peaks were observed at higher m/z values with the IR laser .

Microbes Infect, 2000 Jul, 2(8), 885 - 90
Intranasal, rectal and intraperitoneal immunization with protoxin Cry1Ac from Bacillus thuringiensis induces compartmentalized serum, intestinal, vaginal and pulmonary immune responses in Balb/c mice; Moreno-Fierros L et al.; Recently we discovered that the Cry1Ac protoxin of Bacillus thuringiensis administered to Balb/c mice intraperitoneally (i.p.) or intragastrically is a systemic and intestinal immunogen as potent as cholera toxin . To further characterize the mucosal immunogenicity of Cry1Ac we additionally tried the intranasal (i.n.) and rectal routes and used enzyme-linked immunoassays to determine anti-Cry1Ac antibody responses in the serum as well as in vaginal and tracheobronchial washes and in the fluids of the large and the small intestine . Immunization by the i.p., i.n . and rectal routes induced IgM, IgG and IgA antibodies in all the mucosal surfaces analyzed, but the magnitude and predominant isotype of each response depended on the route used and the mucosal site analyzed . These data extend our findings on the striking mucosal immunogencity of Cry1Ac and provide additional evidence on the compartmentalization of the mucosal immune system.

J Acquir Immune Defic Syndr, 1999 Dec 15, 22(5), 467 - 76
Immunologic responses of HIV-1-infected study subjects to immunization with a mixture of peptide protein derivative-V3 loop peptide conjugates; Rubinstein A et al.; V3 loop peptide sequences from several HIV-1 strains were covalently linked to purified protein derivative (PPD) of Mycobacterium tuberculosis . A mixture of PPD conjugates of V3 loop peptides from six different strains of HIV-1 induced a stronger antibody response than a single V3 peptide-conjugate administered to guinea pigs and humans . Sera from animals immunized with a PPD-six peptide-PPD conjugate neutralized multiple primary-isolate strains of HIV-1 . Potent immune responses were noted only when animals were primed with bacillus Calmette-Guerin (BCG), PPD was covalently bound to the peptides, and PPD was used as the carrier protein . Based on these animal studies, an immunogen consisting of PPD-conjugated V3 loop peptides from five HIV-1 strains was tested in 7 HIV-1 seropositive PPD skin test positive study subjects . Vaccinees exhibited over time a uniform increase in neutralizing antibodies for both laboratory adapted and primary isolates of HIV-1, including strains from multiple clades . In 3 patients with baseline viral loads between 8000 and 12,000 RNA copies/ml, the viral load declined in 2 patients to <400 copies/ml and in 1 patient to 1200 copies/ml without concurrent administration of highly active antiretroviral therapy (HAART).

J Org Chem, 2000 Jul 28, 65(15), 4509 - 14
A novel class of zinc-binding inhibitors for the phosphatidylcholine-preferring phospholipase C from Bacillus cereus; Martin SF et al.; The phospholipase C (PLC) isozymes catalyze the hydrolysis of phospholipids to provide diacylglycerol (DAG) and a phosphorylated headgroup . Because DAG has been implicated in cellular signal transduction cascades in mammalian systems, there has been considerable interest in the development of inhibitors of these enzymes . Toward this end, we have discovered that the cyclic N,N'-dihydroxyureas 6-10 inhibit the phosphatidylcholine preferring PLC from Bacillus cereus (PLCBc) . This class of inhibitors is believed to function by the bidentate chelation of the N,N'-dihydroxyurea array to one or more of the zinc ions at the active site of the enzyme . Because the affinities of these compounds correlate with the pKaS of the N-OH hydroxyl groups, it is apparent that one or both of the hydroxyl groups must be ionized for effective coordination to the zinc ions . It is also apparent that there may be rather strict steric requirements for these inhibitors.

Antonie Van Leeuwenhoek, 2000 May, 77(4), 393 - 9
Prevalence and expression of enterotoxins in Bacillus cereus and other Bacillus spp., a literature review; McKillip JL; Members of the Bacillus genus are ubiquitous soil microorganisms and are generally considered harmless contaminants . However, a few species are known toxin producers, including the foodborne pathogen, B . cereus . This species produces two distinct types of foodborne illness, the emetic (vomit-inducing) syndrome, associated with consumption of toxin in cooked rice dishes, and the diarrheal illness seen occasionally following consumption of contaminated meats, sauces, and certain dairy products . In the latter case, illness results from the production of enterotoxins by vegetative cells in the small intestine of the host . In dairy products, the occurrence of Bacillus spp . is inevitable, and the spore-forming ability of this organism allows it to easily survive pasteurization . Many strains have been shown to grow and produce enterotoxin in dairy products at refrigeration temperatures . Evaluation of toxin gene presence and toxin expression in Bacillus spp . other than B . cereus has not been thoroughly investigated . However, the presence of natural isolates of Bacillus spp . harboring one or more enterotoxin gene(s) and subsequent demonstration of conditions which may support toxin expression holds crucial importance in the food safety arena.

Aquat Toxicol, 2000 Sep 1, 50(3), 221 - 230
Relationships between acid-soluble thiol peptides and accumulated Pb in the green alga Stichococcus bacillaris; Pawlik-Skowro&nacute;ska B; Stichococcus bacillaris, an ubiquitous green microalga accumulated inorganic lead (Pb) from aqueous solutions extra- and intracellularly . In response to Pb uptake acid-soluble thiol peptides (glutathione - GSH and phytochelatins - PC) were synthesized . The proportion of the intracellular Pb uptake by algal cells was low and comprised only 3-6% of the total metal sorption . The intracellular uptake was dependent on external Pb concentration, time of metal exposure and cell metabolism . Pb accumulation in alga was determined by means of 210Pb radiometry . Reduced GSH and PC were determined in algal cells using HPLC with the post-column derivatization with Ellman's reagent . Within the studied concentration range 0.1-20 microM, inorganic lead caused a significant production of induced thiol peptides: PC (n=2-4) and some other unidentified oligopeptides, probably (GluCys)n . The time of appearance and the concentration of individual oligomers of phytochelatins were dependent on the external Pb concentration and time of metal exposure . In algal cells exposed to Pb, significant changes in the GSH level accompanying the formation of the induced thiol peptides were also observed . The GSH level decreased in the cells exposed to the lower (up to 10 microM) studied Pb concentrations or increased in the cells treated with higher (20 microM) Pb concentrations . The thiol groups originated from induced peptides (mainly phytochelatins) followed a stoichiometric relationship 2:1 to the intracellular Pb amounts, however, only at the lowest studied external concentration (0.1 microM) . At higher concentrations (up to 2.5 microM), intracellular Pb concentration was equal or even exceeded (at Pb>2.5 microM) two to three times the level of induced thiols . S . bacillaris accumulated intracellularly by 46% more Pb in light than in dark and the level of induced thiol peptides was significantly higher in the cells exposed to Pb under illumination . The rapid formation of these peptides in S . bacillaris in response to Pb, and their elimination (by about 90%) when algae were placed into the Pb-free solution reveal a tight regulation of GSH and phytochelatin pools in the algal cells exposed to toxic metals . The obtained results suggest that both PCs and GSH are the primary line of defence against the Pb toxicity . Additionally, the induced thiol peptides in S . bacillaris could be a good indicator of intracellular Pb availability and stress at the metal concentrations found in polluted fresh waters.

Int J Cancer, 2000 Sep 15, 87(6), 844 - 52
What are the immunologically active components of bacille Calmette-Guérin in therapy of superficial bladder cancer?
Zlotta AR, Van Vooren JP, Denis O, Drowart A, Daffe M, Lefevre P, Schandene L, De Cock M, De Bruyn J, Vandenbussche P, Jurion F, Palfliet K, Simon J, Schulman CC, Content J, Huygen K.
The subcomponents of bacille Calmette-Guerin (BCG) involved in the mechanism of action of intravesical BCG immunotherapy used for prophylaxis of superficial bladder cancer recurrences have been poorly investigated . We purified various BCG subcomponents and analyzed in vitro their ability to enhance a Th1 polarized immune response as well as to increase lymphocyte-mediated cytotoxicity against bladder tumors . Human peripheral blood mononuclear cells (PBMCs) from healthy purified protein derivative-positive subjects were incubated for 7 days with whole BCG and various fractions (BCG cell wall, plasma membrane, cytosol, purified polysaccharides as glucan or arabinomannan, purified native proteins from BCG culture filtrate, recombinant 22 kDa protein, phosphate transporter PstS-2 and -3 proteins) . IFN-gamma, IL-12, IL-2, and IL-6 production by stimulated PBMCs was compared to unstimulated controls and the phenotype of expanded cells analyzed by flow cytometry (FACS analysis) . A (51)Cr-release assay monitored the cytotoxicity of amplified effector cells against T24 bladder tumor cells . Live BCG and most of its subcomponents (with the exception of cytosol, PstS-2 and -3) significantly enhanced IFN-gamma and IL-12 secretion, expanded CD3(-)CD56(+) cells and the non-MHC-restricted cytotoxicity against bladder tumor cells compared to unstimulated controls (all P < 0.001, t-test) . IL-2 receptor blockage resulted in a clear reduction in the cytotoxic activity of stimulated PBMCs . Numerous BCG subcomponents thus provide positive stimuli for Th1 cell differentiation and enhance in vitro, non-MHC-restricted cytotoxicity against bladder tumor cells . Our findings provide the basis for the therapeutic use of several of these subfractions in experimental animal models bearing bladder tumors .

Biochemistry, 2000 Aug 29, 39(34), 10439 - 47
Control of directionality in nonribosomal peptide synthesis: role of the condensation domain in preventing misinitiation and timing of epimerization; Linne U et al.; Product assembly by nonribosomal peptide synthetases (NRPS) is initiated by starter modules that comprise an adenylation (A) and a peptidyl carrier protein (PCP) domain . Elongation modules of NRPS have in addition a condensation (C) domain that is located upstream of the A domain . They cannot initiate peptide bond formation . To understand the role of domain arrangements and the influence of the domains present upstream of the A domains of the elongation modules of TycB on the initiation and epimerization activities, we constructed a set of proteins derived from the tyrocidine synthetases of Bacillus brevis, which represent several N-terminal truncations of TycB and the first module of TycC . The latter was fused with the thioesterase domain (Te) to give TycC(1)-CAT-Te and to ensure product turnover . TycB(2)(-)(3)-AT.CATE and TycB(3)-ATE, lacking an N-terminal C domain, were capable of initiating peptide synthesis and epimerizing . In contrast, the corresponding constructs with a cognate N-terminal C domain, TycB(2)(-)(3)-T.CATE and TycB(3)-CATE, were strongly reduced in initiation and epimerization . Evidence is also provided that this reduction is due to substrate binding in an enantioselective binding pocket at the acceptor position of the C domains . By using TycB(2)(-)(3)-AT.CATE and TycB(3)-ATE, we were able to turn an elongation module into an initiation module, and to establish an in-trans system for the formation of new di- and tripeptides with recombinant NRPS modules . We also show that epimerization domains of elongation modules can in principle epimerize both aminoacyl-S-Ppant (TycB(3)-ATE) and peptidyl-S-Ppant (TycB(2)(-)(3)-AT.CATE) substrates, although the efficiency for epimerizing the noncognate aminoacyl-S-Ppant substrates appears to be lowered.

Biochemistry, 2000 Aug 29, 39(34), 10385 - 96
The crystal structure of bacillus cereus phosphonoacetaldehyde hydrolase: insight into catalysis of phosphorus bond cleavage and catalytic diversification within the HAD enzyme superfamily; Morais MC et al.; Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and phosphate using Mg(II) as cofactor . The reaction proceeds via a novel bicovalent catalytic mechanism in which an active-site nucleophile abstracts the phosphoryl group from the Schiff-base intermediate formed from Lys53 and phosphonoacetaldehyde . In this study, the X-ray crystal structure of the Bacillus cereus phosphonatase homodimer complexed with the phosphate (product) analogue tungstate (K(i) = 50 microM) and the Mg(II) cofactor was determined to 3.0 A resolution with an R(cryst) = 0.248 and R(free) = 0.284 . Each monomer is made up of an alpha/beta core domain consisting of a centrally located six-stranded parallel beta-sheet surrounded by six alpha-helices . Two flexible, solvated linkers connect to a small cap domain (residues 21-99) that consists of an antiparallel, five-helix bundle . The subunit-subunit interface, formed by the symmetrical packing of the two alpha8 helices from the respective core domains, is stabilized through the hydrophobic effect derived from the desolvation of paired Met171, Trp164, Tyr162, Tyr167, and Tyr176 side chains . The active site is located at the domain-domain interface of each subunit . The Schiff base forming Lys53 is positioned on the cap domain while tungstate and Mg(II) are bound to the core domain . Mg(II) ligands include two oxygens of the tungstate ligand, one oxygen of the carboxylates of Asp12 and Asp186, the backbone carbonyl oxygen of Ala14, and a water that forms a hydrogen bond with the carboxylate of Asp190 and Thr187 . The guanidinium group of Arg160 binds tungstate and the proposed nucleophile Asp12, which is suitably positioned for in-line attack at the tungsten atom . The side chains of the core domain residue Tyr128 and the cap domain residues Cys22 and Lys53 are located nearby . The identity of Asp12 as the active-site nucleophile was further evidenced by the observed removal of catalytic activity resulting from Asp12Ala substitution . The similarity of backbone folds observed in phosphonatase and the 2-haloacid dehalogenase of the HAD enzyme superfamily indicated common ancestry . Superposition of the two structures revealed a conserved active-site scaffold having distinct catalytic stations . Analysis of the usage of polar amino acid residues at these stations by the dehalogenases, phosphonatases, phosphatases, and phosphomutases of the HAD superfamily suggests possible ways in which the active site of an ancient enzyme ancestor might have been diversified for catalysis of C-X, P-C, and P-O bond cleavage reactions.

Int J Clin Pract, 2000 Jun, 54(5), 345 - 7
Refractory Bacillus cereus infection in a neonate; Tuladhar R et al.; Bacillus cereus is a Gram-positive aerobic or facultatively anaerobic spore-forming rod, which usually causes food poisoning . Its recognition as a pathogen in neonates has increased over the past two decades . The clinical course of a neonate (gestation 24 weeks) with B . cereus infection refractory to therapy is described . Death occurred after withdrawal of support following persistently positive blood and bone marrow cultures despite therapy with vancomycin, gentamicin, imipenum, clindamycin, ciprofloxacillin, immunoglobulin and granulocyte colony stimulating factor over a period of 49 days . No obvious focus of sepsis was identified . Contamination from the environment into the hospital and clinics occurs because of the ubiquitous presence of B . cereus . Combination therapy with vancomycin and gentamycin is appropriate for meningitis/severe systemic infections related to most bacillus species . The significance of repeated isolation of B . cereus in neonates with compromised host defences is emphasised.

Anticancer Res, 2000 Jul-Aug, 20(4), 2329 - 38
Molecular targets of guanine nucleotides in differentiation, proliferation and apoptosis; Yalowitz JA et al.; Guanine nucleotides are important substrates for macromolecular synthesis, cell signaling, and integration of metabolic status, and have an evolutionarily conserved role in differentiation, proliferation, and apoptosis . Bacteria, yeast, and mammalian cells are all dependent on an adequate supply of guanylates to maintain proliferation . Depletion of intracellular guanylates, especially by inhibition of de novo synthesis via the IMP dehydrogenase pathway, is a potent signal for inhibition of proliferation, as well as apoptosis . Growth inhibition by depletion of GTP is a conserved pathway from humans to Bacillus . IMPDH expression is downregulated by the p53 tumor suppressor gene . Many inhibitors of IMP dehydrogenase are used as clinical agents . These agents are antivirals (ribavirin), antitumor (tiazofurin {TR}, selenazofurin {SR}, and benzamide riboside {BR}), and immunosuppressants (mycophenolic acid {MPA}) . The biochemical actions of IMP dehydrogenase inhibitors are well known, but correlation with in vivo activities is difficult because the extent of exogenous contributions to the nucleotide metabolic pathways is not fully known . IMPDH inhibitors are biochemically convenient in inhibiting parallel pathways, since excess reactants IMP and 5'-phospho-ribose-1'-pyrophosphate (PRPP) inhibit guanine salvage synthesis . IMPDH activity is a progression-linked key enzyme in tumorigenesis . The antitumor potential of IMPDH inhibitors is therefore particularly high.

Prostate, 2000 Sep 1, 44(4), 279 - 86
Antibacterial activity of human prostasomes; Carlsson L et al.; BACKGROUND: Prostasomes are prostate-derived organelles in semen exhibiting pluripotent properties . The present study deals with their possible antibacterial effects . METHODS: Antibacterial activity was assessed by growth inhibition of bacteria in an incubation medium containing prostasomes, after which the incubate was inoculated on cystine lactose electrolyte deficient agar (CLED) plates . In cases involving Bacillus megaterium, the effects were also documented ultrastructurally with scanning electron microscopy and atomic force microscopy . RESULTS: A dose-dependent growth inhibition was apparent, and a complete inhibition of growth was seen at a prostasome protein concentration of 30 microg/ml with Bacillus megaterium . Ultrastructurally, increasingly irregular contours and a loosening of the smooth surface were observed, combined with a fragmentation of the bacteria . Among 9 other bacterial strains tested, a complete growth inhibition by prostasomes was attained in 3 strains, while the other 6 were unaffected . CONCLUSIONS: Our data suggest that prostasomes, or prostasome-derived proteins, are responsible for the antibacterial effects on Bacillus megaterium and some other bacterial strains . The results may serve as a basis of development of a new class of antibacterial drugs .

J Infect Dis, 2000 Sep, 182(3), 865 - 72 Epub 2000 Aug 17.
Natural history of infection with Bartonella bacilliformis in a nonendemic population; Kosek M et al.; An investigation was performed after an outbreak of bartonellosis in a region of Peru nonendemic for this disorder . Symptoms of acute and chronic bartonellosis were recorded . Serological analysis was performed on 55% of the affected population (554 individuals), 77.5% of whom demonstrated previous infection with Bartonella bacilliformis . The attack rate of Oroya fever was 13.8% (123 cases); the case-fatality rate was 0.7% . The attack rate of verruga peruana was 17.6% . A new specific immunostain was developed and used to confirm the presence of B . bacilliformis in the biopsied skin lesions . Most seropositive individuals (56%) were asymptomatic . The symptoms that were associated with prior infection, as determined by Western blot, included fever (37.2% of the seropositive vs . 17.2% of the seronegative population; P<.001), bone and joint pain (27% vs . 9%; P<.001), headache (27% vs . 12.3%; P <.001), and skin lesions described as verruga peruana (26.8% vs . 4.9%; P<.001) . Our findings suggest that infection with B . bacilliformis causes a broad spectrum of disease that is significantly milder in severity than that frequently reported.

Appl Biochem Biotechnol, 2000 May, 87(2), 95 - 101
Immobilization of alkaliphilic Bacillus sp . cells for xylanase production using batch and continuous culture; Mamo G et al.; Agar-immobilized alkaliphilic Bacillus sp . AR-009 cells were used for xylanase production using batch and continuous culture . In a batch culture, maximum enzyme production was observed after 48 h and remained high up to 72 h . In repeated batch cultivation, immobilized cells produced an appreciable level of xylanase activity in seven consecutive batches without any significant decline in productivity . For continuous xylanase production, immobilized cells were packed in a jacketed glass column and sterile medium was continuously pumped . A stable continuous production of xylanase was observed over a period of 1 mo . The volumetric productivity of the continuous culture was 17-fold higher than the batch culture using free cells.

J Biotechnol, 2000 Jul 14, 80(3), 195 - 202
Rapid degradation of ferulic acid via 4-vinylguaiacol and vanillin by a newly isolated strain of bacillus coagulans; Karmakar B et al.; A new strain Bacillus coagulans BK07 was isolated from decomposed wood-bark, based on its ability to grow on ferulic acid as a sole carbon source . This strain rapidly decarboxylated ferulic acid to 4-vinylguaiacol, which was immediately converted to vanillin and then oxidized to vanillic acid . Vanillic acid was further demethylated to protocatechuic acid . Above 95% substrate degradation was obtained within 7 h of growth on ferulic acid medium, which is the shortest period of time reported to date . The major degradation products, was isolated and identified by thin-layer chromatography, high performance liquid chromatography and 1H-nuclear magnetic resonance spectroscopy were 4-vinylguaiacol, vanillin, vanillic acid and protocatechuic acid.

Infect Immun, 2000 Sep, 68(9), 5269 - 76
Evidence for contribution of tripartite hemolysin BL, phosphatidylcholine-preferring phospholipase C, and collagenase to virulence of Bacillus cereus endophthalmitis; Beecher DJ et al.; Bacillus cereus causes a highly fulminant endophthalmitis which usually results in blindness . We previously concluded that hemolysin BL (HBL), a tripartite necrotizing pore-forming toxin, is a probable endophthalmitis virulence factor because it is highly toxic to retinal tissue in vitro and in vivo . We also determined that B . cereus produces additional retinal toxins that might contribute to virulence . Here we fractionated crude B . cereus culture supernatant by anion-exchange chromatography and found that in vitro retinal toxicity was also associated with phosphatidylcholine-preferring phospholipase C (PC-PLC) . The pure enzyme also caused retinal necrosis in vivo . We showed that phosphatidylinositol-specific PLC and sphingomyelinase were nontoxic and that two hemolysins, cereolysin O and a novel hemolysin designated hemolysin IV, were marginally toxic in vitro . The histopathology of experimental septic endophthalmitis in rabbits mimicked the pathology produced by pure HBL, and both HBL and PC-PLC were detected at toxic concentrations in infected vitreous fluid . Bacterial cells were first seen associated with the posterior margin of the lens and eventually were located throughout the lens cortex . Detection of collagenase in the vitreous humor suggested that infiltration was facilitated by the breakdown of the protective collagen lens capsule by that enzyme . This work supports our conclusion that HBL contributes to B . cereus virulence and implicates PC-PLC and collagenase as additional virulence factors.

