Bacillus

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 Th1-type cytokine production in response to cytoplasmic and secreted antigens from M . bovis BCG . Whereas initial colony-forming unit (CFU) counts of M . intracellulare and M . avium were higher in lungs than in spleen, the opposite was observed for M . scrofulaceum . Mycobacterium intracellulare was the most virulent species and its replication could not be controlled in either mouse strain . It also induced the strongest antibody response . Mycobacterium avium was eliminated in both mouse strains and M . scrofulaceum finally was eliminated in C57BL/6 but multiplied in spleen from BALB/c mice . Significant sustained interleukin-2 and interferon-gamma production towards BCG antigens was only found in M . scrofulaceum infection . As in BCG-vaccination, M . scrofulaceum-infected C57BL/6 mice demonstrated a higher response towards whole BCG culture filtrate, BCG extract and purified antigen 85 complex (Ag85) from BCG than did BALB/c mice . The data suggest that the presence of M . scrofulaceum in the environment may possibly interfere in genetically predisposed subjects with BCG vaccine and its protective efficacy against M . tuberculosis.

J Struct Biol, 1997 Jul, 119(2), 123 - 8
S-layer stabilized solid support lipid bilayers; Wetzer B et al.; This work describes composite structures composed of lipid bilayer or tetraetherlipid monolayer films attached to solid supports with associated crystalline bacterial cell surface layers (S-layers) . The bilayer system was established by making use of the strong chemisorption of a first monolayer of thiolipids (1-octadecanethiol or 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol) on gold and attaching a second monolayer of 1,2-dipalmitoyl-sn-3-phosphatidylethanolamine by the Langmuir Schaefer technique . The tetraetherlipid monolayer was composed of Glycerol-dialkyl-nonitol tetraetherlipid (GDNT) . The monolayer of GDNT exhibits the thickness of a bilayer with hydrophilic headgroups on both sides and a hydrophobic inner part . Isolated S-layer protein from Bacillus sphaericus (CCM2177, which was injected into the subphase of an LB-trough, recrystallized into a coherent monolayer at the solid supported phospholipid bilayer and at the tetraehtherlipid monolayer . The composite lipid/S-layer structures were stable enough to allow lifting from the air-water interface, rinsing in water, and transfer into a scanning force microscope.

Arch Pathol Lab Med, 1997 Jul, 121(7), 724 - 9
Granulomatous prostatitis on needle biopsy; Oppenheimer JR et al.; OBJECTIVE: To assess pathologic findings of granulomatous prostatitis (GP) on needle biopsy . DESIGN: Ninety-four cases of granulomatous prostatitis were culled from 25,852 (incidence 0.36%) consecutive men who underwent needle biopsy; clinical correlations were obtained for 75 . Cases were categorized as nonspecific (NSGP, 77.7%), infectious (IGP, 18.1%), or indeterminate (4.3%) granulomatous prostatitis based on histologic and clinical criteria . SETTING: Consecutive cases from a large commercial laboratory and consultation cases . RESULTS: All cases of IGP had a history of prior bacillus Calmette-Guerin therapy for transitional cell carcinoma . Histologically, 57% of NSGP cases mimicked infection and 4% mimicked cancer . Caseating necrosis was identified in 76% of cases of IGP . Significant numbers of eosinophils were found in 68% of NSGP cases, but in only 12% of IGP cases . In no case was eosinophilia documented in peripheral blood . Multinucleated giant cells were absent or rare in 69% of NSGP cases . Significant numbers of neutrophils were found in 53% of NSGP cases, but in only 29% of IGP cases . At the time of biopsy, cancer was clinically suspected in 55% of cases categorized as NSGP and 73% categorized as IGP . Serum prostate-specific antigen ranged from less than 0.5 ng/mL to 114 ng/mL (mean 12.7 ng/mL) in NSGP and from 0.9 ng/mL to 9.7 ng/mL (mean 4.2 ng/mL) in IGP . Digital rectal exam was abnormal in 69% and 91% of NSGP and IGP cases, respectively . Transrectal ultrasound was abnormal in 77% and 100% of NSGP and IGP cases, respectively . There was no correlation between the extent of core involvement with either clinical impression, prostate-specific antigen levels, transrectal ultrasound, or digital rectal exam . Thirty additional granulomatous prostatitis cases on needle biopsy were obtained from the consultation files of one of the authors . The major difference in this group was a higher percentage of cases histologically mimicking cancer (20%); two cases were misdiagnosed by the referring pathologist as high-grade cancer . CONCLUSIONS: While NSGP is the most common granulomatous prostatitis seen on needle biopsy, bacillus Calmette-Guerin granulomas are not seen infrequently . Lesser known histologic features of NSGP were the frequent finding of neutrophils and eosinophils and infrequent multinucleated giant cells . Granulomatous prostatitis may be clinically indistinguishable from cancer, and NSGP may also histologically mimic carcinoma.

Br J Urol, 1997 Jul, 80(1), 35 - 9
Effects of isoniazid on the proliferation and cytokine production of bladder cancer cells in vitro induced by bacille Calmette-Guérin; Bevers RF et al.; OBJECTIVE: To determine the effects of isoniazid (isonicotinic acid hydrazide), used to reduce the serious side-effects of immunotherapy of superficial bladder cancer with bacille Calmette-Guerin (BCG), on the proliferation and constitutive BCG-induced synthesis of interleukins 6 (IL6) and 8 (IL8) in human bladder cancer cells cultured in vitro . MATERIALS AND METHODS: Three poorly differentiated human cell lines, T24, TCC-SUP and BT-B, were used to study the effect of isoniazid on cell proliferation . Cells were inoculated in tissue culture plates and various concentrations of isoniazid added to the medium . Cell density was then monitored for up to 6 days using a colorimetric assay . To determine the effects of isoniazid on constitutive and BCG-induced cytokine synthesis, cells were cultured in medium containing no additions, BCG, isoniazid or BCG with isoniazid, at various concentrations . Samples of medium were collected regularly for 6 h and the cytokine content (IL6 and IL8) determined using enzyme-linked immunosorbent assays . RESULTS: Continuous incubation of proliferating T24, TCC-SUP and BT-B cells with isoniazid at concentrations of 0-100 micrograms/mL did not affect the rate of proliferation . Unlike TCC-SUP and BT-B cells, T24 cells released more IL6 and IL8 during incubation with BCG . At 6 h after the addition of BCG, the cumulative mean (SD) IL6 and IL8 production of T24 cells was 2.6 (0.1) and 2.3 (0.4) ng per 3 x 10(5) cells, compared with a constitutive level of 0.1 (0.0) and 1.3 (0.2) ng, respectively . There was no significant effect of isoniazid (1-100 micrograms/mL) on either the constitutive or BCG-induced synthesis of IL6 and IL8 in T24 cells . CONCLUSION: Assuming an essential role of (tumour) epithelial cells in the local immune response induced by BCG, these in vitro results suggest that the administration of isoniazid does not interfere with this part of the mechanisms by which BCG operates.

Vet Res Commun, 1997 Jul, 21(5), 375 - 80
An apparent effect of immunopotentiation during late gestation on the postpartum reproductive performance of Nili-Ravi buffaloes (Bubalus bubalis); Qureshi ZI et al.; Thirty-two Nili-Ravi buffaloes were used to determine the effect of prepartum immunopotentiation in late gestation with levamisole hydrochloride (0.5 mg/kg), vitamin E+selenium (vitE-Se) (Etosol-SE, 10 ml intramuscularly) or Bacille Calmette Guerin (BCG) (0.5 ml/animal, subcutaneously) on postpartum reproductive performance . The immunopotentiating treatment was given twice, with treatments one week apart, approximately 80 days prior to the expected date of parturition . Prepartum treatment with vitE-Se or BCG significantly (p < 0.05) reduced the calving to first oestrus interval and the length of the postpartum service period compared to the control group . The uterine involution period was significantly shorter in buffaloes treated with vitE-Se compared to the control group . Levamisole hydrochloride apparently improved the reproductive performance, but this result was not statistically significant.

J Eukaryot Microbiol, 1997 Jul-Aug, 44(4), 314 - 20
Ribosomal RNA analysis indicates a benthic pennate diatom ancestry for the endosymbionts of the dinoflagellates Peridinium foliaceum and Peridinium balticum (Pyrrhophyta); Chesnick JM et al.; The establishment of chloroplasts as cellular organelles in the dinoflagellate, heterokont (stramenopile), haptophyte, and cryptophyte algae is widely accepted to have been the result of secondary endosymbiotic events, that is, the uptake of a photosynthetic eukaryote by a phagotrophic eukaryote . However, the circumstances that promote such associations between two phylogenetically distinct organisms and result in the integration of their genomes to form a single functional photosynthetic cell is unclear . The dinoflagellates Peridinium foliaceum and Peridinium balticum are unusual in that each contains a membrane-bound eukaryotic heterokont endosymbiont . These symbioses have been interpreted, through data derived from ultrastructural and biochemical investigations, to represent an intermediate stage of secondary endosymbiotic chloroplast acquisition . In this study we have examined the phylogenetic origin of the P . foliaceum and P . balticum heterokont endosymbionts through analysis of their nuclear small subunit ribosomal RNA genes . Our analyses clearly demonstrate both endosymbionts are pennate diatoms belonging to the family Bacillariaceae . Since members of the Bacillariaceae are usually benthic, living on shallow marine sediments, the manner in which establishment of a symbiosis between a planktonic flagellated dinoflagellate and a bottom-dwelling diatom is discussed . In particular, specific environmentally-associated life strategy stages of the host and symbiont, coupled with diatom food preferences by the dinoflagellate, may have been vital to the formation of this association.

Clin Exp Immunol, 1997 Jul, 109(1), 157 - 65
Humoral response against heat shock proteins and other mycobacterial antigens after intravesical treatment with bacille Calmette-Guérin (BCG) in patients with superficial bladder cancer; Zlotta AR et al.; Few studies have analysed the antibody response during intravesical BCG immunotherapy for superficial bladder cancer . We have examined the evolution in serum antibody response against several heat shock proteins (hsp), including the recombinant mycobacterial hsp65 and the native protein P64 from BCG, GroEL from Escherichia coli (hsp60 family), recombinant mycobacterial hsp70 and the E . coli DnaK (hsp70 family), against purified protein derivative of tuberculin (PPD) and the AG85 complex of Mycobacterium bovis BCG, as well as against tetanus toxoid in 42 patients with a superficial bladder tumour, 28 treated with six intravesical BCG instillations and 14 patients used as controls . We also analysed the lymphoproliferative response of peripheral blood mononuclear cells against PPD in this population . Data of antibody responses at 6 weeks post BCG were available in all 28 patients, and at 4 month follow up in 17 patients . All patients who demonstrated a significant increase in IgG antibodies against PPD at 4 months follow up had a significant increase already at 6 weeks of follow up . In contrast, IgG antibodies against hsp increased significantly from 6 weeks to 4 months post-treatment . A significant increase in IgG antibodies against PPD, hsp65, P64, GroEL, and hsp70 at 4 months follow up was observed in 10/17, 8/17, 10/17, 4/17 and 8/17 patients . Native P64 protein elicited a higher antibody response than recombinant mycobacterial hsp65 . No increase in antibody response was observed against Dnak from E . coli, against AG85 or tetanus toxoid after BCG therapy . An increase in IgG antibodies against P64 at 4 months follow up compared with pretreatment values was found to be a significant predictor of tumour recurrence (P<0.01) . Further studies with a larger number of patients are needed to confirm the value of the antibody response against P64 as a clinical independent prognostic factor.

J Invertebr Pathol, 1997 Jul, 70(1), 41 - 9
Intramolecular proteolytic cleavage of Bacillus thuringiensis Cry3A delta-endotoxin may facilitate its coleopteran toxicity; Carroll J et al.; The Cry3A delta-endotoxin protein inclusion synthesized by Bacillus thuringiensis subsp . tenebrionis is soluble in alkaline and acid buffer solutions but the toxin precipitates when returned to neutral pH conditions . The midgut pH of susceptible beetle larvae is neutral to slightly acidic, a pH environment in which the Cry3A toxin is insoluble . To investigate this paradox we studied the Cry3A toxin after various proteolytic treatments . In many cases the toxin was cleaved into polypeptides that remained associated under non-denaturing conditions . Interestingly a chymotrypsinized Cry3A product was soluble under neutral pH conditions, retained full activity against susceptible beetle larvae, and exhibited specific binding to Leptinotarsa decemlineata midgut membranes.

