Semin Cutan Med Surg, 1997 Sep, 16(3), 188 - 99 Bacillary angiomatosis and other Bartonella species infections; Wong R et al.; Infections with organisms of the genus Bartonella, for many years important only in South and Central America, have assumed significance in developing countries, especially in conjunction with the advent of the pandemic of the human immunodeficiency virus infection . New molecular and culture techniques have determined that these organisms cause new diseases such as bacillary angiomatosis as well as diseases the etiology of which have been unknown such as cat scratch disease . In this article, the microbiology, pathogenesis, histopathology and clinical manifestations of diseases caused by these organisms are discussed. Protein Sci, 1997 Sep, 6(9), 1937 - 44 Determination of pKa values of the histidine side chains of phosphatidylinositol-specific phospholipase C from Bacillus cereus by NMR spectroscopy and site-directed mutagenesis; Liu T et al.; Two active site histidine residues have been implicated in the catalysis of phosphatidylinositol-specific phospholipase C (PI-PLC) . In this report, we present the first study of the pKa values of histidines of a PI-PLC . All six histidines of Bacillus cereus PI-PLC were studied by 2D NMR spectroscopy and site-directed mutagenesis . The protein was selectively labeled with 13C epsilon 1-histidine . A series of 1H-13C HSQC NMR spectra were acquired over a pH range of 4.0-9.0 . Five of the six histidines have been individually substituted with alanine to aid the resonance assignments in the NMR spectra . Overall, the remaining histidines in the mutants show little chemical shift changes in the 1H-13C HSQC spectra, indicating that the alanine substitution has no effect on the tertiary structure of the protein . H32A and H82A mutants are inactive enzymes, while H92A and H61A are fully active, and H81A retains about 15% of the wild-type activity . The active site histidines, His32 and His82, display pKa values of 7.6 and 6.9, respectively . His92 and His227 exhibit pKa values of 5.4 and 6.9 . His61 and His81 do not titrate over the pH range studied . These values are consistent with the crystal structure data, which shows that His92 and His227 are on the surface of the protein, whereas His61 and His81 are buried . The pKa value of 6.9 corroborates the hypothesis of His82 acting as a general acid in the catalysis . His32 is essential to enzyme activity, but its putative role as the general base is in question due to its relatively high pKa. Anal Biochem, 1997 Aug 15, 251(1), 45 - 9 Determination of the kinetic parameters for phospholipase C (Bacillus cereus) on different phospholipid substrates using a chromogenic assay based on the quantitation of inorganic phosphate; Hergenrother PJ et al.; The kinetic parameters of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLCBc) have been evaluated for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine substrates with a new assay based on the quantitation of inorganic phosphate (Pi) . Treatment of the phosphomonoester product of the PLCBc-catalyzed hydrolysis of these phospholipids with alkaline phosphatase releases Pi . This Pi forms a complex with ammonium molybdate that is then reduced by ascorbic acid to provide a blue molybdenum chromogen with an absorbance maximum at 700 nm . This highly sensitive assay may be used to determine accurately less than 5 nmol of Pi in solution . Performing the assay in 96-well plates provides a rapid and convenient method to evaluate a variety of phospholipids as substrates for PLCBc . The assay has been utilized to ascertain the kinetic constants for the PLCBc-catalyzed hydrolysis of 1,2-dihexanoyl-sn-glycero-3-phosphocholine, 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine, and 1,2-dihexanoyl-sn-glycero-3-phospho-L-serine . It is found that these compounds are substrates for the enzyme with their VmaxS being in the order of phosphatidylcholine > phosphatidylethanolamine > phosphatidylserine. Electrophoresis, 1997 Aug, 18(8), 1384 - 92 'Proteomic contigs' of Mycobacterium tuberculosis and Mycobacterium bovis (BCG) using novel immobilised pH gradients; Urquhart BL et al.; Tuberculosis remains a major health problem throughout the world and the failure of the existing bacille Calmette-Guerin (BCG) vaccine in recent trials has prompted a search for potential replacements . Recent advances in molecular and cell biology have cast doubts on the ability of genetic analysis alone to predict polygenic human diseases and other complex phenotypes and have therefore redirected our attention to proteome studies to complement information obtained from DNA sequencing initiatives . Novel acidic (pH 2.3-5) and basic (pH 6-11) IPG gel gradients were employed in conjunction with commercially available pH 4-7 gradients to significantly increase (fourfold) the number of protein spots previously resolved on two-dimensional (2-D) gels of Mycobacterium species . A total of 772 and 638 protein spots were observed for M . bovis BCG and M . tuberculosis H37Rv, respectively, the latter corresponding to only the pH regions 4-7 and 6-11 . Of interest was the bimodal distribution observed for proteins separated from M . bovis BCG across both M(r) and pH ranges . Some differences in protein expression were observed between these two organisms, contrary to what may have been expected considering the high degree of conservation in gene order and sequence similarity between homologous genes . Further work will be directed towards a more detailed analysis of these differences, so as to allow more accurate diagnosis between vaccination and active tuberculosis . The latter is of major importance to epidemiological studies and for patient management. Biochim Biophys Acta, 1997 Aug 21, 1358(1), 103 - 12 HDL3 binds to glycosylphosphatidylinositol-anchored proteins to activate signalling pathways; Nazih-Sanderson F et al.; Previous studies have indicated that in HepG2 cells HDL3-signalling involves glycosylphosphatidylinositol (GPI) anchored proteins . HDL3-binding to HepG2 cells was found to be enhanced by cellular preincubation with PI-PLC inhibitors and sensitive to a cellular preincubation with exogenous PI-PLC, suggesting that HDL3 binds directly on GPI-anchored proteins to initiate signaling . Moreover HDL3-binding was found to be partly inhibited by antibodies against the HDL-binding protein (AbHBP) . HDL3, when binding to HepG2 cells, promoted the release in the culture medium of a 110 kDa protein that binds AbHBP, while a cellular preincubation with antibodies against the inositol-phosphoglycan (IPG) moiety of GPI-anchor (AbIPG), used to block lipolytic cleavage of the GPI-anchor, inhibits HDL3-induced release of the 110 kDa protein in the culture medium . In {3H}-PC prelabeled HepG2 cells, AbHBP were found to stimulate PC-hydrolysis and DAG generation within 5 min as did HDL3 stimulation . Cellular preincubation with AbIPG was found to inhibit only the HDL3-signal and not the AbHBP-signal, while a prior cellular pretreatment with PI-PLC from Bacillus cereus was found to inhibit the HDL3-and AbHBP-signal . Moreover cellular preincubation with AbHBP for 1 h at 37 degrees C was found to inhibit HDL3-signalling pathways . Our results suggest that in HepG2 cells a 110 kDa protein, which could be HBP, can be anchored to the membrane via GPI, and can function in HDL3-signalling pathways as binding sites. J Biol Chem, 1997 Sep 19, 272(38), 23473 - 6 Proteinase-mediated insect resistance to Bacillus thuringiensis toxins; Oppert B et al.; Two Bacillus thuringiensis (Bt)-resistant strains of the Indianmeal moth, Plodia interpunctella, lack a major gut proteinase that activates Bt protoxins . The absence of this enzyme is genetically linked to larval survival on Bt-treated diets . When considered with previous data supporting the existence of receptor-mediated insect resistance to Bt, these results provide evidence that insect adaptation to these toxins occurs through multiple physiological mechanisms, which complicate efforts to prevent or manage resistance to Bt toxins in insect control programs. Kekkaku, 1997 Aug, 72(8), 499 - 504 {Clinical evaluation on causes of death in patients with pulmonary tuberculosis who died within one year after diagnosing as TB}; Ohse H et al.; We evaluated the cause of death in patients with pulmonary tuberculosis who died within one year after diagnosing as tuberculosis . Of 325 bacillary patients during the past seven years, 43 (13.2%) died within one year . Twenty-three patients (53.5%) died directly of tuberculosis . In this group, 13 patients died in emaciation state . Most of them were aged and under a poor nutritional condition . Some patients died in spite of improvement of tuberculosis . The fact indicates the need to detect tuberculosis as early as possible in elderly persons, and treatment should be initiated immediately . Eight patients died of respiratory failure and their chest X-ray film showed wade-spread tuberculosis . Seven of the patients died in spite of initiating treatment within one month after the onset of symptoms . This fact suggests the importance of regular check up by chest X-ray to detect tuberculosis early . Two patients died of massive hemoptysis . They had an episode of bloody sputum and the laboratory examination showed anemia . On the other hand, 20 patients died due to coexisting diseases unrelated to tuberculosis . Ten patients died of malignant diseases and most of them were lung cancer . Two patients died of hepatic failure possibly caused by the adverse reaction of TB chemotherapy . The interval between the onset of the treatment and death was less than a month, and the fact suggests the need to observe carefully for adverse reactions especially in the early stage of treatment. Appl Environ Microbiol, 1997 Sep, 63(9), 3569 - 76 Cloning, sequencing, and expression of the gene encoding extracellular alpha-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme; Dong G et al.; The gene encoding the hyperthermophilic extracellular alpha-amylase from Pyrococcus furiosus was cloned by activity screening in Escherichia coli . The gene encoded a single 460-residue polypeptide chain . The polypeptide contained a 26-residue signal peptide, indicating that this Pyrococcus alpha-amylase was an extracellular enzyme . Unlike the P . furiosus intracellular alpha-amylase, this extracellular enzyme showed 45 to 56% similarity and 20 to 35% identity to other amylolytic enzymes of the alpha-amylase family and contained the four consensus regions characteristic of that enzyme family . The recombinant protein was a homodimer with a molecular weight of 100,000, as estimated by gel filtration . Both the dimer and monomer retained starch-degrading activity after extensive denaturation and migration on sodium dodecyl sulfate-polyacrylamide gels . The P . furiosus alpha-amylase was a liquefying enzyme with a specific activity of 3,900 U mg-1 at 98 degrees C . It was optimally active at 100 degrees C and pH 5.5 to 6.0 and did not require Ca2+ for activity or thermostability . With a half-life of 13 h at 98 degrees C, the P . furiosus enzyme was significantly more thermostable than the commercially available Bacillus licheniformis alpha-amylase (Taka-therm). Appl Environ Microbiol, 1997 Sep, 63(9), 3419 - 25 Ligand specificity and affinity of BT-R1, the Bacillus thuringiensis Cry1A toxin receptor from Manduca sexta, expressed in mammalian and insect cell cultures; Keeton TP et al.; The Manduca sexta receptor for the Bacillus thuringiensis Cry1Aa, Cry1Ab, and Cry1Ac toxins, BT-R1, has been expressed in heterologous cell culture, and its ligand binding characteristics have been determined . When transfected with the BT-R1 cDNA, insect and mammalian cell cultures produce a binding protein of approximately 195 kDa, in contrast to natural BT-R1 from M . sexia, which has an apparent molecular weight of 210 kDa . Transfection of cultured Spodoptera frugiperda cells with the BT-R1 cDNA imparts Cry1A-specific high-affinity binding activity typical of membranes prepared from larval M . sexta midguts . Competition assays with BT-R1 prepared from larval M . sexta midguts and transiently expressed in cell culture reveal virtually identical affinities for the Cry1Aa, Cry1Ab, and Cry1Ac toxins, clearly demonstrating the absolute specificity of the receptor for toxins of the lepidopteran-specific Cry1A family . BT-R1 therefore remains the only M . sexta Cry1A binding protein to be purified, cloned, and functionally expressed in heterologous cell culture, and for the first time, we are able to correlate the Cry1Aa, Cry1Ab, and Cry1Ac toxin sensitivities of M . sexta to the identity and ligand binding characteristics of a single midgut receptor molecule. Blood, 1997 Sep 1, 90(5), 2047 - 56 Presence in human erythrocyte membranes of a novel form of sialidase acting optimally at neutral pH; Venerando B et al.; The feature of intact human erythrocytes and erythrocyte white ghosts is a unique sialidase activity with acidic optimal pH (acidic sialidase) . The treatment of white ghosts with mildly alkaline isotonic solutions at 37 degrees C, like that used to produce resealed ghosts, is accompanied by the expression, together with the acidic sialidase, of a novel sialidase with a pH optimum of 7.2 (neutral sialidase) that remained masked in the inside-out vesicles prepared from white ghosts . Exhaustive treatment of resealed ghosts with Bacillus Thuringiensis phosphatidylinositol-specific phospholipase C causes an almost complete release of the acidic sialidase, with the neutral enzyme remaining totally unaffected . The treatment of resealed ghosts with 1.2% Triton X-100 resulted in the solubilization of only the neutral sialidase, whereas 3.6% octylglucoside also solubilized the acidic sialidase . The neutral enzyme affected not only the artificial substrate but also any sialoderivatives of a ganglioside, glycoprotein, and oligosaccharide nature; the