Infect Immun, 2000 Sep, 68(9), 4972 - 9
Molecular cloning, sequencing, expression, and characterization of an immunogenic 43-kilodalton lipoprotein of Bartonella bacilliformis that has homology to NlpD/LppB; Padmalayam I et al.; A recombinant clone expressing an immunoreactive antigen of Bartonella bacilliformis was isolated by screening a genomic DNA library with serum from a patient with the chronic verruga phase of bartonellosis . The clone, pBIPIM-17, contained a partial open reading frame that expressed an immunoreactive fusion protein . Subsequent rescreening of the library by plaque hybridization resulted in the isolation of recombinant clones that contain the entire open reading frame . The open reading frame (ORF-401) is capable of encoding a protein of 401 amino acids with a predicted molecular mass of 43 kDa . The deduced amino acid sequence of the encoded protein was found to be highly homologous to a recently identified bacterial lipoprotein (LppB/NlpD) which has been associated with virulence . Evidence has been provided to show that the 43-kDa antigen of B . bacilliformis is a lipoprotein and that it is likely to use the same biosynthetic pathway as other bacterial lipoproteins . This is the first report to date that characterizes a lipoprotein of B . bacilliformis . The immunogenicity of the B . bacilliformis LppB homologue was demonstrated by Western blot analysis using sera from patients with clinical bartonellosis . Sera from patients who had a high titer for Bartonella henselae, the causative agent of bacillary angiomatosis and cat scratch disease, also recognized the recombinant 43-kDa antigen, suggesting that a homologue of this antigen is present in B . henselae . Using a cocktail of synthetic peptides corresponding to predicted major antigenic sites, polyclonal antiserum specific for the LppB homologue of B . bacilliformis was generated . This antiserum did not recognize the NlpD homologue of Escherichia coli or the 43-kDa antigen of B . henselae.

Infect Immun, 2000 Sep, 68(9), 4961 - 7
Antibody and cytokine responses to the cilium-associated respiratory bacillus in BALB/c and C57BL/6 mice; Kendall LV et al.; The cilium-associated respiratory (CAR) bacillus is a gram-negative, gliding bacterium that causes persistent respiratory tract infections in rodents despite histologic and serologic evidence of a marked immune response . To assess humoral immunity and cytokine responses in CAR bacillus disease, 6-week-old female BALB/c and C57BL/6 mice were inoculated intratracheally with 10(5) CAR bacillus organisms . CAR bacillus-specific serum immunoglobulins (immunoglobulin M {IgM}, IgG1, IgG2a, IgG2b, IgG3, and IgA) and local pulmonary cytokines (tumor necrosis factor alpha {TNF-alpha}, gamma interferon {IFN-gamma}, and interleukin-4 {IL-4}) were evaluated by enzyme-linked immunosorbent assay every 7 days for 49 days . BALB/c mice developed CAR bacillus-induced lesions early in the course of disease that became more severe with time . Correlating with increasing disease severity, BALB/c mice had elevations in all antibody isotypes tested, and elevations in pulmonary TNF-alpha, IFN-gamma, and IL-4 . C57BL/6 mice developed mild lesions with mild increases in serum IgM, IgG1, IgG2b, and IgG3 levels and minimally detectable IgG2a and IgA . Cytokine perturbations were not detected in C57BL/6 mice . The persistence of infection in BALB/c mice with vigorous serum antibody responses and increased IFN-gamma and IL-4 responses suggests that humoral immunity and T-cell responses are ineffective at preventing CAR bacillus disease . Furthermore, the lackluster antibody responses and undetectable cytokine responses in C57BL/6 mice suggest that humoral immunity and T-cell responses are not critical in resistance to CAR bacillus-induced disease.

Biochem J, 2000 Sep 1, 350 Pt 2, 477 - 84
New type of starch-binding domain: the direct repeat motif in the C-terminal region of Bacillus sp . no . 195 alpha-amylase contributes to starch binding and raw starch degrading; Sumitani J et al.; The alpha-amylase from Bacillus sp . no . 195 (BAA) consists of two domains: one is the catalytic domain similar to alpha-amylases from animals and Streptomyces in the N-terminal region; the other is the functionally unknown domain composed of an approx . 90-residue direct repeat in the C-terminal region . The gene coding for BAA was expressed in Streptomyces lividans TK24 . Three active forms of the gene products were found . The pH and thermal profiles of BAAs, and their catalytic activities for p-nitrophenyl maltopentaoside and soluble starch, showed almost the same behaviours . The largest, 69 kDa, form (BAA-alpha) was of the same molecular mass as that of the mature protein estimated from the nucleotide sequence, and had raw-starch-binding and -degrading abilities . The second largest, 60 kDa, form (BAA-beta), whose molecular mass was the same as that of the natural enzyme from Bacillus sp . no . 195, was generated by proteolytic processing between the two repeat sequences in the C-terminal region, and had lower activities for raw starch binding and degrading than those of BAA-alpha . The smallest, 50 kDa, form (BAA-gamma) contained only the N-terminal catalytic domain as a result of removal of the C-terminal repeat sequence, which led to loss of binding and degradation of insoluble starches . Thus the starch adsorption capacity and raw-starch-degrading activity of BAAs depends on the existence of the repeat sequence in the C-terminal region . BAA-alpha was specifically adsorbed on starch or dextran (alpha-1,4 or alpha-1,6 glucan), and specifically desorbed with maltose or beta-cyclodextrin . These observations indicated that the repeat sequence of the enzyme was functional in the starch-binding domain (SBD) . We propose the designation of the homologues to the SBD of glucoamylase from Aspergillus niger as family I SBDs, the homologues to that of glucoamylase from Rhizopus oryzae as family II, and the homologues of this repeat sequence of BAA as family III.

J Egypt Soc Parasitol, 2000 Aug, 30(2), 573 - 80
Bacillus thuringiensis var . israelensis (B.t . serotype H-14) against Lucilia sericata third stage larvae; Morsy TA et al.; Bacillus thuringiensis var . israelensis (B.t . H-14) was mixed with minced liver in different concentrations and given to newly moulted third stage larvae of Lucilia sericata . The LC50 was 9 ppm (0.76-1.5) and the slope function was 0.59 . This bacterium which is safe and friendly proved to be effective against the myiasis producing L . sericata larvae.

J Egypt Soc Parasitol, 2000 Aug, 30(2), 377 - 86
Extended effect of Bacillus thuringiensis H-14 on Culex pipiens adults surviving larval treatment; Hafez GA; Three Bacillus thuringiensis H-14 preparations (a laboratory prepared and two commercial formulations namely, Tecknar and Vectobac) were assayed against third instar Culex pipiens larvae in order to trace their toxic activity on larval treatment . The LC50 values were 0.002, 0.02 and 0.04 ppm . respectively . The laboratory strain was tested on the development of Culex pipiens . Larval mortality, pupation, adult emergence and sex ratio showed significant differences as compared to the non-treated group . Toxic action on the treated larvae with laboratory strain of B . thuringiensis H-14 revealed drastic effect on larval mid-gut epithelium and resulted in extended pathological activity in adults, which survived larval treatments . Nervous and reproductive systems were found to be the most damaged systems in female adults.

J Appl Microbiol, 2000 Jul, 89(1), 16 - 23
In vitro cytotoxicity of non-cyt inclusion proteins of a Bacillus thuringiensis isolate against human cells, including cancer cells; Kim HS et al.; A soil isolate designated 90-F-45-14, belonging to Bacillus thuringiensis serovar dakota (H15), was examined for characterization of in vitro cytotoxicity, associated with parasporal inclusion proteins, against human cells . When activated with proteolytic processing, inclusion proteins of the isolate 90-F-45-14 exhibited a moderate cytotoxicity against the human uterus cervix cancer cells (HeLa) with an EC(50) value of 60.8 microg ml(-1), while showing extremely high activities on the human leukaemic T cells (MOLT-4) and the normal T cells with EC(50) values of 0.27 and 0.20 microg ml(-1), respectively . Anti-leukaemic cell activity of the 90-F-45-14 proteins was eight to nine times greater than that of the B . thuringiensis serovar israelensis proteins containing the Cyt1 protein, a broad-spectrum cytolysin . The cytopathy by the 90-F-45-14 proteins was characterized by marked cell-ballooning, while the israelensis proteins induced early breakdown of the cells due to cytolysis . Inclusions of the isolate consisted of five major polypeptides of 170, 103, 73, 40 and 32 kDa . A 100% homology was observed in the sequence of 15 N-terminal amino acids between the proteins of 170 and 103 kDa . There was no N-terminal sequence homology between 90-F-45-14 proteins and the existing Cry/Cyt proteins of B . thuringiensis . Proteolytic processing by proteinase K yielded several proteins with molecular masses ranging from 40 to 28 kDa.

Biosens Bioelectron, 2000 Jan, 14(10-11), 849 - 52
A microfluidic cartridge to prepare spores for PCR analysis; Belgrader P et al.; A prototype cartridge system is described that rapidly disrupts Bacillus spores by sonication, adds PCR reagent to the disrupted spores, and dispenses the mixture into a PCR tube . The total time to automatically process the spores in the cartridge and then detect the spore DNA by real-time PCR was 20 min.

Biosens Bioelectron, 2000 Jan, 14(10-11), 771 - 7
Multi-analyte interrogation using the fiber optic biosensor; Anderson GP et al.; The capabilities of the portable, automated fiber optic biosensor, RAPTOR, have recently been evaluated . Developed to perform rapid fluoroimmunoassays in the field, the RAPTOR was designed to test samples for up to four different target analytes simultaneously . Assay time could be varied from a 3-min rapid screen to a standard 10-min test . A trial of 203 blind samples tested for Staphylococcal enterotoxin B, ricin, Francisella tularensis, and Bacillus globigii has been conducted . Sensitivities obtained were 10, 50 ng/ml, 5 x 10(5), and 5 x 10(4) cfu/ml, respectively.

J Vet Med Sci, 2000 Jul, 62(7), 797 - 800
Sequence of 16S rRNA gene of rat-origin cilia-associated respiratory (CAR) bacillus SMR strain; Kawano A et al.; The 16S rRNA gene of the SMR strain of cilia-associated respiratory (CAR) bacillus, which was isolated from a spontaneously infected rat at our institute, was sequenced . Its 1,521 nucleotides were determined . On the basis of the results of the sequence analysis, the SMR strain was found to be most closely related to members of the Flavobacter/Flexibacter group . This sequence was compared with the previously determined 16S rRNA gene sequences (rat-origin: three; mouse-origin: one; rabbit-origin: one) of CAR bacillus isolates . The SMR strain showed the highest sequence similarity (99.9%) to the rat-origin CARB-NIH strain (Schoeb et al., 1993), and it was concluded that the strains are identical.

Biosci Biotechnol Biochem, 2000 Jul, 64(7), 1379 - 93
The primary structure of the subunit in Bacillus thermoamyloliquefaciens KP1071 molecular weight 540,000 homohexameric alpha-glucosidase II belonging to the glycosyl hydrolase family 31; Kashiwabara S et al.; The gene that coded for the subunit of an molecular weight (Mr) 540,000 homohexameric alpha-glucosidase II (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) produced by Bacillus thermoamyloliquefaciens KP1071 (FERM-P8477) growing at 30 to 66 degrees C was expressed in Escherichia coli HB101 . The resulting homohexameric enzyme had a half-life of 10 min at 80 degrees C . Its purification and characterization showed that the enzyme was identical with the native one except for the latter deleting 7 N-terminal residues found in the former . The primary sequence of the subunit with 787 residues and an Mr of 91,070 deduced from the gene was 24-34% identical to the corresponding sequences of 15 alpha-glucosidases in the glycosyl hydrolase family 31 from 14 eukaryotic origins and the archaeon Sulfolobus solfataricus 98/2 . From the sequence analysis by the neural network method of Rost and Sander {Rost, B . and Sander, C., Proteins: Struct . Funct . Genet., 19, 55-72 (1994)}, we inferred that alpha-glucosidase II might make each subunit of 3 secondary structural regions, i.e., one N-terminal beta region, one central alpha/beta region with two catalytic residues Asp407 and Asp484, and one C-terminal beta region.

Int Dent J, 2000 Apr, 50(2), 103 - 7
Bacterial aerosols in the dental clinic: effect of time, position and type of treatment; Kedjarune U et al.; OBJECTIVES: The objectives of this study were to investigate changes in the concentration of total bacterial aerosols before, during, and after the working period at different positions within the same multichair dental clinics . Also to investigate the contribution to total bacterial aerosols, if any, of the aerosols generated from different types of dental procedures, as well as the environment . METHODS: Air sampling using a Slit-to-Agar air sampler at three positions in a multichair dental clinic, performed three times per day over a three week period before work, during work and after work . The second part of the study, in another multichair dental clinic, was performed before working and during three types of dental procedures . RESULTS: The concentration of total bacterial aerosols and Bacillus sp . in air which circulated in the dental clinic was lower at the end of the day than at the beginning . There was no significant change in the concentration of total bacterial aerosols in different positions in the dental clinic or after the three types of dental treatments . The concentration of Bacillus sp . in air not mainly generated during dental procedures and which may come from an environmental source, was reduced . CONCLUSIONS: This study suggests that the proportions of different types of bacteria in air may change before, during and after dental treatment . Preventive measures may need to be instituted to reduce build up of bacterial aerosols in the dental clinic during non-working periods.

J Chromatogr B Biomed Sci Appl, 2000 Jun 23, 743(1-2), 349 - 56
Extraction in aqueous two-phase systems of alkaline xylanase produced by Bacillus pumilus and its application in kraft pulp bleaching; Bim MA et al.; The aim of this work was to extract and to purify xylanase, produced by Bacillus pumilus from the crude fermentation broth, using aqueous two-phase systems (ATPS) . The xylanase was extracted by partitioning in ATPS composed of phosphate and polyethylene glycol (PEG) . The effect of tie-line length, PEG molecular mass and NaCl concentrations upon the purification factors and yields of xylanase were investigated by statistical design . The best system studied was that containing 22% PEG6000, 10% K2HPO4 and 12% NaCl with a purification factor of 33 and a 98% yield of enzyme activity . This system was also used for continuous extraction in a pulsed caps column . Subsequently, the xylanase from the crude fermentation broth was tested in hardwood kraft pulp bleaching.

Int J Food Microbiol, 2000 Jul 15, 58(3), 203 - 12
Biological variability and exposure assessment; Delignette-Muller ML et al.; Predictive models are now commonly used for exposure assessment, with growth parameters defined for each microbial species . In this study, we tried to take into account microbial growth variability among strains of a single species . Bacillus cereus in pasteurized milk was chosen to illustrate the influence of the biological variability on the outcome of exposure assessment . Each parameter of the exposure assessment (growth parameters, shelf-life conditions) was characterized by a probability distribution describing variability and/or uncertainty . The impact of the intra-species variability on the result of the exposure assessment was then quantified and discussed . Two simple domestic shelf life conditions were tested . The results confirm that the biological variability has a great impact on the accuracy of the result and should not be systematically neglected.

Free Radic Biol Med, 2000 Jun 1, 28(11), 1611 - 8
Oxidative cellular damage associated with transformation of Helicobacter pylori from a bacillary to a coccoid form; Nakamura A et al.; Exposure to unfavorable conditions results in the transformation of Helicobacter pylori, a gastric pathogen, from a bacillary form to a coccoid form . The mechanism and pathophysiological significance of this transformation remain unclear . The generation of the superoxide radical by H . pylori has previously been shown to inhibit the bactericidal action of nitric oxide, the concentration of which is relatively high in gastric juice . With the use of chemiluminescence probes, both the quality and quantity of reactive oxygen species generated by H . pylori have now been shown to change markedly during the transformation from the bacillary form to the coccoid form . The transformation of H . pylori was associated with oxidative modification of cellular proteins, including urease, an enzyme required for the survival of this bacterium in acidic gastric juice . Although the cellular abundance of urease protein increased during the transformation, the specific activity of the enzyme decreased and it underwent aggregation . Specific activities of both superoxide dismutase and catalase in H . pylori also decreased markedly during the transformation . The transformation of H . pylori was also associated with oxidative modification of DNA, as revealed by the generation of 8-hydroxyguanine, and subsequent DNA fragment . These observations indicate that oxidative stress elicited by endogenously generated reactive oxygen species might play an important role in the transformation of H . pylori from the bacillary form to the coccoid form.

Med Klin (Munich), 2000 Jun 15, 95(6), 314 - 20
{Update in infectious diseases . Part I: epidemiology}; Salzberger B et al.; A number of infectious agents has been newly detected in the last 10 years . Climatic changes and migration have been the most important factors in the emergence of new and old infections . Additionally, new methods for the detection of DNA and RNA have played an important role in the detection of agents difficult to culture . Relevant new bacterial pathogens are Bartonella henselae (cat scratch disease, bacillary angiomatosis), Tropheryma whippeli (Whipple's disease) and new Rickettsiae . Newly detected viral pathogens include Sin-nombre virus (pulmonary Hanta virus syndrome), Nipah- and Hendra virus and avian influenza . Bovine spongiform encephalopathy has been transmitted to humans causing the newly described syndrome of variant Creuzfeldt-Jakob disease . The extent of this new epidemic is not yet clear . These trends from the last years clearly indicate, that further new infections and infectious agents will be detected in the future.

Protein Sci, 2000 Jul, 9(7), 1402 - 6
Structural effects of the active site mutation cysteine to serine in Bacillus cereus zinc-beta-lactamase; Chantalat L et al.; Beta-lactamases are involved in bacterial resistance . Members of the metallo-enzyme class are now found in many pathogenic bacteria and are becoming thus of major clinical importance . Despite the availability of Zn-beta-lactamase X-ray structures their mechanism of action is still unclear . One puzzling observation is the presence of one or two zincs in the active site . To aid in assessing the role of zinc content in beta-lactam hydrolysis, the replacement by Ser of the zinc-liganding residue Cys168 in the Zn-beta-lactamase from Bacillus cereus strain 569/H/9 was carried out: the mutant enzyme (C168S) is inactive in the mono-Zn form, but active in the di-Zn form . The structure of the mono-Zn form of the C168S mutant has been determined at 1.85 A resolution . Ser168 occupies the same position as Cys168 in the wild-type enzyme . The protein residues mostly affected by the mutation are Asp90-Arg91 and His210 . A critical factor for the activity of the mono-Zn species is the distance between Asp90 and the Zn ion, which is controlled by Arg91: a slight movement of Asp90 impairs catalysis . The evolution of a large superfamily including Zn-beta-lactamases suggests that they may not all share the same mechanism.

Rev Latinoam Microbiol, 1997 Jan-Jun, 39(1-2), 73 - 81
Purification and partial characterization of a chromate reductase from Bacillus; Campos-Garcia J et al.; A soluble NADH-dependent enzyme capable of reducing hexavalent chromium {Cr(VI)} to the trivalent form {Cr(III)} was purified from chromate-resistant Bacillus QC1-2 . An enriched single protein band of 24 kDa was observed by SDS-PAGE following HPLC ion-exchange and size-exclusion procedures . In the latter step, the chromate reductase showed a molecular mass of 44 kDa, which suggested that the enzyme consists of two subunits of about 24 kDa . Purified chromate reductase displayed optimal activity at a temperature and pH of 37 degrees C and 7.0, respectively . The enzyme showed a Km of 0.35 mM for chromate and a Vmax of 50 nmol Cr(VI) reduced per minute per mg protein.

Clin Exp Immunol, 2000 Aug, 121(2), 216 - 25
Immunotherapy of a human papillomavirus (HPV) type 16 E7-expressing tumour by administration of fusion protein comprising Mycobacterium bovis bacille Calmette-Guérin (BCG) hsp65 and HPV16 E7; Chu NR et al.; Human papillomavirus type 16 (HPV16) infection has been linked to the development of cervical and anal dysplasia and cancer . One hallmark of persistent infection is the synthesis of the viral E7 protein in cervical epithelial cells . The expression of E7 in dysplastic and transformed cells and its recognition by the immune system as a foreign antigen make it an ideal target for immunotherapy . Utilizing the E7-expressing murine tumour cell line, TC-1, as a model of cervical carcinoma, an immunotherapy based on the administration of an adjuvant-free fusion protein comprising Mycobacterium bovis BCG heat shock protein (hsp)65 linked to HPV16 E7 (hspE7) has been developed . The data show that prophylactic immunization with hspE7 protects mice against challenge with TC-1 cells and that these tumour-free animals are also protected against re-challenge with TC-1 cells . In addition, therapeutic immunization with hspE7 induces regression of palpable tumours, confers protection against tumour re-challenge and is associated with long-term survival (> 253 days) . In vitro analyses indicated that immunization with hspE7 leads to the induction of a Th1-like cell-mediated immune response based on the pattern of secreted cytokines and the presence of cytolytic activity following antigenic recall . In vivo studies using mice with targeted mutations in CD8 or MHC class II or depleted of CD8 or CD4 lymphocyte subsets demonstrate that tumour regression following therapeutic hspE7 immunization is CD8-dependent and CD4-independent . These studies extend previous observations on the induction of cytotoxic T lymphocytes by hsp fusion proteins and are consistent with the clinical application of hspE7 as an immunotherapy for human cervical and anal dysplasia and cancer.

Immunology, 2000 Aug, 100(4), 494 - 501
Increased resistance to mycobacterial infection in the absence of interleukin-10; Jacobs M et al.; Interleukin-10 (IL-10) down-regulates T helper type 1 cell and macrophage functions . As IL-10 is induced along with tumour necrosis factor (TNF) and IL-12 in mycobacterial infection, we asked whether endogenous IL-10 plays a role in the antimycobacterial response . We demonstrate here that IL-10-deficient mice eliminate Mycobacterium bovis Calmette-Guerin bacillus faster than wild-type mice . Granulomas are significantly larger, containing more CD-11b- and CD11c-positive antigen-presenting cells and T cells, and the expression of major histocompatibility complex class II and intracellular adhesion molecule-1 is increased . Macrophages in granulomas of IL-10-deficient mice express high levels of TNF, acid phosphatase and inducible nitric oxide synthase (iNOS) . Finally, an increased cutaneous delayed-type hypersensitivity reaction to mycobacterial proteins is further evidence of an augmented cell-mediated immune response . In conclusion, the cell-mediated immunity is enhanced in the absence of IL-10, resulting in a robust granuloma response, which accelerates the clearance of mycobacteria . Therefore, endogenous IL-10 attenuates mycobacterial immunity.