Appl Environ Microbiol, 1997 Jul, 63(7), 2920 - 4
Growth of silicone-immobilized bacteria on polycarbonate membrane filters, a technique to study microcolony formation under anaerobic conditions; Hojberg O et al.; A technique was developed to study microcolony formation by silicone-immobilized bacteria on polycarbonate membrane filters under anaerobic conditions . A sudden shift to anaerobiosis was obtained by submerging the filters in medium which was depleted for oxygen by a pure culture of bacteria . The technique was used to demonstrate that preinduction of nitrate reductase under low-oxygen conditions was necessary for nonfermenting, nitrate-respiring bacteria, e.g., Pseudomonas spp., to cope with a sudden lack of oxygen . In contrast, nitrate-respiring, fermenting bacteria, e.g., Bacillus and Escherichia spp., formed microcolonies under anaerobic conditions with or without the presence of nitrate and irrespective of aerobic or anaerobic preculture conditions.

Appl Environ Microbiol, 1997 Jul, 63(7), 2798 - 801
Biochemical and molecular characterization of the insecticidal fragment of CryV; Sekar V et al.; Two C-terminal deletion constructs were made to study the effect of such deletions on the biological activity of the CryV protein of Bacillus thuringiensis subsp . kurstaki . The results of feeding on neonatal larvae of Ostrinia nubilalis (European corn borer {ECB}) indicated that the 50% lethal dose of the full-length CryV protein was 3.34 micrograms/g of diet (95% fiducial limits, 2.53 to 4.32 micrograms/g of diet) . Removal of 71 amino acids (aa) from the C terminus had little effect on toxicity, whereas deletion of 184 aa abolished the insecticidal activity of the CryV protein completely . Truncations of the full-length CryV protein were also generated with trypsin and the midgut protease of ECB . The proteolytically treated products were characterized by determining their N-terminal amino acid sequences . The CryV protein was found to be cleaved by both proteases through a two-step process . Initially an intermediary form was generated which contained aa 45 of full-length CryV as its N-terminal end . The C-terminal end of this peptide was not experimentally determined . However, analysis of the deduced amino acid sequence of CryV indicated that the C-terminal end of the intermediary form is likely either aa 655 or 659 . Further N-terminal processing of the intermediary form resulted in a protease-resistant core form . The core included aa 156 to aa 655 or 659 . While the intermediary form retained 100% of the ECB larval toxicity, the core form exhibited only approximately 22% of the toxicity of the full-length protein.

Appl Environ Microbiol, 1997 Jul, 63(7), 2792 - 7
Unique regulation of crystal protein production in Bacillus thuringiensis subsp . yunnanensis is mediated by the cry protein-encoding 103-megadalton plasmid; Srinivas G et al.; In sporulating cultures of Bacillus thuringiensis subsp . yunnanensis HD977, two cell types are observed: cells forming only spores and cells forming only crystals . Curing analysis suggested that the crystal proteins are plasmid encoded . Through plasmid transfer experiments, it was established that a 103-MDa plasmid is involved in the crystal production . Conjugal transfer of this plasmid to Cry- recipient cells of Bacillus thuringiensis subsp . kurstaki HD73-26 conferred the ability to produce crystals exclusively on asporogenous cells of the recipient, indicating that the 103-MDa plasmid mediates the unique regulation of Cry protein production . When the dipteran-specific cryIVB gene was introduced into wild-type (Cry+) and Cry- backgrounds of B . thuringiensis subsp . yunnanensis by phage CP51ts45-mediated transduction, similar to all other B . thuringiensis strains, irregular crystals of CryIVB protein were produced by spore-forming cells in both backgrounds . However, the synthesis of the bipyramidal inclusions of B . thuringiensis subsp . yunnanensis was still limited only to asporogenous cells of the transductant . Thus, it appears that the unique property of exclusive crystal formation in asporogenous cells of B . thuringiensis subsp . yunnanensis is associated with the crystal protein gene(s) per se or its cis acting elements . As the crystals in B . thuringiensis subsp . yunnanensis were formed only in asporogenous cells, attempts were made to find out whether crystal formation had any inhibitory effect on sporulation . It was observed that both Cry+ and Cry- strains of B . thuringiensis subsp . yunnanensis (HD977 and HD977-1, respectively) exhibited comparable sporulation efficiencies . In addition, the Cry- B . thuringiensis subsp . kurstaki host (HD73-26) and its Cry+ transconjugant (HD73-26-16), expressing the B . thuringiensis subsp . yunnanensis crystal protein, were also comparable in their sporulation efficiencies, indicating that production of the crystal proteins of B . thuringiensis subsp . yunnanensis does not affect the process of sporulation.

Appl Environ Microbiol, 1997 Jul, 63(7), 2716 - 21
Identification and characterization of a previously undescribed cyt gene in Bacillus thuringiensis subsp . israelensis; Guerchicoff A et al.; Mosquitocidal Bacillus thuringiensis strains show as a common feature the presence of toxic proteins with cytolytic and hemolytic activities, Cyt1Aa1 being the characteristic cytolytic toxin of Bacillus thuringiensis subsp . israelensis . We have detected the presence of another cyt gene in this subspecies, highly homologous to cyt2An1, coding for the 29-kDa cytolytic toxin from B . thuringiensis subsp . kyushuensis . This gene, designated cyt2Ba1, maps upstream of cry4B coding for the 130-kDa crystal toxin, on the 72-MDa plasmid of strain 4Q2-72 . Sequence analysis revealed, as a remarkable feature, a 5' mRNA stabilizing region similar to those described for some cry genes . PCR amplification and Southern analysis confirmed the presence of this gene in other mosquitocidal subspecies . Interestingly, anticoleopteran B . thuringiensis subsp . tenebrionis belonging to the morrisoni serovar also showed this gene . On the other hand, negative results were obtained with the anti-lepidopteran strains B . thuringiensis subsp . kurstaki HD-1 and subsp . aizawai HD-137 . Western analysis failed to reveal Cyt2A-related polypeptides in B . thuringiensis subsp . israelensis 4Q2-72 . However, B . thuringiensis subsp . israelensis 1884 and B . thuringiensis subsp . tenebrionis did show cross-reactive products, although in very small amounts.

J Bacteriol, 1997 Jul, 179(13), 4336 - 41
Cloning and analysis of the first cry gene from Bacillus popilliae; Zhang J et al.; An 80-kDa parasporal crystal protein was detected in protein extracts of sporangia of Bacillus popilliae isolated from a diseased larva of the common cockchafer (Melolontha melolontha L.) . Amino acid analysis of tryptic peptides revealed significant homology to the Cry2Aa endotoxins of Bacillus thuringiensis . The gene cryBP1 (cry18Aa1), which codes for the parasporal crystal protein, was found in a putative cry operon on the bacterial chromosome, which contains at least one further (smaller) open reading frame, orf1 . The 706-amino-acid-long CryBP1 (Cry18Aa1) protein has a predicted molecular mass of 79 kDa and shows about 40% sequence identity to the Cry2 polypeptides of B . thuringiensis . In the light of published observations which suggest that the parasporal crystal proteins of B . popilliae are slightly toxic to their grub hosts, we propose the following survival strategy of B . popilliae . As an obligate pathogen of grubs, B . popilliae germinates in the gut of a grub and the parasporal crystal proteins are released and activated . The activated protein does not cause colloid osmotic lysis but instead damages the gut wall somehow to allow the vegetative cells to enter the hemolymph more easily . By becoming a parasite, B . popilliae can continue to proliferate efficiently while the living grub provides a food supply . This process is in contrast to that of B . thuringiensis, which rapidly kills the insect and is then limited to growth on the larval carcass.

Biophys J, 1997 Jul, 73(1), 500 - 6
Polarized light scattering for rapid observation of bacterial size changes; Van de Merwe WP et al.; The effect of changing growth conditions on the diameter of rod-shaped bacteria was studied in vivo with the use of polarized light scattering . The value of a ratio of scattering matrix elements was measured as a function of scattering angle at various times after nutritional "upshift" for two strains of Escherichia coli cells . The peak locations of the scattering function were calibrated against the diameter for rod-shaped bacteria . The peaks moved toward smaller angles as a function of time after upshift, indicating that the diameter was increasing . Under special conditions, substantial peak shifts occurred within a few minutes of growth condition change, indicating a rapid onset of growth in diameter . The rate of increase of the diameters after upshift was obtained from the angular shift of peak location . This rate was approximately 14 nm/min for E . coli K12 and approximately 9 nm/min for E . coli B/r at 37 degrees C . The rate of diameter increase is smaller at lower temperatures . Experiments with Bacillus megaterium showed that any diameter change after nutritional upshift at 37 degrees C is limited to at most a very small increase, at least for the strain and medium tested.

J Clin Microbiol, 1997 Jul, 35(7), 1667 - 70
Detection of Mycoplasma pulmonis in cilia-associated respiratory bacillus isolates and in respiratory tracts of rats by nested PCR; Schoeb TR et al.; To improve the detection of Mycoplasma pulmonis contamination of isolates of cilia-associated respiratory (CAR) bacillus, we developed a nested PCR method using primers for 16S rRNA gene sequences . Of 140 samples of 16 different CAR bacillus isolates, 73 (52%) were inhibitory in the first PCR, as indicated by the absence of amplicons of the internal control, but only 11 of 140 (7.9%) were inhibitory in the second PCR . Of 27 samples known to contain M . pulmonis, only 12 (44%) were positive in the first PCR, but 25 of 27 (93%) were positive in the second PCR . Nested PCR also detected M . pulmonis in 21 of 61 (34%) CAR bacillus samples from which M . pulmonis could not be cultured and identified 2 additional M . pulmonis-contaminated CAR bacillus isolates . Of 359 respiratory and reproductive tract lavage samples from rats and mice, 35 (9.8%) were inhibitory in the first PCR, but only 15 (4.2%) were inhibitory in the second PCR . Of 72 lavage specimens from rats inoculated with an avirulent, poorly infective M . pulmonis strain, 14 (19%) were positive by nested PCR, but only 2 of 72 (2.8%) were positive by culture . Nested PCR also detected M . pulmonis in 14 of 20 (70%) paraffin sections of lung and trachea from rats and mice inoculated with CAR bacillus isolates known to contain M . pulmonis, whereas single PCR gave no positive results . We conclude that nested PCR is superior to single PCR or culture for detecting M . pulmonis, and that M . pulmonis is present in all but four CAR bacillus isolates in our collection that were from naturally infected rats; the four isolates that were exceptions were obtained from rats from a single colony.

J Urol, 1997 Jul, 158(1), 126 - 7
Transrectal ultrasound appearance of prostatic granulomas secondary to bacillus Calmette-Guerin instillation; Terris MK et al.; PURPOSE: To our knowledge the transrectal ultrasound appearance of prostatic granulomas occurring after intravesical bacillus Calmette-Guerin (BCG) therapy has not been thoroughly described . MATERIALS AND METHODS: A total of 13 men with a history of transitional cell carcinoma of the bladder treated with intravesical BCG underwent transrectal ultrasound followed by prostate biopsy and/or cystoprostatectomy . RESULTS: Of the 13 patients studied 9 (69.2%) had intensely hypoechoic lesions anteriorly in the transition zone of the prostate on ultrasound images . The lesions were histologically proved to be necrotizing granulomas . CONCLUSIONS: Prostatic granulomas secondary to BCG instillation appear as distinct, intensely hypoechoic anterior lesions within the transition zone of the prostate . Prostatic adenocarcinoma arising in the transition zone is usually not visible and would not be easily confused with granulomas . However, since transitional cell carcinoma involving the prostate can appear hypoechoic in the transition zone, transrectal or transurethral tissue sampling may be indicated.