acidic enzyme did not affect sialoglycoproteins . Erythrocyte endogenous gangliosides were hydrolyzed by both sialidases, whereas the endogenous sialoglycoproteins responded to only the neutral enzyme . It was definitely proved that the acidic sialidase is located on the outer erythrocyte membrane surface, so presumably the neutral enzyme has the same location . It could be that the newly discovered neutral sialidase has a physiologic role in the releasing of sialic acid from erythrocytes during the erythrocyte aging process, leading to eventual phagocytosis by macrophages. J Microencapsul, 1997 Sep-Oct, 14(5), 627 - 38 Production of BCG alginate-PLL microcapsules by emulsification/internal gelation; Esquisabel A et al.; A biocompatible emulsification method for microencapsulation of live cells and enzymes within a calcium alginate matrix applied to Bacillus Calmette-Guerin (BCG) has been developed . Small-diameter alginate beads (microcapsules) were formed via internal gelation of an alginate solution emulsified within vegetable oil . Five different oils (sesame, sweet almond, perhydrosqualene, camomile and jojoba) were used . The rheological analysis of the oils showed a Newtonian behaviour, with viscosities = 30.0, 37.7, 51.2, 59.3 and 67.1 mPa.s for perhydrosqualene, jojoba, camomile, sesame and sweet almond oil respectively . The particle size of the microcapsules obtained ranged from 30.3 microns for the microcapsules prepared with sweet almond oil to 57.0 microns for those made with perhydrosqualene . The mean particle diameter obtained was found to be dependent on the viscosity of the oil employed, according to the equation: phi (micron) = 76.6-0.628 eta (mPa.s) (r2 = 0.943) . The encapsulated BCG was identified by the Difco TB stain set K, followed by observation under optical microscopy . Freeze-drying of the microcapsules was carried out to ensure their stability during storage . Two batches of microcapsules (those prepared with sesame and jojoba oil) and four types of cryoprotectors (glucose, trehalose, mannitol and sorbitol), at three concentration levels (5, 10 and 20% w/v) were studied . The parameters evaluated were particle size, physical appearance, reconstitution of lyophilizates and microscopical evaluation . For both batches of microcapsules the best results were obtained with trehalose 5%, showing particle sizes of 42.1 microns in the case of the microcapsules prepared with sesame oil, and of 45.3 microns for those prepared with jojoba. Indian J Med Res, 1997 Aug, 106, 174 - 97 Biological control of malaria vectors; Das PK et al.; A comprehensive review is presented of the potentiality of biocontrol agents viz . entomophagus bacteria (Bacillus thuringiensis var . israelensis and Bacillus sphaericus), fungi, microsporidians, predators and parasites against malaria vectors in the field condition . Unlike insecticides, these control agents are host specific and safer to the environment . However, barring fishes which are being used in certain situations, other biocontrol agents have not yet reached the operational stage . Two spore forming bacteria B . thuringiensis var . israelensis and B . sphaericus have been extensively tested against malaria vectors in the field . Though they are effective in suppressing anopheline larval population, their recycling capacity and availability of toxin hearing spores on the water surface are limited . Therefore, there is a need for developing improved formulations through bio-engineering techniques for enhancing their residual activity and availability of spores for anopheline larvae which feed mostly on the water surface . The biocontrol potentiality of other agents in the field condition is yet to be explored fully . The use of biocontrol agents for malaria control also poses certain operational constraints in view of the vastness of the anopheline breeding habitats and less acceptance for their use in domestic environments . However, there is a scope for using these biocontrol agents in conjunction with other control methods in integrated control programmes. Indian J Med Res, 1997 Aug, 106, 149 - 63 Urban malaria vector biology; Hati AK; One of the main reasons for the set-back in the urban malaria control programme is the peculiar biobehaviour of the principal urban malaria vector Anopheles stephensi . Certain relevant facts such as incrimination as the vector of malaria, sibling or biological species, resting habitat, manlanding behaviour, seasonal prevalence, blood meal analysis, longevity, parity status, daily survival and mortality rates of adults, breeding habitats and vertical distribution of larvae of An . stephensi have been discussed . Determination of density of the vector using various parameters and their relation to malaria endemicity in an urban situation have been reviewed . An . stephensi has become resistant to DDT, HCH, malathion and propoxur in many places in India . Hence for control source reduction, use of predators such as fish and biolarvicides such as Bacillus thuringiensis var israelensis H14 and B . sphaericus, personal protection, i.e., use of appropriate clothing, bed nets, indigenous repellents, etc., information, education and communication (IEC) are to be stressed. Indian J Malariol, 1997 Mar, 34(1), 25 - 36 Field evaluation of Bacillus sphaericus, H5a5b and B . thuringiensis var . israelensis, H-14 against the Bancroftian filariasis vector Culex quinquefasciatus, Say in Chennai, India; Kar I et al.; Fortnightly application of Bacillus sphaericus (strain B101, serotype H5a5b) and B . thuringiensis var . israelensis (strain 164, serotype H-14) in two different waterways of Chennai @ 1 g/sq m surface area has resulted in significant reduction in both immature and adult densities of Culex quinquefasciatus Say . The use of these biolarvicides as biocontrol agents is suggested in the urban areas to control mosquitoes in general. J Biol Chem, 1997 Sep 12, 272(37), 23094 - 103 The phosphatidyl-myo-inositol anchor of the lipoarabinomannans from Mycobacterium bovis bacillus Calmette Guérin . Heterogeneity, structure, and role in the regulation of cytokine secretion; Nigou J et al.; Lipoarabinomannans are major mycobacterial antigens capable of modulating the host immune response; however, the molecular basis underlying the diversity of their immunological properties remain an open question . In this study a new extraction and purification approach was successfully applied to isolate ManLAMs (lipoarabinomannans with mannosyl extensions) from bacillus Calmette Guerin leading to the obtention of two types of ManLAMs namely parietal and cellular . Structurally, they were found to differ by the percentage of mannooligosaccharide caps, 76 and 48%, respectively, and also, thanks to a new analytical method, by the structure of the phosphatidyl-myo-inositol anchor lipid moiety . A novel fatty acid in the mycobacterium genus assigned to a 12-O-(methoxypropanoyl)-12-hydroxystearic acid was the only fatty acid esterifying C-1 of the glycerol residue of the parietal ManLAMs, while the phosphatidyl unit of the cellular ManLAMs showed a large heterogeneity due to a combination of palmitic and tuberculostearic acid . Finally, parietal and cellular ManLAMs were found to differentially affect interleukin-8 and tumor necrosis factor-alpha secretion from human dendritic cells . We show that parietal but not cellular ManLAMs were able to stimulate tumor necrosis factor-alpha secretion from dendritic cells . From these studies we propose that the 1-{12-O-(methoxypropanoyl)-12-hydroxystearoyl}-sn-glycerol part is the major cytokine-regulating component of the ManLAMs . It seems likely that modification of the ManLAM lipid part, which may occur in hostile environments, could regulate macrophagic mycobacterial survival by altering cytokine stimulation. J Biol Chem, 1997 Sep 12, 272(37), 23011 - 6 Thermal conversion from low- to high-activity forms of catalase I from Bacillus stearothermophilus; Kobayashi C et al.; Catalase I from Bacillus stearothermophilus has the interesting property of increasing its enzyme activity on heating . It was confirmed that after heating at 70 degrees C for 10 min or 65 degrees C for 20 min, almost all the enzyme molecules were converted irreversibly to the activated form . The increase in kcat from 1400 to 3930 s-1 and the decrease in Km for H2O2 from 4.4 to 2.7 mM by heat activation indicate changes in the kinetic property of the enzyme molecule . Therefore, it follows that catalase I has two active forms, a high-activity form and a low-activity form . The heat activation process followed the first-order kinetics with an activation enthalpy (DeltaH*) of 191 kJ/mol while the heat denaturation process had a DeltaH* of 545 kJ/mol . The CD spectra of the two enzyme forms had small but marked differences . The conversion of the low-activity form to the high-activity form was an endothermic process with a Tm of 56 degrees C, which is much lower than that of the heat denaturation (Tm = 76 degrees C), and the enthalpy change for the transition was only 5% of that for the denaturation . It has to be noted that the high-activity form of the enzyme was converted back to a low-activity form through the process of denaturation, refolding, and reconstitution with heme . In addition, the newly obtained low-activity form was brought to a high-activity form by heating . These results suggest that the native state of catalase I has two active conformations that are roughly the same but not identical and are separated by a high energy barrier. J Bacteriol, 1997 Sep, 179(17), 5625 - 7 A nuclear genetic lesion affecting Saccharomyces cerevisiae mitochondrial translation is complemented by a homologous Bacillus gene; Kim SI et al.; A novel Bacillus gene was isolated and characterized . It encodes a homolog of Saccharomyces cerevisiae Pet112p, a protein that has no characterized relative and is dispensable for cell viability but required for mitochondrial translation . Expression of the Bacillus protein in yeast, modified to ensure mitochondrial targeting, partially complemented the phenotype of the pet112-1 mutation, demonstrating a high degree of evolutionary conservation for this as yet unidentified component of translation. Biophys J, 1997 Sep, 73(3), 1147 - 59 Molecular dynamics study of time-correlated protein domain motions and molecular flexibility: cytochrome P450BM-3; Arnold GE et al.; Time-correlated atomic motions were used to characterize protein domain boundaries from atomic coordinates generated by molecular dynamics simulations . A novel application of the dynamical cross-correlation matrix (DCCM) analysis tool was used to help identify putative protein domains . In implementing this new approach, several DCCM maps were calculated, each using a different coordinate reference frame from which protein domain boundaries and protein domain residue constituents could be identified . Cytochrome P450BM-3, from Bacillus megaterium, was used as the model protein in this study . The analyses indicated that the simulated protein comprises three distinct domain regions; in contrast, only two protein domains were identified in the original crystal structure report . Specifically, the DCCM analyses showed that the F-G helix region was a separate domain entity and not a part of the alpha domain, as previously designated . The simulations demonstrated that the domain motions of the F-G helix region effected both the size and shape of the enzyme active site, and that the dynamics of the F-G helix domain could possibly control access of substrate to the binding pocket. Infect Immun, 1997 Sep, 65(9), 3947 - 50 Activation of CD8 T cells with specificity for mycobacterial heat shock protein 60 in Mycobacterium bovis bacillus Calmette-Guérin-vaccinated mice; Zugel U et al.; Heat shock protein 60 (hsp60)-specific CD8 T cells lysed Mycobacterium bovis BCG-infected macrophages in vitro and adoptively transferred protection against mycobacterial infection . Moreover, CD8 T cells with this hsp60 specificity were activated in vivo by BCG vaccination . Our studies suggest there is participation of hsp60-specific CD8 T cells in BCG-induced immunity. Infect Immun, 1997 Sep, 65(9), 3672 - 9 Coccoid forms of Helicobacter pylori are the morphologic manifestation of cell death; Kusters JG et al.; Helicobacter pylori can transform from its normal helical bacillary morphology to a coccoid morphology . Since this coccoid form cannot be cultured in vitro, it has been speculated that it is a dormant form potentially involved in the transmission of H . pylori and in a patient's relapse after antibiotic therapy . In this study we determined the effects of aging, temperature, aerobiosis, starvation, and antibiotics on the morphologic conversion rate and culturability of H . pylori . Aerobiosis and the addition of a bactericidal antibiotic to the culture medium resulted in the highest conversion rate . During the conversion to coccoid forms, the cultures always lost culturability at the stage where 50% of the organisms were still in bacillary form; this result indicated that culturability and coccoid morphology are two separate but related entities . Independent of the conditions used to induce the conversion into coccoids, the morphological conversion was accompanied by several marked antigenic and ultrastructural changes . Also, both the total amounts and the integrity of RNA and DNA were significantly reduced in coccoid forms . With the potential-sensitive probe diOC(5)-3, a clear loss of membrane potential in coccoid forms was observed . Inhibition of protein or RNA synthesis by the addition of bacteriostatic antibiotics did not prevent the conversion to coccoid forms but resulted in an increased conversion rate . Hence, we conclude that conversion of H . pylori from the bacillary to the coccoid form is a passive process that does not require protein synthesis . Our data suggest that the coccoid form of H . pylori is the morphologic