PDA J Pharm Sci Technol, 2000 May-Jun, 54(3), 172 - 92
A comparison of the MicroCount Digital System to plate count and membrane filtration methods for the enumeration of microorganisms in water for pharmaceutical purposes; Marino G et al.; The enumeration of microorganisms in water for pharmaceutical purposes using the MicroCount Digital System (Millipore Corporation, Bedford, MA) was compared to the USP-recommended Pour Plate and Membrane Filtration Count methods . A study, using a pure culture of Buckholderia cepacia, ATCC#25416, showed that the accuracy, precision, reproducibility and linearity of the MicroCount ATP Bioluminescence System was equivalent to or better than the traditional methods . When the MicroCount System was used to monitor purified water and water for injection taps in a pharmaceutical plant over a month, comparable counts to the traditional methods were obtained within 24 hours compared to 48 to 72 hours with the other methods . The effectiveness of the memory device used for the isolation of colonies for characterization was demonstrated by comparing the number and pattern of the positive wells in the MicroCount plates with the isolation of colonies on the microbial count agar plates . The recovery on agar plates, although slightly higher, was not statistically different to the MicroCount plates . The predominated microorganisms isolated using all three methods were Ralstonia pickettii, Bacillus sphaericus, Stenotrophomonas maltophia, and a Staphylococcus species.

Biochem J, 2000 Aug 15, 350 Pt 1, 321 - 8
Purification and characterization of Ak.1 protease, a thermostable subtilisin with a disulphide bond in the substrate-binding cleft; Toogood HS et al.; Ak.1 protease, a thermostable subtilisin isolated originally from Bacillus st . Ak.1, was purified to homogeneity from the Escherichia coli clone PB5517 . It is active against substrates containing neutral or hydrophobic branched-chain amino acids at the P(1) site, such as valine, alanine or phenylalanine . The K(m) and k(cat) of the enzyme decrease with decreasing temperature, though not to the same degree with all substrates, suggesting that specificity changes with temperature . The protease is markedly stabilized by Ca(2+) ions . At 70 degrees C, a 10-fold increase in Ca(2+) concentration increases the half-life by three orders of magnitude . Ak.1 protease is stabilized by Ca(2+) to a greater extent than is thermitase . This may be due, in part, to the presence of an extra Ca(2+)-binding site in Ak.1 protease . Other metal ions, such as Sr(2+), increase the thermostability of the enzyme, but to a significantly lower degree than does Ca(2+) . The structure of the protease showed the presence of a disulphide bond located within the active-site cleft . This bond influences both enzyme activity and thermostability . The disulphide bond appears to have a dual role: maintaining the integrity of the substrate-binding cleft and increasing the thermostability of the protease . The protease was originally investigated to determine its usefulness in the clean-up of DNA at high temperatures . However, it was found that this protease has a limited substrate specificity, so this application was not explored further.

Biochem J, 2000 Aug 15, 350 Pt 1, 275 - 82
Membrane pore architecture of a cytolytic toxin from Bacillus thuringiensis; Promdonkoy B et al.; To investigate the membrane pore structure of Cyt2Aa1 toxin from Bacillus thuringiensis, 14 single-cysteine substitutions of the toxin were constructed . Five of these mutants (L172C, V186C, L189C, E214C and L220C) yielded characteristic products when processed by proteinase K; other mutants were degraded by this enzyme . Mutants that yielded characteristic proteolysed products and wild-type toxin were labelled with polarity-sensitive acrylodan (6-acryloyl-2-dimethylaminonaphthalene) at the thiol group of cysteine residues . A green-blue shift in the emission spectra was observed with all labelled toxins on transfer from an aqueous solution into a solution containing membranes or liposomes from red blood cells . These results suggested that the label moved into the hydrophobic environment of the membrane or became buried within hydrophobic regions of the protein oligomers . Digestion of membrane-bound labelled toxin with proteinase K did not cause a significant decrease in emission intensity from any of the labelled mutants . This suggests that L172C, V186C, L189C, E214C and L220C are inserted into the membrane and are therefore protected from proteolysis . In contrast, a marked decrease in emission intensity was observed when membrane-bound labelled wild-type toxin was digested with proteinase K . This suggests that Cys-19 does not insert into the membrane . Fluorimetric analysis of delipidated pore complexes suggests that L172C, V186C, L189C and E214C point towards the lipid in the membrane, whereas L220C is either within the hydrophobic environment of the protein oligomers or exposed to the membrane lipids . Most of the Cys-19 from wild-type molecules is enclosed within the hydrophobic pockets of the protein oligomers.

Am J Physiol Lung Cell Mol Physiol, 2000 Aug, 279(2), L216 - 23
Surfactant protein A enhances mycobacterial killing by rat macrophages through a nitric oxide-dependent pathway; Weikert LF et al.; Surfactant-associated protein A (SP-A) is involved in surfactant homeostasis and host defense in the lung . We have previously demonstrated that SP-A specifically binds to and enhances the ingestion of bacillus Calmette-Guerin (BCG) organisms by macrophages . In the current study, we investigated the effect of SP-A on the generation of inflammatory mediators induced by BCG and the subsequent fate of ingested BCG organisms . Rat macrophages were incubated with BCG in the presence and absence of SP-A . Noningested BCG organisms were removed, and the release of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide were measured at varying times . TNF-alpha and nitric oxide production induced by BCG were enhanced by SP-A . In addition, SP-A enhanced the BCG-induced increase in the level of inducible nitric oxide synthase protein . Addition of antibodies directed against SPR210, a specific macrophage SP-A receptor, inhibited the SP-A-enhanced mediator production . BCG in the absence of SP-A showed increased growth over a 5-day period, whereas inclusion of SP-A dramatically inhibited BCG growth . Inhibition of nitric oxide production blocked BCG killing in the presence and absence of SP-A . These results demonstrate that ingestion of SP-A-BCG complexes by rat macrophages leads to production of inflammatory mediators and increased mycobacterial killing.

Genetics, 2000 Aug, 155(4), 1693 - 9
Bacillus thuringiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans; Marroquin LD et al.; The protein toxins produced by Bacillus thuringiensis (Bt) are the most widely used natural insecticides in agriculture . Despite successful and extensive use of these toxins in transgenic crops, little is known about toxicity and resistance pathways in target insects since these organisms are not ideal for molecular genetic studies . To address this limitation and to investigate the potential use of these toxins to control parasitic nematodes, we are studying Bt toxin action and resistance in Caenorhabditis elegans . We demonstrate for the first time that a single Bt toxin can target a nematode . When fed Bt toxin, C . elegans hermaphrodites undergo extensive damage to the gut, a decrease in fertility, and death, consistent with toxin effects in insects . We have screened for and isolated 10 recessive mutants that resist the toxin's effects on the intestine, on fertility, and on viability . These mutants define five genes, indicating that more components are required for Bt toxicity than previously known . We find that a second, unrelated nematicidal Bt toxin may utilize a different toxicity pathway . Our data indicate that C . elegans can be used to undertake detailed molecular genetic analysis of Bt toxin pathways and that Bt toxins hold promise as nematicides.

Biochemistry, 2000 Aug 8, 39(31), 9099 - 107
Structural analysis of a chimeric bacterial alpha-amylase . High-resolution analysis of native and ligand complexes; Brzozowski AM et al.; Several chimeric alpha-amylases genes were constructed by an in vivo recombination technique from the Bacillus amyloliquefaciens and Bacillus licheniformis genes . One of the fusion amylases (hereafter BA2), consisting of residues 1-300 from B . amyloliquefaciens and 301-483 from B . licheniformis, has been extensively studied by X-ray crystallography at resolutions between 2.2 and 1.7 A . The 3-dimensional structure of the native enzyme was solved by multiple isomorphous replacement, and refined at a resolution of 1.7 A . It consists of 483 amino acids, organized similarly to the known B . lichiniformis alpha-amylase structure {Machius et al . (1995) J . Mol . Biol . 246, 545-559}, but features 4 bound calcium ions . Two of these form part of a linear cluster of three ions, the central ion being attributed to sodium . This cluster lies at the junction of the A and B domains with one calcium of the cluster structurally equivalent to the major Ca(2+) binding site of fungal alpha-amylases . The third calcium ion is found at the interface of the A and C domains . BA2 contains a fourth calcium site, not observed in the B . licheniformis alpha-amylase structure . It is found on the C domain where it bridges the two beta-sheets . Three acid residues (Glu261, Asp328, and Asp231) form an active site similar to that seen in other amylases . In the presence of TRIS buffer, a single molecule of TRIS occupies the -1 subsite of the enzyme where it is coordinated by the three active-center carboxylates . Kinetic data reveal that BA2 displays properties intermediate to those of its parents . Data for crystals soaked in maltooligosaccharides reveal the presence of a maltotriose binding site on the N-terminal face of the (beta/alpha)(8) barrel of the molecule, not previously described for any alpha-amylase structure, the biological function of which is unclear . Data for a complex soaked with the tetrasaccharide inhibitor acarbose, at 1.9 A, reveal a decasaccharide moiety, spanning the -7 to +3 subsites of the enzyme . The unambiguous presence of three unsaturated rings in the (2)H(3) half-chair/(2)E envelope conformation, adjacent to three 6-deoxypyranose units, clearly demonstrates synthesis of this acarbose-derived decasaccharide by a two-step transglycosylation mechanism.

Curr Microbiol, 2000 Jul, 41(1), 11 - 4
Biodegradation of phenanthrene by a Bacillus species; Doddamani HP et al.; A bacterial strain capable of utilizing phenanthrene as sole source of carbon was isolated from soil and identified as a Bacillus sp . The organism also utilized naphthalene, biphenyl, anthracene, and other aromatic compounds as growth substrates . The organism degraded phenanthrene through the intermediate formation of 1-hydroxy-2-naphthoic acid, which was further metabolized via o-phthalate by a protocatechuate pathway, as evidenced by oxygen uptake and enzymatic studies.

Appl Microbiol Biotechnol, 2000 Jun, 53(6), 668 - 73
Investigating expression systems for the stable large-scale production of recombinant L-leucine-dehydrogenase from Bacillus cereus in Escherichia coli; Ansorge MB et al.; The established Escherichia coli expression vectors ptrc99a, pKK223-3, pPLlambda, pAsk75, pRA95, and pRA96, which differ in copy number, mode of induction, selection marker, and use of par sequences for stabilization, were investigated for the stable expression of recombinant L-leucine dehydrogenase from Bacillus cereus with a view to large-scale production . Best results were achieved with pIET98, a runaway-replication system derived from pRA96 . Expression of L-leucine dehydrogenase was controlled by its constitutive B . cereus promoter and depended on host strain, cultivation temperature, induction time, and media composition . After cell cultivation at 30 degrees C and shifting to 41 degrees C to induce plasmid replication, E . coli BL21{pIET98} yielded 200 U LeuDH/mg protein, which corresponds to >50% of the soluble cell protein . Continuous cultivation in a semisynthetic high-cell-density medium verified structural and segregational stability over 100 generations in the absence of a selection pressure.

Appl Microbiol Biotechnol, 2000 Jun, 53(6), 640 - 5
Efficient secretory production of alkaline phosphatase by high cell density culture of recombinant Escherichia coli using the Bacillus sp . endoxylanase signal sequence; Choi JH et al.; New secretion vectors containing the Bacillus sp . endoxylanase signal sequence were constructed for the secretory production of recombinant proteins in Escherichia coli . The E . coli alkaline phosphatase structural gene fused to the endoxylanase signal sequence was expressed from the trc promoter in various E . coli strains by induction with IPTG . Among those tested, E . coli HB101 showed the highest efficiency of secretion (up to 25.3% of total proteins) . When cells were induced with 1 mM IPTG, most of the secreted alkaline phosphatase formed inclusion bodies in the periplasm . However, alkaline phosphatase could be produced as a soluble form without reduction of expression level by inducing with less (0.01 mM) IPTG, and greater than 90% of alkaline phosphatase could be recovered from the periplasm by the simple osmotic shock method . Fed-batch cultures were carried out to examine the possibility of secretory protein production at high cell density . Up to 5.2 g/l soluble alkaline phosphatase could be produced in the periplasm by the pH-stat fed-batch cultivation of E . coli HB101 harboring pTrcS1PhoA . These results demonstrate the possibility of efficient secretory production of recombinant proteins in E . coli by high cell density cultivation.

Biomed Pharmacother, 2000 Jun, 54(5), 268 - 73
Use of xenogenized (modified) tumor cells for treatment in experimental tumor and in human neoplasia; Ben-Efraim S et al.; The need to modify tumor cells in order to render them more "immunogenic" was based on the assumption that normal, nonmodified tumor cells are non- or weakly immunogenic and as such are unable to raise an efficient protective immune response . Various methods for "xenogenization" (modification of tumor cells) were suggested: induction of new foreign antigens, treatment with either chemicals or enzymes and use of mutagens . Xenogenized tumor cells by their coupling to proteins, and use of chemicals like DTIC (5-{3,3-dimethyl- 1-triazeno}-imidazole-4-carboxamide), TZC (8-carbamoyl-3-methyl-imidazo{5, 1-d}- 1,2,3,5-tetrazin-4 {3H}-one 8-carbamoyl-3-{2-chloroethyl} imidazole {5,1 -d}- 1,2,3,5-tetrazin-4{3H}-one) and antiemetic drugs, were tested in experimental models of murine leukemia . Non-tumorigenic clones, xenogenization with DNA hypomethylating agents, aryl-triazine derivatives and DTIC were evaluated for their induction of protective immune response in murine lymphoma . Murine plasmacytoma cells were used for immunization after treatment with glutaraldehyde . Viral modifications of tumor cells were evaluated for their ability to induce a protective tumor response in model systems of rat fibrosarcoma, liver metastatic rat tumor cells, lymphoid tumor cells and hamster tumor cells . In the case of human cancer, attempts were reported to use DNP-conjugated melanoma cells, mutagenic triazine compounds, an autologous colon tumor cell bacillus Calmette-Guerin (BCG) vaccine and genetically engineered vaccines for immunization . The general conclusion drawn from experimental tumor models and for human cancer is, that although modified tumor cells were found to be partially effective in experimental models, it is still necessary to provide more data in order to determine the effective use of xenogenized human tumor cells for immunotherapy.

Biosci Biotechnol Biochem, 2000 Jun, 64(6), 1210 - 6
Molecular cloning, overproduction and characterization of the Bacillus cereus IMP dehydrogenase; Kim SI et al.; The gene of IMP dehydrogenase of Bacillus cereus ts-4, a temperature-sensitive mutant of B . cereus JCM 2152, was subcloned and its sequence was analyzed . A B . cereus ts-4 DNA fragment of 2,065 bp containing the entire impdh gene and flanking regions was sequenced . The fragment contained an open reading frame of 1,527 bp encoding 509 amino acids with a calculated molecular mass of 55,390 Da . The impdh sequence of JCM 2152 was also analyzed by TA cloning using PCR products amplified with primers from B . cereus ts-4 impdh gene . The gene amplified by PCR was expressed in Escherichia coli using a pET17 x b expression plasmid . The N-terminal amino acid sequence of the overproduced enzyme was identified as Met-Trp-Glu-Ser-Lys-Phe-Val-Lys-Glu-Gly-Leu-Thr-Phe-AspAsp-Val-Leu -Leu-Val- Pro . The overproduced enzyme was eluted at a molecular mass of about 225 kDa by gel filtration . The molecular mass of the subunit was estimated to be 56 kDa by SDS-PAGE . The overproduced enzyme was active against IMP, IDP, and ITP, and showed the highest activity at pH 9.5 . These properties of the recombinant enzyme were almost identical to those of IMP dehydrogenase of B . cereus.

Zhonghua Yi Xue Za Zhi, 1998 Jul, 78(7), 540 - 3
{Effects of selective induceble nitric oxide synthase inhibitor on immunological hepatic injury in rat}; Zhang G et al.; OBJECTIVE: To investigate the role and induced regulation of nitric oxide (NO) in rat immunological hepatic injury, and the effect of selective inhibitor on induceble nitric oxide synthase (iNOS) . METHODS: The intracellular NOS gene expression in the immune damaged hepatocytes was determined by in situ PCR technique, and NO concentration and lactate dehydrogenase (LDH) activity in culture supernatant were measured . RESULTS: Administration of bacille calmette guerin (BCG, 15, 30, 50 mg/rat, i.v.) alone or BCG with the inflammatory cytokines mixture (CM), including IL-1 beta, IFN-gamma, TNF-alpha and lipopolysacchride (LPS) significantly increased NO production and LDH release in culture medium(P < 0.05) . NO production was enhanced with hepatic injury degree in direct proportion, and with BCG dose in inverse proportion . Under immunological stimuli condition, hepatocytes NOS mRNA mainly expressed an induceble and soluble isoform (iNOS) in cytoplasm . Aminoguanidine (AG), a selective iNOS inhibitor, inhibited NO production and LDH release (83.4% and 36.0%, P < 0.05), but the transcription inhibitor actinomycin D enhanced LDH level in the medium (25.5%, P < 0.05) . CONCLUSION: NO produced by immunological stimuli participates in rat hepatic injury mechanism.

FEMS Microbiol Ecol, 2000 Jul 1, 33(1), 35 - 39
Insecticidal toxin from Bacillus thuringiensis is released from roots of transgenic Bt corn in vitro and in situ; Saxena D et al.; The insecticidal toxin encoded by the cry1Ab gene from Bacillus thuringiensis was released in root exudates from transgenic Bt corn during 40 days of growth in soil amended to 0, 3, 6, 9, or 12% (v/v) with montmorillonite or kaolinite in a plant growth room and from plants grown to maturity in the field . The presence of the toxin in rhizosphere soil was determined by immunological and larvicidal assays . No toxin was detected in any soils from isogenic non-Bt corn or without plants . Persistence of the toxin was apparently the result of its binding on surface-active particles in the soils, which reduced the biodegradation of the toxin . The release of the toxin could enhance the control of insect pests or constitute a hazard to nontarget organisms, including the microbiota of soil, and increase the selection of toxin-resistant target insects.

J Clin Microbiol, 2000 Aug, 38(8), 2943 - 8
Characterization of Bartonella clarridgeiae flagellin (FlaA) and detection of antiflagellin antibodies in patients with lymphadenopathy; Sander A et al.; Cat scratch disease (CSD) is a frequent clinical outcome of Bartonella henselae infection in humans . Recently, two case reports indicated Bartonella clarridgeiae as an additional causative agent of CSD . Both pathogens have been isolated from domestic cats, which are considered to be their natural reservoir . B . clarridgeiae and B . henselae can be distinguished phenotypically by the presence or absence of flagella, respectively . Separation of the protein content of purified flagella of B . clarridgeiae by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis indicated that the flagellar filament is mainly composed of a polypeptide with a mass of 41 kDa . N-terminal sequencing of 20 amino acids of this protein revealed a perfect match to the N-terminal sequence of flagellin (FlaA) as deduced from the sequence of the flaA gene cloned from B . clarridgeiae . The flagellin of B . clarridgeiae is closely related to flagellins of Bartonella bacilliformis and several Bartonella-related bacteria . Since flagellar proteins are often immunodominant antigens, we investigated whether antibodies specific for the FlaA protein of B . clarridgeiae are found in patients with CSD or lymphadenopathy . Immunoblotting with 724 sera of patients suffering from lymphadenopathy and 100 healthy controls indicated specific FlaA antibodies in 3.9% of the patients' sera but in none of the controls . B . clarridgeiae FlaA is thus antigenic and expressed in vivo, providing a valuable tool for serological testing . Our results further indicate that B . clarridgeiae might be a possible etiologic agent of CSD or lymphadenopathy . However, it remains to be clarified whether antibodies to the FlaA protein of B . clarridgeiae are a useful indicator of acute infection.

J Med Entomol, 2000 Jul, 37(4), 534 - 40
Laboratory selection for resistance to Bacillus sphaericus in Culex quinquefasciatus (Diptera: Culicidae) from California, USA; Wirth MC et al.; A previously untreated field population of Culex quinquefasciatus Say, collected near Bakersfield, CA, was subjected to intensive laboratory selection with the bacterial insecticide Bacillus sphaericus Neide (strain 2362) at a level producing 95% mortality . Resistance rapidly appeared and resistance levels increased such that fourth instars of generation 12 were able to survive a concentration of B . sphaericus that was 7,000 times higher than the median lethal concentration (LC50) of the susceptible reference colony . Similar resistance levels were detected in first instars . Cross-resistance in the selected colony was detected toward B . sphaericus strains 1593 and 2297, but little or no cross-resistance was observed toward B . sphaericus strains IAB59 or ISPC5 (= WHO 2173) . Cross-resistance also was not detected toward the bacterial insecticide Bacillus thuringiensis subsp . israelensis, toward a recombinant strain expressing both B . thuringiensis subsp . israelensis and B . sphaericus (strain 1593) toxins, toward individual or multiple toxins from B . thuringiensis subsp . israelensis, or toward conventional synthetic insecticides . Genetic analysis revealed that B . sphaericus resistance was inherited as a recessive trait and controlled by a single major locus . These data are discussed in relation to cases of field resistance toward this biopesticide in the Cx . pipiens (L.) complex.