J Urol, 1997 Jul, 158(1), 62 - 7
The treated natural history of high risk superficial bladder cancer: 15-year outcome; Cookson MS et al.; PURPOSE: The long-term outcome of patients with high risk superficial bladder cancer is unknown . We report the results of 15 years of followup of high risk patients treated initially with aggressive local therapy, including transurethral resection alone or combined with intravesical bacillus Calmette-Guerin . MATERIALS AND METHODS: Between 1978 and 1981, 86 high risk patients enrolled in a randomized study of transurethral resection alone or with intravesical bacillus Calmette-Guerin for superficial bladder cancer . Of these patients 81% had diffuse carcinoma in situ and 44% had stage T1 tumors before entry into the study . Patients were followed until death (61%) or until the present time (median followup 184 months) . RESULTS: Disease stage progressed in 46 patients (53%) and 31 (36%) eventually underwent cystectomy for progression (28) or refractory carcinoma in situ (3), while 18 (21%) had upper tract tumors at a median of 7.3 years . The 10 and 15-year disease specific survival rates were 70 and 63%, respectively . At 15 years 34% of patients overall were dead of bladder cancer, 27% were dead of other causes and 37% were alive, including 27% with an intact functioning bladder . CONCLUSIONS: Despite aggressive local therapy patients with high risk superficial bladder cancer are at lifelong risk for development of stage progression and upper tract tumors . A third of patients are at risk for death from bladder cancer, justifying careful and vigilant long-term followup . These results support the use of initial aggressive local therapy in patients with high risk superficial bladder cancer.

J Biomed Mater Res, 1997 Summer, 38(2), 115 - 9
Autosterilization of biodegradable implants by injection molding process; Konig C et al.; Sterilization of degradable implants by standard procedures may damage the parts due to the labile chemical nature of the polymers . This study examined whether the injection molding process used for the production of polymeric parts may itself sterilize the implant due to high temperature, pressure, and shear forces applied . Poly-D,L-lactic acid (PDLLA) and poly-L-lactic acid (PLLA) granules were contaminated with thermoresistant spores of Bacillus stearothermophilus (>10(5) spores/g) . Sterile and contaminated granules of both polymers were injection molded and tested for sterility . All 27 samples produced with sterile PDLLA and processed at 120 degrees C and all 18 samples produced with sterile PLLA at 200 degrees C remained sterile after injection molding and handling . However, in five out of 28 PDLLA samples and in one out of 26 PLLA samples produced with contaminated material, spores had survived the process . In conclusion, the injection molding process could not reliably sterilize parts produced with polylactic acid granules that were heavily contaminated with thermoresistant organisms . However, the number of viable spores was significantly reduced by more than 99.99% . Thus, the injection molding process might allow the autosterilization of parts produced with raw material that is not heavily contaminated.

Curr Microbiol, 1997 Jul, 35(1), 40 - 3
Investigation of mosquito-specific larvicidal activity of a soil isolate of Bacillus thuringiensis serovar canadensis; Ishii T et al.; The LC50 value of alkali-solubilized parasporal inclusion proteins of a Diptera-specific strain, belonging to Bacillus thuringiensis serovar canadensis, was 2.4 microg/ml for larvae of the mosquito, Aedes aegypti . A significant loss in larvicidal activity occurred when solubilized inclusion proteins were treated with A . aegypti larval gut extract, silkworm (Bombyx mori) larval gut juice, and the proteinase K . Approximately 90% of the larvicidal activity was destroyed upon treatment with proteases in 30 min . The parasporal inclusion was composed of major proteins of 65, 53, and 28 kDa and some other minor proteins . Proteolysis profiles showed that the 65-kDa major protein is highly sensitive to proteases . Purification experiments with DEAE-Toyopearl column chromatography revealed that the 65-kDa protein is responsible for the mosquitocidal activity of this strain . The LC50 value of the purified protein was 5.4 microg/ml.

Curr Microbiol, 1997 Jul, 35(1), 1 - 8
Characterization of a cry2A gene cloned from an isolate of Bacillus thuringiensis serovar sotto; Sasaki J et al.; A cry2A-type gene, designated as cry2(SKW), was cloned from Bacillus thuringiensis serovar sotto SKW01-10.2-06, and some unique features of the gene were revealed . The cry2(SKW) gene encoded a polypeptide of 635 residues with a predicted molecular mass of 71,137 Da . Cry2(SKW) had 95.4% identity with Cry2Aa in amino acid sequence and was two residues longer than Cry2Aa . Two open reading frames (ORFs), designated as orf1 and orf2, were present upstream of the cry2(SKW) and showed high homology with the corresponding ORFs in the cry2Aa operon . The Orf2 from SKW01-10.2-06 contained a region of repeated sequences . However, unlike Cry2Aa, Cry2(SKW) formed the cuboidal crystalline inclusions when the cry2(SKW) gene was expressed in an acrystalliferous B . thuringiensis strain in the absence of the upstream ORFs . Furthermore, Cry2(SKW) was less toxic to a lepidopteran species, Bombyx mori, than Cry2Aa in spite of high homology between the two proteins.

FEBS Lett, 1997 Jun 30, 410(2-3), 397 - 402
Restriction of intramolecular movements within the Cry1Aa toxin molecule of Bacillus thuringiensis through disulfide bond engineering; Schwartz JL et al.; Disulfide bridges were introduced into CrylAa, a Bacillus thuringiensis lepidopteran toxin, to stabilize different protein domains including domain I alpha-helical regions thought to be involved in membrane integration and permeation . Bridged mutants could not form functional ion channels in lipid bilayers in the oxidized state, but upon reduction with beta-mercaptoethanol, regained parental toxin channel activity . Our results show that unfolding of the protein around a hinge region linking domain I and II is a necessary step for pore formation . They also suggest that membrane insertion of the hydrophobic hairpin made of alpha-helices 4 and 5 in domain I plays a critical role in the formation of a functional pore.

J Biol Chem, 1997 Jun 20, 272(25), 15729 - 33
Cyclodextrins are not the major cyclic alpha-1,4-glucans produced by the initial action of cyclodextrin glucanotransferase on amylose; Terada Y et al.; The initial action of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from an alkalophilic Bacillus sp . A2-5a on amylose was investigated . Synthetic amylose was incubated with purified CGTase then terminated in the very early stage of the enzyme reaction . When the reaction mixture was treated with glucoamylase and the resulting glucoamylase-resistant glucans were analyzed with high performance anion exchange chromatography, cyclic alpha-1,4-glucans, with degree of polymerization ranging from 9 to more than 60, in addition to well known alpha-, beta-, and gamma-cyclodextrin (CD), were detected . The time-course analysis revealed that larger cyclic alpha-1, 4-glucans were preferentially produced in the initial stage of the cyclization reaction and were subsequently converted into smaller cyclic alpha-1,4-glucans and into the final major product, beta-CD . CGTase from Bacillus macerans also produced large cyclic alpha-1, 4-glucans except that the final major product was alpha-CD . Based on these results, a new model for the action of CGTase on amylose was proposed, which may contradict the widely held view of the cyclization reaction of CGTase.

Gene, 1997 Jun 19, 192(2), 271 - 81
Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element; Chong S et al.; A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step . The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag . The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol (beta-ME) or cysteine at low temperatures and over a broad pH range . A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column . The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column . No exogenous proteolytic cleavage is needed . Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.

Eur J Biochem, 1997 Jun 15, 246(3), 756 - 62
Sequence and expression of an Eisenia-fetida-derived cDNA clone that encodes the 40-kDa fetidin antibacterial protein; Lassegues M et al.; Fetidins are 40-kDa and 45-kDa hemolytic and antibacterial glycoproteins present in the coelomic fluid of the earthworm Eisenia fetida andrei . By screening a cDNA library with a polyclonal antifetidin serum, we have cloned a cDNA that encoded the 40-kDa fetidin . The clone contains an insert of 1.44 kb encoding a protein of 34 kDa, which corresponds to the size of deglycosylated fetidins . The recombinant protein inhibits Bacillus megaterium growth . Restriction fragment polymorphisms were observed on Southern blots and correspond to a known protein polymorphism . The sequence of the cDNA contains a peroxidase signature and fetidins from earthworm coelomic fluid have peroxidase activity . The 40-kDa and 45-kDa fetidins therefore represent two related polymorphic defence factors in invertebrates.

Eur J Biochem, 1997 Jun 15, 246(3), 652 - 7
Aminopeptidase N from Bombyx mori as a candidate for the receptor of Bacillus thuringiensis Cry1Aa toxin; Yaoi K et al.; Cry1Aa toxin-binding proteins from the midgut brush border membrane vesicles of Bombyx mori, a toxin-susceptible silkworm, were analyzed to find candidates for the toxin receptors . Ligand blotting showed that Cry1Aa toxin bound to a 120-kDa protein . A part of the 120-kDa protein was solubilized from the membrane vesicles with phosphatidylinositol-specific phospholipase C, resulting in a 110-kDa protein which therefore may be linked to a glycosyl-phosphatidylinositol anchor . The 120-kDa and 110-kDa Cry1Aa toxin-binding proteins were solubilized with detergent or pohosphatidylinositol-specific phospholipase C, respectively, and purified using anion-exchange chromatography . Scatchard plot analysis for the specific binding of purified 110-kDa protein to Cry1Aa toxin yielded a Kd value of 7.6 nM, which was similar to that for the binding of intact brush border membrane vesicles to the toxin . N-terminal and internal amino acid sequences of the 120-kDa and 110-kDa proteins showed high degrees of similarity to those of aminopeptidase N, a putative Cry1Ac toxin receptor, reported in Manduca sexta and Heliothis virescens . On this basis, the 120-kDa Cry1Aa toxin-binding protein from B . mori was identified as a member of the aminopeptidase family.

J Immunol, 1997 Jun 15, 158(12), 5685 - 91
Evidence for self and nonself peptide partial agonists that prolong clonal survival of mature T cells in vitro; Matsushita S et al.; When examining the effects of peptide analogues without proliferation-inducing activity on three human CD4+ T cell clones with distinct TCRbeta recognizing a nonself mycobacterial bacillus Calmette-Guerin a (BCGa) peptide fragment (EEYLILSARDVLAVVSK)/HLA-DR14 complex, we found that 1) stimulation of T cells with a one-residue-substituted analogue or a minimally homologous self peptide fragment derived from human connexin 26 (IMILVVAAKEVWGDEQA) can prolong the in vitro survival of T cells, in a clone specific-manner; 2) this prolongation is associated with the up-regulation of Bcl-xL without proliferation; and 3) these peptide-clone combinations are capable of inducing lymphokine secretion . Thus, peptide partial agonism may play a role in the survival of not only thymocytes but also mature T cells, in the absence of wild-type ligands.

J Biotechnol, 1997 Jun 13, 55(2), 101 - 12
Secretion of authentic 20-kDa human growth hormone (20K hGH) in Escherichia coli and properties of the purified product; Uchida H et al.; Using Bacillus amyloliquefaciens neutral protease gene (npr), we have constructed a secretion system of 20-kDA human growth hormone (20K hGH) in E . coli . The secretion-signal region from npr was modified inserting a fragment coding a 2Lys-5Leu cluster . In this system we found that co-expression of glutathione reductase remarkably increased accumulation level of 20K hGH in periplasm and confirmed that secreted 20K hGH was correctly processed . The recombinant 20K hGH was highly purified and subjected to analyses of physicochemical properties and biological activities which are still unclear and controversial due to difficulty in preparing the sample with authentic structure . The secreted recombinant product had authentic disulfide linkages and showed molecular weight of 20,270.5 +/- 3.7 (theoretical value, 20,269.9) . The results suggest that the recombinant 20K hGH is a full agonist on rat growth promotion and lipolysis stimulation in isolated rat adipose tissues . In particular, the lipolysis-stimulating activity of 20K hGH was distinct as compared with that of 22K hGH under physiological concentration . Cell proliferation activity via prolactin-receptor in Nb-2 lymphoma was obviously low as compared with that of 22K hGH.