manifestation of bacterial cell death. Infect Immun, 1997 Sep, 65(9), 3644 - 7 Mechanism of nitric oxide-dependent killing of Mycobacterium bovis BCG in human alveolar macrophages; Nozaki Y et al.; We demonstrated that products of the L-arginine-dependent pathway of human alveolar macrophages (AM) effectively kill the Mycobacterium bovis bacillus Calmette-Guerin (BCG) in vitro . The formation of products was triggered by inoculation with BCG itself . Many reports have shown that activated rodent AM could produce an amount of nitric oxide (NO) sufficient to suppress the growth of mycobacteria . However, there have been no definitive results as to whether human AM might have the NO-dependent mechanism for the killing of mycobacteria . Therefore, we have undertaken some experiments to answer this question . Immunofluorescence assays showed an increased production of inducible nitric oxide synthase (iNOS) and peroxynitrite in BCG-inoculated AM from patients with pulmonary fibrosis . Reverse transcriptase-PCR also revealed the higher expression of iNOS-coding mRNA . Colony assays demonstrated that these human AM effectively killed BCG in their cytoplasm . However, treatment of AM with N(G)-monomethyl-L-arginine monoacetate resulted in markedly reduced killing activity . These results clearly show that BCG-induced NO and its reactive product with the oxygen radical peroxynitrite could play an important role in the killing of BCG in human AM. J Invertebr Pathol, 1997 Sep, 70(2), 136 - 42 Bacillus thuringiensis delta-Endotoxin Binding Sites in Two Lepidoptera, Wiseana spp . and Epiphyas postvittana Simpson RM, Burgess EPJ, Markwick NP. Proteins from the midguts of the light-brown apple moth Epiphyas postvittana (Walker) and the porina caterpillars Wiseana cervinata (Walker), W . copularis (Meyrick), and W . jocosa (Meyrick) which bind the Bacillus thuringiensis delta-endotoxin Cry1Ba were characterized using Cry1Ba labeled with Bolton and Hunter reagent . A comparison of two iodine labeling techniques on the toxicity of B . thuringiensis delta-endotoxins showed that labeling using chloramine-T substantially decreased Cry1Ba toxicity against the light-brown apple moth E . postvittana, whereas labeling using the Bolton and Hunter reagent had no effect on the toxicity of either Cry1Ac or Cry1Ba to this insect . The characteristics of Cry1Ac binding sites from E . postvittana midguts were determined by competitive binding assays using toxin labeled with 125I by both methods . The difference in values of binding site characteristics found by the two methods was shown to be caused by modification of the toxin by the conditions of chloramine-T labeling . The relationship between number and affinity of the binding sites and the toxicity of the delta-endotoxins is discussed. J Invertebr Pathol, 1997 Sep, 70(2), 113 - 20 Suitability of 30 Agricultural Products and By-Products as Nutrient Sources for Laboratory Production of Bacillus thuringiensis subsp . aizawai (HD133) Morris ON, Kanagaratnam P, Converse V V. Bacillus thuringiensis subsp . aizawai (HD133) was grown in culture media in which dextrose was a common carbon source and 30 different agricultural products and by-products were tested as the main nitrogen sources . These products included legumes, cereals, animal proteins, leaf proteins, yeasts, oilseeds, tubers, and casamino acid . Of the 30 products tested, cottonseed meal, defatted soy flour, and corn gluten meal were the most efficient substrates for the production of spore-crystal biomass and endotoxin potency . The carbohydrate/nitrogen ratios for these additives ranged from 0.3 to 0.5 and the glutamic acid content of their proteins from 9.2 to 16.0% . There was no close relationship between the estimates of the amounts of endotoxin produced and the potency of the product when fed to bertha armyworm, Mamestra configurata. Arch Biochem Biophys, 1997 Sep 1, 345(1), 79 - 87 Active site analysis of P450 enzymes: comparative magnetic circular dichroism spectroscopy; Andersson LA et al.; Recent structural studies indicate that the substrate- and O2-binding distal pocket of the P450 enzymes are not identical . Thus, P450terp (CYP108) from the alpha-terpineol-metabolizing Pseudomonad differs from P450cam (CYP-101) (C . A . Hasemann et al., J . Mol . Biol . 236, 1169, 1994) . In contrast, the distal pockets of P450terp and P450BMP (CYP102 heme domain; Bacillus megaterium) are more closely similar, including novel hydrogen-bonding interactions between the distal H2O ligand and the I helix (C . A . Hasemann et al., Structure, 3, 41-62, 1995) . To evaluate the significance of these differences, we have compared solution magnetic circular dichroism (MCD) spectra of P450terp with spectra of other P450 enzymes (e.g., P450cam, P450BMP, P450BM-3holo, and P450BM1), as well as with spectra of chloroperoxidase and NO synthase . Spectra of native P450terp are more similar to those of P450BMP and those of mammalian P450LM-2 than to those of P450cam . Upon substrate-binding, the MCD spectra of ferric P450terp and all other thiolate-ligated heme systems examined to date display a strong Soret band that is distinctly unique relative to the typical Soret MCD pattern(s) of catalases or other 5-coordinate ferric heme systems . This intense negative MCD feature thus appears diagnostic for cysteinate-linked ferric hemes . In the case of ferrous P450s, the intensity of the Soret-region MCD trough varies between substrate-bound and substrate-free enzymes (despite the fact that the substrate is NOT in direct contact with the heme moiety) . A novel finding of particular interest is the clear spectral shifts of the Soret MCD band between the substrate-bound and substrate-free forms of ferrous-CO-P450terp . No such observation has been made previously . Furthermore, the band positions for BOTH types of P450terp are red-shifted from known bands of ferrous-CO-P50cam . These data thus indicate a surprising sensitivity of MCD spectra to active-site polarity and to H2O occupancy, concurring with reports of distal pocket effects on CO-binding rates and equilibrium constants . Comparative analysis of the spectral properties of P450terp with MCD spectra of other P450 enzymes, as well as with chloroperoxidase and NO synthase, demonstrates both the expected similarities and the significant differences that reflect active-site structural features . The detailed spectral analysis of P450terp relative to other P450 enzymes presented herein includes the first observation of a substrate-induced spectral shift for a ferrous-CO-P450 . Furthermore, testable structural predictions for P450-BM-1 and for the novel NO synthase enzyme (neither of which has been crystallized to date) are made herein . This work thus provides insights into structurally defined P450s and may also lead to understanding of other P450 enzymes. Biopolymers, 1997 Sep, 42(3), 319 - 36 Substrate binding and catalytic mechanism in phospholipase C from Bacillus cereus: a molecular mechanics and molecular dynamics study; da Graca Thrige D et al.; For the first time a consistent catalytic mechanism of phospholipase C from Bacillus cereus is reported based on molecular mechanics calculations . We have identified the position of the nucleophilic water molecule, which is directly involved in the hydrolysis of the natural substrate phosphatidylcholine, in phospholipase C . This catalytically essential water molecule, after being activated by an acidic residue (Asp55), performs the nucleophilic attack on the phosphorus atom in the substrate, leading to a trigonal bipyramidal pentacoordinated intermediate (and structurally similar transition state) . The subsequent collapse of the intermediate, regeneration of the enzyme, and release of the products has to involve a not yet identified second water molecule . The catalytic mechanism reported here is based on a series of molecular mechanics calculations . First, the x-ray structure of phospholipase C from B cereus including a docked substrate molecule was subjected to a stepwise molecular mechanics energy minimization . Second, the location of the nucleophilic water molecule in the active site of the fully relaxed enzyme-substrate complex was determined by evaluation of nonbonded interaction energies between the complex and a water molecule . The nucleophilic water molecule is positioned at a distance (3.8 A) from the phosphorus atom in the substrate, which is in good agreement with experimentally observed distances . Finally, the stability of the complex between phospholipase C, the substrate, and the nucleophilic water molecule was verified during a 100 ps molecular dynamics simulation . During the simulation the substrate undergoes a conformational change, but retains its localization in the active site . The contacts between the enzyme, the substrate, and the nucleophilic water molecule display some fluctuations, but remain within reasonable limits, thereby confirming the stability of the enzyme-substrate-water complex . The protocol developed for energy minimization of phospholipase C containing three zinc ions located closely together at the bottom of the active site cleft is reported in detail . In order to handle the strong electrostatic interactions in the active site realistically during energy minimization, delocalization of the charges from the three zinc ions was considered . Therefore, quantum mechanics calculations on the zinc ions and the zinc-coordinating residues were carried out prior to the molecular mechanics calculations, and two different sets of partial atomic charges (MNDO-Mulliken and AMI-ESP) were applied . After careful assignment of partial atomic charges, a complete energy minimization of the protein was carried out by a stepwise procedure without explicit solvent molecules . Energy minimization with either set of charges yielded structures, which were very similar both to the x-ray structure and to each other, although using AMI-ESP partial atomic charges and a dielectric constant of 4, yielded the best protein structure. Semin Surg Oncol, 1997 Sep-Oct, 13(5), 335 - 41 Treatment of superficial bladder cancer with intravesical chemotherapy; Badalament RA et al.; Proper care of patients with superficial bladder cancer requires the assessment of multiple factors, including an understanding of the natural history of this disease, accurate clinical staging, and the expected efficacy of each drug . The pharmacology of intravesical mytomycin C is discussed in detail, as many of this drug's pharmacological principles are applicable to all intravesical chemotherapeutic agents, including doxorubicin, thiotepa, bacillus Calmette-Guerin, epirubicin, and ethoglucid . The bladder wall, bladder cavity, chemical properties of intravesical chemotherapeutic agents, and tumor considerations are discussed . Suggestions based on pharmacological studies are presented to optimize the efficacy of intravesical chemotherapy. J Urol, 1997 Sep, 158(3 Pt 1), 812 - 3 Palliative effect of intravesical bacillus Calmette-Guerin in elderly patients with advanced bladder carcinoma; Holmang S et al.; PURPOSE: Intravesical bacillus Calmette-Guerin (BCG) was used to palliate severe local symptoms in patients with invasive carcinoma . MATERIALS AND METHODS: Four patients with unresectable bladder carcinoma who were unfit for radical cystectomy because of age and poor performance status were treated with a 6-week course of BCG followed by monthly instillations . RESULTS: Urgency and frequency were reduced in 3 patients and the improvement lasted for 9 to 19 months . All 4 patients ultimately died of bladder carcinoma . CONCLUSIONS: The results of palliative BCG treatment were encouraging, but further experience is necessary. Biochemistry, 1997 Aug 26, 36(34), 10498 - 505 Mutational analysis of potential zinc-binding residues in the active site of the enterococcal D-Ala-D-Ala dipeptidase VanX; McCafferty DG et al.; VanX, one of the five proteins required for the vancomycin-resistant phenotype in clinically pathogenic Enterococci, is a zinc-containing d-Ala-d-Ala dipeptidase . To identify potential zinc ligands and begin defining the active site residues, we have mutated the 2 cysteine, 5 histidine, and 4 of the 28 aspartate and glutamate residues in the 202 residue VanX protein . Of 10 mutations, 3 cause inactivation and greater than 90% loss of zinc in purified enzyme samples, implicating His116, Asp123, and His184 as zinc-coordinating residues . Homology searches using the 10 amino acid sequence SxHxxGxAxD, in which histidine and aspartate residues are putative zinc ligands, identified the metal coordinating ligands in the N-terminal domain of the murine Sonic hedgehog protein, which also exhibits an architecture for metal coordination identical to that observed in thermolysin from Bacillus thermoproteolyticus . Furthermore, this 10 amino acid consensus sequence is found in the Streptomyces albus G zinc-dependent N-acyl-d-Ala-d-Ala carboxypeptidase, an enzyme catalyzing essentially the same d-Ala-d-Ala dipeptide bond cleavage as VanX, suggesting equivalent mechanisms and zinc catalytic site architectures . VanX residue Glu181 is analogous to the Glu143 catalytic base in B . thermoproteolyticus thermolysin, and the E181A VanX mutant has no detectable dipeptidase activity, yet maintains near-stoichiometric zinc content, a result consistent with the participation of the residue as a catalytic base. Biochemistry, 1997 Aug 19, 36(33), 10089 - 97 Allosteric activation of phosphatidylinositol-specific phospholipase C: specific phospholipid binding anchors the enzyme to the interface; Zhou C et al.; Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis exhibits 'interfacial activation' toward the water-soluble substrate myo-inositol 1,2-(cyclic)phosphate {Zhou et al . (1997) Biochemistry 36, 347-355} . The activation of PI-PLC enzyme is optimal with PC or PE interfaces . NMR experiments (TRNOE and 31P line width analyses) were carried out to