Curr Microbiol, 2000 Sep, 41(3), 214 - 9
Bacillus thuringiensis delta-endotoxin proteins show a correlation in toxicity and short circuit current inhibition against Helicoverpa zea; Karim S et al.; Pesticidal activity of Bacillus thuringiensis delta-endotoxins, Cry1Aa, Cry1Ab, Cry1Ac, and Cry2A, was determined by using the force-feeding bioassay method to 4(th) instar larvae of Helicoverpa zea . H . zea was susceptible to Bt toxins in the order Cry1Ac > Cry1Ab > Cry1Aa > Cry2A with 63.60, 89.04, 159.65, and 375.78 ng/larvae respectively . The abilities of selected Bacillus thuringiensis toxins to inhibit short circuit current (I(SC)) in midgut epithelia of H . zea were also investigated by voltage clamp assay . The voltage-clamp studies were conducted on isolated midguts, measuring the inhibition of short circuit current (I(SC)) by activated toxin . A Cry1Aa toxin dilution of 33.3 and 500 ng/ml resulted in inhibition of I(SC) of -2.29 microA/min (lag time 15 min) and -4.48 microA/min (lag time, 2 min) respectively . The Cry1Ab dilution of 25 ng/ml inhibited I(SC) to -1.39 microA/min, a lag time of 14 min, and 333.3 ng/ml dilution resulted in decay of I(SC) -2.49 microA/min, lag time 1 min respectively . The Cry1Ac lower dilution 16.7 ng/ml inhibited I(SC) to -1.39 microA/min, lag time 4 min, and a high dilution 333.3 ng/ml decay I(SC) to -2.44 microA/min, lag time 1 min . The inhibition of I(SC) (-1.10 microA/min, lag time 25) at lower dilution (33.3 ng/ml) and high dilution (500 ng/ml), decay (-2.38 microA/min, lag time 5 min), showed a correlation between toxin concentration and inhibitory response with Cry2A toxin . The lag time decreased with increasing concentration of toxin applied, which is additional evidence of dose response besides direct correlation of toxicity assays and I(SC).

Curr Microbiol, 2000 Sep, 41(3), 187 - 91
Hyper-production of insecticidal crystal protein (delta-endotoxin) by Bacillus thuringiensis var . israelensis is not related to sporulation-specific biochemical functions; Bhattacharya PR; Hypertoxic mutant strains of Bacillus thuringiensis var . israelensis were isolated by mutagenesis of the parent strain . The correlation, if any, between hyper-production of insecticidal crystal protein (delta-endotoxin) by hypertoxic mutant strains of Bacillus thuringiensis var . israelensis and sporulation-specific biochemical functions was studied . No increase in sporulation-specific biochemical markers was observed in the hypertoxic mutant strains . Asporogenous mutants of hypertoxic mutant strains blocked at different stages of sporulation were isolated, and larvicidal activity was studied . The hypertoxic parent strains and the sporulation-deficient, hypertoxic mutant strains showed almost identical larvicidal activity . Therefore, the increased production of toxin is not related to sporulation-specific biochemical changes.

J Food Prot, 2000 Jul, 63(7), 926 - 9
Growth of Bacillus cereus on solid media as affected by agar, sodium chloride, and potassium sorbate; Stecchini ML et al.; The effect of two independent variables: microstructure, as modified by the agar content (1.0, 4.0, 7.0%), and water activity (a(w)), as modified by the NaCl content (0.5, 2.5, 4.5%), in the absence or in the presence of potassium sorbate (0.0; 2,000 ppm) on Bacillus cereus growth on solid media was studied . The time to visible growth (TVG) and the radial growth rate (RGR) of colonies were evaluated . TVG was not affected by microstructure and K-sorbate, although when a(w) was reduced, TVG tended to increase . RGR depended on linear effects of microstructure and a(w) variables and their interaction . When K-sorbate was added to cultural media, RGR was reduced significantly . However, in the presence of K-sorbate, RGR was found to change only when a(w) vas varied.

FEMS Microbiol Lett, 2000 Aug 1, 189(1), 55 - 9
Hemin-dependent growth and hemin binding of Bartonella henselae; Sander A et al.; Bartonella henselae causes cat-scratch disease and bacillary angiomatosis peliosis . The bacteria reside in erythrocytes of asymptomatic cats, which represent the natural reservoir for this pathogen . B . henselae is usually grown on blood-enriched media . Growth experiments on Brucella medium without blood demonstrated that heme compounds are essential for the growth of B . henselae and can completely substitute the addition of blood components . The heme precursor protoporphyrin IX alone, or in combination with FeCl(2) or FeCl(3), as well as transferrin or lactoferrin did not support growth, indicating that B . henselae cannot synthesize heme itself . Hemin supported growth even when free iron was chelated, indicating that hemin is also used as an iron source . Binding assays showed that hemin starvation increased the binding capacity of B . henselae for hemin, providing evidence that the bacteria carry a specific hemin uptake system, which might be regulated by hemin.

FEBS Lett, 2000 Jul 7, 476(3), 194 - 7
Kinetic analysis of the reaction catalyzed by chitinase A1 from Bacillus circulans WL-12 toward the novel substrates, partially N-deacetylated 4-methylumbelliferyl chitobiosides; Honda Y et al.; The kinetic behavior of chitinase A1 from Bacillus circulans WL-12 was investigated using the novel fluorogenic substrates, N-deacetylated 4-methylumbelliferyl chitobiosides {GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)(2)-UMB (4)}, and the results were compared with those obtained using 4-methylumbelliferyl N, N'-diacetylchitobiose {(GlcNAc)(2)-UMB (1)} as the substrate . The chitinase did not release the UMB moiety from compound 4, but successfully released UMB from the other substrates . k(cat)/K(m) values determined from the releasing rate of the UMB moiety were: 145.3 for 1, 8.3 for 2, and 0.1 s(-1) M(-1) for 3 . The lack of an N-acetyl group at subsite (-1) reduced the activity to a level 0.1% of that obtained with compound 1, while the absence of the N-acetyl group at subsite (-2) reduced the relative activity to 5.7% . These observations strongly support the theory that chitinase A1 catalysis occurs via a 'substrate-assisted' mechanism . Using these novel fluorogenic substrates, we were able to quantitatively evaluate the recognition specificity of subsite (-2) toward the N-acetyl group of the substrate sugar residue . The (-2) subsite of chitinase A1 was found to specifically recognize an N-acetylated sugar residue, but this specificity was not as strict as that found in subsite (-1).

Clin Infect Dis, 2000 Jul, 31(1), 131 - 5 Epub 2000 Jul 25.
Bartonella quintana and urban trench fever; Ohl ME et al.; Contemporary Bartonella quintana infections have emerged in diverse regions of the world, predominantly involving socially disadvantaged persons . Available data suggest that the human body louse Pediculus humanus is the vector for transmission of B . quintana . Descriptions of the clinical manifestations associated with contemporary B . quintana infections have varied considerably and include asymptomatic infection, a relapsing febrile illness, headache, leg pain, "culture-negative" endocarditis, and, in human immunodeficiency virus-infected persons, bacillary angiomatosis . Laboratory diagnosis is most convincing when B . quintana is isolated in blood culture, but growth often takes 20-40 days; problems exist with both sensitivity and specificity of serological assays . On the basis of available information, use of doxycycline, erythromycin, or azithromycin to treat B . quintana infections is recommended . Treatment of uncomplicated B . quintana bacteremia for 4-6 weeks and treatment of B . quintana endocarditis (in a person who does not undergo valve surgery) for 4-6 months are recommended, with the addition of a bactericidal agent (such as a third-generation cephalosporin or an aminoglycoside) during the initial 2-3 weeks of therapy for endocarditis.

Curr Opin Pulm Med, 2000 Jul, 6(4), 259 - 66
The use of adenosine deaminase and adenosine deaminase isoenzymes in the diagnosis of tuberculous pleuritis; Perez-Rodriguez E et al.; The bacillary population described in tuberculous pleuritis is small, and its most likely pathogenetic mechanism is essentially immunologic . This explains why, until now, the diagnostic identification of tuberculous pleuritis (TP) has been based on the presence of granulomas in pleural biopsy . Correcting this diagnostic deficiency through other parameters related to the specific pathogenetic mechanism has been widely studied . The determination of the levels of adenosine deaminase (ADA) in pleural fluid offers high performance in its discriminating capacity to identify TP (sensitivity 87 to 100%, specificity 81 to 97%) . Adenosine deaminase expresses the sum of two isoenzymes (ADA1 and ADA2) . ADA1 is ubiquitous in all cells, including lymphocytes and monocytes, whereas ADA2 is found only in monocytes . Analysis and determination of these isoenzymes have shown that ADA in TP increases particularly at the expense of ADA2 and that the ADA1 /ADAp activity ratio improves performance in terms of sensitivity, specificity, and efficacy (100%, 92 to 97%, and 98%, respectively) in correcting all false-negative and false-positive results except 1 to 9% of nonlymphoproliferative malignancies . Only the high performance of ADA in the identification of TP allows it to be assumed that pleural biopsy can be obviated, especially in patients aged less than 35 years of age or having a lymphocyte-to-neutrophil proportion of more than 0.75 in regions of high prevalence . Quick determination and low cost justify its routine use in exudates . The ADA1 /ADAp activity ratio improves performance even more and could be used in cases with uncertain diagnoses or in regions with low prevalence of tuberculosis.

Diabetes, 2000 Jul, 49(7), 1106 - 15
Aberrant macrophage cytokine production is a conserved feature among autoimmune-prone mouse strains: elevated interleukin (IL)-12 and an imbalance in tumor necrosis factor-alpha and IL-10 define a unique cytokine profile in macrophages from young nonobese diabetic mice; Alleva DG et al.; Cytokines derived from macrophages (Mo) play a critical role in the development of type 1 diabetes in the nonobese diabetic (NOD) mouse . Based on earlier findings from lupus-prone strains of inherent cytokine defects in Mo , NOD Mo were evaluated for intrinsically dysregulated cytokine production with the potential to initiate or exacerbate disease . Endotoxin-activated peritoneal Mo from young prediseased NOD mice produced interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha levels similar to those of Mo from a panel of control strains but reduced compared with the congenic diabetes-resistant NOR strain . IL-6 and IL-10 production were similar in NOD and NOR Mo, indicating that reduction in NOD IL-1 and TNF-alpha expression was selective . Nevertheless, the ratio of TNF-alpha and IL-10 production, a stringent index of normal Mo function, distinguished NOD from all normal strains . The most striking feature of NOD Mo, however, was their substantially elevated IL-12 production . This response was induced not only by endotoxin but also by bacillus Calmette-Guerin (BCG) and CD40 ligand and was associated with (and likely caused by) the enhanced and prolonged expression of p40 mRNA . Moreover, NOD Mo IL-12 expression appeared to be near maximally induced by lipopolysaccharide (LPS) alone, because it was only slightly enhanced by the addition of gamma-interferon, a stimulus that substantially elevated LPS-induced IL-12 production in Mo from normal strains . Accompanied by a unique profile of TNF-alpha and IL-10, the dramatic elevation of IL-12 expression by NOD Mo reflects intrinsic defects of the innate immune system with the potential to initiate and propagate the pathogenic autoreactive T-helper type 1 response characteristic of type 1 diabetes.

J Dairy Sci, 2000 Jul, 83(7), 1659 - 63
Johne's disease and milk: do consumers need to worry?
Stabel JR.
Mycobacterium paratuberculosis, an acid-fast bacillus that causes enteritis in ruminants, has been suggested as an etiological agent of Crohn's disease in humans . The mode of transmission is unclear; however, some evidence suggests that humans may become infected via contaminated milk . Currently, it is not known whether commercial pasteurization effectively kills M . paratuberculosis in contaminated raw milk . Using a laboratory-scale pasteurizer unit designed to simulate the high-temperature, short-time method (72 degrees C, 15 sec) currently used by commercial dairies, we previously demonstrated that treatment of raw milk inoculated with 10(4) to 10(6) cfu of M . paratuberculosis/ml reduced numbers to an undetectable level . However, M . paratuberculosis is an intracellular pathogen that resides within the macrophages of the host and evades destruction . We subsequently performed further experiments examining heat treatment of milk inoculated with mammary gland macrophages containing ingested M . paratuberculosis . Heat treatment of these samples under high-temperature, short-time conditions demonstrated that the macrophage does not protect the organism because we were unable to recover any viable M . paratuberculosis from the samples . Conversely, other researchers have demonstrated that a residual population of M . paratuberculosis may survive heat treatment of milk . In addition, a recent news report stated that viable M . paratuberculosis organisms have been cultured from retail-ready milk in Ireland . A summary of past and current studies concerning this issue along with a discussion of methodologies used to recover M . paratuberculosis from experimentally inoculated milk will be presented in this paper.

J Infect, 2000 May, 40(3), 287 - 90
Infective endocarditis and septic embolization with Ochrobactrum anthropi: case report and review of literature; Mahmood MS et al.; Ochrobactrum anthropi, previously known as CDC group Vd, is an aerobic, Gram-negative bacillus of low virulence that occasionally causes human infection . We describe a case of infective endocarditis with O . anthropi complicated by septic embolization . A review of all the literature reported cases of O . anthropi infection is presented and categorized into 'Central line related', 'Transplant related' and "Other pyogenic infections" . Mortality appears to be related to the underlying disease state, rather than the organism.

Can J Microbiol, 1999 Oct, 45(10), 816 - 25
Activation and fragmentation of Bacillus thuringiensis delta-endotoxin by high concentrations of proteolytic enzymes; Pang AS et al.; Commercial enzymes and insect gut juice at various concentrations were used to digest Bacillus thuringiensis subsp . sotto Cry1Aa protoxin and examine the fragmentation pattern and effect on insecticidal activity . Trypsin at both high (5 mg/mL) and low (0.05 mg/mL) concentrations converted protoxin to toxin with no difference in insecticidal activity against Bombyx mori larvae . In both cases, the toxin protein had an apparent M(r) of 58.4 kDa (SDS-PAGE) . Active toxin of identical M(r) was also produced with low concentrations of Pronase and subtilisin, but at high concentration, it was degraded into two protease-resistant fragments of apparent M(r) 31.8 and 29.6 kDa, and exhibited no insecticidal activity . Sequencing data established the primary cleavage site to be in domain II, the receptor-binding region of the toxin, in an exposed loop between two beta-sheet strands . Fragmentation was not observed, however, when the digests were analyzed by native protein techniques, but rather the toxin molecule appeared to be intact . The amount of activated toxin produced by Choristoneura fumiferana gut juice was markedly reduced when the gut-juice concentration was increased from 1 to 50% and correlated with a loss in insecticidal activity . However, no lower M(r) protease-resistant fragments were evident in the SDS-PAGE of these digests.

J Biol Chem, 2000 Oct 13, 275(41), 31635 - 40
Cold adaptation of a mesophilic subtilisin-like protease by laboratory evolution; Wintrode PL et al.; Enzymes isolated from organisms native to cold environments generally exhibit higher catalytic efficiency at low temperatures and greater thermosensitivity than their mesophilic counterparts . In an effort to understand the evolutionary process and the molecular basis of cold adaptation, we have used directed evolution to convert a mesophilic subtilisin-like protease from Bacillus sphaericus, SSII, into its psychrophilic counterpart . A single round of random mutagenesis followed by recombination of improved variants yielded a mutant, P3C9, with a catalytic rate constant (k(cat)) at 10 degrees C 6.6 times and a catalytic efficiency (k(cat)/K(M)) 9.6 times that of wild type . Its half-life at 70 degrees C is 3.3 times less than wild type . Although there is a trend toward decreasing stability during the progression from mesophile to psychrophile, there is not a strict correlation between decreasing stability and increasing low temperature activity . A first generation mutant with a >2-fold increase in k(cat) is actually more stable than wild type . This suggests that the ultimate decrease in stability may be due to random drift rather than a physical incompatibility between low temperature activity and high temperature stability . SSII shares 77 . 4% identity with the naturally psychrophilic protease subtilisin S41 . Although SSII and S41 differ at 85 positions, four amino acid substitutions were sufficient to generate an SSII whose low temperature activity is greater than that of S41 . That none of the four are found in S41 indicates that there are multiple routes to cold adaptation.

Cad Saude Publica, 1996 Oct, 12(4), 497 - 505
{Obstacles to compliance with treatment for Hansen's disease}; Bakirtzief Z; This research project aimed at identifying some of the factors related to leprosy patient compliance with the multidrug treatment regimen . The methodological framework of the Social Representations Theory was used . Two groups of patients were interviewed: compliant and non-compliant with treatment and those coming from two different health services . We observed a common understanding about treatment in the various interviews, expressed as a metaphor to describe the treatment experience: the figure of a battle in which the bacillus is portrayed as a threat, the patient as a victim, the medication as a weapon and the health professional as a hero or saint . Still, the medication is represented as being both good and bad for the patient's well-being . Finally, quality of the physician-patient relationship appeared to be the main difference between the two groups of subjects studied.

Cad Saude Publica, 1996 Oct, 12(4), 473 - 482
{Integrated control of the filariasis vector with community participation in an urban area of Recife, Pernambuco, Brazil}; Regis L et al.; A pilot study for the control of Bancroftian filariasis transmission was developed in two areas of Recife, Brazil, where microfilaraemic prevalence was 10% in 1991 . Mass treatment with diethylcarbamazine (DEC) using low and spaced doses was employed in both areas . In one such instance, DEC therapy was associated with vector control using physical measures and periodic treatment of Culex breeding sites with the entomopathogen Bacillus sphaericus . The vector population density, reaching 60 - 120 Culex/room /night before the intervention, was drastically reduced to 4 - 16 Culex/room/night, and maintained at this level for more than two years . Actions to engage the local school community in the vector control process were implemented, and as a consequence several classroom and extracurricular activities were put into practice, culminating with the effective participation of a team of students called the "Vector Vigilantes", in the application of control measures against the vector . The enthusiastic involvement of schoolteachers and students pointed to schools as a place amenable to programs such as this.

Am J Respir Crit Care Med, 2000 Jul, 162(1), 232 - 9
Synthetic oligodeoxynucleotides inhibit IgE induction in human lymphocytes; Fujieda S et al.; Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs have the capacity to stimulate T-helper (Th)1-type responses in mice . Th1 cytokines are known to act as downregulators of IgE production . In this study we investigated whether synthetic ODNs inhibited IgE production in human peripheral blood mononuclear cells (PBMC) from normal donors stimulated with interleukin (IL)-4 plus anti-CD40 monoclonal antibody (mAb) in vitro . Thirty-mer single-stranded ODNs were randomly selected from the complementary DNA encoding the MPB-70 of Mycobacterium bovis Bacillus Calmette-Guerin . Two ODNs, containing CGTACG or AACGTT inhibited IgE production by human PBMC . When other oligonucleotides were substituted in a portion of the sequence of the core or flanking oligonucleotides in the ODN containing CGTACG, ODNs containing NACGTTCG or A/CTCGTTCG sequences specifically inhibited IgE production by human PBMC in vitro . The inhibition of IgE production by certain ODNs was mediated by both interferon (IFN)-gamma and IL-12, since the ODN-induced suppression was blocked by the addition of anti-IFN-gamma or anti-IL-12 mAb . Also, the ODNs inhibited induction of epsilon germline transcripts by IL-4 . Our findings indicate that synthetic ODNs appear to be candidates for the treatment of IgE-dependent allergic disease in humans.

Proc R Soc Lond B Biol Sci, 2000 Jun 22, 267(1449), 1177 - 84
Host-plant diversity of the European corn borer Ostrinia nubilalis: what value for sustainable transgenic insecticidal Bt maize?
Bourguet D, Bethenod MT, Trouve C, Viard F.
The strategies proposed for delaying the development of resistance to the Bacillus thuringiensis toxins produced by transgenic maize require high levels of gene flow between individuals feeding on transgenic and refuge plants . The European corn borer Ostrinia nubilalis (Hubner) may be found on several host plants, which may act as natural refuges . The genetic variability of samples collected on sagebrush (Artemisia sp.), hop (Humulus lupulus L.) and maize (Zea mays L.) was studied by comparing the allozyme frequencies for six polymorphic loci . We found a high level of gene flow within and between samples collected on the same host plant . The level of gene flow between the sagebrush and hop insect samples appeared to be sufficiently high for these populations to be considered a single genetic panmictic unit . Conversely, the samples collected on maize were genetically different from those collected on sagebrush and hop . Three of the six loci considered displayed greater between-host-plant than within-host-plant differentiation in comparisons of the group of samples collected on sagebrush or hop with the group of samples collected on maize . This indicates that either there is genetic isolation of the insects feeding on maize or that there is host-plant divergent selection at these three loci or at linked loci . These results have important implications for the potential sustainability of transgenic insecticidal maize.

J Econ Entomol, 2000 Jun, 93(3), 1011 - 6
Effects of transgenic Bacillus thuringiensis maize grain on B . thuringiensis-susceptible Plodia interpunctella (Lepidoptera: Pyralidae); Giles KL et al.; Percentage survivorship, developmental time, adult body length, and sex ratio of Plodia interpunctella (Hubner) reared on field-produced grain from sixteen cultivars of maize, Zea mays L., including several transgenic Bacillus thuringiensis (Bt) Berliner hybrids and selected non-Bt isolines, were evaluated under laboratory conditions . Compared with isolines, development was delayed and survivorship reduced for P . interpunctella reared on grain from transgenic hybrids with the CaMV/35s promoter that express Cry1Ab protein . Similarly, compared with non-Bt hybrids, a transgenic hybrid with the CaMV/35s promoter that expresses Cry9C protein delayed development, decreased survivorship, and caused reductions in adult body length of P . interpunctella . In contrast, no significant differences in P . interpunctella developmental times or survivorship were observed between transgenic hybrids with the PEPC promoter expressing Cry1Ab and their isolines . Additionally, developmental time, survivorship, and adult body length were similar between P . interpunctella reared on a transgenic hybrid with the CaMV/35s promoter expressing Cry1Ac and non-Bt hybrids . Our data demonstrate that transgenic Bt maize grain, especially grain from hybrids with the CaMV/35s promoter expressing Cry1Ab or Cry9C, can significantly affect B . thuringiensis-susceptible P . interpunctella populations up to 4 or 5 mo after harvest.