J Biol Chem, 1997 Jun 13, 272(24), 15106 - 12
Comparative enzymatic properties of GapB-encoded erythrose-4-phosphate dehydrogenase of Escherichia coli and phosphorylating glyceraldehyde-3-phosphate dehydrogenase; Boschi-Muller S et al.; GapB-encoded protein of Escherichia coli and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) share more than 40% amino acid identity . Most of the amino acids involved in the binding of cofactor and substrates to GAPDH are conserved in GapB-encoded protein . This enzyme shows an efficient non-phosphorylating erythrose-4-phosphate dehydrogenase activity (Zhao, G., Pease, A . J., Bharani, N., and Winkler, M . E . (1995) J . Bacteriol . 177, 2804-2812) but a low phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity, whereas GAPDH shows a high efficient phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity and a low phosphorylating erythrose-4-phosphate dehydrogenase activity . To identify the structural factors responsible for these differences, comparative kinetic and binding studies have been carried out on both GapB-encoded protein of Escherichia coli and GAPDH of Bacillus stearothermophilus . The KD constant of GapB-encoded protein for NAD is 800-fold higher than that of GAPDH . The chemical mechanism of erythrose 4-phosphate oxidation by GapB-encoded protein is shown to proceed through a two-step mechanism involving covalent intermediates with Cys-149, with rates associated to the acylation and deacylation processes of 280 s-1 and 20 s-1, respectively . No isotopic solvent effect is observed suggesting that the rate-limiting step is not hydrolysis . The rate of oxidation of glyceraldehyde 3-phosphate is 0.12 s-1 and is hydride transfer limiting, at least 2000-fold less efficient compared with that of erythrose 4-phosphate . Thus, it can be concluded that it is only the structure of the substrates that prevails in forming a ternary complex enzyme-NAD-thiohemiacetal productive (or not) for hydride transfer in the acylation step . This conclusion is reinforced by the fact that the rate of oxidation for erythrose 4-phosphate by GAPDH is 0.1 s-1 and is limited by the acylation step, whereas glyceraldehyde 3-phosphate acylation is efficient and is not rate-determining (>/=800 s-1) . Substituting Asn for His-176 on GapB-encoded protein, a residue postulated to facilitate hydride transfer as a base catalyst, decreases 40-fold the kcat of glyceraldehyde 3-phosphate oxidation . This suggests that the non-efficient positioning of the C-1 atom of glyceraldehyde 3-phosphate relative to the pyridinium of the cofactor within the ternary complex is responsible for the low catalytic efficiency . No phosphorylating activity on erythrose 4-phosphate with GapB-encoded protein is observed although the Pi site is operative as proven by the oxidative phosphorylation of glyceraldehyde 3-phosphate . Thus the binding of inorganic phosphate to the Pi site likely is not productive for attacking efficiently the thioacyl intermediate formed with erythrose 4-phosphate, whereas a water molecule is an efficient nucleophile for the hydrolysis of the thioacyl intermediate . Compared with glyceraldehyde-3-phosphate dehydrogenase activity, this corresponds to an activation of the deacylation step by >/=4.5 kcal.mol-1 . Altogether these results suggest subtle structural differences between the active sites of GAPDH and GapB-encoded protein that could be revealed and/or modulated by the structure of the substrate bound . This also indicates that a protein engineering approach could be used to convert a phosphorylating aldehyde dehydrogenase into an efficient non-phosphorylating one and vice versa.

FEBS Lett, 1997 Jun 9, 409(2), 242 - 6
Inverse gene expression of prostacyclin and thromboxane synthases in resident and activated peritoneal macrophages; Kuwamoto S et al.; Prostacyclin and thromboxane A2 produced from prostaglandin H2 are known to be important modulators with opposite biological activities . To examine possible roles of these prostanoids in immune responses, we have studied the gene expression of prostacyclin synthase (PGIS) and thromboxane synthase (TXS) in murine resident macrophages or in macrophages elicited with casein or bacillus Calmette-Guerin (BCG) . Northern blot analyses showed that the PGIS mRNA was expressed in a decreasing order in the resident, and casein- and BCG-elicited macrophages . In contrast, the TXS mRNA was expressed in an increasing order in the resident, and casein- and BCG-elicited macrophages . On the other hand, the mRNA for cyclooxygenase-2, which produces PGH2 and participates in the production of prostanoids in inflammation, was expressed in both the resident and BCG-elicited macrophages but barely in the casein-elicited cells . In situ hybridization analysis showed that the expression of mRNAs for PGIS and TXS was ascribable not only to the alteration of the expression levels of both mRNAs in the each macrophage but also to the changes in subpopulations of the cells expressing these mRNAs . These observations suggested that the inverse gene expression of PGIS and TXS in macrophages contributes to immune responses by modulating the relative levels of prostacyclin and thromboxane A2.

Protein Eng, 1997 Jun, 10(6), 627 - 34
High-resolution crystal structure of M-protease: phylogeny aided analysis of the high-alkaline adaptation mechanism; Shirai T et al.; M-protease is a subtilisin-family serine protease produced by an alkaliphilic Bacillus sp . strain . Optimal enzymatic activity of the protein occurs at pH 12.3 . The crystal structure of M-protease (space group P2(1)2(1)2(1), a = 62.3, b = 75.5, c = 47.2 A) has been refined to a crystallographic R-factor of 17.2% at 1.5 A resolution . The alkaline adaptation mechanism of the enzyme was analyzed . Molecular phylogeny construction was used to determine the amino acid substitutions that occurred during the high-alkaline adaptation process . This analysis revealed a decrease in the number of negatively charged amino acids (aspartic acid and glutamic acid) and lysine residues and an increase in arginine and neutral hydrophilic amino acids (histidine, asparagine and glutamine) residues during the course of adaptation . These substitutions increased the isoelectric point of M-protease . Some of the acquired arginine residues form hydrogen bonds or ion pairs to combine both N- and C-terminal regions of M-protease . The substituted residues are localized to a hemisphere of the globular protein molecule where positional shifts of peptide segments, relative to those of the less alkaliphilic subtilisin Carlsberg, are observed . The biased distribution and interactions caused by the substituted residues seem to be responsible for stabilization of the conformation in a high-alkaline condition.

Jpn J Antibiot, 1997 Jun, 50(6), 525 - 31
Bactericidal effect of ecabet sodium on clarithromycin- and metronidazole-resistant clinical isolates of Helicobacter pylori; Shibata K et al.; Ecabet sodium showed a bactericidal effect on Helicobacter pylori NCTC 11637 which is susceptible to antimicrobial agents (Antimicrob . Agents Chemother, 39: 1295-1299, 1995) . In the present study, we investigated the bactericidal effect of ecabet sodium on clarithromycin- and metronidazole-resistant strains of clinical isolates of H . pylori under acidic conditions . In a buffer supplemented with urea at pH 3.0, ecabet sodium decreased the number of viable cells in both isolates . In the morphological study, ecabet sodium changed the isolates from the bacilliform to the horseshoe-shaped form and denatured the cytoplasm . Ecabet sodium also showed the bactericidal effect on both isolates in buffers at pH 4.0 and 5.0, and the bactericidal effect was dependent on pH, i.e., the lower the pH, the greater the effect . These results suggest that the susceptibility of H . pylori to antimicrobial agents does not affect the bactericidal effect of ecabet sodium on H . pylori.

Scand J Urol Nephrol, 1997 Jun, 31(3), 317 - 8
Epididymo-orchitis and Reiter's disease . Two infrequent complications after intravesical bacillus Calmette-Guérin therapy; Hansen CP et al.; Two cases of adverse reactions to intravesical treatment with bacillus Calmette-Guerin are reported . The first patient presented with a severe suppurating epididymo-orchitis three months after his last treatment . The second patient developed Reiter's disease with arthritis of one knee and conjunctivitis 1 week after the fourth instillation.

J Am Mosq Control Assoc, 1997 Jun, 13(2), 158 - 63
Field trial of Bacillus sphaericus strain B-101 (serotype H5a, 5b) against filariasis and Japanese encephalitis vectors in India; Yadav RS et al.; A large-scale operational field trial was conducted from June 1993 to October 1994 to evaluate the efficacy of Bacillus sphaericus (strain B-101, serotype H5a,5b) for control of the vectors of filariasis (Culex quinquefasciatus) and Japanese encephalitis (Cx . tritaeniorhynchus and Cx . vishnui) in Rourkela city . Application of B . sphaericus, when sprayed at 1 g/m2 in storm drains, wastewater pools, abandoned masonry tanks, peripheral paddy fields, ditches, and other small water collections and at 4 g/m2 in domestic septic tanks, significantly reduced larval and pupal counts (P < 0.0001) and significantly reduced the percentage of habitats containing larvae (3rd-4th instars) (P < 0.0001) as compared with routine antilarval measures . This in turn resulted in a reduction in the indoor density of disease vectors in particular and a reduction in mosquito nuisance in general . The trial demonstrated that B . sphaericus has good potential for use against disease vectors and mosquito breeding in polluted as well as clean waters.

J Am Mosq Control Assoc, 1997 Jun, 13(2), 140 - 4
Phlebotomine sand fly control using bait-fed adults to carry the larvicide Bacillus sphaericus to the larval habitat; Robert LL et al.; Sugar meals of plant origin are an important component of the sand fly diet . We show that sugar solution baits have potential as vehicles for phlebotomine sand fly control . In the laboratory, adult Phlebotomus duboscqi Neveu-Lemaire and Sergentomyia schwetzi (Adler, Theodor, and Parrot) that have consumed an aqueous sucrose solution containing Bacillus sphaericus Neide toxins and are subsequently eaten by larvae produce significant larval death (P < 0.01) . In the field, when vegetation near animal burrows and eroded termite mounds was sprayed with sucrose solution with or without incorporation of the larval toxicant B . sphaericus, 40% of female sand flies fed in situ . Dispersing B . sphaericus-carrier sand flies caused significant larval mortality (P < 0.01) in resting and breeding sites in animal burrows 10-30 m from the sprayed vegetation for 2-12 wk posttreatment . Also, adult sand fly populations breeding and resting inside animal burrows were significantly reduced (P < 0.01) following direct application of the sucrose/B . sphaericus solution to the burrow entrances . This control effect lasted 4-10 wk post-treatment . The effect was not seen for sand fly populations breeding and resting inside eroded termite mounds . This approach may be useful for the application of biological control agents against phlebotomine sand flies in biotypes where larvae and adults use the same habitats.

Vaccine, 1997 Jun, 15(8), 834 - 8
Protection against tuberculosis by a plasmid DNA vaccine; Lowrie DB et al.; Past attempts to use fractions of mycobacteria as an alternative to BCG have given disappointing results . The availability of cloned genes and suitable vectors has now opened a new avenue in which individual mycobacterial protein antigens are synthesised within transfected mammalian cells . In an ex vivo transfection approach with a retroviral vector we found that even a single antigen (hsp65) could evoke strong protection when expressed as a transgene and that expression of protection was largely a function of antigen specific cytotoxic T cells . We now find that intramuscular injection of plasmid DNA expressing the antigen from either a viral or a murine promoter can also give protection equivalent to Bacillus Calmette-Guerin (BCG) . Plasmids expressing some other mycobacterial antigens, hsp70, 36 kDa and 6 kDa, are also effective, suggesting that this approach may lead to a new vaccine.

Comp Biochem Physiol B Biochem Mol Biol, 1997 Jun, 117(2), 253 - 7
Channelling of deoxyribose moiety of exogenous DNA into carbohydrate metabolism: role of deoxyriboaldolase; Sgarrella F et al.; In bacteria, the addition of (deoxy)nucleosides or (deoxy)ribose to the growth medium causes induction of enzymes involved in their catabolism, leading to the utilisation of the pentose moiety as carbon and energy source . In this respect, deoxyriboaldolase appears the key enzyme, allowing the utilisation of deoxyribose 5-P through glycolysis . We observed that not only deoxynucleosides, but also DNA added to the growth medium of Bacillus cereus induced deoxyriboaldolase; furthermore, the switch of the culture from aerobic to anaerobic conditions caused a further increase in enzyme activity, leading to a more efficient channelling of deoxyribose 5-P into glycolysis, probably as a response to the low energy yield of the sugar fermentation . In eukaryotes, the catabolism of (deoxy)nucleosides is well known . However, the research in this field has been mainly devoted to the salvage of the bases formed by the action of nucleoside phosphorylases, whereas the metabolic fate of the sugar moiety has been largely neglected . Our results indicate that the deoxyriboaldolase activity is present in the liver of several vertebrates and in a number of cell lines . We discuss our observations looking at the nucleic acids not only as informational molecules, but also as a not negligible source of readily usable phosphorylated sugar.