investigate the interaction of PI-PLC with activator amphiphiles . These studies showed that the enzyme had high affinity for phosphatidylcholine (or PE) molecules with dissociation constants of 0.5 and 0.3 mM for diC6PC and diC7PC, respectively . TRNOE cross-peaks of bound PC were confirmed to represent intramolecular relaxation pathways using partially perdeuterated PC molecules consistent with a single molecule binding tightly . The large activation by a PC interface can be explained by a single PC molecule binding specifically to PI-PLC and anchoring the enzyme-lipid complex to the interface . Other interfaces, such as micellar diC8PS, can activate PI-PLC about 2-3-fold; however, the monomers of these detergents showed little affinity for the enzyme as measured by TRNOE or 31P NMR line widths . The 3.6-fold activation produced by polymerized vesicles of 1,2-bis{12-(lipoyloxy)dodecanoyl}-sn-glycero-3-phosphocholine (compared to the 15-fold activation generated by nonpolymerized PC vesicles) was comparable to the nonspecific activation of other detergents . This confirmed that single-PC molecule binding was allosteric and anchored the enzyme in the interface . The conformation of interfacially activated enzyme is discussed in term of the stabilization of a critical surface loop and helix B observed with weak intensity in the X-ray crystal structure. FEBS Lett, 1997 Aug 18, 413(2), 339 - 43 The catalytic domain of dihydrolipoyl acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus . Expression, purification and reversible denaturation; Allen MD et al.; A sub-gene encoding the catalytic (acetyltransferase) domain (E2pCD) comprising residues 173-427 of the dihydrolipoyl acetyltransferase (E2p) chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was expressed in Escherichia coli . The product assembled to form the characteristic icosahedral (60-mer) core structure with full catalytic activity . The Km values for dihydrolipoamide and acetyl-CoA were 1.2 mM and 13 microM, respectively . Dissociation of the icosahedral E2pCD into monomers by exposure to guanidine hydrochloride and the subsequent reassociation by gradual removal of the denaturing agent demonstrated the ability of the polypeptide chain to fold and reassemble in the absence of chaperonins. FEMS Microbiol Lett, 1997 Aug 15, 153(2), 419 - 24 Marginal cross-resistance to mosquitocidal Bacillus thuringiensis strains in Cry11A-resistant larvae: presence of Cry11A-like toxins in these strains; Cheong H et al.; Culex quinquefasciatus mosquito larvae resistant to the Cry11A toxin showed marginal cross-resistance to the multiple toxin crystals from B . thuringiensis subsp . israelensis and also to toxin crystals from three other mosquitocidal strains, i.e . B . thuringiensis subsp . fukuokaensis, subsp . jegathesan, and subsp . kyushuensis . Cross-resistance patterns of the Cry11A-resistant larvae to mosquitocidal strains of B . thuringiensis together with the immunological screening using antisera raised against Cry11A indicated the presence of Cry11A-like toxins in these strains and could be used as a screening tool for the identification of novel toxins . The Cry11A-resistant larvae had significantly less resistance to the Cry11B toxin from B . thuringiensis subsp . jegathesan . The occurrence of cytolytic toxins in all of these mosquitocidal strains partially explains the marginal cross-resistance observed with multiple toxin crystals since each of these crystals also contains cytolytic toxins. Biochemistry, 1997 Aug 12, 36(32), 9927 - 34 Trapping and characterization of the reaction intermediate in cyclodextrin glycosyltransferase by use of activated substrates and a mutant enzyme; Mosi R et al.; Cyclodextrin glycosyltransferases (CGTases) catalyze the degradation of starch into linear or cyclic oligosaccharides via a glycosyl transfer reaction occurring with retention of anomeric configuration . They are also shown to catalyze the coupling of maltooligosaccharyl fluorides . Reaction is thought to proceed via a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate . This intermediate can be trapped by use of 4-deoxymaltotriosyl alpha-fluoride (4DG3alphaF) . This substrate contains a good leaving group, fluoride, thus facilitating formation of the intermediate, but cannot undergo the transglycosylation step since the nucleophilic hydroxyl group at the 4-position is missing . When 4DG3alphaF was reacted with wild-type CGTase (Bacillus circulans 251), it was found to be a slow substrate (kcat = 2 s-1) compared with the parent glycosyl fluoride, maltotriosyl alpha-fluoride (kcat = 275 s-1) . Unfortunately, a competing hydrolysis reaction reduces the lifetime of the intermediate precluding its trapping and identification . However, when 4DG3alphaF was used in the presence of the presumed acid/base catalyst mutant Glu257Gln, the intermediate could be trapped and analyzed because the first step remained fast while the second step was further slowed (kcat = 0.6 s-1) . Two glycosylated peptides were identified in a proteolytic digest of the inhibited enzyme by means of neutral loss tandem mass spectrometry . Edman sequencing of these labeled peptides allowed identification of Asp229 as the catalytic nucleophile and provided evidence for a covalent intermediate in CGTase . Asp229 is found to be conserved in all members of the family 13 glycosyl transferases. FEBS Lett, 1997 Aug 4, 412(3), 587 - 91 Simultaneous production of the 34-kDa and 40-kDa proteins from Bacillus thuringiensis subsp . thompsoni is required for the formation of inclusion bodies; Rang C et al.; Cooperation of two crystal proteins from Bacillus thuringiensis subsp . thompsoni . strain HnC was shown to be essential for the formation of inclusion bodies . Expression of the operon containing the 34-kDa and 40-kDa protein genes from HnC in a B . thuringiensis crystal minus strain resulted in the formation of inclusion bodies identical to those from strain HnC . Interruption of one of the genes in the operon led to the lack of inclusion body and to low production of the remaining protein . Absence of inclusion body and low rate of protein production were also observed when both genes were simultaneously expressed but on different vectors . To show a cooperative effect in the formation of the inclusion body, both proteins must be produced from the same transcript. FEBS Lett, 1997 Aug 4, 412(3), 501 - 5 The D13C variant of Bacillus schlegelii 7Fe ferredoxin is an 8Fe ferredoxin as revealed by 1H-NMR spectroscopy; Aono S et al.; The N-terminal cluster binding motif Cys8XXXXXXXCys16....Cys49 of Bacillus schlegelii 7Fe ferredoxin, which provides the ligands to the {Fe3S4}+ cluster, was modified by the mutation Asp13 --> Cys . The mutant D13C is expressed in Escherichia coli as an 8Fe ferredoxin, with NMR properties similar to those of clostridial-type ferredoxins . The full assignment of the hyperfine shifted resonances indicates that Cys13 serves as ligand to the new fourth iron atom in the N-terminal cluster despite the atypical binding sequence CysXXXXCysXXCys....Cys . The C alpha-C beta-S-Fe dihedral angles of all cysteine ligands to the two {Fe4S4}2+ clusters of the D13C variant are similar to those observed in other 8Fe and 4Fe ferredoxins. Eur J Biochem, 1997 Aug 1, 247(3), 754 - 61 Binding kinetics of Bacillus sphaericus binary toxin to midgut brush-border membranes of Anopheles and Culex sp . mosquito larvae; Silva-Filha MH et al.; Direct-binding assays and homologous-competition assays were used to identify specific binding between the radiolabelled toxin of Bacillus sphaericus and brush-border membrane fractions (BBMF) from Anopheles gambiae and Anopheles stephensi, obtained from whole larvae preparations . In both species, the toxin bound to a single class of receptors . BBMF of A . gambiae had the highest binding affinity for the toxin of the species tested, with a dissociation constant (Kd) of 30 +/- 15 nM and a maximum receptor concentration of 5 +/- 1 pmol/mg . Toxin binding to A . gambiae BBMF was compared with that to BBMF from B . sphaericus-susceptible (IP) and B . sphaericus-resistant (SPHAE) Culex pipiens populations . BBMF toxin binding was slower in A . gambiae than in the C . pipiens populations . The BBMF of the B . sphaericus-resistant population of C . pipiens had an association profile that was similar to the susceptible population, despite of the lack of susceptibility in vivo . No relationship between toxicity and irreversibility of toxin binding was detected . On the contrary, toxin dissociation from BBMF was fast and almost complete in BBMF of all species studied. Am J Trop Med Hyg, 1997 Aug, 57(2), 174 - 9 Bartonellosis in Ecuador: serosurvey and current status of cutaneous verrucous disease; Amano Y et al.; Human bartonellosis is a classically biphasic disease caused by infection with the alpha-2 Proteobacteria Bartonella bacilliformis, which is phylogenetically related to the etiologic agents of cat scratch disease, bacillary angiomatosis, and trench fever . In Ecuador, typical bartonellosis has remained endemic for the past century in highland provinces near the Peruvian border . During the past six years, public health officials have noted an increasing number of atypical cases in which monophasic verrucous cutaneous disease is the only clinical manifestation . Epidemiologic, immunologic, histopathologic, and molecular biological studies have confirmed the presence of sporadic, atypical bartonellosis in residents of the lowland province of Manabi, where archeologic evidence exists of bartonellosis in pre-Colombian times . Between 1987 and 1995, 11 cases of cutaneous bartonellosis were investigated and serologic studies were done on 224 persons from five villages, two lowland and three highland . In the lowland village of Pajan in the province of Manabi, there was a 21% seropositivity proportion in contacts of index cases . These combined data suggest that bartonellosis is significantly under-reported due to the existence of mild clinical disease, possibly associated with less virulent bacterial strains, which are now disseminating or re-emerging in previously disease-free areas. Vaccine, 1997 Aug, 15(11), 1214 - 7 Inhibition of multiplication of Mycobacterium leprae in mouse foot pads by immunization with ribosomal fraction and culture filtrate from Mycobacterium bovis BCG; Matsuoka M et al.; Immunization of mice with the ribosomal fraction from ruptured Mycobacterium bovis Bacillus Calmette-Guerin (BCG) and the culture filtrate reduced remarkably the multiplication of Mycobacterium leprae in the foot pads of mice . This is the first reported case of the protective activity against M . leprae multiplication in mice of the BCG ribosomal fraction and culture filtrate . The inhibition was more evident with the culture filtrate than with the ribosomal fraction . When the ribosomal proteins separated from ribosomal RNA were injected into mice, only slight inhibition was observed . Ribosomal RNA alone did not inhibit at all, in contrast to the conclusion reported by Youmans and Youmans. Biochem Mol Biol Int, 1997 Aug, 42(5), 901 - 8 Involvement of an endogenous metalloprotease in the activation of protoxin in Bacillus thuringiensis subsp . kurstaki; Kumar NS et al.; Insecticidal crystal proteins harvested from sporulated cultures of Bacillus thuringiensis subsp . kurstaki contain the protoxin (Mr 132 kDa) and minor amounts of toxin (66 kDa) . The proteolytic processing of 132 kDa protoxin to an active 66 kDa toxin is brought about by exogenous proteases or larval gut enzymes . Under denaturing/reducing conditions this conversion is also mediated by endogenous protease(s) of the producer organism . This endogenous protease is identified as a metalloprotease as the activation process is inhibited by ethylenediamine tetraacetic acid at 2 mM concentration. Cutis, 1997 Aug, 60(2), 101 - 2 Submandibular abscess caused by Eikenella corrodens; Heymann WR et al.; Eikenella corrodens is a slow-growing, facultative, anaerobic, gram-negative bacillus that is part of the normal oral flora and is found in dental plaque . It has become increasingly recognized as a pathogen in nonimmunocompromised and immunocompromised hosts . A case of a submandibular abscess due to Eikenella corrodens, which was successfully treated by administration of cefuroxime accompanied by incision and drainage, is presented . Dermatologists need to be aware of this pathogen is the evaluation of suppurative lesions of the head and neck. J Appl Microbiol, 1997 Aug, 83(2), 243 - 7 A non-intrusive method for the measurement of water vapour sorption by bacterial spores; Rubel GO; Single particle levitation (SPL) is used to measure the sorption and desorption of water vapour from microparticles comprising of Bacillus spores . Water gain is determined from increases in the weight-balancing levitation voltages . Spore water isotherms are compared to values previously reported using bulk gravimetric methods . Two salient differences are found between the SPL and bulk data: (1) greater osmotic swelling is observed in the SPL data; and (2) the SPL data exhibit open loop hysteresis while bulk data exhibit closed loop hysteresis. J Dairy Sci, 1997 Aug, 80(8), 1546 - 53 Effect of storage temperatures and ingredients on growth of Bacillus cereus in coffee creamers; Feijoo SC et al.; Growth of Bacillus cereus ATCC 33018 was evaluated in half and half (10.5% fat), whipping cream (30% fat), and nondairy creamer (7.5% fat) . Samples were inoculated with approximately 10 vegetative cells/ml or 100 spores/ml and were subsequently stored at 4, 7, 23 and 32 degrees C . Within 9 h at 32 degrees C and 11 h at 23 degrees C, in both half and half and whipping cream, vegetative cells and spores reached population levels that can cause foodborne illness . No growth occurred in any product stored at 4 or 7 degrees C . Sodium stearoyl lactylate, a fatty acid derivative that is used as an emulsifier, inhibited