J Econ Entomol, 2000 Jun, 93(3), 993 - 9
Performance of transgenic corn hybrids in Missouri for insect control and yield; Barry BD et al.; The efficacy of Bacillus thuringiensis-transformed corn (Zea mays L.) hybrids compared with comparable nontransformed corn hybrids for controlling first- and second-generation European corn borer, Ostrinia nubilalis (Hubner), and second-generation southwestern corn borer, Diatraea grandiosella Dyar, was determined . Yield comparisons were obtained from the same plots of corn hybrids . Both generations of European and the second-generation of southwestern corn borer were effectively controlled, but the Bt hybrids varied in degree of control . Hybrids from Ciba Seeds, DEKALB, and Mycogen had more European corn borer tunneling than those from Novartis or Cargill, and this was generally ascribed to different transgenic events . The Bt-transformed hybrids had virtually no leaf-feeding damage and less tunneling than the non-Bt corn hybrids . Some Bt corn hybrids had no tunneling, whereas other Bt hybrids had a small amount of tunneling . All of the non-Bt hybrids had significant leaf-feeding damage and stalk tunneling from both insects . Only three live European corn borer larvae (stunted) were found in the Bt corn hybrids while splitting stalks to assess tunnel length . When insect damage was significant, and in some evaluations where damage was not significant, differences in yields among hybrids were observed . No significant insect population differences were observed for five genera of beneficial insects for Bt versus non-Bt corn hybrids . Corn hybrids that have been transformed with the Bt gene provide an effective means of control for corn borers and efforts to reduce the likelihood of development of borer resistance are warranted.

J Econ Entomol, 2000 Jun, 93(3), 963 - 70
Susceptibility of Plutella xylostella (L.) (Lepidoptera: Plutellidae) populations in Mexico to commercial formulations of Bacillus thuringiensis; Diaz-Gomez O et al.; Populations of diamondback moth, Plutella xylostella (L.), sampled from commercial fields of crucifers in three states of Mexico, were tested for susceptibility to commercial formulations of Bacillus thuringiensis subsp . kurstaki (Berliner) (Dipel 2X), B . thuringiensis subsp . aizawai (XenTari), delta endotoxin Cry 1C (MC), and CryIA(c) (MVP), and a mixture of B . thuringiensis subsp . kurstaki and subsp . aizawai (Agree) . Leaf-dip bioassays confirmed variation in susceptibility of up to 13-fold for MVP, 12-fold for Dipel 2X, sevenfold for XenTari, fivefold for Agree, and less than fivefold for MC . Comparisons with previously published data indicate that at least the 12-fold variation in Dipel 2X would result in significant differences in control in the field . Based on the LC99 values observed for the products, we propose discriminating concentrations for each product . To ensure continued performance in the field we suggest that a resistance monitoring program be implemented to detect any changes in susceptibility to B . thuringiensis products and specific toxins and that their use be restricted to one generation per crop and that they be rotated with other groups of insecticides . Furthermore, we suggest enforcement of a crucifer host-free period and the development and implementation of cultural and biological control strategies to reduce overall population pressure so that fewer insecticidal treatments will be needed.

J Econ Entomol, 2000 Jun, 93(3), 937 - 48
Seed mixtures as a resistance management strategy for European corn borers (Lepidoptera: Crambidae) infesting transgenic corn expressing Cry1Ab protein; Davis PM et al.; Dispersal of neonate European corn borers, Ostrinia nubilalis (Hubner), in seed mixtures of transgenic corn expressing Cry1Ab protein (Bt+) and nontransgenic corn (Bt-) was evaluated in a 2-yr field study . The main objective was to determine if larval dispersal limits the effectiveness of seed mixtures as a resistance management strategy . Mixtures evaluated included (1) all Bt+ plants, (2) every fifth plant Bt- with remaining plants Bt+, (3) every fifth plant Bt+ with remaining plants Bt-, and (4) all Bt- plants . The transformation events MON 802 (B73 BC1F2 x Mol7) and MON 810 (B73 BC1F1 x Mo17), which express the Cry1Ab endotoxin isolated from Bacillus thuringiensis subsp . kurstaki, were used as the sources of Bt+ seed in 1994 and 1995, respectively (YieldGard, Monsanto, St . Louis, MO) . At corn growth stage V6-V8, subplots within each mixture (15-20 plants each) were infested so that every fifth plant in mixtures 1 and 4, every Bt- plant in mixture 2, and every Bt+ plant in mixture 3 received two egg masses . Larval sampling over a 21-d period indicated increased neonate dispersal off of Bt+ plants, reduced survival of larvae that dispersed from Bt+ plants to Bt- plants, and a low incidence of late-instar movement from Bt- plants to Bt+ plants . Computer simulations based on mortality and dispersal estimates from this study indicate that seed mixtures will delay the evolution of resistant European corn borer populations compared with uniform planting of transgenic corn . However, resistant European corn borer populations likely will develop faster in seed mixes compared with separate plantings of Bt and non-Bt corn.

J Econ Entomol, 2000 Jun, 93(3), 931 - 6
Assessment of insecticide resistance after the outbreak of diamondback moth (Lepidoptera: Plutellidae) in California in 1997; Shelton AM et al.; During an outbreak of the diamondback moth, Plutella xylostella (L.), in California in 1997, nine populations were collected from the major broccoli areas throughout the state . Populations were assayed for their susceptibility to currently used materials (Bacillus thuringiensis subsp . kurstaki, permethrin, and methomyl) and to newer materials that had not yet been commercially used in California (spinosad, emamectin benzoate, and chlorfenapyr) . For the currently used insecticides, elevated levels of resistance were seen only with permethrin and seven of the nine populations had tolerance ratios (TR) of > 100 . With the newer chemistries, TR values were all < 15 . To compare potential cross-tolerance, TR values of the currently used insecticides were compared with TR values of the newer insecticides . There were significant relationships found between: methomyl and emamectin benzoate, methomyl and spinosad, and permethrin and spinosad . Further biochemical studies are needed to confirm the actual mechanisms that lead to these relationships and field tests are needed to determine what impact, if any, such TR levels would have on control in the field . These data indicate that resistance to at least one of the commonly used insecticides (permethrin) may have played a role in the outbreak during 1997 . However, other factors may have been at least equally important . The winter of 1996-1997 was warmer than normal, and during the period from February through August of 1997 the amount of rainfall was < 50% of normal . Hot and dry conditions are known to be conducive to outbreaks of P . xylostella . These data add to an overall knowledge about the geographic variation of resistance in P . xylostella populations within the United States . They also serve as a baseline for monitoring changes in susceptibility to these newer insecticides and can also help explain the occurrence of outbreaks caused by factors other than insecticide resistance.

J Econ Entomol, 2000 Jun, 93(3), 925 - 30
Development of diagnostic concentrations for monitoring Bacillus thuringiensis resistance in European corn borer (Lepidoptera: Crambidae); Marcon PC et al.; Two candidate diagnostic concentrations of the Cry1Ab and Cry1Ac toxins from Bacillus thuringiensis corresponding to the LC99 and EC99 (effective concentration that causes 99% growth inhibition) for European corn borer, Ostrinia nubilalis (Hubner), were determined based on previously obtained baseline data . Validation experiments using field-collected European corn borer populations from across North America showed that for Cry1Ab, a concentration corresponding to the upper limit of the 95% confidence interval of the LC99, produced mortality > 99% for all populations tested . However, for Cry1Ac, adjustments and further validation are probably necessary . Development of B . thuringiensis resistance monitoring programs that rely on diagnostic techniques are discussed.

J Econ Entomol, 2000 Jun, 93(3), 788 - 94
Impact of insecticides and surfactant on lettuce physiology and yield; Haile FJ et al.; Insecticides are used extensively on lettuce, Lactuca sativa L., grown in southwestern Arizona because of heavy insect pressure that can potentially reduce lettuce productivity . Multiple sprays are made per season to manage these insects in lettuce . One of the major concerns related to extensive insecticide applications in lettuce is the potential subtle impact of insecticides that may reduce lettuce photosynthesis and yield . We conducted field and greenhouse experiments to examine the impact of multiple insecticides and surfactant spray applications on lettuce photosynthesis and yield . Lettuce was planted in the field in 1998, insecticides and surfactant were applied, and lettuce gas-exchange and dry weights were determined . Treatments were arranged in a split-plot consisting of insecticides as main plot and surfactant as subplot treatments in a randomized complete block design with four replications . Photosynthetic rates of lettuce were significantly reduced by endosulfan, methomyl, acephate, and surfactant at seedling stage 4 h and 2 d after the spray application was made . However, the reduction in lettuce photosynthesis by these insecticides and surfactant was only transient, and lettuce photosynthesis recovered 5 d after the spray application was made . Photosynthetic rates were not altered by zeta-cypermethrin, emamectin benzoate, and spinosad at the seedling stage . Insecticides or surfactant (Kinetic, a nonionic surfactant) did not significantly affect lettuce photosynthesis after rosette formation . In addition, lettuce dry weight was not significantly altered . These studies suggest that lettuce photosynthesis may be susceptible to some insecticides at the seedling stage . Consequently, we found that biorational insecticides, introduced to manage insect pests in lettuce, have no influence on lettuce physiology at the seedling stage, unlike the chlorinated hydrocarbons, organophosphates, or carbamates tested in this study . In a greenhouse study, we found that lettuce photosynthesis and yield were not altered by Bacillus thuringiensis application . Our results indicate that B . thuringiensis and the newer insecticides, particularly biorationals, can be used to manage lettuce insect pests without significantly altering lettuce gas-exchange and yield.

J Econ Entomol, 2000 Jun, 93(3), 763 - 8
Effect of insecticides on the diamondback moth (Lepidoptera: Plutellidae) and its parasitoid Diadegma insulare (Hymenoptera: Ichneumonidae); Hill TA et al.; Studies were conducted to evaluate the toxicity of insecticides to adult Diadegma insulare (Cresson) and its host the diamondback moth, Plutella xylostella (L.) . Leaf-dip and direct-dip bioassays for diamondback moth larvae and residual bioassays for adults of diamondback moth and D . insulare were used to assess mortalities . Larval mortalities at field rates were significantly higher with carbaryl, permethrin, spinosad, and tebufenozide when compared with Bacillus thuringiensis, or imidacloprid in the larval-dip bioassay 72 h after treatment . In the leaf-dip and residual bioassays, both permethrin and spinosad caused 100% mortalities to diamondback moth larvae and adults, respectively, 72 h after treatment . Of all the materials tested, only B . thuringiensis and tebufenozide were not toxic to D . insulare 24 h after treatment . Spinosad was not toxic to D . insulare 30 min after treatment . However, 100% mortality was observed 8 h after treatment.

J Econ Entomol, 2000 Jun, 93(3), 713 - 20
Laboratory and field evaluations of two Bacillus thuringiensis formulations, Novodor and Raven, for control of cottonwood leaf beetle (Coleoptera: Chrysomelidae); Coyle DR et al.; Laboratory and field experiments were conducted to determine the efficacy of two Bacillus thuringiensis Berliner formulations, Novodor and Raven, for controlling cottonwood leaf beetle, Chrysomela scripta F . (Coleoptera: Chrysomelidae) . In laboratory bioassays, larvae or adults were added to petri dishes containing Populus x euramericana Guinier 'Eugenei' foliage that had been treated with distilled water (control) or one of the commercial Bt formulations at either high or low label rates . Survival was recorded on a 24-h basis, and leaf area consumed was measured at the conclusion of all trials . Significant differences from the control in mortality and leaf area consumption resulted in the Novodor and Raven treatments for all life stages tested; however, adults were better able to withstand the effects of B . thuringiensis toxins than were the immatures . Early- and late instar C . scripta populations were monitored in the field (1998 and 1999) after treatment with either water or various concentrations of one of the commercial Bt formulations . Significant mortality resulted with all concentrations and for all life stages tested compared with the control (tap water) . The commercial formulations also were tested under plantation conditions as part of a long-term defoliation study . Both Novodor and Raven reduced cottonwood leaf beetle defoliation damage after a single application, giving high efficacy for control of cottonwood leaf beetle under the conditions and concentrations evaluated.

J Econ Entomol, 2000 Jun, 93(3), 690 - 6
Cotton boll abscission and yield losses associated with first-instar bollworm (Lepidoptera: Noctuidae) injury to nontransgenic and transgenic Bt cotton; Gore J et al.; Field tests were conducted in northeastern Louisiana to determine the effects of infestations by Helicoverpa zea (Boddie) on cotton bolls of varying ages . First instars were caged on bolls of nontransgenic ('Deltapine 5415') or transgenic Bacillus thuringiensis Berliner variety kurstaki (Bt) ('NuCOTN 33B') cotton from 29 June to 11 August during 1997 and 1998 . Deltapine 5415 bolls that accumulated 179 (7.2 d), 281 (11.2 d), and 253 (10.1 d) heat units beyond anthesis were safe from bollworm-induced abscission at 72 h after infestation, 7 d after infestation, and at the time of harvest, respectively . NuCOTN 33B bolls that accumulated 157 (6.3 d), 185 (7.4 d), and 180 (7.2 d) heat units beyond anthesis were safe from bollworm-induced abscission at 72 h after infestation, 7 d after infestation, and at the time of harvest, respectively . Bollworm larvae reduced seedcotton weights of Deltapine 5415 bolls that accumulated between 58.5 (2.3 d) and 350.5 (14.0 d) heat units beyond anthesis . Seedcotton weights of NuCOTN 33B bolls that accumulated between 0 and 281 (11.2 d) heat units beyond anthesis were reduced by bollworm injury . Deltapine 5415 and NuCOTN 33B bolls that accumulated 426.5 (17.1 d) and 299.5 (12.0 d) heat units beyond anthesis, respectively, before infestation were not injured by first-instar bollworm larvae . These data provide information about late-season insecticide termination strategies for bollworms on nontransgenic and transgenic Bt-cotton . This, in turn, will help pest managers determine when insecticides are no longer economical during the late season.

J Econ Entomol, 2000 Jun, 93(3), 680 - 9
Sublethal acute and chronic exposure of Colorado potato beetle (Coleoptera: Chrysomelidae) to the delta-endotoxin of Bacillus thuringiensis; Costa SD et al.; Sublethal exposure of Colorado potato beetle, Leptinotarsa decemlineata (Say), larvae to the delta-endotoxin of Bacillus thuringiensis variety tenebrionis (Berliner) caused a dose-dependent reduction in feeding and weight gain when tested in a leaf disk bioassay . The highest doses of chronic (continuous-lower concentration) exposure resulted in peak foliage consumption on day 1 as compared with peak consumption on days 3 and 4 when exposure was acute (24-h higher concentration) . Dose and exposure regimen interacted significantly in their effects on the extension of development . When development time was analyzed separately for each exposure regimen, only acute exposure caused significant delays in development that extended through to adult eclosion . The efficiency of conversion of ingested material to biomass (ECI) declined significantly with both exposure regimens . The lethal and most sublethal effects of exposure to delta-endotoxin were not cumulative, in that similar total doses, whether delivered acutely or chronically, produced different effects . Female adults that survived acute and chronic exposure to delta-endotoxin as larvae had significantly reduced weight and longevity, and tended to produce fewer eggs (45 and 44% reductions in acute and chronic exposures, respectively) when compared with control adults . The intrinsic rate of increase (r) and net reproductive rate (R0) also appeared to be reduced.

J Econ Entomol, 2000 Jun, 93(3), 667 - 72
Toxicity of insecticides for control of freshwater Culex annulirostris (Diptera: Culicidae) to the nontarget shrimp, Caradina indistincta (Decapoda: Atyidae); Brown MD et al.; Laboratory evaluations were conducted in southeastern Queensland, Australia, to determine the toxicities of two organophosphate compounds (temephos and pirimiphos-methyl), an insect growth regulator (s-methoprene), and an entomopathogenic bacterium (Bacillus thuringiensis variety israelensis de Barjac {B.t.i.}) to Culex annulirostris (Skuse), an Australian freshwater mosquito vector of arboviruses, and to Caradina indistincta Calman, a co-habiting nontarget shrimp species . S-methoprene and B.t.i . were safest for Cx . annulirostris control with lethal dose ratios (LC95 nontarget/LC95 target) of 3,300 and 846,000, respectively . In contrast, lethal dose ratios for temephos and pirimiphos-methyl were 0.05 and 0.00005, respectively, suggesting that they are environmentally unsuitable . Based on their high lethal dose ratios, s-methoprene and B.t.i . are recommended for control of larval Cx . annulirostris in Australian freshwater habitats.

J Econ Entomol, 2000 Jun, 93(3), 613 - 22
Field evaluation of soybean engineered with a synthetic cry1Ac transgene for resistance to corn earworm, soybean looper, velvetbean caterpillar (Lepidoptera: Noctuidae), and lesser cornstalk borer (Lepidoptera: Pyralidae); Walker DR et al.; A transgenic line of the soybean 'Jack', Glycine max (L.) Merrill, expressing a synthetic cry1Ac gene from Bacillus thuringiensis variety kurstaki (Jack-Bt), was evaluated for resistance to four lepidopteran pests in the field . Jack-Bt and genotypes serving as susceptible and resistant controls were planted in field cages and artificially infested with larvae of corn earworm, Helicoverpa zea (Boddie), and velvetbean caterpillar, Anticarsia gemmatalis (Hubner), in 1996, 1997, and 1998, and also with soybean looper, Pseudoplusia includens (Walker), in 1996 . Susceptible controls included Jack (1996-1998), 'Cobb' (1996), and Jack-HPH (1996) . GatIR 81-296 was used as the resistant control in all 3 yr . Compared with untransformed Jack, Jack-Bt showed three to five times less defoliation from corn earworm and eight to nine times less damage from velvetbean caterpillar . Defoliation of GatIR 81-296 was intermediate between that of Jack and Jack-Bt for corn earworm, and similar to that of Jack for velveltbean caterpillar . Jack-Bt exhibited significant, but lower resistance to soybean looper . Jack-Bt also showed four times greater resistance than Jack to natural infestations of lesser cornstalk borer, Elasmopalpus lignosellus (Zeller), in conventional field plots at two locations in 1998 . Data from these experiments suggest that expression of this cry1Ac construct in soybean should provide adequate levels of resistance to several lepidopteran pests under field conditions.

Heart Lung, 2000 Jul-Aug, 29(4), 306 - 8
Necrotizing fasciitis caused by Aeromonas hydrophila; Minnaganti VR et al.; Aeromonas Hydrophila is a gram-negative bacillus commonly found in soil, sewage, and fresh or brackish water in many parts of the United States . In healthy people, the most common clinical manifestations attributed to Aeromonas are diarrhea and soft tissue infections . In people with suppressed immune systems or liver disease, A hydrophila can cause meningitis, endocarditis, peritonitis, hemolytic-uremic syndrome, or septicemia . We present the first known case of fulminant necrotizing fasciitis from A hydrophila that is not associated with trauma, liver disease, or immunosuppression.

Plant Foods Hum Nutr, 2000, 55(2), 111 - 8
Aerobic spore-forming bacteria and chemical composition of some Nigerian fermented soup condiments; Sanni AI et al.; A total of 97 strains of spore-forming Bacillus were isolated from 45 samples of three Nigerian fermented condiments, obtained from retail markets located in Southwestern Nigeria . The isolates were identified as B . subtilis (33%), B . pumilus (19%), B . licheniformis (22%), B . brevis (9%), B . megaterium (12%) and B . polymyxa (5%) . The microbial load of the condiments showed that the average count of spore-formers was between 107 to 109 cfu/g . The moisture contents of iru, ugba and ogiri were 57.18%, 46.32% and 42.34%, respectively, while the protein contents were 18.26%, 17.17% and 17.96% . The percentage fat was 29.88%, 40.25% and 44.14% for iru, ugba and ogiri . The ash content ranged from 5.8 to 6.1%; a 0.1% titratable acidity and pH values above 7.0 were obtained for the three condiments.

Cell Immunol, 2000 Jun 15, 202(2), 103 - 12
Induction of apoptosis in bacillus Calmette-Guérin-activated T cells by transforming growth factor-beta; Mendez-Samperio P et al.; In view of the critical role played by bacillus Calmette-Guerin (BCG) in the development and functional activation of protective T cells against tuberculosis, it has become important to understand the mechanisms by which cytokines regulate BCG-mediated immune responses . There is evidence that cytokine-mediated suppression of T cell function by mechanisms, including apoptosis, may reduce host resistance in tuberculosis . However, it is unclear whether cytokine-mediated suppression of antigen-responsive T cells through apoptotic mechanisms may be operating during human cellular activation induced by BCG . Here we present evidence, for the first time, that treatment of BCG-activated T cells with transforming growth factor-beta (TGF-beta) induces cellular apoptosis . These results were further supported by the fact that treatment of cells with a blocking mAb directed to TGF-beta significantly inhibited the percentage of apoptosis induced by TGF-beta . Interestingly, TGF-beta-mediated death of BCG-activated T cells in cultures containing interleukin (IL)-12 was observed . Moreover, our results demonstrated the induction of apoptosis by TGF-beta in BCG-activated T cells cultured in the presence of exogenous IL-12 . In addition, our data indicated that TGF-beta significantly inhibited both BCG-induced cell growth determined by thymidine uptake and BCG-induced IFN-gamma secretion . Finally, TGF-beta-induced apoptosis in BCG-activated T cells correlated inversely with BCG-induced IFN-gamma secretion . Taken together, these findings indicate that TGF-beta induces apoptosis in human T cells activated with BCG and at the same time suggest that loss of BCG-reactive T cells through apoptotic mechanisms could contribute to an increased susceptibility to Mycobacterium tuberculosis infection .

Urol Int, 2000, 64(4), 229 - 32
Nephrogenic adenoma of the bladder after intravesical bacillus Calmette-Guérin treatment; Kilciler M et al.; A 60-year-old female patient was subjected to transurethral resection of transitional cell carcinoma of the bladder and was given intravesical bacillus Calmette-Guerin treatment for 6 weeks . The control cytoscopy performed after 6 months revealed a polypoid lesion at the trigon and the lesion was resected . The pathological examination of the specimen showed no evidence of cancer but the presence of a metaplastic lesion that was nephrogenic adenoma .