Plant Mol Biol, 1997 Jun, 34(3), 485 - 96
Specific sequence modifications of a cry3B endotoxin gene result in high levels of expression and insect resistance; Iannacone R et al.; Solanum melongena (eggplant) cv . Picentia and the wild species Solanum integrifolium were transformed with both a wild type (wt) and four mutagenized versions of Bacillus thuringiensis (Bt) gene Bt43 belonging to the cry3 class . The Bt gene was partly modified in its nucleotide sequence by replacing four target regions (W: +1 to +170; X: +592 to +1057; Y: +1203 to +1376; Z: +1376 to +1984) with synthetic fragments obtained by polymerase chain reaction amplification of crude oligonucleotides . The synthetic Bt genes were designed to avoid, in their modified regions, sequences such as ATTTA sequence, polyadenylation sequences and splicing sites, which might destabilize the messenger RNA . Furthermore, the codon usage was improved for a better expression in the plant system . The amino acid composition was not altered . Four versions of the modified Bt gene were obtained, BtE, BtF, BtH and BtI, with a nucleotide subtitution percentage of 8.2, 8.6, 14, and 16%, respectively, in comparison to the wt gene Bt43 . Modified versions contained different subsets of substituted regions: BtE-W + Z, BtF - Y + Z, BtH-X + Y + Z, BtI - W + X + Y + Z . In the final modified version (BtI), overall guanine+cytosine was increased from the 34.1% of the wt gene to 45.5%, and most of the destabilizing sequences were eliminated . Transgenic plants obtained with the more modified versions, BtH and BtI, were fully resistant to Leptinotarsa decemlineata Say first- and third-instar larvae, while Bt43 wt, BtE and BtF genotypes did not cause mortality and did not affect larval development.

J Vector Ecol, 1997 Jun, 22(1), 43 - 51
Efficacy of a granular formulation of Bacillus sphaericus against Culex quinquefasciatus and Anopheles gambiae in West African countries; Skovmand O et al.; The efficacy of a sustained released granular formulation of Bacillus sphaericus strain 2362 was compared to a flowable concentrate in containers, cesspools, and water ponds . Duration of control was dependent on formulation, dosage, exposure to sun, site, recycling, and target mosquito larvae . In a series of container tests with repeated additions of fourth-instar Culex quinquefasciatus larvae exposed to 0.3 or 3.0 g/m2 when cadavers were not removed, more than 95 percent control was obtained for two and four days in containers that were exposed to the sun with sewage water treated with the flowable concentrate compared to four and seven days for those treated with the granule . In sun-exposed containers with sewage water, control persisted for two days for the flowable concentrate at both dosages and one and six days for the granule at 0.3 g/m2 and 3.0 g/m2, respectively . Compared to the above tests, more than seven weeks control was obtained with 0.3 g/m2 of the flowable concentrate in closed containers where larvae were added weekly . In closed containers without weekly addition of larvae, the control was 15 percent when larvae were added five weeks after the treatment . Spore counts at the surface and bottom of the containers with lids showed an increase in number of spores at the surface where larvae were added weekly and a rapid decline where they were not . Spore counts at the bottom showed settling in both cases, but to a much higher level where larvae were added weekly . Nearly 100 percent control of Cx, quinquefasciatus larvae was obtained for at least 16 days in cesspools in Yaounde, Cameroon, treated with the granule at 3.0 g/m2 compared to approximately nine days for the flowable concentrate . At 0.3 g/m2, the duration of this reduction was five days for both products . Nearly 100 percent control of Anopheles gambiae was obtained in sun-exposed water ponds near the village Kotiokh, Senegal, for at least 15 days with the granule at 3.0 g/m2 compared to just five days for the flowable concentrate at the same dosage.

Biosci Biotechnol Biochem, 1997 Jun, 61(6), 1022 - 3
Inactivation of Bacillus spores by the supercritical carbon dioxide micro-bubble method; Ishikawa H et al.; Bacillus spores were effectively inactivated by the supercritical (SC) CO2 micro-bubble method . The micro-bubble SC CO2 treatment of B . cereus, B . subtilis, B . megaterium, B . polymyxa, and B . coagulans at 40 degrees C and 30 MPa for 30 min produced greater reduction (about 3 log cycles of reduction) than a similar treatment without a filter . The SC CO2 treatment of B . polymyxa, B . cereus, and B . subtilis spores at 45 degrees C, 50 degrees C, respectively, and 30 MPa for 60 min resulted in a 6-log cycle reduction of survival . The SC CO2 treatment under the foregoing conditions should offer higher efficiency than that of heat treatment at 100 degrees C for 60 min . In addition, the SC CO2 treatment (30 MPa, 60 degrees C, 30 min) of B . polymyxa and B . cereus spores also produced a 6-log cycle reduction.

Eur J Biochem, 1997 Jun 1, 246(2), 539 - 47
General structure/function properties of microbial methionyl-tRNA synthetases; Schmitt E et al.; Alignment of the sequences of methionyl-tRNA synthetases from various microbial sources shows low levels of identities . However, sequence identities are clustered in a limited number of sites, most of which contain peptide patterns known to support the activity of the Escherichia coli enzyme . In the present study, site-directed mutagenesis was used to probe the role of these conserved residues in the case of the Bacillus stearothermophilus methionyl-tRNA synthetase . The B . stearothermophilus enzyme was chosen in this study because it can be produced as an active truncated monomeric form, similar to the monomeric derivative of E . coli methionyl-tRNA synthetase produced by mild proteolysis . The two core enzyme molecules share only 27% identical residues . The results allowed the identification of the binding sites for ATP, methionine and tRNA, as well as that responsible for the tight binding of the zinc ion to the enzyme . It is concluded that the thermostable synthetase adopts a three-dimensional folding very similar to that of the E . coli one . Therefore, the two methionyl-tRNA synthetase sequences, although significantly different, maintain a common scaffold with the functionally important residues exposed at constant positions . Sequence alignments suggest that the above conclusion can be generalized to the known methionyl-tRNA synthetases from various sources.

J Can Dent Assoc, 1997 Jun, 63(6), 448 - 53
Effectiveness of salt versus glass bead sterilizers; Haddad AJ et al.; Microorganisms can be removed from dental instruments by various methods, including treatment in salt and glass bead sterilizers . However, no rigorous, controlled, in vivo or in vitro studies have been performed to verify the respective efficiencies of these methods . The goals of this study were to determine if the positioning of instruments at the centre or edge of a salt sterilizer results in differential sterilization effectiveness, and to compare the effectiveness of salt sterilizers relative to glass bead sterilizers . Autoclaved number 60 reamers were contaminated by plunging them to the handle in a commercial Bacillus stearothermophilus spore suspension . They were then sterilized for different periods of time and at different positions in the sterilizers . Each experiment included positive and negative controls . The results showed that better sterilization is achieved at the edge of the chamber than at the centre, and that salt sterilizers are more effective than glass bead sterilizers for a given period of time (15 seconds) in the sterilizer.

Lett Appl Microbiol, 1997 Jun, 24(6), 438 - 40
A high homology exists in N-terminal amino acid sequences of delta-endotoxins between Lepidoptera-specific and Coleoptera-specific Bacillus thuringiensis strains; Wasano N et al.; The 130 kDa parasporal inclusion proteins of the Lepidoptera-specific Bacillus thuringiensis reference strains for four H serovars were examined for sequences of 14 N-terminal amino acids . The four sequences fell into a single category, MNRNNQNEYEVIDA, and were dissimilar to any of the established sequences for Lepidoptera- and/or Diptera-killing crystal proteins . They were highly related to the reported sequence of the 130 kDa protein of the strain Buibui (serovar japonensis), which is specific for scarabaeid coleopterans.

Am J Infect Control, 1997 Jun, 25(3), 283 - 6
Benefit of two-step PPD testing of new employees at a New York City hospital; Sepkowitz KA et al.; BACKGROUND: Recent concern about nosocomial transmission of tuberculosis has led hospitals to scrutinize employee tuberculin conversion rates . The Centers for Disease Control and Prevention recommends two-step testing of new employees to limit the booster phenomenon . The cost of such a program and its subsequent yield have not recently been examined . METHODS: Employee health records were retrospectively reviewed of persons hired from 1993 and 1994 at St . Clare's Hospital in New York City, all of whom received two-step testing at time of initial employment . RESULTS: Of 262 new employees, 107 (41%) had positive tuberculin results on initial testing . The results of 15 (9.7%) of the remaining 155 patients became positive on two-step testing administered 1 week later . Persons with a positive second test result were significantly more likely to be male or foreign born or to have received previous bacille Calmette-Guerin vaccination . Identification of these 15 persons and exclusion of them from probable subsequent conversion prevented an almost 50% increase in the annual conversion rate at our hospital, from 3.2% to 4.7% . CONCLUSION: Two-step tuberculin testing is an essential means of identifying persons with a baseline positive tuberculin test result, thus allowing accurate reporting of subsequent employee tuberculin conversions.

Am J Infect Control, 1997 Jun, 25(3), 229 - 35
Status of tuberculosis infection control programs at Texas hospitals, 1989 through 1991; Manangan LP et al.; BACKGROUND: Paralleling the resurgence of tuberculosis (TB) in the United States, the reported number of persons with TB in Texas increased by 33% during 1985 through 1992, the third largest rise among all the states . This increase prompted us to survey hospitals in Texas to determine their degree of compliance with recommendations in the Centers for Disease Control and Prevention TB guidelines . METHODS: In April 1992, we mailed a voluntary questionnaire about TB infection control practices, health care worker tuberculin skin testing procedures, and Mycobacterium tuberculosis laboratory methods to a convenience sample of hospitals in Texas . RESULTS: Of 180 hospitals surveyed, 151 (83%) returned completed questionnaires . Of these, 90 (60%) were nonteaching community hospitals; 28 (19%) were teaching community hospitals; 13 (9%) were university-affiliated hospitals; and 20 (13%) were other hospitals . The number of hospitals to which patients with TB were admitted increased from 98 (65%) in 1989 to 122 (81%) in 1991 . Respondent hospitals had a mean of 183 acute care beds (median 100, range 5 to 999), 6 acid-fast bacillus isolation rooms (median 2, range 0 to 57) and 7.5 admissions/year of patients with TB (median 2, range 0 to 202) . Of hospitals responding to specific questions, 20% (27/137) admitted patients with multidrug-resistant TB, 18% (25/140) reported not having any acid-fast bacillus isolation rooms, and 28% (35/125) had no rooms meeting all of the Centers for Disease Control and Prevention criteria for acid-fast bacillus isolation (negative air pressure, > or = 6 air changes per hour, and air directly vented to the outside) . The tuberculin skin test conversions among health care workers rose from 246 (0.6%) in 1989 to 547 (0.9%) in 1991 . CONCLUSION: Although the number of Texas hospitals admitting patients with TB increased during 1989 through 1991, many facilities still did not have infection control practices consistent with the 1992 Centers for Disease Control and Prevention TB guidelines.

J Appl Microbiol, 1997 Jun, 82(6), 677 - 82
The use of Bacillus diarrhoeal enterotoxin (BDE) detection using an ELISA technique in the confirmation of the aetiology of Bacillus-mediated diarrhoea; Tan A et al.; A commercially available ELISA kit was used for the detection of Bacillus diarrhoeal enterotoxin (BDE) in a variety of foods and faeces . The ability of isolates of Bacillus spp., including Bacillus cereus, to produce BDE in Brain Heart Infusion broth containing 0.1% glucose was also checked by use of the kit . Results show that 29 out of 31 B . cereus isolates were enterotoxigenic . Foods positive for performed BDE were always contaminated with > 10(5) B . cereus cfu g-1, but not all foods contaminated with large numbers of B . cereus were positive for BDE . Bacillus sp., other than one isolate which closely resembled B . subtilis, were negative for BDE production . Criteria for the confirmation of Bacillus-mediated diarrhoea should now include reports of symptoms and incubation periods consistent with the diarrhoeal form of food-poisoning by Bacillus spp., together with the results of tests for enterotoxigenicity of the Bacillus isolate, and detection of BDE in either the food and/or faeces.