growth of spores and vegetative cells in the nondairy creamers stored at either 32 or 23 degrees C. J Virol Methods, 1997 Aug, 67(1), 1 - 4 A simple and efficient method for purification of prawn baculovirus DNA; Yang F et al.; A new method for isolation of prawn baculovirus and subsequent extraction of viral DNA was developed . No density gradient centrifugation, ultracentrifugation or phenol-chloroform extraction steps were involved . Phenylmethylsulfonyl fluoride (PMSF) was used to prevent proteinase degradation, DNase and RNase were used to degrade prawn DNA and RNA respectively . The nucleocapsid was a bacilliform virion, about 58 62 nm in width and 300-350 nm in length as observed by transmission electron microscopy . Intact viral DNA was obtained by lysing nucleocapsids with guanidine hydrochloride and degrading protein with proteinase K . As the viral DNA was digested with restriction endonuclease and separated by electrophoresis, restriction fragments were clearly shown on the agarose gel . The size of the DNA was estimated approximately to be 290 kb . The virus which appeared to be a prawn baculovirus was named prawn white spot baculovirus (PWSBV) due to the white spots which appeared on the inside surface of the crust of infected prawns. Microbiology, 1997 Aug, 143 ( Pt 8), 2807 - 15 Heat shock response and groEL sequence of Bartonella henselae and Bartonella quintana; Haake DA et al.; Transmission of Bartonella species from ectoparasites to the mammalian host involves adaptation to thermal and other forms of stress . In order to better understand this process, the heat shock response of Bartonella henselae and Bartonella quintana was studied . Cellular proteins synthesized after shift to higher temperatures were intrinsically labelled with {25S}methionine and analysed by gel electrophoresis and fluorography . The apparent molecular masses of three of the major heat shock proteins produced by the two Bartonella species were virtually identical, migrating at 70, 60 and 10 kDa . A fourth major heat shock protein was larger in B . quintana (20 kDa) than in B . henselae (17 kDa) . The maximum heat shock response in B . quintana and B . henselae was observed at 39 degrees C and 42 degrees C, respectively . The groEL genes of both Bartonella species were amplified, sequenced and compared to other known groEL genes . The phylogenetic tree based on the groEL alignment places B . quintana and B . henselae in a monophyletic group with Bartonella bacilliformis . The deduced amino acid sequences of Bartonella GroEL homologues contain signature sequences that are uniquely shared by members of the Gram-negative alpha-purple subdivision of bacteria, which live within eukaryotic cells . Recombinant His6-GroEL fusion proteins were expressed in Escherichia coli to generate specific rabbit antisera . The GroEL antisera were used to confirm the identity of the 60 kDa Bartonella heat shock protein . These studies provide a foundation for evaluating the role of the heat shock response in the pathogenesis of Bartonella infection. Microbiology, 1997 Aug, 143 ( Pt 8), 2537 - 47 Diversity and differential distribution of IS231, IS232 and IS240 among Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides; Leonard C et al.; Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides are very closely related bacteria, generally considered as subspecies of B . cereus sensu lato . Different transposable elements have been isolated from B . thuringiensis, including IS231, IS232 and IS240 and their variants . The distribution of these three insertion sequences (IS) within the B . cereus group has been investigated in 90 strains of B . thuringiensis (representing 61 serovars), in 30 reference strains of B . cereus and in 33 strains of B . mycoides . Since these IS elements are delimited by well-conserved and specific inverted repeats, the use of primers corresponding to these ends allowed their amplification by PCR . The results showed that IS231 is the most abundant element in the three taxa, whereas IS232 is apparently exclusively associated with B . thuringiensis . Hybridization and Dral RFLP analysis of the PCR products confirmed and extended knowledge of the heterogeneity previously observed among iso-IS231 elements . Moreover, a similar diversity was observed among iso-IS240 elements . This contrasted with the relative homogeneity displayed by iso-IS232 elements . No specific association appeared to exist between any particular iso-element and a specific strain or serotype. J Am Acad Dermatol, 1997 Aug, 37(2 Pt 2), 303 - 4 An ulcerated lesion at the BCG vaccination site during the course of Kawasaki disease; Kuniyuki S et al.; We describe a bacillus Calmette-Guerin (BCG) granuloma that occurred during the course of Kawasaki disease . A 12-month-old male infant with Kawasaki disease had an erythematous indurated plaque with prominent necrotic ulceration at the BCG vaccination site on the left upper arm . Histologic study showed a granulomatous reaction consisting of epithelioid histiocytes, lymphoid cells, and Langhans-type giant cells . No evidence of mycobacterial infection was obtained . The lesion healed completely within 2 weeks without administration of antituberculous agents . We believe that the granulomatous reaction occurred as a result of hypersensitivity to proteins in the BCG vaccine, which appeared after the onset of Kawasaki disease. Protein Expr Purif, 1997 Aug, 10(3), 365 - 72 Cloning, overexpression, refolding, and purification of the nonspecific phospholipase C from Bacillus cereus; Tan CA et al.; Bacillus cereus secretes a nonspecific phospholipase C (PLC) that catalyzes the hydrolysis of phospholipids to yield diacylglycerol and a phosphate monoester . B . cereus PLC has been overexpressed with its signal sequence in Escherichia coli using a T7 expression system . The expressed enzyme formed intracellular inclusion bodies which were solubilized in the presence of 8 M urea . Renaturation was initiated by gradual removal of urea and addition of zinc ions . The signal peptide was specifically cleaved by a protease, clostripain, added when the urea concentration was 1.5 M . Factors that led to protein reaggregation included rapid removal of urea, use of Tris instead of barbital buffer, and presence of the signal peptide when the urea concentration was below 1.5 M . The folded protein was purified by Q-Sepharose Fast flow chromatography to yield a preparation > 99% pure . The final yield of active enzyme was 30-40 mg per liter of culture . The recombinant PLC exhibited biochemical and kinetic properties identical to those of extracellularly produced PLC from B . cereus . Site-specific mutagenesis of Asn-134 was carried out as a test of the general effectiveness of the refolding procedure. Proteins, 1997 Aug, 28(4), 580 - 5 Crystals of cytochrome c-553 from Bacillus pasteurii show diffraction to 0.97 A resolution; Benini S et al.; We report here the purification and characterization of a c-type cytochrome present in the soluble fraction of the gram-positive, alkaliphilic, and highly ureolytic soil bacterium Bacillus pasteurii . The cytochrome is acidic (pI = 3.3), has a molecular mass of 9.5 kDa, and appears to dimerize in 150 mM ionic strength solution . The electronic spectrum is typical of a low-spin hexa-coordinated heme iron . Crystals of the protein in the oxidized state were grown by vapor diffusion at pH 5, by using 3.2 M ammonium sulfate as precipitant . Diffraction data at ultrahigh resolution (0.97 A) and completeness (99.9%) have been collected under cryogenic conditions, by using synchrotron radiation . The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with cell constants a = 37.14, b = 39.42, c = 44.02 A, and one protein monomer per asymmetric unit . Attempts to solve the crystal structure by ab initio methods are in progress. J Bacteriol, 1997 Aug, 179(16), 5000 - 8 Nucleotide sequence and characterization of the cryptic Bacillus thuringiensis plasmid pGI3 reveal a new family of rolling circle replicons; Hoflack L et al.; The complete nucleotide sequence of plasmid pGI3 from Bacillus thuringiensis subsp . thuringiensis H1.1 . was obtained . Although this 11,365-bp molecule contained at least 11 putative open reading frames (ORFs), extensive database searches did not reveal any homologous sequences with the exception of ORF6, which displayed similarity to the largest ORF of pSTK1, a 1,883-bp cryptic plasmid isolated from Bacillus stearothermophilus . Deletion analysis to determine the pGI3 minimal replicon revealed that ORF6 is the rep gene . Replication occurred via a single-stranded DNA (ssDNA) intermediate, as demonstrated by S1 treatment and Southern hybridization in nondenaturating conditions . Interestingly, however, no homology was found between the pGI3 (ORF6) and pSTK1 (ORF3) rep genes and those from other single-stranded DNA plasmids, nor was there any DNA similarity to the double-strand origins of replication characterized so far, indicating that pGI3 and pSTK1 form another, new family of ssDNA plasmids . PCR analysis revealed that the pGI3 rep gene is largely distributed among B . thuringiensis strains but can also be found in B . cereus and B . mycoides strains, albeit at a lower frequency . Finally, segregation experiments performed with B . subtilis and B . thuringiensis showed that the pGI3 derivatives, including the minimal replicon, were segregationally stable at temperatures suitable for B . thuringiensis growth (<43 degrees C). Am J Clin Pathol, 1997 Aug, 108(2), 202 - 9 Immunologic response to Bartonella henselae as determined by enzyme immunoassay and Western blot analysis; Litwin CM et al.; Bartonella henselae is now regarded as the etiologic agent of cat-scratch disease and a cause of bacillary angiomatosis . We examined the human immune response to Bartonella henselae infection using a newly developed enzyme immunoassay (EIA) and a Western blot procedure using outer-membrane proteins . The EIA showed 98.6% and 91.4% agreement with an indirect fluorescence method (IFA) for detection of IgM and IgG antibodies, respectively . By using Western blot analysis, reactivity to an 8-kd band showed significant correlation with positive results by the IgM IFA and EIA . In contrast, reactivity to 209-, 208.5-, 208-, 116-, and 80-kd bands was identified only in positive IgG IFA serum samples . The EIA and Western blot should be useful tests in determining the antibody response to B . henselae infection and may also be important in determining the critical epitopes in the host-parasite interaction of this organism. Biochem Mol Med, 1997 Aug, 61(2), 214 - 28 Isolation and partial characterisation of insulin-mimetic inositol phosphoglycans from human liver; Caro HN et al.; Extracts of human liver were found to contain activities which copurified and coeluted with the two major subtypes of mediators (type A and type P) isolated from insulin-stimulated rat liver . The putative type A mediator from human liver inhibited cAMP-dependent protein kinase from bovine heart, decreased phosphoenolypyruvate carboxykinase mRNA levels in rat hepatoma cells, and stimulated lipogenesis in rat adipocytes . The putative type P mediator stimulated bovine heart pyruvate dehydrogenase phosphatase . Both fractions were able to stimulate proliferation of EGFR T17 fibroblasts and the type A was able to support growth in organotypic cultures of chicken embryo cochleovestibular ganglia . Both activities were resistant to Pronase treatment and the presence of carbohydrates, phosphate, and free-amino groups were confirmed in the two fractions . These properties are consistent with the structure/ function characteristics of the type A and P inositolphosphoglycans (IPG) previously characterized from rat liver . Further, the ability of the human-derived mediators to interact with rat adipocytes and bovine-derived metabolic enzymes suggests similarity in structure between the mediators purified from different species . Galactose oxidase-susceptible membrane-associated glycosylphosphatidylinositols (GPI) have been proposed to be the precursors of IPG . GPI was purified from human liver membranes followed by treatment with galactose oxidase and reduction with NaB3H4 . Serial t.l.c . revealed three radiolabeled bands which comigrated with the putative GPI precursors found in rat liver . These galactose-oxidase-reactive lipidic compounds, however, were only partially susceptible to hydrolysis with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis and were resistant to glycosylphosphatidylinositol-specific phospholipase C from Trypanosoma brucei . These data indicate that IPG molecules with insulin-like biological activities are present in human liver. FEMS Microbiol Lett, 1997 Aug 1, 153(1), 221 - 6 Analysis of CcpA mutations defective in carbon catabolite repression in Bacillus megaterium; Kraus A et al.; Five mutations in ccpA of Bacillus megaterium with impaired functions were analysed for carbon catabolite repression . The phenotypes support the hypothesis that CcpA assumes a PurR/LacI fold . The completely inactive mutants CcpA119GE and CcpA326am cause alterations which are incompatible with that fold . A mutation with reduced activity, CcpA81GE, affects a site that would be partially surface exposed and may interfere with structure formation or cofactor binding . A mutation in the putative hinge alpha-helix, CcpA52AE, is negative transdominant over wild-type ccpA . The mutant CcpA38am is inactive, although reduced amounts of wild-type size protein are produced. Appl Environ Microbiol, 1997 Aug, 63(8), 3254 - 60 Cloning and characterization of a cytolytic and mosquitocidal delta-endotoxin from Bacillus thuringiensis subsp . jegathesan; Cheong H et al.; A cytolytic toxin gene encoding a 30.1-kDa Cyt2Bb1 toxin protein from B . thuringiensis subsp . jegathasan was cloned employing a limited-growth PCR screening method with forward and reverse oligonucleotide primers designed from