J Urol, 2000 Aug, 164(2), 526 - 31
Murine IL-2 secreting recombinant Bacillus Calmette-Guerin augments macrophage-mediated cytotoxicity against murine bladder cancer MBT-2; Yamada H et al.; PURPOSE: This study was to establish a more effective anti-cancer immunomodulating agent by constructing recombinant (r) Bacillus Calmette-Guerin (BCG) secreting alpha-antigen (alpha-Ag) fused murine (m) interleukin (IL)-2, and to study its biological activity on cell-mediated cytotoxicity against murine bladder cancer cell, MBT-2, in vitro . MATERIALS AND METHODS: pSO246 plasmid vector ligated with mIL-2 gene was introduced into BCG by electroporation . Thioglycollate-elicited murine peritoneal exudate cells (PEC) were stimulated in vitro with parental BCG or rBCG and their cytotoxic activity and the cytokine production was studied . Cytokines were assayed by an enzyme-linked immunosorbent assay (ELISA) and L929 bioassay . Cytotoxicity was measured by 51Cr releasing assay . RESULTS: rBCG (alpha-Ag-IL-2) secreted functional IL-2 and augmented more efficient cytotoxicity to MBT-2 and cytokines such as IL-12, tumor necrosis factor and interferon (IFN)-gamma in PEC than parental BCG did . rBCG (alpha-Ag) had the same activity as BCG . Anti-IL-2 antibody reduced rBCG (alpha-Ag-IL-2)-mediated cytotoxicity and IFN-gamma production . Exogenous IL-2 also enhanced BCG-mediated cytotoxicity, but 100 times more IL-2 was required to express the same activity as rBCG (alpha-Ag-IL-2) . Anti-IL-12 neutralizing antibody and the depletion of T cells and NK cells reduced IFN-gamma production by PEC stimulated with rBCG (alpha-Ag-IL-2), suggesting that T cells, NK cells and IL-12 participate in the enhancement of IFN-gamma production . CONCLUSIONS: rBCG secreting IL-2 showed significant antitumor activity and cytokine production and this will be a promising agent for bladder cancer patient to reduce both clinical dose and side effects of BCG for immunotherapy.

J Urol, 2000 Aug, 164(2), 349 - 51
Fluorescence detection of flat bladder carcinoma in situ after intravesical instillation of hypericin; D'Hallewin MA et al.; PURPOSE: We determined the sensitivity and specificity of detecting flat bladder carcinoma in situ through fluorescent detection after intravesical hypericin instillations . MATERIALS AND METHODS: The study included 40 patients, of whom 26 presented with macroscopic visible tumor, 9 had a positive cytology without visible tumor and 5 underwent cystoscopy after bacillus Calmette-Guerin instillations (4) or radiotherapy (1) . We instilled 40 ml . of a 8 microM . solution of hypericin intravesically for at least 2 hours . Fluorescence excitation with blue light was effective up to 16 hours after termination of the instillation . RESULTS: All visible papillary tumors showed red fluorescence . In addition, 134 flat fluorescent areas were detected . Analysis of 281 biopsies from flat bladder wall indicated 93% sensitivity and 98.5% specificity for detecting carcinoma in situ . Visible lesions resulting from radiotherapy, chemotherapy or immunotherapy did not show any fluorescent signs and, therefore, did not induce false-positive readings . There were no signs of photobleaching during inspection and resection . CONCLUSIONS: We report a simple yet comprehensive endoscopic method for early detection of bladder cancer, including carcinoma in situ . Hypericin induced fluorescence has a high sensitivity and specificity for detection of bladder transitional cell carcinoma, papillary and flat carcinoma in situ . When carcinoma in situ is suspected, this technique is highly recommended.

J Chromatogr A, 2000 Jun 2, 880(1-2), 233 - 42
Determination of aldehydes in food by high-performance liquid chromatography with biosensor coupling and micromembrane suppressors; Schultheiss J et al.; A high-performance liquid chromatography system for the determination of aldehydes in food was developed incorporating an Cation MicroMembrane Suppressor (CMMS) and enzyme reactors packed with VA-Epoxy on which aldehyde dehydrogenase from bakers yeast and NADH oxidase from Bacillus licheniformis were immobilized . The method was based on the principle that the separation efficiency of HPLC is combined with the sensitivity of electrochemical detection and the specificity of enzymes . Main attention was directed to the determination of 5-hydroxymethyl-2-furaldehyde and 2-furaldehyde, the occurrence of which is an indication of quality deterioration in several food products . The efficiency of the method has been shown by the analysis of honey, coffee and related beverages, refreshments, sherry, port, dry fruits and breakfast cereals.

Cell Mol Life Sci, 2000 May, 57(5), 828 - 33
Novel insecticidal toxins from nematode-symbiotic bacteria; ffrench-Constant RH et al.; The current strategy of using transgenic crops expressing insecticidal protein toxins is placing increasing emphasis on the discovery of novel toxins, beyond those already derived from the bacterium Bacillus thuringiensis . Here we review the cloning of four insecticidal toxin complex (tc) encoding genes from a different bacterium Photorhabdus luminescens and of similar gene sequences from Xenorhabdus nematophilus . Both these bacteria occupy the gut of entomopathogenic nematodes and are released into the insect upon invasion by the nematode . In the insect the bacteria presumably secrete these insecticidal toxins, as well as a range of other antimicrobials, to establish the insect cadaver as a monocultural breeding ground for both bacteria and nematodes . In this review, the protein biochemistry and structure of the tc encoding loci are discussed in relation to their observed toxicity and histopathology . These toxins may prove useful as alternatives to those derived from B . thuringiensis for deployment in insect-resistant transgenic plants.

Microbiol Immunol, 2000, 44(5), 389 - 93
Sequence of the gene encoding an alkaline serine proteinase of Bacillus pumilus TYO-67; Aoyama M et al.; The complete nucleotide sequence of the gene encoding an alkaline serine proteinase (aprP) of Bacillus pumilus TYO-67 was determined . The sequence analysis showed an open reading frame of 1,149 bp (383 amino acids) that encoded a signal peptide consisting of 29 residues and a propeptide of 79 residues . The deduced 3 amino acid residues, D32, H64, and S221, were identical with 3 essential amino acids in the catalytic center of subtilases . The sequence around these residues revealed that APRP was a new member of the true subtilisin subgroup of the subtilisin family . The highest homology was found in subtilisin NAT at 64.4% in the DNA sequence . The residue S189 of APRP was different from those of other subtilases.

Yi Chuan Xue Bao, 2000, 27(3), 270 - 7
{Expression of Bacillus thuringiensis (Bt) crystal toxin gene in the chloroplast of tobacco}; Zhang ZL et al.; The 3.5 kb wild-type Bt Cry I A(c) gene and its 3' truncated forms (2.1 kb, 1.8 kb) were placed under the control of plastid expression signals consisting of the strong light-induced psbA promoter and its 3' untranslated region with the aadA cassette (Prrn, aadA and psbA3') as a selectable marker . The resulting vectors pBT3, pBT8 and pBT22 also contain flanking tobacco plastid DNA homology regions to direct insertion of the Bt transgene into the tobacco plastid genome between psbA and trnK by homologous recombination . Transformed plastid genomes were selectively amplified by growing the cells on spectinomycin medium . Several independently transformed lines were obtained at last . The results of Southern and Western blot demonstrated that these three kinds of Bt genes had been introduced into tobacco plants, and their filial generations are resistant to spectinomycin . Insecticidal activity assay with transgenic tobacco leaves indicate that some plants have strong toxicity to cotton bollworm . This is the first report in China that Bt gene has been introduced and successfully expressed in the chloroplast of higher plants.

Immunology, 2000 Jun, 100(2), 194 - 202
In vitro T-cell activation of monocyte-derived macrophages by soluble messengers or cell-to-cell contact in bovine tuberculosis; Liebana E et al.; The macrophage plays a dual role in tuberculosis, promoting not only protection against mycobacteria, but also survival of the pathogen . Macrophages inhibit multiplication of mycobacteria but also act in concert with lymphocytes through presentation of antigens to T cells . Studies in animal and human infections have suggested a correlation of in vitro growth rates of mycobacteria with in vivo virulence, using uracil uptake to assess mycobacterial metabolism . This study found that blood-derived, non-activated bovine macrophages were capable of controlling Mycobacterium bovis bacillus Calmette-Gurin growth for up to 96 hr, but were permissive to intracellular growth of virulent M . bovis . The present investigation compared the in vitro modulation of these macrophage activities by cytokine-rich T-cell supernatants or cell-to-cell contact . On the one hand, treatment of cultured monocytes with mitogen-produced T-cell supernatants promoted morphological changes suggestive of an activation status, enhanced the antigen presentation capabilities of monocytes and up-regulated major histocompatibility complex class II expression . However, this activation was not associated with enhanced anti-M . bovis activity . On the other hand, incubation of infected monocytes with T-cell populations resulted in proportionally increased inhibition of M . bovis uracil uptake . This inhibition was also seen using cells from uninfected animals and indicated the necessity for cell-to-cell contact to promote antimycobacterial capability.

Rev Argent Microbiol, 2000 Apr-Jun, 32(2), 71 - 6
{Inhibition of Bacillus coagulans growth in laboratory media and in fruit purees}; Cerrutti P et al.; The growth of two strains of B . coagulans was inhibited in laboratory media at pH < or = 4.5, and at water activity (aw) levels of 0.96 for B . coagulans NRS 609 and 0.95 for B . coagulans ATCC 803 . The growth of both strains was also inhibited in apple and strawberry purees (pH = 3.5) stored at 37 degrees C for over two months . B . coagulans was able to grow in banana puree (pH approximately equal to 5.0) but acidification of the puree at pH = 3.5 was enough to prevent growth . The addition of up to 3,000 ppm vainillin ("natural" preservative) or 1,000 ppm potassium sorbate (traditional preservative) at pH higher than the inhibitory level previously determined could not prevent growth of B . coagulans in laboratory or in fruits, but 100 ppm lysozyme retarded growth in laboratory media at different pH levels (from 4.5 to 6.7) and in banana puree . As lysozyme showed to be effective at pH < or = 6.7, it might be used to prevent growth of B . coagulans at an eventual increment of pH during storage.

J Asthma, 2000 Jun, 37(4), 329 - 34
Heat-killed Mycobacterium bovis-bacillus Calmette Guerin-suppressed total serum IgE response in ovalbumin-sensitized newborn mice; Bakir M et al.; To determine the impact of bacillus Calmette Guerin (BCG) vaccination on IgE production in ovalbumin (OVA)-sensitized newborn mice, four groups (I, II, III, IV) of BALB/c mice were immunized on the first day of life with live BCG, killed BCG, BCG diluent, and saline, respectively . No injection was applied to mice in group V (control) . All mice except group V were sensitized and challenged with OVA in the fourth and sixth weeks, respectively, and serum total IgE levels were determined at 8 weeks, 2 weeks after the second OVA challenge . IgE levels of all groups were significantly higher than the control group except for group II (p = 0.95) . Mice in group II showed significantly lower IgE values than group IV and I (p = 0.007 and p = 0.003, respectively) . We concluded that heat-killed BCG may downregulate IgE response to OVA in newborn mice.

Clin Diagn Lab Immunol, 2000 Jul, 7(4), 625 - 34
Parasporin, a human leukemic cell-recognizing parasporal protein of Bacillus thuringiensis; Mizuki E et al.; An unusual property, human leukemic cell-recognizing activity, associated with parasporal inclusions of a noninsecticidal Bacillus thuringiensis soil isolate was investigated, and a protein (named parasporin in this study) responsible for the activity was cloned . The parasporin, encoded by a gene 2,169 bp long, was a polypeptide of 723 amino acid residues with a predicted molecular weight of 81, 045 . The sequence of parasporin contained the five conserved blocks commonly found in B . thuringiensis Cry proteins; however, only very low homologies (<25%) between parasporin and the existing classes of Cry and Cyt proteins were detected . Parasporin exhibited cytocidal activity only when degraded by proteases into smaller molecules of 40 to 60 kDa . Trypsin and proteinase K activated parasporin, while chymotrypsin did not . The activated parasporin showed strong cytocidal activity against human leukemic T cells (MOLT-4) and human uterus cervix cancer cells (HeLa) but not against normal T cells.

Clin Diagn Lab Immunol, 2000 Jul, 7(4), 617 - 24
Differentiation of Bartonella species by a microimmunofluorescence assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western immunoblotting; Liang Z et al.; Bartonella species can be differentiated by microimmunofluorescence assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting with murine polyclonal antisera to Bartonella henselae, B . quintana, B . elizabethae, and B . bacilliformis . A pairwise comparison on the basis of SDS-PAGE protein profiles demonstrated similarity values for proteins of different Bartonella species ranging from 28.6 to 86.4% . Antigenic relationships revealed by immunoblotting with murine antisera were equivalent to those of proteins observed by SDS-PAGE . A dendrogram obtained on the basis of protein bands of SDS-polyacrylamide gels showed that Bartonella species could be divided into three groups . B . bacilliformis was distinct from all other Bartonella species; B . grahamii, B . taylorii, B . doshiae, and B . vinsonii formed a cluster, as did B . henselae, B . quintana, B . elizabethae, and B . clarridgeiae . These relationships were consistent with those revealed by parsimony trees derived from 16S rRNA and gltA gene sequencing . SDS-PAGE analysis showed that 120-, 104-, 85-, 71-, 54-, 47-, 40-, 33-, 30-, and 19-kDa proteins were present in all species, with the 54-kDa protein being the most dominant . Proteins with a molecular mass of less than 54 kDa allow the differentiation of species and are a possible target for future species-specific antibodies and antigens.

Drugs, 2000 Jun, 59(6), 1217 - 21
Development of an asthma vaccine: research into BCG; Scanga CB et al.; Asthma is an atopic disorder characterised by the activation and recruitment of eosinophils to the lung resulting in chronic swelling and inflammation of the airways . Allergic disorders such as atopic asthma and dermatitis have been increasingly prevalent in developed countries, and the inverse correlation between exposure to major diseases such as tuberculosis and atopy prevalence has been reported . Intranasal administration of Mycobacterium bovis-Bacillus Calmette-Guerin (BCG) has been demonstrated to suppress airway eosinophilia in a model of atopic asthma . This immunomodulation is attributed to the ability of interferon (IFN)-gamma produced by BCG-specific T(H)1 lymphocytes to inhibit the development of lung T(H)2 responses such as airway eosinophilia . The mechanism of IFNgamma-induced inhibition is yet to be defined, but could involve activation of macrophages, direct suppression of developing T(H)2 lymphocytes, or altered dendritic cell activation and antigen presentation . Mycobacteria such as BCG and certain mycobacterial fractions are strong inducers of a T(H)1 immune response . The effectiveness of BCG in inhibiting atopic airway eosinophilia suggests its potential as a useful therapeutic agent in the treatment of atopic asthma.

Eur J Biochem, 2000 Jul, 267(14), 4520 - 8
Mechanism of product chain length determination for heptaprenyl diphosphate synthase from Bacillus stearothermophilus; Hirooka K et al.; A member of the medium-chain prenyl diphosphate synthases, Bacillus stearothermophilus heptaprenyl diphosphate synthase, catalyzes the consecutive condensation of isopentenyl diphosphate with allylic diphosphate to produce (all-E)-C35 prenyl diphosphate as the ultimate product . We previously showed that the product specificity of short-chain prenyl diphosphate synthases is regulated by the structure around the first aspartate-rich motif (FARM) . The FARM is also conserved in a subunit of heptaprenyl diphosphate synthase, component II', which suggests that the structure around the FARM of component II' regulates the elongation . To determine whether component II' regulates the product chain length by a mode similar to that of the short-chain prenyl diphosphate synthases, we replaced a bulky amino acid at the eighth position before the FARM of component II', isoleucine 76, by glycine and analyzed the product specificity . The mutated enzyme, I76G, can catalyze condensations of isopentenyl diphosphate beyond the native chain length of C35 . Moreover, two mutated enzymes of A79Y and S80F, which have a single replacement to the aromatic residue at the fourth or the fifth position before the FARM, mainly yielded a C20 product . These results strongly suggest that a common mechanism controls the product chain length of both short-chain and medium-chain prenyl diphosphate synthases and that, in wild-type heptaprenyl diphosphate synthase, the prenyl chain can grow on the surface of the small residues at positions 79 and 80, and the elongation is precisely blocked at the length of C35 by isoleucine 76.

J Exp Med, 2000 Jul 3, 192(1), 117 - 22
Interferon gamma eliminates responding CD4 T cells during mycobacterial infection by inducing apoptosis of activated CD4 T cells; Dalton DK et al.; In Mycobacterium bovis Bacille Calmette-Guerin (BCG)-infected wild-type mice, there was a large expansion of an activated (CD44(hi)) splenic CD4 T cell population followed by a rapid contraction of this population to normal numbers . Contraction of the activated CD4 T cell population in wild-type mice was associated with increased apoptosis of activated CD4 T cells . In BCG-infected interferon (IFN)-gamma knockout (KO) mice, the activated CD4 T cell population did not undergo apoptosis . These mice accumulated large numbers of CD4(+)CD44(hi) T cells that were responsive to mycobacterial antigens . Addition of IFN-gamma to cultured splenocytes from BCG-infected IFN-gamma KO mice induced apoptosis of activated CD4 T cells . IFN-gamma-mediated apoptosis was abolished by depleting adherent cells or Mac-1(+) spleen cells or by inhibiting nitric oxide synthase . Thus, IFN-gamma is essential to a regulatory mechanism that eliminates activated CD4 T cells and maintains CD4 T cell homeostasis during an immune response.

Am J Pathol, 2000 Jul, 157(1), 37 - 42
Osteopontin expression correlates with clinical outcome in patients with mycobacterial infection; Nau GJ et al.; Osteopontin (OPN) is a protein that is expressed in chronic inflammatory diseases including tuberculosis, and its deficiency predisposes to more severe mycobacterial infections in mice . However, no reports have identified altered OPN expression in, or correlated these alterations to, infections in humans . The data presented herein identify alterations in the tissue expression of OPN protein and describe an inverse correlation between these levels and disease progression after inoculation of Mycobacterium bovis bacillus Calmette-Guerin vaccine in humans . Patients with regional adenitis and good clinical outcomes had abundant OPN in infected lymph nodes . This pattern of OPN accumulation was also observed in patients infected by M . avium-intracellulare . In contrast, patients with disseminated infection and histologically ill-defined granulomas had no significant osteopontin accumulation in infected lymph nodes; these patients had either deficiencies in the interferon-gamma receptor 1 or idiopathic immune defects . The level of OPN protein expression was inversely correlated with disseminated infection and, of particular interest, with death of the patient . We conclude that osteopontin expression correlates with an effective immune and inflammatory response when humans are challenged by a mycobacterial infection and that osteopontin contributes to human resistance against mycobacteria.

Syst Appl Microbiol, 2000 Apr, 23(1), 25 - 30
Cloning and characterization of a Bacillus thuringiensis serovar higo gene encoding a novel class of the delta-endotoxin protein, Cry27A, specifically active on the Anopheles mosquito; Saitoh H et al.; A novel gene encoding a 98-kDa mosquitocidal delta-endotoxin protein, designated Cry27A, was cloned from a Bacillus thuringiensis serovar higo strain . The Cry27A protein contained the five sequence blocks of amino acids commonly conserved in most B . thuringiensis Cry proteins . Relatively high homologies, ranging from 43.0% to 84.4%, existed between the Cry27A protein and several established classes of mosquitocidal Cry proteins (Cry4A, Cry10A, Cry19A, Cry19B, and Cry20A) in the sequence of 51 N-terminal amino acids . The complete sequence of this protein, however, showed low levels (<40%) of amino acid identity to those of the known Cry proteins . Although the expression level of the cry27A gene was low in the transformants under the control of its own promoter, the use of the cyt1A promoter resulted in high-level expression of the gene, leading to the formation of inclusions . The expressed Cry27A protein showed larvicidal activity highly specific for Anopheles stephensi, but lacked the toxicity against Culex pipiens molestus and Aedes aegypti . The results suggest that the Cry27A protein is responsible for the Anopheles-preferential toxicity of the B . thuringiensis serovar higo strain.

Lab Invest, 2000 Jun, 80(6), 901 - 14
Correction of defective host response to Mycobacterium bovis BCG infection in TNF-deficient mice by bone marrow transplantation; Jacobs M et al.; Tumour necrosis factor-alpha (TNF) plays a central role in the recruitment and activation of mononuclear cells in mycobacterial infection . In the absence of type 1 TNF receptor, Mycobacterium bovis Bacillus Calmette-Guerin (BCG) infection of mice is not contained, leading to fatal disease . Because type 1 TNF receptor binds both TNF and lymphotoxin-a, we used TNF-deficient mice to determine the specific role of TNF in the host resistance to BCG infection . The bacterial burden of the lungs of TNF-deficient mice was substantially increased and the mice succumbed to pneumonia between 8 and 12 weeks with a defective granuloma response . Atypical granulomas developed by 4 weeks expressing low levels of MHC class II, intracellular adhesion molecule (ICAM-1), CD11b and CD11c . Macrophages showed little signs of activation and had low levels of acid phosphatase activity and inducible nitric oxide synthase (INOS) expression . Despite the defective cellular recruitment, the chemokines, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1alpha), were increased in broncho-alveolar lavage fluid of TNF-deficient mice . The defective host response was corrected by the transplantation of normal bone marrow cells into irradiated TNF-deficient mice . These results demonstrate that TNF derived from hemopoietic cells rather than from mesenchymal origin are essential for a normal host response to BCG infection . Furthermore, TNF dependent expression of adhesion molecules may be essential for the recruitment of mononuclear cells for the formation of bactericidal BCG granulomas.