No To Shinkei, 1997 Jun, 49(6), 537 - 40
{Cerebral infarction due to bacterial emboli associated with methamphetamine abuse}; Imanishi M et al.; The number of stimulant-drug addicts has recently been on the rise again, and they are being increasingly encountered in the emergency room . There are also frequent reports of cerebrovascular disorders complicating drug toxicity . These cerebrovascular disorders have included subarachnoid hemorrhage, intracranial hematoma, and a few cases of cerebral infarction . Here, we report the case of a 37-year-old male with drug toxicity, consciousness disorder, and hyperthermia . He was in a coma with a temperature of 43.1 degrees C and blood pressure of 58/35 mmHg when brought to our hospital . His condition worse rapidly deteriorated, and he died the same day . Cerebral infarction caused by gram-positive bacillus embolism, not necrotizing angiitis, was found at autopsy . Because drug addicts, especially stimulant-drug addicts, tend to inject themselves drug under unsanitary conditions, the possibility of this type of complication is always present . This is the first such case ever reported, and is therefore regarded as a rare complication of stimulant-drug intoxication.

Mod Pathol, 1997 Jun, 10(6), 524 - 9
Myocarditis in Whipple's disease: an unsuspected cause of symptoms and sudden death; Mooney EE et al.; Whipple's disease (WD) is an uncommonly diagnosed infection caused by the recently characterized bacillus, Tropheryma whippelii . The association of WD with pericarditis and endocarditis is widely recognized, although less attention has been paid to the myocardium as a site of disease . Although the disease was uniformly fatal before antibiotic therapy, current treatment usually results in cure . We report two patients whose deaths were directly related to cardiac involvement by WD and whose underlying disease escaped diagnosis for years . The first, a 60-year-old white woman, suffered a cardiovascular collapse, and lymphocytic myocarditis was demonstrated at autopsy . The second, a 48-year-old black man, had a lengthy history of progressive cardiac failure that terminated in arrhythmia . Extensive myocardial fibrosis, with lymphocytic and granulomatous inflammation, was demonstrated at autopsy . The presence of T . whippelii was confirmed by electron microscopic examination in both cases and by polymerase chain reaction in one . Patients with WD might harbor an undiagnosed lymphocytic or granulomatous myocarditis, and this diagnosis should be considered in the evaluation of cardiac failure.

Clin Infect Dis, 1997 Jun, 24(6), 1252 - 5
Cerebral relapse of sarcoidlike Whipple's disease; Peters FP et al.; Whipple's disease, an infection with the recently identified intracellular bacillus Tropheryma whippelii, is a systemic disorder that can be life threatening when untreated . In a few patients, the signs and symptoms of the disease are similar to those of sarcoidosis, and this illness is referred to as sarcoidlike Whipple's disease . This variant must be recognized because patients with sarcoidlike Whipple's disease must be treated with antibiotics instead of corticosteroids, which would be indicated for patients with true sarcoidosis . We describe a 53-year-old man who had sarcoidlike Whipple's diseases with polyvisceral granulomatous dissemination that was treated with procaine penicillin G and streptomycin followed by doxycycline . His condition initially improved . However, during his 4-month course of treatment he developed a cerebral relapse; this relapse was successfully treated with ceftriaxone and cefixime.

Clin Infect Dis, 1997 Jun, 24(6), 1139 - 46
Disseminated bacille Calmette-Guérin disease after vaccination: case report and review; Talbot EA et al.; The attenuated bacille Calmette-Guerin (BCG) vaccine is administered to prevent tuberculosis . Complications of vaccination are uncommon . We report a new case of disseminated BCG disease and review 27 additional cases identified from a review of > 5,000 reports published between 1980 and 1996 . Twenty-four of the 28 total cases were associated with an immune deficiency, including nine cases of AIDS . Seventy-one percent of the cases occurred in children younger than 2 years old . Sixty-eight percent of the patients were male . About one-half of the patients were vaccinated in a developed nation, but 85% of the cases were reported from a developed nation . Response to therapy was poor, with an overall mortality rate of 71% . We made two new observations . Disseminated BCG disease has historically been a disease of infants, but cases now occur in adults and older children coinfected with human immunodeficiency virus . Cases also occur after revaccination of individuals who were anergic following the initial administration of BCG vaccine . Disseminated BCG disease is an uncommon but devastating complication of vaccination that should be considered in the appropriate clinical setting . Immunocompromised infants and patients with late-stage AIDS are at greatest risk and respond poorly to standard therapies.

J Bacteriol, 1997 Jun, 179(12), 3892 - 8
Evidence that the N-terminal part of the S-layer protein from Bacillus stearothermophilus PV72/p2 recognizes a secondary cell wall polymer; Ries W et al.; The S-layer of Bacillus stearothermophilus PV72/p2 shows oblique lattice symmetry and is composed of identical protein subunits with a molecular weight of 97,000 . The isolated S-layer subunits could bind and recrystallize into the oblique lattice on native peptidoglycan-containing sacculi which consist of peptidoglycan of the A1gamma chemotype and a secondary cell wall polymer with an estimated molecular weight of 24,000 . The secondary cell wall polymer could be completely extracted from peptidoglycan-containing sacculi with 48% HF, indicating the presence of phosphodiester linkages between the polymer chains and the peptidoglycan backbone . The cell wall polymer was composed mainly of GlcNAc and ManNAc in a molar ratio of 4:1, constituted about 20% of the peptidoglycan-containing sacculus dry weight, and was also detected in the fraction of the S-layer self-assembly products . Extraction experiments and recrystallization of the whole S-layer protein and proteolytic cleavage fragments confirmed that the secondary cell wall polymer is responsible for anchoring the S-layer subunits by the N-terminal part to the peptidoglycan-containing sacculi . In addition to this binding function, the cell wall polymer was found to influence the in vitro self-assembly of the guanidinium hydrochloride-extracted S-layer protein . Chemical modification studies further showed that the secondary cell wall polymer does not contribute significant free amino or carboxylate groups to the peptidoglycan-containing sacculi.

Am J Med Sci, 1997 Jun, 313(6), 372 - 6
Use of the bacille Calmette-Guérin vaccination for the prevention of tuberculosis: renewed interest in an old vaccine; Cohn DL; The reemergence of tuberculosis, including the impact of HIV infection and multidrug-resistant tuberculosis, have renewed interest in the bacille Calmette-Guerin (BCG) vaccine . During the past 7 decades, numerous studies have shown variable efficacy of BCG vaccination, ranging from 0% to 80% . The BCG vaccine is more likely to prevent disseminated forms of tuberculosis in children than pulmonary tuberculosis in adolescents or adults . Bacille Calmette-Guerin vaccination is recommended in asymptomatic children with or at risk for HIV infection, but it rarely may cause disseminated BCG infection and should not be used in persons with symptomatic HIV infection or AIDS . In healthcare workers with exposure to Mycobacterium tuberculosis, including multidrug-resistant tuberculosis, BCG vaccination generally is not recommended . Revaccination with BCG does not confer more benefit than initial vaccination, and repeat vaccinations should be discontinued . With recent advances in technology and a better understanding of the immunopathogenesis of tuberculosis, efforts to develop a more potent and specific vaccine need to be pursued . If a more effective vaccine against tuberculosis is developed, vaccination can be expected to have an additional impact on global tuberculosis control in conjunction with current strategies of case detection, treatment of disease, and preventive therapy.

Am J Med Sci, 1997 Jun, 313(6), 364 - 71
In vitro cellular and cytokine responses to mycobacterial antigens: application to diagnosis of tuberculosis infection and assessment of response to mycobacterial vaccines; Lein AD et al.; Mycobacterial infection leads to the development of specific cell-mediated immune responses that have been measured clinically by assessing delayed-type hypersensitivity with Mantoux skin testing . Several characteristics of Mantoux skin testing for tuberculosis infection can make the procedure inaccurate, inconvenient, and sometimes misleading . It is also a poor predictor of immunity to tuberculosis in bacille Calmette-Guerin vaccinees, yet decisions to revaccinate often are based on skin test responses after initial immunization . Skin testing with other mycobacterial antigens has similar limitations . In vitro assessment of cellular immunity to mycobacteria offers multiple, potential advantages over skin testing and has become technically feasible in recent years . Measurement of the effector functions that comprise cell-mediated immunity (eg, cytokine secretion and cytotoxicity) rather than cutaneous delayed-type hypersensitivity responses is more likely to reflect meaningfully specific mycobacterial immunity and, therefore, provide a means for determining mycobacterial immunity after immunization . Eliminating the variability in placement and interpretation inherent in skin testing could provide a more stable foundation for comparative studies in populations and improve decision making for individuals . Finally, in vitro testing permits the use of discrete mycobacterial antigens instead of crude protein preparations, allowing greater specificity in the detection of infection as well as assessment of responses to defined candidate vaccine antigens . Several studies have compared skin testing with in vitro proliferation of lymphocytes stimulated by mycobacterial antigens for the detection of Mycobacterium tuberculosis infection . Preliminary veterinary and human studies suggest that in vitro assessment of gamma-interferon production in response to mycobacterial antigens can be used to detect prior infection with organisms of the M tuberculosis complex . Streamlined techniques for in vitro testing of cellular immunity may allow its practical adoption in the clinical setting and lead to its use as a replacement for Mantoux skin testing.

Appl Environ Microbiol, 1997 Jun, 63(6), 2311 - 7
Cloning of the nprA gene for neutral protease A of Bacillus thuringiensis and effect of in vivo deletion of nprA on insecticidal crystal protein; Donovan WP et al.; The nprA gene, encoding Bacillus thuringiensis neutral protease A, was cloned by the use of gene-specific oligonucleotides . The size of neutral protease A deduced from the nprA sequence was 566 amino acids (60,982 Da) . The cloned nprA gene was partially deleted in vitro, and the deleted allele, designated nprA3, was used to construct an nprA3 strain (neutral protease A-deficient strain) of B . thuringiensis . Growth and sporulation of the nprA3 strain were similar to those of an isogenic nprA+ strain, although the extracellular proteolytic activity of the nprA3 strain was significantly less than that of the nprA+ strain . The nprA3 strain produced insecticidal crystal proteins that were more stable than those of the isogenic nprA+ strain after solubilization in vitro, and sporulated cultures of the nprA3 strain contained higher concentrations of full-length insecticidal crystal proteins than did those of its isogenic counterpart . The absence of neutral protease A did not affect the insecticidal activity of a lepidopteran-specific crystal protein of B . thuringiensis . These results indicate that crystal protein stability and yield may be improved by deletion of specific proteases from B . thuringiensis.

J Clin Microbiol, 1997 Jun, 35(6), 1533 - 5
Use of pulsed-field gel electrophoresis to investigate a pseudo-outbreak of Bacillus cereus in a pediatric unit; Liu PY et al.; Bacillus cereus is a well-known cause of food poisoning . It also causes rare systemic infections, usually in immunocompromised patients . Dissemination of this species in hospitals had been reported . Most of these episodes were pseudo-outbreaks and were usually secondary to equipment or environmental contamination . We report here on the use of pulsed-field gel electrophoresis (PFGE) to analyze a pseudo-outbreak of B . cereus in a pediatric unit . Different restriction endonucleases had been tested, and SmaI was found to give the best result for PFGE . Among the 26 clinical isolates of B . cereus and the type strain of the species, 15 distinct PFGE patterns were distinguished . PFGE after DNA macrorestriction with SmaI could clearly differentiate between the epidemiologically related isolates and the unrelated isolates . Because the same epidemic strain of B . cereus was isolated from the settle plates which were exposed near the outlet of the ventilation system, the source of this pseudo-outbreak was suspected to be the unit's air filtration system . This is one of the first reports of the application of PFGE to the study of B . cereus, and this method is useful for epidemiological investigation.