N-terminal amino acid sequences of native and trypsin-cleaved protein, respectively . The expressed protein showed little cross-reactivity to the antibody raised against the Cyt1Aa protein . Unlike Cyt1Aa and Cyt2Aa expression, there was little or no visible crystal inclusion formation under microscopic observation . The amino acid sequence alignment indicated 31 and 66% identity to Cyt1Aa and Cyt2Aa, respectively . The sequence alignment for five known cytolytic proteins indicated three highly conserved regions, two in the loop regions between alpha-helices and beta-sheets and one in the loop region between beta-sheets 5 and 6 . beta-Blocks 4 to 7 are also conserved, not only structurally but also among the amino acids in the hydrophobic faces . Mosquitocidal activity assays indicated that the Cyt2Bb toxin had less toxicity than Cyt1Aa and had about 600-times-lower toxicity than the wild-type whole toxin crystal . However, both the Cyt2Bb and the Cyt1Aa toxin showed comparable levels of hemolytic activity. Appl Environ Microbiol, 1997 Aug, 63(8), 3139 - 43 Identification and detection of Bacillus sporothermodurans spores in 1, 10, and 100 milliliters of raw milk by PCR; Herman LM et al.; A PCR method was developed to detect spores of Bacillus sporothermodurans in 1, 10, and 100 ml of raw milk . Two primers were derived from a unique sequence after subtractive hybridization of B . sporothermodurans DNA with DNA of MB 397, a not yet identified spore-forming bacterium isolated from raw milk, closely related to B . sporothermodurans . Specific identification was proven on a large collection of Bacillus strains and on strains from relevant taxa . The detection of B . sporothermodurans in raw milk is based on activation, germination, and outgrowth of the spores, followed by PCR identification . Spores from 10 and 100 ml were concentrated by centrifugation after chemical extraction of the milk components . The total test takes 28 h . The detection limits are 9, 0.4, and 0.22 CFU/ml for 1, 10, and 100 ml, respectively. Appl Environ Microbiol, 1997 Aug, 63(8), 2997 - 3002 PCR-based approach for detection of novel Bacillus thuringiensis cry genes; Juarez-Perez VM et al.; A two-step strategy, named exclusive PCR or E-PCR, has been developed to overcome the main limitation of PCR, which is the detection of already-known sequences only . This strategy allows the ability to detect and further clone and sequence genes for which no specific primers are available and in which a variable region exists between two conserved regions . This approach has been applied to Bacillus thuringiensis cryI genes by the use of mixtures of degenerate and specific primers recognizing well-known sequences . The first step allows the accurate identification of already-characterized cryI genes by the use of three primers . During the second step, the same sets of primers are used to exclude known sequences and to positively detect cryI genes unrecognized by any specific primer . The method, as well as its application to detect, clone, and sequence a novel cryIB gene, is described in this article. Int Arch Allergy Immunol, 1997 Aug, 113(4), 400 - 8 Modulation of protective and pathological immunity in mycobacterial infections; Ottenhoff TH et al.; Mycobacterial infections represent major problems to global health care . Tuberculosis is feared particularly because of its high mortality rates whereas in leprosy the occurrence of immunopathology, particularly nerve damage, is a major problem since the bacillus itself is relatively harmless . Thus, both effective vaccination strategies as well as novel immunomodulating regimens are warranted for the control of morbidity and mortality in mycobacterial diseases . Since CD4+ Th1 cells and type-1 cytokines play a key role both in protective immunity and immunopathology in mycobacterial infections, we here describe new pharmacological and cytokine-based strategies to regulate Th1 immunity. Arch Biochem Biophys, 1997 Aug 1, 344(1), 37 - 42 Arginase of Bacillus brevis Nagano: purification, properties, and implication in gramicidin S biosynthesis; Kanda M et al.; An arginase {EC 3.5.3.1} was purified to homogeneous state from a gramicidin S-producing Bacillus brevis Nagano . The enzyme has a molecular weight of about 180,000 on gel filtration . The subunit molecular weight is 32,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the enzyme is hexameric . The optimum pH is found near 10.0 . Mn2+ is essential for its activity and Fe2+, Co2+, Ni2+, and Mg2+ cannot replace Mn2+ . The enzyme is highly specific for L-arginine with a K(m) value of 12.8 mM for L-arginine, which is similar to that of liver-type arginase in ureotelic animals . B . brevis arginase is apparently induced by the addition of L-arginine to the glutamate medium . The increased formation of L-ornithine, a constituent amino acid of gramicidin S, by arginase may be involved in the accelerated production of gramicidin S by B . brevis in the presence of L-arginine in the growth medium. J Bacteriol, 1997 Aug, 179(15), 4689 - 98 Characterization of a gene cluster for glycogen biosynthesis and a heterotetrameric ADP-glucose pyrophosphorylase from Bacillus stearothermophilus; Takata H et al.; A chromosomal region of Bacillus stearothermophilus TRBE14 which contains genes for glycogen synthesis was cloned and sequenced . This region includes five open reading frames (glgBCDAP) . It has already been demonstrated that glgB encodes branching enzyme (EC 2.4.1.18 {H . Takata et al., Appl . Environ . Microbiol . 60:3096-3104, 1994}) . The putative GlgC (387 amino acids {aa}) and GlgD (343 aa) proteins are homologous to bacterial ADP-glucose pyrophosphorylase (AGP {EC 2.7.7.27}): the sequences share 42 to 70% and 20 to 30% identities with AGP, respectively . Purification of GlgC and GlgD indicated that AGP is an alpha2beta2-type heterotetrameric enzyme consisting of these two proteins . AGP did not seem to be an allosteric enzyme, although the activities of most bacterial AGPs are known to be allosterically controlled . GlgC protein had AGP activity without GlgD protein, but its activity was lower than that of the heterotetrameric enzyme . The GlgA (485 aa) and GlgP (798 aa) proteins were shown to be glycogen synthase (EC 2.4.1.21) and glycogen phosphorylase (EC 2.4.1.1), respectively . We constructed plasmids harboring these five genes (glgBCDAP) and assayed glycogen production by a strain carrying each of the derivative plasmids on which the genes were mutated one by one . Glycogen metabolism in B . stearothermophilus is discussed on the basis of these results. J Infect Dis, 1997 Aug, 176(2), 478 - 84 Widespread dissemination of a drug-susceptible strain of Mycobacterium tuberculosis; Friedman CR et al.; In New York City, a large proportion of new tuberculosis cases has been caused by 1 drug-susceptible strain (called C strain) of Mycobacterium tuberculosis . Between 1991 and 1994, among >600 tuberculosis patients consecutively identified in four large hospitals in the city, 54 with C strain, 69 with non-C cluster pattern strains, and 42 with noncluster pattern strains were studied . Susceptibility to reactive nitrogen intermediates (RNI) of selected isolates was compared . In a case-control analysis, 51% of patients with C strain, 28% with non-C cluster strains (P < .05), and 14% with noncluster strains (P < .01) were found to be injection drug users . C strain but not 13 other unrelated isolates were resistant to RNI . Injection drug use may provide a selective pressure for an RNI-resistant tubercle bacillus to emerge, which may give the organism a biologic advantage and explain the widespread dissemination of C strain M . tuberculosis within the city. J Clin Microbiol, 1997 Aug, 35(8), 2068 - 71 Mycobacterial growth and bacterial contamination in the mycobacteria growth indicator tube and BACTEC 460 culture systems; Cornfield DB et al.; The BACTEC 460 system currently provides the most rapid detection of mycobacterial growth, but the system is radiometric and requires needles to inoculate specimens through the bottle's septum . The Mycobacteria Growth Indicator Tube (MGIT) system has a liquid medium, like the BACTEC system, and does not require needles when inoculating specimens . We compared mycobacterial growth from 510 specimens in the two systems . Average time to acid-fast bacillus (AFB) detection and identification to the species level was less with the BACTEC system, but this result was statistically significant only for AFB detection in specimens containing Mycobacterium avium-M . intracellulare complex . The contamination rate with MGIT was 29%; the BACTEC rate was 5% . To investigate MGIT contamination, we initiated a second study with changes in specimen processing . The MGIT contamination rate was reduced to 12%; the BACTEC rate was not significantly affected (5.5%) . The most likely explanation for the contamination in MGIT is the richness of its medium compared to the BACTEC medium . Cost analysis for the two systems in a laboratory that processes 4,500 specimens a year is presented . The data suggest that the BACTEC 460 and the MGIT systems are approximately equivalent in cost and ability to support the growth of AFB . The MGIT system appears safer and easier to use and was preferred by laboratory personnel, but it cannot currently be used for blood specimens or antituberculosis susceptibility testing. J Urol, 1997 Aug, 158(2), 646 - 52 Genetically regulated response to intravesical bacillus Calmette Guerin immunotherapy of orthotopic murine bladder tumor; Kadhim SA et al.; PURPOSE: Genetically regulated host response to intravesical Bacillus Calmette Guerin (BCG) immunotherapy was assessed using the murine bladder tumor MM45T in Bcgr and Bcgs inbred congenic strains of mice . MATERIALS AND METHODS: Tumor detection and monitoring of treatment response to BCG was carried out using magnetic resonance imaging (MRI) of BALB/c (Bcgs allele) and BALB/c . CD2 (CD2) (Bcgr allele) mice implanted orthotopically with MM45T tumor cells . Intravesical BCG instillation (3 doses per week for 3 weeks) was used as prophylaxis against tumor implantation in both Bcgr and Bcgs strains and as definitive treatment against MRI-confirmed established tumors . Tumors implanted in both strains of untreated mice served as controls . Intravesical injection of BCG was also performed in established heterotopic subcutaneous tumors in both strains . Immunologic response in all groups was assessed by flow cytometric analysis of the bladder irrigation fluid cell composition, measuring CD4+ (helper/inducer) and CD8+ (cytotoxic/ suppressor) cell subsets . RESULTS: Intralesional injection of BCG into established heterotopic tumors showed growth inhibition in the Bcgs strain but not in the Bcgr strain . Intravesical BCG treatment against established orthotopic tumors showed significant tumor regression in the Bcgs strain compared to control but there was no effect in the Bcgr strain . CONCLUSION: The differential anti-tumor activity of BCG in the Bcgs and Bcgr congenic murine strains supports the notion that Bcg gene-controlled responsiveness to BCG innoculation determines, at least partially, the host response to immunotherapy . These results have potential clinical significance in patient selection for intravesical therapy for bladder cancer. Curr Microbiol, 1997 Aug, 35(2), 71 - 6 Isolation of Bacillus megaterium mutants that produce high levels of heterologous protein, and their use to construct a highly mosquitocidal strain; England DF et al.; A xylose-regulated plasmid expression system for producing high levels of recombinant proteins in Bacillus megaterium has recently been described {Appl Microbiol Biotechnol 35:594, 1991} . Using an antibiotic resistance protein as the expressed protein, we have been able to select mutant plasmids that produce increased levels of heterologous protein . The mutant plasmids show increased segregational stability and have lost the ability to be transformed into Escherichia coli . The same selection protocol has been used to isolate a mutant strain producing high levels of the Bacillus sphaericus mosquitocidal binary toxin . This strain shows toxicity to Culex quinquefasciatus larvae that is comparable toB . sphaericus 2362 and higher than a B . megaterium strain with the original expression plasmid . This approach may be generally useful for high-level regulated protein expression in B . megaterium. FEBS Lett, 1997 Jul 28, 412(2), 270 - 6 Ion channels formed in planar lipid bilayers by Bacillus thuringiensis toxins in the presence of Manduca sexta midgut receptors; Schwartz JL et al.; A purified, GPI-linked receptor complex isolated from Manduca sexta midgut epithelial cells was reconstituted in planar lipid bilayers . CryIAa, CryIAc and CryIC, three Bacillus thuringiensis insecticidal proteins, formed channels at much lower doses (0.33-1.7 nM) than in receptor-free membranes . The non-toxic protein CryIB also formed channels, but at doses exceeding 80 nM . The channels of CrylAc, the most potent toxin against M . sexta, rectified the passage of cations . All other toxin channels displayed linear current-voltage relationships . Therefore, reconstituted Cry receptors catalyzed channel formation in phospholipid membranes and, in two cases, were involved in altering their biophysical properties. Lancet, 1997 Jul 19, 350(9072), 169 - 72 Control of tuberculosis by community health workers in Bangladesh; Chowdhury AM et al.; BACKGROUND: Tuberculosis remains a major public-health problem in Bangladesh, despite national efforts to improve case identification and treatment compliance . In 1984, BRAC (formerly the Bangladesh Rural Advancement Committee), a national, non-governmental organisation, began an experimental tuberculosis-control programme in one thana (subdistrict) . Community health workers screened villagers for chronic cough and collected sputum samples for acid-fast bacillus (AFB) microscopy (phase one) . Positive patients received 12 months of directly observed therapy . Phase two (1992-94) included another nine thanas and, in phase three (1995), eight more thanas were included . From 1995, the treatment was an 8-month oral regimen . METHODS: In 1995-96, we analysed all programme data from 1992 to 1995 . First we analysed phases two (12-month therapy) and three (8-month therapy) separately for proportion cured, died, treatment, failed, defaulted, migrated, and referred . Second, we did a cross-sectional survey of tuberculosis cases in more than 9000 randomly selected households in two phase-two thanas and one non-programme thana, and analysed the follow-up of all patients treated in the programme thanas . FINDINGS: In the phase-two analysis, 3497 (90%) of 3886 cases identified had accepted 12-month treatment . In phase three, all of 1741 identified cases accepted the 8-month regimen . 