Biosci Biotechnol Biochem, 2000 May, 64(5), 1079 - 81
Use of Bacillus brevis for synthesis and secretion of Des-B30 single-chain human insulin precursor; Koh M et al.; A synthetic gene encoding a single chain human insulin precursor {B-chain (1-29)-A-chain} linked to the C-terminal lysine of human epidermal growth factor (1-28) (EGF-SCI) was constructed . This gene was expressed using Bacillus brevis . EGF-SCI was isolated from the supernatant of the culture broth . Treatment of EGF-SCI with lysyl endopeptidase resulted in the formation of des-B30 human insulin . The identification of the formed des-B30 human insulin was made by the measurement of molecular weight and amino acid analysis . The binding coefficient to anti-human insulin antibody was comparable to that of human insulin.

Microbiology, 2000 Jul, 146 ( Pt 7), 1727 - 34
Serine and alanine racemase activities of VanT: a protein necessary for vancomycin resistance in Enterococcus gallinarum BM4174; Arias CA et al.; Vancomycin resistance in Enterococcus gallinarum results from the production of UDP-MurNAc-pentapeptide{D-Ser} . VanT, a membrane-bound serine racemase, is one of three proteins essential for this resistance . To investigate the selectivity of racemization of L-Ser or L-Ala by VanT, a strain of Escherichia coli TKL-10 that requires D-Ala for growth at 42 degrees C was used as host for transformation experiments using plasmids containing the full-length vanT from Ent . gallinarum or the alanine racemase gene (alr) of Bacillus stearothermophilus: both plasmids were able to complement E . coli TKL-10 at 42 degrees C . No alanine or serine racemase activities were detected in the host strain E . coli TKL-10 grown at 30, 34 or 37 degrees C . Serine and alanine racemase activities were found almost exclusively (96%) in the membrane fraction of E . coli TKL-10/pCA4(vanT): the alanine racemase activity of VanT was 14% of the serine racemase activity in both E . coli TKL-10/pCA4(vanT) and E . coli XL-1 Blue/pCA4(vanT) . Alanine racemase activity was present mainly (95%) in the cytoplasmic fraction of E . coli TKL-10/pJW40(alr), with a trace (1.6%) of serine racemase activity . Additionally, DNA encoding the soluble domain of VanT was cloned and expressed in E . coli M15 as a His-tagged polypeptide and purified: this polypeptide also exhibited both serine and alanine racemase activities; the latter was approximately 18% of the serine racemase activity, similar to that of the full-length, membrane-bound enzyme . N-terminal sequencing of the purified His-tagged polypeptide revealed a single amino acid sequence, indicating that the formation of heterodimers between subunits of His-tagged C-VanT and endogenous alanine racemases from E . coli was unlikely . The authors conclude that the membrane-bound serine racemase VanT also has alanine racemase activity but is able to racemize serine more efficiently than alanine, and that the cytoplasmic domain is responsible for the racemase activity.

Microbiology, 2000 Jul, 146 ( Pt 7), 1585 - 91
The acetyl xylan esterase of Bacillus pumilus belongs to a family of esterases with broad substrate specificity; Degrassi G et al.; The Bacillus pumilus gene encoding acetyl xylan esterase (axe) was identified and characterized . The axe gene was expressed and the recombinant enzyme produced in Escherichia coli was purified and characterized . The recombinant enzyme displayed similar properties to the acetyl xylan esterase (AXE) purified from B . pumilus . The AXE primary structure was 76% identical to the cephalosporin C deacetylase of B . subtilis, and 40% to two recently identified AXEs from Thermoanaerobacterium and Thermotoga maritima . These four proteins are of similar size and represent a new family of esterases having a broad substrate specificity . The recombinant AXE was demonstrated to have activity on several acetylated substrates, including on cephalosporin C.

Yi Chuan Xue Bao, 1999, 26(6), 715 - 20
{Integration via Tn10 and expression of Bti cryIVA gene in the chromosome DNA of flourescent Pseudomonas bacterial strains}; Liu GQ et al.; cryIVA gene of the Bacillus thuringiensis subsp . israelensis was subcloned into the Tn10 of a suicide transposon plasmid vector and a transposon plasmid pLF97A carrying cryIVA was constructed . By electroporation and transposition, cryIVA gene integrated into chromosome DNA of F.P.DE2 . Thus the genetic engineered bacterial strain, F.P.DE202, was constructed . Southern blotting revealed that this integration was happened in different loci of the chromosome . Western blotting demonstrated that cryIVA was expressed in the engineered strain, and that the expressed product possesed the pesticidal ability against the third-instar larva of Bradysia odoriphaga.

Clin Infect Dis, 2000 Jun, 30 Suppl 3, S309 - 15
Safety and immunogenicity of a five-dose series of inactivated Mycobacterium vaccae vaccination for the prevention of HIV-associated tuberculosis; Waddell RD et al.; Five doses of inactivated Mycobacterium vaccae vaccine were administered intradermally to 22 human immunodeficiency virus (HIV)-infected patients (11 bacille Calmette-Guerin {BCG}-positive and 11 BCG-negative) in Zambia whose CD4 lymphocyte counts were >/=200 cells/mm(3) . HIV viral load and lymphocyte proliferation responses were compared for vaccine recipients and 22 HIV-infected control patients (11 BCG-positive and 11 BCG-negative) . Immunization was safe and well tolerated in all patients, and induration at the vaccine site decreased from dose 1 to dose 5 . A transient decrease in HIV viral load was observed in BCG-positive vaccine recipients after dose 3 but not after subsequent doses . Median lymphocyte stimulation indices to M . vaccae were 6.0 in vaccine recipients and 2.3 in control patients (P<.001) . Stimulation indices were >/=3.0 in 19 vaccine recipients (86%) and 7 control patients (32%; P=.001) . A 5-dose series of vaccination with inactivated M . vaccae is safe in HIV-infected patients and induces lymphocyte proliferation responses to the vaccine antigen . M . vaccae vaccine is a candidate for the prevention of tuberculosis in HIV infection.

Clin Infect Dis, 2000 Jun, 30 Suppl 3, S299 - 301
Comparison of the protective efficacy of bacille calmette-Guérin vaccination against aerosol challenge with Mycobacterium tuberculosis and Mycobacterium bovis; Williams A et al.; The aim of this study was to compare protection by bacille Calmette-Guerin (BCG) vaccination against aerosol challenge with either Mycobacterium bovis or Mycobacterium tuberculosis in guinea pigs . Animals were challenged 5 weeks after vaccination with 10 or 100 lung lesion-forming units (lfu) of M . bovis or M . tuberculosis . Four weeks after challenge, numbers of lung lesions and counts of viable mycobacteria in spleens were high in saline-immunized animals . In contrast, BCG vaccination resulted in fewer lung lesions; after challenge with 10 and 100 lfu, the reduction was greater in animals infected with M . bovis (mean number of lesions, 1.17 and 31.2, respectively; P<.02) than in those infected with M . tuberculosis (mean number of lesions, 16.2 and 75.8, respectively) . No mycobacteria were recovered from spleens of BCG-vaccinated animals after challenge with 10 lfu of M . bovis, whereas 4 of 6 animals had detectable spleen mycobacterial counts after challenge with M . tuberculosis . Collectively, the results suggest that BCG vaccination may confer greater protection against challenge with M . bovis than challenge with M . tuberculosis.

Clin Infect Dis, 2000 Jun, 30 Suppl 3, S291 - 8
Toward the development of diagnostic assays to discriminate between Mycobacterium bovis infection and bacille Calmette-Guérin vaccination in cattle; Vordermeier HM et al.; A scientific review of the recent sharp increase in bovine tuberculosis in Great Britain has concluded that the development of a cattle vaccine holds the best prospect for long-term disease control . It is important to develop a diagnostic test that differentiates between vaccinated and Mycobacterium bovis-infected animals, to ensure that test-and-slaughter control strategies can continue alongside vaccination . The mycobacterial antigens ESAT-6, MPB64, and MPB83 are expressed at high levels in M . bovis but are expressed at low levels or not at all in bacille Calmette-Guerin (BCG) Pasteur . Promiscuous bovine T cell epitopes of these antigens were identified and formulated into a peptide cocktail . This cocktail and a cocktail composed of recombinant forms of the 3 antigens was able to distinguish cattle infected with virulent M . bovis from those vaccinated with BCG and from those sensitized to avian tuberculin in lymphocyte transformation and interferon-gamma assays.

Clin Infect Dis, 2000 Jun, 30 Suppl 3, S283 - 7
Vaccination of mice and cattle with plasmid DNA encoding the Mycobacterium bovis antigen MPB83; Chambers MA et al.; A scientific review of bovine tuberculosis in Great Britain has concluded that the development of a cattle vaccine holds the best prospect for long-term disease control . Recent reports of successful DNA vaccination against Mycobacterium tuberculosis in small animal models have raised the possibility of using a similar strategy to produce vaccines against Mycobacterium bovis infection in cattle . To test this possibility, BALB/c mice were immunized with DNA encoding the M . bovis antigen MPB83 . The mice responded to vaccination with a mixed IgG1/IgG2a response to the antigen and were protected from intravenous challenge with virulent M . bovis to a similar extent as those vaccinated with bacille Calmette-Guerin . The immunogenicity of the DNA vaccine in cattle was tested, after having established that DNA encoding MPB83 was immunogenic and elicited protective immunity in mice . In these studies, vaccinated animals had strong proliferative responses to MPB83.

Clin Infect Dis, 2000 Jun, 30 Suppl 3, S271 - 5
Ethical issues in tuberculosis vaccine trials; Snider DE Jr; Bacille Calmette-Guerin (BCG) vaccines are widely used, even though estimates of efficacy have ranged from zero to 80% . BCG is a relatively safe vaccine, but it can cause disseminated infection, especially in immunocompromised hosts . Thus, the development of a more reliably efficacious and safer vaccine is important to the control of tuberculosis . The testing of any new vaccine in human populations presents a number of ethical challenges that must be addressed . These include (1) the appropriateness of conducting such trials in developing countries; (2) the use of a BCG-vaccinated population as the control group; (3) the provision of tuberculin skin-test screening and preventive therapy to study participants; (4) the involvement of various "communities" in the trial(s); (5) the structure and process of ethical review; (6) establishing an effective method of obtaining informed consent; and (7) the roles and responsibilities of researchers and others in ensuring that trial results are available to the study population after the trial ends.

Clin Infect Dis, 2000 Jun, 30 Suppl 3, S262 - 5
Applying experiences from trials of bacille Calmette-Guérin vaccine; Wilson ME; Bacille Calmette-Guerin (BCG) vaccine, a live vaccine developed to prevent tuberculosis (TB), has been given to billions of persons over more than 7 decades . Studies of the efficacy of BCG vaccine have had widely divergent results, underscoring the complexity of the biology and immunology of TB . The long duration of TB infection, the heterogeneity of its clinical expression, and lack of inexpensive, reliable markers of infection and disease have made it difficult to study the impact of a vaccine, especially in resource-poor areas . A meta-analysis of data from trials of BCG vaccine found that studies conducted at sites that are a greater distance from the equator are associated with better vaccine efficacy, a finding that needs fuller study . BCG vaccine trials with higher validity scores showed higher rates of protection . Ongoing changes, including human immunodeficiency virus infection and demographic shifts, should be considered when developing trials of future vaccines . Analyses of past studies of BCG vaccine can identify sources of variation that may guide the design of studies of new vaccines . Rigorous study design and new tools are needed if studies are to provide clear, useful answers about new vaccines.

Clin Infect Dis, 2000 Jun, 30 Suppl 3, S257 - 61
In vitro measurement of protective mycobacterial immunity: antigen-specific expansion of T cells capable of inhibiting intracellular growth of bacille Calmette-Guérin; Worku S et al.; We investigated the ability of T cells expanded with mycobacterial antigens from healthy purified protein derivative-reactive donors and bacille Calmette-Guerin (BCG)-vaccinated volunteers to inhibit intracellular growth of BCG . Peripheral blood mononuclear cells were incubated for 7 days with mycobacterial whole lysate, live BCG, tetanus toxoid as control antigen, or medium alone . Autologous monocytes were separated by plastic adherence, allowed to mature for 6 days, and infected with BCG before serving as target cells . Expanded effector cells were cocultured with target cells for 72 h . Cocultures were then treated with 0.2% saponin to lyse infected monocytes and release intracellular BCG . Quantities of viable BCG present in these lysates were studied by colony-forming unit counting and radiometric labeling . We reproducibly found that lymphocytes expanded with mycobacterial whole lysate or live BCG significantly inhibited the intracellular growth of BCG, compared with lymphocytes expanded with tetanus toxoid or rested in medium . In addition, BCG vaccination enhanced the ability of T cells to inhibit intracellular mycobacterial growth in 3 of 5 volunteers . This assay may be useful for estimates of protective immunity induced by tuberculosis vaccines in human trials.

Clin Infect Dis, 2000 Jun, 30 Suppl 3, S250 - 3
Simple, practical ways to assess the protective efficacy of a new tuberculosis vaccine; Comstock GW; There is strong evidence that tuberculin sensitivity cannot be used to evaluate the efficacy of different strains of bacille Calmette-Guerin (BCG) . For identifying efficacious strains of BCG and evaluating candidates for new vaccines, the best method is a randomized trial . Simple trials in which newborns would be vaccinated with new and old vaccines in alternate years could demonstrate which vaccine was the better.

Clin Infect Dis, 2000 Jun, 30 Suppl 3, S233 - 42
A proposed national strategy for tuberculosis vaccine development; Ginsberg AM; The global tuberculosis epidemic causes approximately 5% of deaths worldwide . Despite recent concerted and largely successful tuberculosis control efforts, the incidence of tuberculosis in the United States remains 74-fold higher than the stated elimination goal of <1 case per million population by the year 2010 . Current bacille Calmette-Guerin vaccines, although efficacious in preventing extrapulmonary tuberculosis in young children, have shown widely variable efficacy in preventing adult pulmonary tuberculosis, confound skin test screening, and are not recommended for use in the United States . The Advisory Council for Elimination of Tuberculosis recently stated that tuberculosis would not be eliminated from the United States without a more effective vaccine . Recent scientific advances have created unprecedented opportunity for tuberculosis vaccine development . Therefore, members of the broad tuberculosis research and control communities have recently created and proposed a national strategy, or blueprint, for tuberculosis vaccine development, which is presented here.

Clin Infect Dis, 2000 Jun, 30 Suppl 3, S223 - 8
Veterinary tuberculosis vaccine development; Griffin JF; Tuberculosis caused by Mycobacterium bovis in domestic livestock and wildlife is a significant problem in many countries worldwide . Wildlife reservoirs of tuberculosis confound programs for tuberculosis eradication from domestic livestock . Successful vaccination against tuberculosis in domestic animals or wildlife could contribute to tuberculosis eradication . Bacille Calmette-Guerin (BCG) has been used as the prototype vaccine for domestic livestock and wildlife . The majority of studies have been carried out with BCG-vaccinated animals challenged experimentally with M . bovis . Although protection against disease has been evident in all these studies, protection against infection has rarely occurred . Results obtained with BCG vaccination of cattle, deer, ferrets, opossums, and rabbits are presented here and highlight the need for appropriate animal models for vaccination and control of the variables that influence the efficacy of BCG vaccine . Refinement of the existing animal models is essential for the advancement of tuberculosis vaccine research of relevance to animals and humans.

Clin Infect Dis, 2000 Jun, 30 Suppl 3, S217 - 22
Mucosal bacille calmette-Guérin vaccination of humans inhibits delayed-type hypersensitivity to purified protein derivative but induces mycobacteria-specific interferon-gamma responses; Hoft DF et al.; We conducted a placebo-controlled, double-dose-escalation trial of oral bacille Calmette-Guerin (BCG) vaccination in 48 healthy volunteers . Seven of 32 BCG recipients became purified protein derivative (PPD)-positive after dose 1, and only 1 remained positive after dose 2, which suggests that oral BCG has inhibitory effects on delayed-type hypersensitivity (DTH) responses . Ten of the original placebo recipients and 11 oral BCG recipients were recruited to return for an intradermal BCG booster vaccination . Five of 10 original placebo recipients developed PPD responses >/=10 mm, but none of 11 oral BCG recipients developed PPD induration after they received an intradermal BCG booster (P<.05; Fisher's exact test) . These results document persistent inhibitory effects of oral BCG vaccination on mycobacteria-specific DTH responses . Despite inhibition of DTH, oral BCG induced significant increases in mycobacteria-specific interferon (IFN)-gamma responses in peripheral blood mononuclear cells . More detailed studies of cytokine and homing molecule expression indicated that differential mucosal versus cutaneous trafficking may explain the dissociation between IFN-gamma and DTH responses.

Clin Infect Dis, 2000 Jun, 30 Suppl 3, S210 - 2
A nonhuman primate model for preclinical testing of new tuberculosis vaccines; McMurray DN; Nonhuman primates appear to have significant advantages over conventional laboratory animals in terms of modeling pulmonary tuberculosis for purposes of vaccine evaluation . Primates are quite susceptible to infection by the aerosol route, develop a humanlike disease, exhibit antigen-induced T lymphocyte reactivity both in vitro and in vivo, and can be protected quite effectively by bacille Calmette-Guerin vaccination . There are fewer than a dozen published studies of experimental tuberculosis in primates, and all of the available data on the response of primates to vaccination have been generated in rhesus monkeys (Macaca mulatta) . There have been no modern immunologic studies of primate tuberculosis . Thus, responses to tuberculosis vaccines in primates are only minimally characterized, and much additional baseline work remains to be done before the responses to new vaccines can be placed in the proper biological context.

J Biol Chem, 2000 Oct 27, 275(43), 33272 - 9
Inhibitory serpins from wheat grain with reactive centers resembling glutamine-rich repeats of prolamin storage proteins . Cloning and characterization of five major molecular forms; Ostergaard H et al.; Genes encoding proteins of the serpin superfamily are widespread in the plant kingdom, but the properties of very few plant serpins have been studied, and physiological functions have not been elucidated . Six distinct serpins have been identified in grains of hexaploid bread wheat (Triticum aestivum L.) by partial purification and amino acid sequencing . The reactive centers of all but one of the serpins resemble the glutamine-rich repetitive sequences in prolamin storage proteins of wheat grain . Five of the serpins, classified into two protein Z subfamilies, WSZ1 and WSZ2, have been cloned, expressed in Escherichia coli, and purified . Inhibitory specificity toward 17 proteinases of mammalian, plant, and microbial origin was studied . All five serpins were suicide substrate inhibitors of chymotrypsin and cathepsin G . WSZ1a and WSZ1b inhibited at the unusual reactive center P(1)-P(1)' Gln-Gln, and WSZ2b at P(2)-P(1) Leu-Arg-one of two overlapping reactive centers . WSZ1c with P(1)-P(1)' Leu-Gln was the fastest inhibitor of chymotrypsin (k(a) = 1.3 x 10(6) m(-1) s(-1)) . WSZ1a was as efficient an inhibitor of chymotrypsin as WSZ2a (k(a) approximately 10(5) m(-1) s(-1)), which has P(1)-P(1)' Leu-Ser-a reactive center common in animal serpins . WSZ2b inhibited plasmin at P(1)-P(1)' Arg-Gln (k(a) approximately 10(3) m(-1) s(-1)) . None of the five serpins inhibited Bacillus subtilisin A, Fusarium trypsin, or two subtilisin-like plant serine proteinases, hordolisin from barley green malt and cucumisin D from honeydew melon . Possible functions involving interactions with endogenous or exogenous proteinases adapted to prolamin degradation are discussed.

Curr Opin Plant Biol, 2000 Aug, 3(4), 329 - 35
Mechanisms, ecological consequences and agricultural implications of tri-trophic interactions; Agrawal AA; Recent research bridging mechanistic and ecological approaches demonstrates that plant attributes can affect herbivores, natural enemies of herbivores, and their interaction . Such effects may be genetically variable among plants and/or induced in individual plants by herbivore attack, and are mediated by primary plant attributes (i.e . nutritional quality and physical structure) and defense-related products (i.e . secondary chemicals and plant volatiles), and may be modified by human activity (e.g . by the introduction of Bacillus thuringiensis) . The study of tri-trophic interactions is important in order to understand natural species interactions and to manipulate these interactions in pest control.

Am J Surg, 2000 Feb 1, 179(2 Suppl 1), 45 - 50
Role of aztreonam in the treatment of nosocomial pneumonia in the critically ill surgical patient; Boucher BA; In 1995 the American Thoracic Society issued an official consensus statement on the treatment of hospital-acquired pneumonia (HAP) . Classes of antimicrobials included in the list of antimicrobials deemed to be suitable for the empiric treatment of severe HAP were the aminoglycosides, quinolones, antipseudomonal penicillins, carbapenems, and beta-lactam/beta-lactamase inhibitor combinations . Aztreonam, a monobactam, was also listed and is unique among these agents based on its spectrum of activity being limited to the gram-negative bacillary bacteria combined with an excellent safety profile . This review focuses on the role of aztreonam in the treatment of nosocomial pneumonia in the critically ill patient.A review of the literature was performed using PubMed and secondary literature sources as to the clinical efficacy of aztreonam in the treatment of lower respiratory tract infections as well as its pharmacokinetic and safety profiles . An analysis of aztreonam's potential pharmacoeconomic advantages compared with other agents was also performed.Numerous studies have documented that aztreonam has effectiveness that is equal or superior to that of other suitable antibiotics in the treatment of nosocomial pneumonia . Its excellent safety profile makes it a particularly attractive agent compared with the aminoglycosides . Considering the potential costs of bacterial resistance from the use of broader-spectrum alternatives, a case can be made that aztreonam is a pharmacoeconomically sound choice as well.