J Urol, 1997 Jun, 157(6), 2090 - 4
The efficacy of intravesical Tice strain bacillus Calmette-Guerin in the treatment of interstitial cystitis: a double-blind, prospective, placebo controlled trial; Peters K et al.; PURPOSE: Interstitial cystitis is a debilitating bladder disease of unknown etiology with no cure . A recent report suggested that bacillus Calmette-Guerin (BCG) may be effective in the treatment of interstitial cystitis . A randomized, prospective, double-blind, placebo controlled trial to evaluate the safety and efficacy of intravesical BCG in treating interstitial cystitis was done . MATERIALS AND METHODS: Patients meeting the National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases criteria for interstitial cystitis received 6 weekly instillations of Tice strain BCG or placebo . Periodic questionnaires, voiding diaries and cystometrograms were obtained . A total of 30 evaluable subjects was enrolled in the study with a mean followup of 8 months (range 6 to 13) . Based on an exit questionnaire a responder was defined as one who rated the interstitial cystitis symptoms as moderately improved or better . RESULTS: A 60% BCG response rate was noted, compared to a 27% placebo response rate . Minimum voided volume and quality of life improved in the BCG group compared to placebo . Adverse events were similar in each group, mostly irritative in nature, and no significant systemic events were noted . CONCLUSIONS: Intravesical Tice strain BCG appears to be safe and efficacious in the treatment of interstitial cystitis . Additional studies must be performed to confirm the results of this pilot study.

Curr Microbiol, 1997 Jun, 34(6), 348 - 53
Distribution of the insertion element IS240 among Bacillus thuringiensis strains; Rosso ML et al.; The presence of IS240 was investigated in 69 Bacillus thuringiensis (Bt) strains including strains from serotype H1 to H45 and additional strains with known Dipteran larvae toxicity . Restriction digests of total DNA and PCR products obtained with a single 16-bases primer corresponding to the IS240 inverted repeated sequence were hybridized with the IS240A element . The results indicate that 67% of the Bt strains tested, including all known mosquitocidal strains, possess at least one IS240-related element . PCR experiments indicate that IS240 represents a family of insertion sequences with several variants.

J Mol Biol, 1997 May 30, 269(1), 142 - 53
The refined crystal structure of Bacillus cereus oligo-1,6-glucosidase at 2.0 A resolution: structural characterization of proline-substitution sites for protein thermostabilization; Watanabe K et al.; The crystal structure of oligo-1,6-glucosidase (dextrin 6-alpha-glucanohydrolase, EC 3.2.1.10) from Bacillus cereus ATCC7064 has been refined to 2.0 A resolution with an R-factor of 19.6% for 43,328 reflections . The final model contains 4646 protein atoms and 221 ordered water molecules with respective root-mean-square deviations of 0.015 A for bond lengths and of 3.166 degrees for bond angles from the ideal values . The structure consists of three domains: the N-terminal domain (residues 1 to 104 and 175 to 480), the subdomain (residues 105 to 174) and the C-terminal domain (residues 481 to 558) . The N-terminal domain takes a (beta/alpha)8-barrel structure with additions of an alpha-helix (N alpha6') between the sixth strand Nbeta6 and the sixth helix N alpha6, an alpha-helix (N alpha7') between the seventh strand Nbeta7 and the seventh helix N alpha7 and three alpha-helices (N alpha8', N alpha8" and N alpha8'" between the eighth strand Nbeta8 and the eighth helix N alpha8 . The subdomain is composed of an alpha-helix, a three-stranded antiparallel beta-sheet, and long intervening loops . The C-terminal domain has a beta-barrel structure of eight antiparallel beta-strands folded in double Greek key motifs, which is distorted in the sixth strand Cbeta6 . Three catalytic residues, Asp199, Glu255 and Asp329, are located at the bottom of a deep cleft formed by the subdomain and a cluster of the two additional alpha-helices N alpha8' and N alpha8" in the (beta/alpha)8-barrel . The refined structure reveals the locations of 21 proline-substitution sites that are expected to be critical to protein thermostabilization from a sequence comparison among three Bacillus oligo-1,6-glucosidases with different thermostability . These sites lie in loops, beta-turns and alpha-helices, in order of frequency, except for Cys515 in the fourth beta-strand Cbeta4 of the C-terminal domain . The residues in beta-turns (Lys121, Glu208, Pro257, Glu290, Pro443, Lys457 and Glu487) are all found at their second positions, and those in alpha-helices (Asn109, Glu175, Thr261 and Ile403) are present at their N1 positions of the first helical turns . Those residues in both secondary structures adopt phi and phi values favorable for proline substitution . Residues preceding the 21 sites are mostly conserved upon proline occurrence at these 21 sites in more thermostable Bacillus oligo-1,6-glucosidases . These structural features with respect to the 21 sites indicate that the sites in beta-turns and alpha-helices have more essential prerequisites for proline substitution to thermostabilize the protein than those in loops . This well supports the previous finding that the replacement at the appropriate positions in beta-turns or alpha-helices is the most effective for protein thermostabilization by proline substitution.

J Mol Biol, 1997 May 30, 269(1), 1 - 9
Species-specific tRNA recognition in relation to tRNA synthetase contact residues; Nair S et al.; In spite of variations in the sequences of tRNAs, the genetic code (anticodon trinucleotides) is conserved in evolution . However, non-anticodon nucleotides which are species specific are known to prevent a given tRNA from functioning in all organisms . Conversely, species-specific tRNA contact residues in synthetases should also prevent cross-species acylation in a predictable way . To address this question, we investigated the relatively small tyrosine tRNA synthetase where contacts of Escherichia coli tRNA(Tyr) with the alpha2 dimeric protein have been localized by others to four specific sequence clusters on the three-dimensional structure of the Bacillus stearothermophilus enzyme . We used specific functional tests with a previously not-sequenced and not-characterized Mycobacterium tuberculosis enzyme and showed that it demonstrates species-specific aminoacylation in vivo and in vitro . The specificity observed fits exactly with the presence of the clusters characteristic of those established as important for recognition of E . coli tRNA . Conversely, we noted that a recent analysis of the tyrosine enzyme from the eukaryote pathogen Pneumocystis carinii showed just the opposite species specificity of tRNA recognition . According to our alignments, the sequences of the clusters diverge substantially from those seen with the M . tuberculosis, B . stearothermophilus and other enzymes . Thus, the presence or absence of species-specific residues in tRNA synthetases correlates in both directions with cross-species aminoacylation phenotypes, without reference to the associated tRNA sequences . We suggest that this kind of analysis can identify those synthetase-tRNA covariations which are needed to preserve the genetic code . These co-variations might be exploited to develop novel antibiotics against pathogens such as M . tuberculosis and P . carinii.

J Biol Chem, 1997 May 30, 272(22), 14420 - 5
Human tyrosyl-tRNA synthetase shares amino acid sequence homology with a putative cytokine; Kleeman TA et al.; To test the hypothesis that tRNATyr recognition differs between bacterial and human tyrosyl-tRNA synthetases, we sequenced several clones identified as human tyrosyl-tRNA synthetase cDNAs by the Human Genome Project . We found that human tyrosyl-tRNA synthetase is composed of three domains: 1) an amino-terminal Rossmann fold domain that is responsible for formation of the activated E.Tyr-AMP intermediate and is conserved among bacteria, archeae, and eukaryotes; 2) a tRNA anticodon recognition domain that has not been conserved between bacteria and eukaryotes; and 3) a carboxyl-terminal domain that is unique to the human tyrosyl-tRNA synthetase and whose primary structure is 49% identical to the putative human cytokine endothelial monocyte-activating protein II, 50% identical to the carboxyl-terminal domain of methionyl-tRNA synthetase from Caenorhabditis elegans, and 43% identical to the carboxyl-terminal domain of Arc1p from Saccharomyces cerevisiae . The first two domains of the human tyrosyl-tRNA synthetase are 52, 36, and 16% identical to tyrosyl-tRNA synthetases from S . cerevisiae, Methanococcus jannaschii, and Bacillus stearothermophilus, respectively . Nine of fifteen amino acids known to be involved in the formation of the tyrosyl-adenylate complex in B . stearothermophilus are conserved across all of the organisms, whereas amino acids involved in the recognition of tRNATyr are not conserved . Kinetic analyses of recombinant human and B . stearothermophilus tyrosyl-tRNA synthetases expressed in Escherichia coli indicate that human tyrosyl-tRNA synthetase aminoacylates human but not B . stearothermophilus tRNATyr, and vice versa, supporting the original hypothesis . It is proposed that like endothelial monocyte-activating protein II and the carboxyl-terminal domain of Arc1p, the carboxyl-terminal domain of human tyrosyl-tRNA synthetase evolved from gene duplication of the carboxyl-terminal domain of methionyl-tRNA synthetase and may direct tRNA to the active site of the enzyme.

Int J Cancer, 1997 May 29, 71(5), 851 - 7
Effects of bacillus Calmette-Guerin and interferon-alpha-2B on human bladder cancer in vitro; Zhang Y et al.; The cytolytic and anti-proliferative effects of bacillus Calmette-Guerin (BCG) and/or interferon-alpha-2b (IFN-alpha-2b) on 5 human bladder carcinoma cell lines, RT4, RT112, MGH, SD and J82, were determined . The cell lines showed different sensitivities to BCG and IFN-alpha-2b . BCG had direct dose-dependent cytolytic and anti-proliferative effects on MGH, J82 and SD (grade 3 cell lines), whereas RT4 and RT112 (grades 1 and 2, respectively) were less sensitive . Surprisingly, higher concentrations of BCG enhanced cell growth of RT4 . IFN-alpha-2b also had cytolytic and anti-proliferative effects on all 5 cell lines . Thus, the RT4 and RT112 cell lines that were not sensitive to BCG were highly sensitive to IFN-alpha-2b . A combination of BCG and IFN-alpha-2b had additive anti-proliferative effects on MGH, J82 and RT112 . Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) production by these 5 cell lines was measured after stimulation with BCG and/or IFN-alpha-2b, by ELISA immunoassays . Production of IL-6 and TNF-alpha was significantly increased in MGH and J82 cell lines by the combination of BCG and IFN alpha-2b . The enhanced cytolytic and anti-proliferative effects of the combination of BCG and IFN-alpha-2b may be related to the induction of cytokines.

Lancet, 1997 May 24, 349(9064), 1513 - 5
Treatment of multidrug-resistant pulmonary tuberculosis with interferon-gamma via aerosol; Condos R et al.; BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) is associated with substantial morbidity, despite drug therapy . Interferon-gamma, a cytokine produced mainly by CD4 T lymphocytes, can activate alveolar macrophages, important effector cells in host immunity against Mycobacterium tuberculosis . We investigated safety and tolerability of aerosolised interferon-gamma in patients with MDR-TB, and assessed its efficacy in terms of sputum-smear grades . METHODS: We did an open-label trial of aerosol interferon-gamma given to five patients with smears and cultures positive for pulmonary MDR-TB, despite documented adherence to therapy . The patients received aerosol interferon-gamma 500 micrograms three times a week for 1 month . Safety and tolerability were assessed, and, as well as routine clinical assessments, sputum samples for smear and culture were collected at entry and weekly . Computed tomography scans of the chest were done at baseline and after therapy ended . FINDINGS: Interferon-gamma was well tolerated by all patients . In all five, bodyweight stabilised or increased . Sputum acid-fast-bacillus smears became negative in all patients, and the time to positive culture increased (from 17 to 24 days, not significant), which suggested that the mycobacterial burden had decreased . The size of cavitary lesions was reduced in all patients, 2 months after treatment had ended . INTERPRETATION: Preliminary data suggest that aerosol interferon-gamma is a well-tolerated treatment that may be useful as adjunctive therapy in patients with MDR-TB who are otherwise not responding well to therapy.

Int J Food Microbiol, 1997 May 20, 36(2-3), 227 - 9
Toxinogenicity of heat-resistant fungi detected by a bio-assay; Pieckova E et al.; The ciliostatic activity of exo- and endometabolites of 125 (100%) heat-resistant fungal isolates was evaluated in vitro by a bioassay on tracheal tissue cultures of 1 day old chicks . For 11 (9%) of the isolates investigated chloroform-extractable secondary metabolites were detected in the medium . For 29 (23%) they were detected in mycelium and spores and for 25 (20%) they were detected in the medium, mycelium and spores at the same time . Especially Dichotomomyces cejpii and Eupenicillium baarnense strains displayed ciliostatic activity . Extracts of Gilmaniella humicola, Talaromyces avellaneus and Talaromyces bacillisporus isolates were not found ciliostatic in our experiment . The chloroform-extractable metabolites of Neosartorya fischeri ATCC 96469 and T . avellaneus ATCC 96465 strains had no ciliostatic activity, either, but G . humicola ATCC 96467 produced ciliostatic metabolites detected in the medium, the mycelia and the spores.