2833 (81.0%) and 1496 (85.9%) in phases two and three, respectively, were cured; 336 (9.6%) and 133 (7.6%) died . The relapse rate 2 or more years after treatment was discontinued was higher than the early relapse rate . The drop-out rate was 3.1% . In the cross-sectional survey, the prevalence of tuberculosis in the two programme thanas was half of that in the comparison thana, where only government services were available (0.07 vs 0.15 per 100 {corrected}) . INTERPRETATION: The BRAC tuberculosis-control programme has successfully achieved high rates of case detection and treatment compliance, with a cure rate of at least 85% and a drop-out rate of 3.1% . The prevalence survey suggested that at least half of all existing cases had been detected by the programmePIP: In 1984 the Bangladesh Rural Advancement Committee (BRAC), a national nongovernmental organization, began an experimental tuberculosis control program in 1 thana (subdistrict) . In phase 1 community health workers screened villagers for chronic cough and collected sputum samples for acid-fast bacillus (AFB) microscopy . Positive patients underwent a 12-month therapy . Phase 2 during 1992-94 included 9 other thanas, and in phase 3 in 1995 8 more thanas were included . Between 1984 and 1994 the treatment regimen consisted of 30 streptomycin injections on alternate days for 2 months and 300 mg of isoniazid and 150 mg of thiacetazone daily for 12 months . From 1985 the treatment was an 8-month oral regimen of isoniazid, pyrazinamide, ethambutol, and rifampicin daily for 2 months, then isoniazid and thiacetazone daily for 6 months . During 1995-96 program data were analyzed from 1992-95 . First the 12-month therapy of phase 2 and the 8-month therapy of phase 3 were analyzed separately for proportion cured, deceased, treatment failed, defaulted, migrated, and referred . Then a cross-sectional survey of TB cases was carried out in more than 9000 randomly selected household in 2 phase-2 thanas and 1 nonprogram thana . The follow-up of all patients in the program thanas was analyzed . In the phase-2 analysis 3497 (90%) of 3886 cases identified had accepted the 12-month treatment . In phase 3 all of 1741 identified cases accepted the 8-month treatment regimen . 2833 (81%) and 1496 (85.9%) in phases 2 and 3, respectively, were cured; 336 (9.6%) and 133 (7.6%) died . 2 or more years after treatment was concluded, the relapse rate was higher than the early relapse rate . The drop-out rate was 3.1% . In the cross-sectional survey the prevalence of TB in the 2 program thanas was half of that in the comparison thana, where only government services were available: 0.07 vs . 0.15 per 1000 . High rates of case detection and treatment compliance were achieved, with a cure rate of at least 85% and a low drop-out rate . At least half of all existing cases had been detected by the program . Biochem Biophys Res Commun, 1997 Jul 18, 236(2), 402 - 6 Biotin synthase, a new member of the family of enzymes which uses S-adenosylmethionine as a source of deoxyadenosyl radical; Guianvarc'h D et al.; The fact that biotin synthase, from Escherichia coli and Bacillus sphaericus, requires S-adenosylmethionine and a reducing system led us to postulate that this synthase could belong to the family of enzymes which use S-adenosylmethionine as a source of deoxyadenosyl radical, namely pyruvate formate-lyase, lysine 2,3-aminomutase, and anaerobic ribonucleotide reductase . We describe here experiments with S-{2,8-(3)H} adenosylmethionine and S-adenosyl-{methyl-3H}methionine which allowed the identification and quantification of the expected cleavage products, deoxyadenosine, and methionine . They are formed in equimolar amounts, in a ratio close to 3 with respect to the biotin produced . We postulate a mechanism involving the homolytic cleavage of two C-H bonds which should consume two equivalents of S-adenosylmethionine . The observed excess of S-adenosylmethionine consumption is attributed to abortive processes. Hosp Pract (Off Ed), 1997 Jul 15, 32(7), 73 - 6, 81-4, 86 Management after exposure to tuberculosis; Jordan TJ et al.; The increased incidence of tuberculosis-coupled with the emergence of mycobacterial strains resistant to the most effective drugs-has highlighted the importance of identifying transmission and preventing active disease . Skin test conversion can document infection, except in most patients vaccinated with bacille Calmette-Guerin . Prophylactic medication is effective, but not without complications. Nucleic Acids Res, 1997 Jul 15, 25(14), 2854 - 60 Plant ribosome shunting in vitro; Schmidt-Puchta W et al.; It has been proposed that cauliflower mosaic virus 35S RNA with its 600 nt long leader uses an unusual translation process (the translational shunt) . A wheat germ in vitro translation assay was used to improve the study of this mechanism . Deletions, the introduction of stable stem-loop structures, and the inhibitory effect of antisense oligonucleotides on gene expression were used to determine the roles of various parts of the leader . It was found that the 5'- and 3'-ends of the leader are absolutely required for translation whereas the middle part is apparently dispensable . These results confirm the data already reported from transient expression experiments with protoplasts . However, the in vitro data suggest in contrast to protoplast experiments that only two relatively short regions at both ends, approximately 100 nt each, are required . The in vitro system provides tools for further studying the shunt model at the molecular level and for examining the involvement of proteins in this mechanism . Shunting was also found to occur with the rice tungro bacilliform virus leader . As wheat is neither a host plant of cauliflower mosaic virus nor rice tungro bacilliform virus, the shunt seems to be host independent, a finding that deviates from earlier studies in protoplasts. Cell Immunol, 1997 Jul 10, 179(1), 41 - 7 Cyclic AMP analogue as a triggering signal for the induction of nitric oxide synthesis in murine peritoneal macrophages; Park YC et al.; To understand the role of cAMP during macrophage activation, we investigated the effects of various cAMP analogues in the induction of nitric oxide (NO) synthesis in murine peritoneal macrophages . Intracellular cAMP modulators such as N6,O2'-dibutyryl cyclic AMP (DB-cAMP), 8-bromo-cAMP, or 8-chloro-cAMP had no effect on NO synthesis by themselves, whereas cAMP analogues in combination with interferon-gamma (IFN-gamma) had a marked cooperative induction of NO synthesis in a dose-dependent manner . This increase in NO synthesis was reflected as an increased amount of inducible NO synthase mRNA, as determined by Northern blotting . To find the point in the signaling pathways of macrophage activation at which cAMP is involved, we carried out several of the following experiments . Although DB-cAMP showed synergistic action with rIFN-gamma to induce NO synthesis when the cells were treated with DB-cAMP after or with simultaneous treatment with rIFN-gamma, there is no synergistic induction of NO synthesis when the cells were treated with DB-cAMP 6 hr before treatment with rIFN-gamma . In addition, when phorbol 12-myristate 13-acetate (PMA), which is known to provide a triggering signal in the induction of NO synthesis in murine macrophages, was added to the cells 6 hr after the treatment with DB-cAMP, PMA showed no synergistic cooperation with DB-cAMP . On the other hand, DB-cAMP alone induced the release of NO to the incubation medium from bacillus Calmette-Guerin-infected peritoneal macrophages just as lipopolysaccharide (LPS) did . However, DB-cAMP, unlike LPS, decreased the secretion of tumor necrosis factor-alpha from IFN-gamma-treated macrophages . Based on the results obtained in this study, we suggest that cAMP analogue could give a "triggering" signal which might be different from one given by LPS in the production of NO by primed macrophages. Biochemistry, 1997 Jul 8, 36(27), 8401 - 12 Functional interactions in cytochrome P450BM3: flavin semiquinone intermediates, role of NADP(H), and mechanism of electron transfer by the flavoprotein domain; Murataliev MB et al.; Cytochrome P450BM3 is a self-sufficient soluble fatty acid hydroxylase from Bacillus megaterium utilizing tightly bound FAD and FMN cofactors to transfer reducing equivalents from NADPH to the heme active site . Active-inactive transitions of cytochrome P450BM3 were exploited to identify catalytic intermediates of the enzyme . Shortly upon reduction by NADPH, a two-electron reduced active P450BM3 is formed with two flavin semiquinones, anionic and neutral, present simultaneously . P450BM3 inactivated by NADPH has a three-electron reduced flavoprotein domain . NADPH is unable to reduce P450BM3 rapidly unless the flavoprotein domain is fully oxidized . During steady-state hydroxylation of a poor substrate, tetradecanol, the flavoprotein reduction state does not exceed two, with two flavin semiquinones, anionic and neutral, present . Absorbance and EPR spectroscopic characterization of both anionic and neutral flavin semiquinone is presented . NADPH and NADH were compared as electron donors for P450BM3-catalyzed fatty acid hydroxylation and cytochrome c and heme iron reduction . The Km for NADH of 3-5 mM is about 3000 times higher than the Km of 1-1.5 microM for NADPH . Although NADH can support cytochrome c reduction and fatty acid hydroxylation with the rates as high as 22 and 13 s-1, respectively, these turnover numbers are only about 20% of those observed with NADPH . The results suggest that nucleotide binding plays an important role in catalysis by controlling electron-transfer properties of the flavin cofactors . In W574G and G570D mutant P450BM3 enzymes that are deficient in FMN, NADP+ binding stabilizes fully reduced FAD . P450BM3 catalyzes single-turnover and steady-state laurate hydroxylation with near stoichiometric product formation at NADPH concentrations below that of the enzyme . A mechanism of electron transfer by the flavoprotein domain of P450BM3 is proposed with the reduction state of the flavoprotein domain cycling in a 0-2-1-0 sequence . We also propose that an interaction of bound NADP+ with anionic FAD semiquinone is essential for splitting a pair of electrons that are then transferred in two one-electron transfer steps to the heme catalytic site. FEBS Lett, 1997 Jul 7, 411(1), 27 - 31 Phage display of Bacillus thuringiensis CryIA(a) insecticidal toxin; Marzari R et al.; The display of proteins or peptides on the surface of filamentous phages or phagemids has been shown to be a very powerful technology for the rescue of specific binders from large combinatorial libraries, as well as to select derivatives of known proteins with altered binding properties . The Bacillus thuringiensis (Bt) crystal proteins are a large family of insecticidal toxins which bind to receptors found on the brush border of larval midgut cells, different crystal toxins having different larval specificities . Here we describe the display of different CryIA(a) toxin regions on the surface of phagemids using the display vector pHEN1, the purpose being the identification of toxin sequences suitable for mutagenesis and selection using phage display . We show that CryIA(a) domain II, in which the receptor binding activity is located, is efficiently displayed as well as being secreted as soluble protein into the periplasm of bacterial cell . This forms the basis of a simple means for the modification of toxin specificity and the selection of toxin proteins with novel or expanded host ranges. J Mol Biol, 1997 Jul 4, 270(1), 111 - 22 The role of Glu73 of barnase in catalysis and the binding of barstar; Schreiber G et al.; Barnase, a small extracellular ribonuclease from Bacillus amyloliquefaciens and its intracellular inhibitor barstar have co-evolved to bind tightly and rapidly . Barnase has also evolved to be catalytically active . The active site of barnase and its binding site for barstar use the same subset of amino acids . The exception is Glu73 (the general base in catalysis), which although located at the centre of the binding site, is separated by three ordered water molecules from barstar . We examined in this work the contribution of Glu73 to both catalysis and barstar binding . Truncation mutants of the general base (Glu73 --> Ala or Ser) retain a residual RNase activity of about 0.3% while mutants with larger hydrophobic replacements (Glu 73 --> Trp or Phe) have virtually no catalytic activity . This, and binding data of 3'-GMP with the different barnase mutants suggest that the loss in activity results from the elimination of the general base, which can be substituted to some extent by water or other polar side-chains in truncation mutants . All of the Glu73 mutations lead to a weakening of the free energy of complex formation with barstar by 1.4 to 3.0 kcal/mol (including Gln) . This is surprising, since Glu73 does not interact directly with barstar and there is an electrostatic repulsion between Glu73 on barnase and the negatively