Biochem Biophys Res Commun, 2000 May 27, 272(1), 218 - 23
Noninsecticidal parasporal proteins of a Bacillus thuringiensis serovar shandongiensis isolate exhibit a preferential cytotoxicity against human leukemic T cells; Lee DW et al.; A Bacillus thuringiensis isolate, 89-T-34-22, belonging to the serovar shandongiensis (H22) produced noninsecticidal and nonhemolytic proteins crystallizing into irregular-shaped parasporal inclusions . The proteins showed in vitro cytotoxicity to human cells, including cancer cells, only when activated by protease treatment . The human leukemic T (MOLT-4) cells were > 100 times more susceptible than HeLa and normal T cells to the proteins of 89-T-34-22 . The cytotoxicity was dose dependent and the median effective concentration for the MOLT-4 was 3.5 microg/ml . The cytopathy induced by the 89-T-34-22 proteins was characterized by remarkable condensation of the nucleus and cell-ballooning . Five major parasporal proteins of 89-T-34-22, with molecular masses in the range of 16-160 kDa, shared no similarity with the previously reported proteins in terms of the N-terminal sequence.

Arch Environ Contam Toxicol, 2000 Aug, 39(2), 177 - 82
Effects of sustained-release methoprene and a combined formulation of liquid methoprene and Bacillus thuringiensis israelensis on insects in salt marshes; Lawler SP et al.; Aquatic insects are an important component of the food web in salt marshes, therefore it is necessary to test whether pesticides used to control mosquitoes in salt marshes are safe for nontarget insects . We tested the nontarget effects of a combined formulation (duplex) of Bacillus thuringiensis israelensis (B.t.i.) and liquid methoprene (an insect development regulator) or sustained-release methoprene pellets (Altosid(R) pellets) by applying these materials to replicated salt marsh ponds at maximum label rates . Untreated ponds served as controls . We measured effects of the pesticides by rearing immature mosquitoes (Aedes dorsalis) and water boatmen (Trichocorixa reticulata) in predator-exclusion cages and by monitoring uncaged populations of invertebrates using replicated sweep-net samples . Both pesticides killed caged mosquitoes, and the activity of the Altosid(R) pellets continued through 99 days . There were no detectable effects of either pesticide on the survival or maturation of T . reticulata, or on abundances of uncaged invertebrates . The long-term activity of the pellets could help minimize mosquito abatement activity in salt marshes where there are breeding birds or endangered species . However, other studies suggest that this advantage needs to be balanced against the risks that sustained-release formulations could lead to development of resistance in mosquitoes or that initially undetected nontarget effects could build over time.

Electrophoresis, 2000 May, 21(9), 1740 - 5
A proteomic analysis of secreted proteins from xylan-induced Bacillus sp . strain K-1; Chu PW et al.; The expression level of extracellular proteins in an alkaliphilic bacterium, Bacillus sp . strain K-1, grown in a xylan-containing medium, is significantly increased when compared with that grown in the nonxylan culture medium . A proteomic approach has been efficiently applied to separate and characterize these differentially expressed secretory proteins . Eight prominent protein spots were identified and subjected to N-terminal amino acid sequencing . The results show that three spots share considerable similarity with the xylanolytic enzymes and that two spots share considerable similarity with the GltC regulatory protein and 3-dehydroquinate dehydratase, respectively . In addition, the three other proteins show little similarity with the known proteins in the database . In conclusion, our results demonstrate that the proteomic approach is a highly efficient method to rapidly study the differential expression of the secreted proteins by Bacillus sp . strain K-1 grown under xylan-induced condition.

J Biol Chem, 2000 Oct 6, 275(40), 31115 - 20
Substrate specificity in the highly heterogeneous M4 peptidase family is determined by a small subset of amino acids; de Kreij A et al.; The members of the M4 peptidase family are involved in processes as diverse as pathogenicity and industrial applications . For the first time a number of M4 family members, also known as thermolysin-like proteases, has been characterized with an identical substrate set and a uniform set of assay conditions . Characterization with peptide substrates as well as high performance liquid chromatography analysis of beta-casein digests shows that the M4 family is a homogeneous family in terms of catalysis, even though there is a significant degree of amino acid sequence variation . The results of this study show that differences in substrate specificity within the M4 family do not correlate with overall sequence differences but depend on a small number of identifiable amino acids . Indeed, molecular modeling followed by site-directed mutagenesis of one of the substrate binding pocket residues of the thermolysin-like proteases of Bacillus stearothermophilus converted the catalytic characteristics of this variant into that of thermolysin.

Nucleic Acids Res . 2000 Jun 1;28(11):E56.
BtrI, a novel restriction endonuclease, recognises the non-palindromic sequence 5'-CACGTC(-3/-3)-3'; Degtyarev SK et al.; The recognition sequence and cleavage positions of a new restriction endonuclease BTR:I isolated from Bacillus stearothermophilus SE-U62 have been determined . BTR:I belongs to a rare type IIQ of restriction endonucleases, which recognise non-palindromic nucleotide sequences and cleave DNA symmetrically within them.

Hepatology, 2000 Jul, 32(1), 56 - 65
Human placenta sphingomyelinase, an exogenous acidic pH-optimum sphingomyelinase, induces oxidative stress, glutathione depletion, and apoptosis in rat hepatocytes; Garcia-Ruiz C et al.; Ceramide has been identified as a putative lipid messenger that mediates diverse cellular processes including cell death . Since glutathione (GSH) depletion is known to sensitize cells to many cytotoxic agents and as a result of the reported regulation of neutral sphyngomyelinase (NSMase) by GSH, the present study compared the role of individual SMases in the induction of oxidative stress, regulation of cellular GSH, and apoptosis of rat hepatocytes . Exposure of cultured rat hepatocytes to exogenous Bacillus cereus sphingomyelinase (bSMase), a neutral SMase, or human placenta sphingomyelinase (hSMase), an acidic SMase (ASMase), generated similar ceramide levels in a dose-dependent manner . However, whereas bSMase increased hepatocellular GSH levels, hSMase depleted GSH stores, an effect that was prevented by monensin and mannose 6-phosphate (M-6-P), suggesting that exogenous hSMase enters hepatocytes by endocytosis and is delivered to an endosomal/lysosomal acidic compartment . Interestingly, despite the differential effect of either SMases on cell GSH levels, both bSMase and hSMase increased gamma-glutamylcysteine synthetase heavy-subunit chain (gamma-GCS-HS) mRNA levels . Consistent with these findings on GSH regulation, hSMase, but not bSMase, generated reactive oxygen species (ROS), being accompanied by mitochondrial depolarization, suggesting that hSMase targeted mitochondria, leading to oxidative stress . Accordingly, hepatocytes displayed a selective sensitivity to hSMase in contrast to bSMase exposure, and depletion of GSH stores enhanced susceptibility to hSMase as a result of potentiation of ROS formation and caspase 3 activation . Thus, these findings reveal the ability of ASMase to induce oxidative stress as a result of the targeting of mitochondria, and that GSH depletion sensitizes hepatocytes to the ASMase-induced apoptosis.

Biotechniques, 2000 Jun, 28(6), 1214 - 9
Purification and reconstitution of an integral membrane protein, the photoreaction center of Rhodobacter sphaeroides, using synthetic sugar esters; Peters H et al.; Detergents are indispensable reagents for the extraction and solubilization of integral membrane proteins, but their removal from a reconstituted phospholipid-protein complex is usually desirable . In this paper, we describe a novel method in which the synthetic sugar esters 6-O-octanoyl-beta-D-glucose (OG) or 6-O-octanoyl-beta-D-mannose (OM) are used as detergents for both the isolation and the rapid reconstitution of the photosynthetic reaction center protein of Rhodobacter sphaeroides . Following solubilization of the reaction center with OG or OM and reconstitution of this protein in liposomes, a convenient removal of these detergents was achieved within less than two hours by hydrolytic cleavage of the sugar esters using immobilized lipases . Best results were achieved with lipase from Bacillus sp . immobilized on silica gel.

Eur J Biochem, 2000 Jul, 267(13), 4068 - 74
Toxic lactonic lipopeptide from food poisoning isolates of Bacillus licheniformis; Mikkola R et al.; Toxins from three Bacillus licheniformis strains connected to a fatal food poisoning were isolated and their structures elucidated . Toxins were purified from methanol extracts of the B . licheniformis biomass using boar sperm cells as the toxicity indicator . The HPLC purified toxins showed protonated masses m/z 1007, 1021 and 1035 in MALDI-TOF-MS . The toxins isolated from the strains of different origins contained the same three components of which and each had a same amino-acid residues L-Gln, L-Leu, D-Leu, L-Val, L-Asp, D-Leu and L-Ile in that order . Toxins were identified as lichenysin A, a cyclic lactonic heptalipopeptide in which the main 3-hydroxy fatty acids are 13-15 carbons in length . We showed that the toxins from food and food poisoning isolates of B . licheniformis were identical to lichenysin A both in the structure and in the toxic symptoms induced to boar spermatozoa . Confocal laser scanning microscopy showed that the acrosome and the plasma membrane of boar spermatozoa were the targets of lichenysin A toxicity.

Indian J Exp Biol, 1999 Dec, 37(12), 1157 - 66
Mycobacterial proteins--immune targets for antituberculous subunit vaccine; Dhiman N et al.; Cellular and humoral immunity induced by Mycobacterium tuberculosis has led to identification of newer vaccine candidates, but despite this, many questions concerning the protection against tuberculosis remain unanswered . Recent progress in this field has centered on T cell subset responses and cytokines that these cells secrete . There has been a steady progress in identification and characterization of several classes of major mycobacterial proteins which includes secretory/export proteins, cell wall associated proteins, heat shock proteins and cytoplasmic proteins . The protein antigens are now believed to represent the key protective immunity inducing antigens in the bacillus . In this review, various mycobacterial protein antigens of vaccination potential are compared for their efficacy in light of current immunological knowledge.

Arch Ophthalmol, 2000 Jun, 118(6), 803 - 6
Effects of intravitreal corticosteroids in the treatment of Bacillus cereus endophthalmitis; Liu SM et al.; OBJECTIVE: To investigate whether intravitreal corticosteroid therapy reduces the extent of inflammatory intraocular tissue damage caused by Bacillus cereus endophthalmitis . METHODS: New Zealand white rabbits were inoculated with 1 x 10(6) B cereus organisms and randomized to receive no treatment (control eyes; n=14), intravitreal vancomycin hydrochloride (n=13), or a combination of intravitreal vancomycin and dexamethasone sodium phosphate (n=13) after 24 hours . The eyes were examined and graded for clinical signs of infection and inflammation on days 7 and 14, followed by enucleation for histopathologic analysis . RESULTS: Both treated groups had significantly less clinical sequelae than controls on day 7 . By day 14, eyes given combination treatment had significantly less clinically graded corneal (P=.03) and conjunctival (P=.007) inflammation than eyes treated with vancomycin . Histopathologic analysis revealed a significant decrease in inflammatory changes between all treated eyes and controls at day 14 . The only statistically significant difference between eyes given combination treatment and eyes given vancomycin alone was in the retina (P=.03) . CONCLUSIONS: Intravitreal corticosteroids may enhance the recovery from B cereus endophthalmitis when given in conjunction with intravitreal antibiotics . The beneficial effect of corticosteroids is noted clinically, but not histologically, by day 14 after single-dose treatment in rabbits . CLINICAL RELEVANCE: This study provides evidence that the use of intravitreal corticosteroids with antibiotics for the treatment of B cereus endophthalmitis may lead to an improvement compared with the use of antibiotics alone . Arch Ophthalmol . 2000;118:803-806

J Mol Biol, 2000 Jun 30, 300(1), 1 - 10
Crystal structure of a novel germination protease from spores of Bacillus megaterium: structural arrangement and zymogen activation; Ponnuraj K et al.; The DNA in the core of spores of Bacillus species is saturated with a group of small, acid-soluble proteins (SASP) that protect DNA from a variety of harsh treatments and play a major role in spore resistance and long-term spore survival . During spore germination, SASPs are rapidly degraded to amino acids and this degradation is initiated by a sequence-specific protease called germination protease (GPR), which exhibits no obvious mechanistic or amino acid sequence similarity to any known class of proteases . GPR is synthesized during sporulation as an inactive tetrameric zymogen termed P(46), which later autoprocesses to a smaller form termed P(41), which is active only during spore germination . Here, we report the crystal structure of P(46) from Bacillus megaterium at 3.0 A resolution and the fact that P(46) monomer adopts a novel fold . The asymmetric unit contains two P(46) monomers and the functional tetramer is a dimer of dimers, with an approximately 9 A channel in the center of the tetramer . Analysis of the P(46) structure and site-directed mutagenesis studies have provided some insight into the mechanism of zymogen activation as well as the zymogen's lack of activity and the inactivity of P(41) in the mature spore .

Biochim Biophys Acta, 2000 Jun 15, 1479(1-2), 185 - 95
The role of amino acid residues in the active site of a midgut microvillar aminopeptidase from the beetle Tenebrio molitor; Cristofoletti PT et al.; Aminopeptidases are major enzymes in the midgut microvillar membranes of most insects and are targets of insecticidal Bacillus thuringiensis crystal delta-endotoxins . Sequence analysis and substrate specificity studies showed that these enzymes resemble mammalian aminopeptidase N, although information on the organization of their active site is lacking . The effect of pH at different temperatures on the kinetic parameters of Tenebrio molitor (Coleoptera) larval aminopeptidase showed that enzyme catalysis depend on a deprotonated (pK 7.6; DeltaH degrees (ion), 7.6 kJ/mol) and a protonated (pK 8.2; DeltaH degrees (ion), 16.8 kJ/mol) group . 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide and diethylpyrocarbonate inactivate the enzyme by modifying a pK 5.8 carboxylate and a imidazole group, respectively, with a reaction order around 1 . Tetranitromethane changes the K(m) of the enzyme without affecting its V(max) by modifying a phenol group . The presence of a competitive inhibitor decrease the inactivation reaction rates in all these cases . EDTA inactivation of the aminopeptidase is affected by pH and temperature suggesting the involvement in metal binding of at least one deprotonated imidazole group (pK 5.8, DeltaH degrees (ion), 20 kJ/mol) . The data support the hypothesis that T . molitor aminopeptidase catalysis depends on a catalytic metal and on a carboxylate and a protonated imidazole group, whereas substrate binding relies in one phenol and one carboxylate groups . The insect aminopeptidase shares common features with mammalian aminopeptidase N, although differing in details of substrate binding and in residues directly involved in catalysis.

Enzyme Microb Technol, 2000 Jul 1, 27(1-2), 95 - 99
Electroelution as a simple and fast protein purification method: isolation of an extracellular xylanase from Bacillus sp . CCMI 966; Sa-Pereira P et al.; An efficient and simple modified method of electroelution is described that can be used as a time-saving method for eluting multiple protein bands . Provided that the proteins are highly expressed, they can be purified rapidly and without requiring any prior knowledge of the protein characteristics . A xylanase excreted by Bacillus sp . CCMI 966 was purified directly from the polyacrylamide gel . Some of the properties of this enzyme are presented . It had an unusually apparent high molecular mass of 340kDa, as determined by native PAGE . The specific activity of the purified xylanase was 137 U/mg.

Enzyme Microb Technol, 2000 Jul 1, 27(1-2), 26 - 32
Immobilization/stabilization on Eupergit C of the beta-galactosidase from B . circulans and an alpha-galactosidase from Aspergillus oryzae; Hernaiz MJ et al.; Two synthetically useful glycosidases, the beta-galactosidase from Bacillus circulans and an alpha-galactosidase from Aspergillus oryzae have been immobilized on Eupergit C . The immobilized enzymes retain high catalytic activity and show increased thermal stability compared with the free enzymes.

Luminescence, 2000 May-Jun, 15(3), 153 - 7
Activation of peripheral phagocytes in BCG-vaccinated subjects; Vuotto ML et al.; The role of polymorphonuclear leukocytes (PMNs) in the immune defence against intracellular bacteria has long been neglected . Only recently have studies begun to address this issue . In this study the behavior of peripheral PMNs in Bacillus Calmette-Guerin (BCG) vaccinated subjects was investigated . Twenty healthy and purified protein derivative-negative adults were studied before, and two and four months after, BCG administration . Luminol-amplified chemiluminescence (CL) emission was evaluated in whole blood phagocytes using a soluble stimulus, such as phorbol mirystate acetate, or particulates such as zymosan opsonized with homologous (OZH) or autologous (OZA) serum . Specific IgG, IgA and IgM against antigen -60 by ELISA, total immunoglobulin, C3 and C4 components of complement, were assessed by immunochemical tests . The results revealed a late heightened production of reactive oxygen intermediates in vaccinated subjects in presence of OZA and OZH . Our findings confirm that the role of PMNs and their mediators in immunoregulation of intracellular diseases needs to be re-evaluated .

N Engl J Med, 2000 Jun 22, 342(25), 1887 - 92
Randomized, controlled trials, observational studies, and the hierarchy of research designs; Concato J et al.; BACKGROUND: In the hierarchy of research designs, the results of randomized, controlled trials are considered to be evidence of the highest grade, whereas observational studies are viewed as having less validity because they reportedly overestimate treatment effects . We used published meta-analyses to identify randomized clinical trials and observational studies that examined the same clinical topics . We then compared the results of the original reports according to the type of research design . METHODS: A search of the Medline data base for articles published in five major medical journals from 1991 to 1995 identified meta-analyses of randomized, controlled trials and meta-analyses of either cohort or case-control studies that assessed the same intervention . For each of five topics, summary estimates and 95 percent confidence intervals were calculated on the basis of data from the individual randomized, controlled trials and the individual observational studies . RESULTS: For the five clinical topics and 99 reports evaluated, the average results of the observational studies were remarkably similar to those of the randomized, controlled trials . For example, analysis of 13 randomized, controlled trials of the effectiveness of bacille Calmette-Guerin vaccine in preventing active tuberculosis yielded a relative risk of 0.49 (95 percent confidence interval, 0.34 to 0.70) among vaccinated patients, as compared with an odds ratio of 0.50 (95 percent confidence interval, 0.39 to 0.65) from 10 case-control studies . In addition, the range of the point estimates for the effect of vaccination was wider for the randomized, controlled trials (0.20 to 1.56) than for the observational studies (0.17 to 0.84) . CONCLUSIONS: The results of well-designed observational studies (with either a cohort or a case-control design) do not systematically overestimate the magnitude of the effects of treatment as compared with those in randomized, controlled trials on the same topic.

J Mol Biol, 2000 May 26, 299(1), 255 - 79
Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase; Joshi MD et al.; The pH optima of family 11 xylanases are well correlated with the nature of the residue adjacent to the acid/base catalyst . In xylanases that function optimally under acidic conditions, this residue is aspartic acid, whereas it is asparagine in those that function under more alkaline conditions . Previous studies of wild-type (WT) Bacillus circulans xylanase (BCX), with an asparagine residue at position 35, demonstrated that its pH-dependent activity follows the ionization states of the nucleophile Glu78 (pKa 4.6) and the acid/base catalyst Glu172 (pKa 6.7) . As predicted from sequence comparisons, substitution of this asparagine residue with an aspartic acid residue (N35D BCX) shifts its pH optimum from 5.7 to 4.6, with an approximately 20% increase in activity . The bell-shaped pH-activity profile of this mutant enzyme follows apparent pKa values of 3.5 and 5.8 . Based on 13C-NMR titrations, the predominant pKa values of its active-site carboxyl groups are 3.7 (Asp35), 5.7 (Glu78) and 8.4 (Glu172) . Thus, in contrast to the WT enzyme, the pH-activity profile of N35D BCX appears to be set by Asp35 and Glu78 . Mutational, kinetic, and structural studies of N35D BCX, both in its native and covalently modified 2-fluoro-xylobiosyl glycosyl-enzyme intermediate states, reveal that the xylanase still follows a double-displacement mechanism with Glu78 serving as the nucleophile . We therefore propose that Asp35 and Glu172 function together as the general acid/base catalyst, and that N35D BCX exhibits a "reverse protonation" mechanism in which it is catalytically active when Asp35, with the lower pKa, is protonated, while Glu78, with the higher pKa, is deprotonated . This implies that the mutant enzyme must have an inherent catalytic efficiency at least 100-fold higher than that of the parental WT, because only approximately 1% of its population is in the correct ionization state for catalysis at its pH optimum . The increased efficiency of N35D BCX, and by inference all "acidic" family 11 xylanases, is attributed to the formation of a short (2.7 A) hydrogen bond between Asp35 and Glu172, observed in the crystal structure of the glycosyl-enzyme intermediate of this enzyme, that will substantially stabilize the transition state for glycosyl transfer . Such a mechanism may be much more commonly employed than is generally realized, necessitating careful analysis of the pH-dependence of enzymatic catalysis.






What Is Functional Genomics?, What Is Bioassay?, What Is Botulism?, What Is Activated Sludge?, What Is Genome?, e, Microbe, n, Bacterium, n, Microbes, r, Bacteriology, i, Microorganism, e, Staphylococcus aureus, e, Bacteriological, r, Escherichia coli, o, Acinetobacter, o, Escherichia coli, e, Yeast, e, Morganella, e, Culture medium, e, Clostridia, c, Clostridia, i, Escherichia coli, s, Antimicrobial, e, S. cerevisiae, n, Streptococcal, n, Lactobacillus, r, Fermentations, i, S. cerevisiae, i, Wastewater, o, Cholera, c, Escherichia coli, c, Biological reactors




 

   Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology
Anaerobic Microbiology
Antimicrobial Susceptibility
Artificial Atmosphere
Bioassay of Antibiotics
Biofilm Microbiology
Bioreactor Technology
Biotechnology
Cell Biology
Clinical Microbiology
Environmental Microbiology
Experiments with Yeast		 Fermentation
Food Microbiology
Functional Genomics
Gene Technology
Growth Media Development
Growth Rate and Lag Time
Industrial Microbiology
Medical/Pharmaceutical Field
Microbiological Assay
Microbiological Research
Microbiology of Cosmetics

go to a specific theme...

 Military Microbiology
Molecular Microbiology
Mutagenicity and Genotoxicity
Oral Microbiology
Patents
Postantibiotic Studies
Soil Microbiology
Spore Microbiology
Veterinary Microbiology
Waste/Wastewater Treatment
Water Microbiology
Wine Microbiology

 


 

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

 

 

Last modified: May 25, 2005