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