charged binding surface of barstar . A newly developed method of constructing double mutant cycles between multiple mutations at the same site appears to pinpoint a favourable interaction between Glu73 and one of its nearest neighbours in barstar, Asp39 . The coupling energy between those residues is presumably indirect: the carboxylate of Glu73 organizes neighbouring positively charged groups in barnase, Lys27, Arg83, and Arg87 to interact with Asp39 in barstar . This emphasizes that an apparent interaction between a pair of residues as measured with double mutant cycles is the sum of their direct and indirect interactions. Eur J Clin Microbiol Infect Dis, 1997 Jul, 16(7), 487 - 506 Current knowledge of Bartonella species; Maurin M et al.; Bartonella species are now considered emerging pathogens . Of the 11 currently recognized species, four have been implicated in human disease, although only two have been encountered in Europe . Bartonella quintana infections are now being diagnosed among the urban homeless and deprived, manifesting as trench fever, and Bartonella henselae has been shown to be the causative agent of cat scratch disease . Both species also cause a variety of HIV-associated infections, including bacillary anglomatosis . However, perhaps the most significant presentation of bartonellae infection is culture-negative endocarditis . The epidemiologies of Bartonella infections are poorly understood; most Bartonella henselae infections are probably acquired from infected cats, either directly by contact with a cat or indirectly via fleas . No animal reservoir has been implicated for Bartonella quintana; however, infection can be transmitted via the human body louse . Diagnosis of Bartonella infections can be made using histological or microbiological methods . The demonstration of specific antibodies may be useful in some instances, although certainly not in all . Cultivation of Bartonella is difficult, as the bacteria are extremely fastidious . Polymerase chain reaction-based or immunological methods for the detection of bartonella in infected tissues have proven useful . Clinical relapse is often associated with Bartonella infections despite a wide range of prescribed regimens . Only aminoglycosides display in vitro bactericidal activity against intracellular Bartonella species; therefore, they are recommended for treatment of Bartonella infections. Microbiol Res, 1997 Jul, 152(2), 199 - 208 Production, purification and characterization of Bacillus lipase; el-Shafei HA et al.; The lipolytic activities in the supernatant fractions of Bacillus cereus and Bacillus coagulans cultures were investigated . Aeration, agitation, different media, emulsified oils, inoculum size and phase of growth affected lipase production . Aeration was essential for lipase production (air: medium ration 4:1) and produced the highest activity . The lipolytic activity reached a maximum level after incubation for two days with continuous agitation . It was also elevated by the presence of either olive oil or tributyrin and with lesser extent in the presence of castor oil . The enzyme levels were drastically reduced in the presence of animal fat, cotton seed oil, margarine or glycerol . The extracellular lipase enzyme from Bacillus cereus was purified with 46.2% overall recovery thought too steps, an acetone precipitation of the whole supernatant and purification by gel filtration on sephadex G-100 . The efficiency of the purification process was evaluated through the polyacrylamide gel electrophoresis . The enzyme has an optimum pH 7.5 at the optimum incubation temperature of 40 degrees C . It is stable and retains its full activity after heating at 40-50 degrees C for 30 min . The activity is lost completely at 80 degrees C. Ecotoxicol Environ Saf, 1997 Jul, 37(2), 152 - 62 Cell integrity markers for in vitro evaluation of cytotoxic responses to bacteria-containing commercial insecticides; Tayabali AF et al.; Toxicity of two commercial "BT" products, containing Bacillus thuringiensis subsp . kurstaki spores (Btk) and associated parasporal inclusion body proteins, was tested in vitro using two unrelated lepidoperan cell lines and several markers of cell integrity (morphology, quantification of loss of adherence and electron transport (redox) activity, and degradation of nuclear DNA, actin, hsp-70, and beta-tubulin) . With doses of 10(-7), 10(-5), and 10(-3) International Units (IU)/target cell, these markers measured exposure-dependent effects closely linked to cell death, which occurred rapidly once Btk spores germinated, unless inhibited by antibiotic . Derivation of marker half-lives (HL50) revealed that temperature critically affected product performance . Between 34 and 37 degrees C, HL50 was < or = 5 hr, but dose discrimination between 10(-5) and 10(-3) IU was poor . At temperatures less than 34 degrees C, the resolution between different HL50s and doses increased in a manner directly relating to published data obtained from in vivo BT-spore-induced LD50 assays . It was concluded that BT product toxification is complex, essentially enabled by an autobiotransformation process in which dose-response lag is affected by temperature-dependent temporal expression of spore germination and critical buildup of vegetative cells and byproduct toxicants . The in vitro dosimetry assays described here are potentially useful for obtaining mechanistic toxicologic data and in vivo relevant quantifications of subingredient activities in various commercial BT formulations as well as in other microbe-based biotechnology products. Skeletal Radiol, 1997 Jul, 26(7), 431 - 3 Sternal abscess due to Bartonella (Rochalimaea) henselae in a renal transplant patient; Bruckert F et al.; Bartonella henselae, previously called Rochalimaea henselae, is the causative agent of cat scratch disease (CSD) in immunocompetent subjects and bacillary angiomatosis in immunocompromised ones . Bone lesions are common in bacillary angiomatosis, but not in CSD . We present the case of a patient with a renal transplant treated by immunosuppressive therapy who developed a sternal abscess with a histological pattern of CSD . The CT pattern was that of a lytic bone lesion with adjacent fluid collection . The diagnosis was made on the basis of a polymerase chain reaction amplification performed on bone material . Bartonella henselae is a newly described bacteria that causes CSD in a normal host and bacillary angiomatosis in immunocompromised patients . We report a case of an osteolytic lesions of the sternum with adjacent fluid collection related to CSD, which occurred in a patient with a renal transplant. New Microbiol, 1997 Jul, 20(3), 253 - 76 Effect of treated sewage on the water quality and phytoplankton populations of Lake Manzala (Egypt) with emphasis on biological assessment of water quality; el-Naggar ME et al.; The effect of treated sewage on the quality of water and phytoplankton populations of Lake Manzala was studied with emphasis on use of algae to monitor water pollution as part of a search for a biological assessment of water quality . Lake Manzala is situated at the northern part of the Nile-delta, Egypt . Disposal of treated sewage into Lake Manzala appeared to have differential effects on water quality and phytoplankton populations . Marked seasonal and local variations were observed for the physical and chemical characteristics of water . 157 species of algae were identified, 59 Chlorophyta, 37 Bacillariophyta, 30 Cyanophyta (Cyanobacteria), 28 Euglenophyta, one Pyrrhophyta and 2 Cryptophyta . Distribution and abundance of these algal divisions were found to differ at different sampling stations . Qualitative and quantitative growth of each algal division displayed great seasonal variations . The phytoplankton standing crop was mainly due to the contribution of Bacillariophyta whereas the species composition is dependent mainly on Chlorophyta . A great parallelism was noted between the quality of water samples based upon the chemical and physical investigations and their quality based upon the biological indices . Compound eutrophication index indicated that the nature of the investigated water ranged between eutrophic and hypereutrophic conditions . Diversity index values indicated that the water in the study area was of a moderate level of pollution . Saprobic index and saprobic quotient revealed the presence of beta- to alpha-mesosaprobic forms of algae. Biosci Biotechnol Biochem, 1997 Jul, 61(7), 1146 - 9 Synthesis of glycosyl-trehaloses by cyclomaltodextrin glucanotransferase through the transglycosylation reaction; Kurimoto M et al.; Cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus produced a series of glycosyl-trehaloses through the transglycosylation reaction with cyclomaltohexaose as the glycosyl donor and trehalose as its acceptor . After beta-amylase treatment, five species of glycosyl-trehaloses were isolated by column chromatography . After chemical and enzymatic analyses, it was concluded that these oligosaccharides were alpha-maltosyl alpha-D-glucopyranoside, alpha-maltotriosyl alpha-D-glucopyranoside, alpha-maltosyl alpha-maltoside, alpha-maltotriosyl alpha-maltoside, and alpha-maltotriosyl alpha-maltotrioside . These were not hydrolyzed by salivary amylase, artificial gastric juice, or pancreatic amylase, however they were hydrolyzed by enzymes of the small intestine. Biosci Biotechnol Biochem, 1997 Jul, 61(7), 1126 - 32 Further studies on thermal denaturation of pyruvate dehydrogenase complex from Bacillus stearothermophilus; Hiromasa Y et al.; Thermally induced changes in pyruvate dehydrogenase complex (PDC) from B . stearothermophilus were examined mainly at temperatures from 60 degrees to 70 degrees C . Accompanied by inactivation of pyruvate decarboxylase, light scattering decreased, and ANS fluorescence increased . These changes including the inactivation were approximately first-order reactions, and the values of rate constants were greatly dependent on temperature . Chromatographic studies showed that any polypeptides were in associated forms and that final products were aggregates (> 230S) and an assembly (48S) smaller than PDC . The aggregates and assembly were rich in decarboxylase and lipoate acetyltransferase, respectively . It was suggested that, during the thermal denaturation, a decarboxylase was dissociated from PDC and immediately involved in aggregates. Biosci Biotechnol Biochem, 1997 Jul, 61(7), 1118 - 20 Galactosyl transfer onto p-nitrophenyl beta-D-glucoside using beta-D-galactosidase from Bacillus circulans; Murata T et al.; beta-D-Gal-(1-->4)-beta-D-Glc-OC6H4NO2-p and its isomers (beta-D-Gal-(1-->3)-beta-D-Glc-OC6H4NO2-p and beta-D-Gal-(1-->6)-beta-D-Glc-OC6H4NO2-p) were synthesized from lactose and beta-D-Glc-OC6H4NO2-p, using transglycosylation by the beta-D-galactosidase from Bacillus circulans . This reaction was efficient enough for us to do a one-pot preparation of galactosyl-glucoside from lactose . The order of the production of the transfer products was (1-->4) > > (1-->3) > (1-->6) in the initial stage of the reaction, and the same relationship was observed for the hydrolytic rate toward the three galactosyl-glucosides . The production of (1-->4)- and (1-->3)-linkages greatly decreased during the subsequent reaction and much more of the (1-->6)- than of the (1-->4)- and (1-->3)-transfer products was found in the later stage of the reaction. Lett Appl Microbiol, 1997 Jul, 25(1), 1 - 4 Factors regulating production of alpha-galactosidase from Bacillus sp . JF2; Li X et al.; Certain factors affecting the production of cell-associated alpha-galactosidase by Bacillus sp . JF2 were investigated . The intention was to maximize alpha-galactosidase activity of potential commercial application, by consecutive optimization of growth media and conditions . The highest alpha-galactosidase activity was obtained when grown on melibiose, whereas sucrose inhibited the production of alpha-galactosidase, alpha-Galactosidase production was optimally active at pH 7.5 and 55 degrees C . It was identified that a soy effluent stream could be used as the best carbon source for alpha-galactosidase by Bacillus sp . JF2. J Lipid Mediat Cell Signal, 1997 Jul, 16(3), 171 - 87 A new phospholipase A2 inhibitor, unrelated to substrate analogues: kinetic characterization of the inhibition of secretory phospholipases A2 by PMS 832; Binisti C et al.; Starting from a series of compounds which were known to be PAF antagonists, we have synthesized molecules that are good inhibitors of PLA2s of groups I or II, with IC50 in the micromolar range (Binisti et al., 1997) . In this report we investigate the mechanism of inhibition of bovine and porcine pancreatic phospholipases A2 (group I), and platelet lysate phospholipase A2 (group II) by one of these compounds, 1-(4'-methoxybenzoyl)-2-n-tridecylpiperazine (PMS 832) . We show that PMS 832 behaves as a reversible, competitive inhibitor, with Ki values of 4.1 +/- 1.2 and 1.5 +/- 0.4 microM for porcine pancreatic phospholipase A2 and platelet lysate phospholipase A2, respectively . PMS 832 failed to inhibit platelet activation induced by several agonists and was also found to be inactive towards phospholipase C from Bacillus cereus, indicating a high specificity for phospholipase A2 inactivation . Thus, PMS 832 and its derivatives could serve as interesting tools to investigate the role of extracellular phospholipases A2 in inflammatory processes, and may be useful in the development of new anti-inflammatory agents. Scand J Immunol, 1997 Jul, 46(1), 16 - 26 Cross-reactive immune responses against Mycobacterium bovis BCG in mice infected with non-tuberculous mycobacteria belonging to the MAIS-Group; Lozes E et al.; Two bacillus Calmette-Guerin (BCG)-susceptible mouse strains, BALB/c and C57BL/6, were infected intravenously with Mycobacterium intracellulare, M . avium or M . scrofulaceum and monitored during 3 months for mycobacterial replication and antibody and