Biochem Biophys Res Commun, 1997 May 19, 234(2), 485 - 8
A thermostable D-hydantoinase isolated from a mesophilic Bacillus sp.AR9; Sharma R et al.; A thermostable hydantoinase has been characterized from a mesophilic Bacillus sp.AR9 . The hydantoinase produced by this Bacillus sp.AR9 is strictly D-specific and is constitutively produced with high yields (4500 U/ml) in this strain . The enzyme is not only alkalo- and thermostable but has a pH and temperature optimum of 9.5 and 65 degrees C, respectively, which is advantageous for the bioconversion of DL-5-monosubstituted-hydantoin derivatives . The enzyme has a half life of 80 minutes at 70 degrees C and loses only 33% of its activity in 4 hr at 60 degrees C . The enzyme has a broad substrate specificity with a maximum of 100% with hydantoin and about 26% with dihydrouracil . Co+ + ions enhance the activity of the enzyme by more than 60%.

J Mol Biol, 1997 May 16, 268(4), 739 - 59
A crystallographic comparison between mutated glyceraldehyde-3-phosphate dehydrogenases from Bacillus stearothermophilus complexed with either NAD+ or NADP+; Didierjean C et al.; Mutations have been introduced in the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus in order to convert its cofactor selectivity from a specificity towards NAD into a preference for NADP . In the B-S mutant, five mutations (L33T, T34G, D35G, L187A, P188S) were selected on the basis of a sequence alignment with NADP-dependent chloroplastic GAPDHs . In the D32G-S mutant, two of the five mutations mentioned above (L187A, P188S) have been used in combination with another one designed from electrostatic considerations (D32G) . Both mutants exhibit a dual-cofactor selectivity at the advantage of either NAD (B-S) or NADP (D32G-S) . In order to analyse the cofactor-binding site plasticity at the molecular level, crystal structures of these mutants have been solved, when complexed with either NAD+ (D32G-Sn, resolution 2.5 A, R = 13.9%; B-Sn, 2.45 A, 19.3%) or NADP+ (D32G-Sp, 2.2 A, 19.2%; B-Sp, 2.5 A, 14.4%) . The four refined models are very similar to that of the wild-type GAPDH and as expected resemble more closely the holo form than the apo form . In the B-S mutant, the wild-type low affinity for NADP+ seems to be essentially retained because of repulsive electrostatic contacts between the extra 2'-phosphate and the unchanged carboxylate group of residue D32 . Such an antideterminant effect is not well compensated by putative attractive interactions which had been expected to arise from the newly-introduced side-chains . In this mutant, recognition of NAD+ is slightly affected with respect to that known on the wild-type, because mutations only weakly destabilize hydrogen bonds and van der Waals contacts originally present in the natural enzyme . Thus, the B-S mutant does not mimic efficiently the chloroplastic GAPDHs, and long-range and/or second-layer effects, not easily predictable from visual inspection of three-dimensional structures, need to be taken into account for designing a true "chloroplastic-like" mutant of cytosolic GAPDH . In the case of the D32G-S mutant, the dissociation constants for NAD+ and NADP+ are practically reversed with respect to those of the wild-type . The strong alteration of the affinity for NAD+ obviously proceeds from the suppression of the two wild-type hydrogen bonds between the adenosine 2'- and 3'-hydroxyl positions and the D32 carboxylate group . As expected, the efficient recognition of NADP+ is partly promoted by the removal of intra-subunit electrostatic repulsion (D32G) and inter-subunit steric hindrance (L187A, P188S) . Another interesting feature of the reshaped NADP+-binding site is provided by the local stabilization of the extra 2'-phosphate which forms a hydrogen bond with the side-chain hydroxyl group of the newly-introduced S188 . When compared to the presently known natural NADP-binding clefts, this result clearly demonstrates that an absolute need for a salt-bridge involving the 2'-phosphate is not required to switch the cofactor selectivity from NAD to NADP . In fact, as it is the case in this mutant, only a moderately polar hydrogen bond can be sufficient to make the extra 2'-phosphate of NADP+ well recognized by a protein environment.

J Biol Chem, 1997 May 16, 272(20), 13006 - 12
Mutational analysis of the major loop of Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases . Effects on protein stability and substrate binding; Pons J et al.; The carbohydrate-binding cleft of Bacillus licheniformis 1,3-1, 4-beta-D-glucan 4-glucanohydrolase is partially covered by the surface loop between residues 51 and 67, which is linked to beta-strand-(87-95) of the minor beta-sheet III of the protein core by a single disulfide bond at Cys61-Cys90 . An alanine scanning mutagenesis approach has been applied to analyze the role of loop residues from Asp51 to Arg64 in substrate binding and stability by means of equilibrium urea denaturation, enzyme thermotolerance, and kinetics . The DeltaDeltaGU between oxidized and reduced forms is approximately constant for all mutants, with a contribution of 5.3 +/- 0.2 kcal.mol-1 for the disulfide bridge to protein stability . A good correlation is observed between DeltaGU values by reversible unfolding and enzyme thermotolerance . The N57A mutant, however, is more thermotolerant than the wild-type enzyme, whereas it is slightly less stable to reversible urea denaturation . Mutants with a <2-fold increase in Km correspond to mutations at residues not involved in substrate binding, for which the reduction in catalytic efficiency (kcat/Km) is proportional to the loss of stability relative to the wild-type enzyme . Y53A, N55A, F59A, and W63A, on the other hand, show a pronounced effect on catalytic efficiency, with Km > 2-fold and kcat < 5% of the wild-type values . These mutated residues are directly involved in substrate binding or in hydrophobic packing of the loop . Interestingly, the mutation M58A yields an enzyme that is more active than the wild-type enzyme (7-fold increase in kcat), but it is slightly less stable.

Eur J Biochem, 1997 May 15, 246(1), 193 - 9
The lantibiotic mersacidin inhibits peptidoglycan biosynthesis at the level of transglycosylation; Brotz H et al.; The lantibiotic mersacidin has been previously reported to interfere with bacterial peptidoglycan biosynthesis, {Brotz, H., Bierbaum, G., Markus, A., Molitor, E . & Sahl, H.-G . (1995) Antimicrob . Agents Chemother . 39, 714-719} . Here, we focus on the target reaction and describe a mersacidin-induced accumulation of UDP-N-acetylmuramoyl-pentapeptide, indicating that inhibition of peptidoglycan synthesis occurs after the formation of cytoplasmic precursors . In vitro studies involving a wall-membrane particulate fraction of Bacillus megaterium KM demonstrated that mersacidin did not prevent the synthesis of lipid II {undecaprenyl-diphosphoryl-N-acetylmuramoyl-(pentapeptide)-N-ac ety lglucosamine} but specifically the subsequent conversion of this intermediate into polymeric nascent glycan strands by transglycosylation . Comparison with other inhibitors of transglycosylation shows that the effective concentration of mersacidin in vitro is in the range of that of the glycopeptide antibiotic vancomycin but 2-3 orders of magnitude higher than that of the competitive enzyme inhibitor moenomycin . The analogy to the glycopeptides may hint at an interaction of mersacidin with the peptidoglycan precursor rather than with the enzyme . Unlike vancomycin however, mersacidin inhibits peptidoglycan formation from UDP-N-acetylmuramoyl-tripeptide and is active against Enterococcus faecium expressing the vanA resistance gene cluster . This indicates that the molecular target site of mersacidin differs from that of vancomycin and that no cross-resistance exists between the two antibiotics.

Cancer, 1997 May 15, 79(10), 1987 - 94
Allium sativum (garlic) treatment for murine transitional cell carcinoma; Riggs DR et al.; BACKGROUND: Currently, immunotherapy with Bacillus Calmette-Guerin (BCG) is the most effective treatment for superficial bladder carcinoma, but treatment-related toxicity may limit its use in some patients . Alternative treatments are needed for patients who fail to respond to BCG immunotherapy . Allium sativum (AS), or garlic, is known to have a broad range of biologic activities, including immune stimulation and reported antitumor activity . For these reasons, the authors conducted a series of experiments designed to explore the possible therapeutic effects of AS in the MBT2 murine bladder carcinoma model . METHODS: C3H/HeN mice were randomized prior to initiation of each experimental protocol . Mice received 1 x 10(3) MBT2 cells in 0.1 mL RPMI-1640, administered subcutaneously into the right thigh, on Day 0 of the experiment . AS was injected at the site of tumor transplantation on Day 1 and at 2- to 7-day intervals up to Day 28 . To evaluate the effects of oral AS in this model, treatment was initiated 30 days prior to tumor inoculation and continued for 30 days after tumor inoculation . Animals in all experiments were followed for tumor incidence, tumor growth, and survival . RESULTS: In the initial experiments, subcutaneous AS significantly reduced tumor volume compared with the saline control (P < 0.05) . Unfortunately, treatment-related death was also observed, requiring reduction in the total dose of AS . Animals that received 5 weekly immunizations of AS (5 mg, 5 mg, 1 mg, 1 mg, and 1 mg; cumulative dose = 13 mg) had significantly reduced tumor incidence, tumor growth, and increased survival when compared with animals that received the saline control . No treatment-related deaths were observed with this treatment schedule . To determine whether systemic AS administration might be effective, orally administered AS was tested at doses of 5 mg, 50 mg, and 500 mg per 100 mL of drinking water . Mice that received 50 mg oral AS had significant reductions in tumor volume (P < 0.05) when compared with animals that received the saline control, and mice that received 500 mg oral AS had significant reductions in both tumor volume and mortality (P < 0.05) . CONCLUSIONS: The significant antitumor efficacy of subcutaneous and oral AS warrants further investigation and suggests that AS may provide a new and effective form of therapy for transitional cell carcinoma of the bladder.

Curr Opin Ophthalmol, 1997 Jun, 8(3), 32 - 8
Endophthalmitis after penetrating ocular trauma; Reynolds DS et al.; Endophthalmitis following penetrating eye injuries has a relatively poor prognosis due to the underlying eye trauma and the frequency of more virulent organisms such as Bacillus species . Risk factors for infection include 1) retained intraocular foreign body, 2) a rural injury setting, 3) delay in primary wound closure, and 4) disruption of the crystalline lens . Although endophthalmitis is difficult to distinguish from traumatic changes, recognition of early clinical signs of endophthalmitis, such as hypopyon, vitritis, or retinal periphlebitis, is important and early treatment is recommended . Comprehensive prophylactic antibiotic treatment at the time of injury repair combined with timely diagnostic vitrectomy and injection of intravitreal antibiotics when infection is suspected may significantly improve visual acuity outcomes following penetrating injuries . Treatment includes intravitreal, periocular, and systemic antibiotics . Intravitreal and periocular corticosteroids are also recommended . Recent and past literature supporting these recommendations, as well as the authors' specific prevention and treatment protocols for post-traumatic endophthalmitis, is included in this review.

J Mol Biol, 1997 May 2, 268(2), 482 - 93
The stability and dynamics of ribosomal protein L9: investigations of a molecular strut by amide proton exchange and circular dichroism; Lillemoen J et al.; Nuclear magnetic resonance and circular dichroism experiments were used to investigate the stability and dynamic aspects of ribosomal protein L9 from Bacillus stearothermophilus in solution . This unusually shaped protein, with its two widely spaced RNA-binding domains linked by a connecting helix, has been hypothesized to serve as a "molecular strut", most likely playing a role in ribosome assembly and/or maintaining the catalytically active conformation of ribosomal RNA . Protection factors for amide proton exchange were quantitatively measured in an extensive series of NMR experiments, providing probes of the stability and dynamics of localized regions of the protein . Results show that each of the two RNA-binding domains contains a highly stable core . The exposed central helix that connects the two domains is helical in solution, albeit not rigid, a result that is supported by amide proton protection factors, circular dichroism measurements, and carbon-13 and proton chemical shift index values . A conserved glycine and lysine-rich loop in the N-terminal domain is ordered and quite stable, a surprising result, since this loop had been presumed to be disordered in the original crystallographic analysis . Interestingly, the most dynamic parts of the protein are the regions that contain the likely RNA-binding residues in each of the two domains . The present results add further support to the notion that the L9 protein plays an architectural role within the ribosome, with the central helix serving as a molecular strut, or perhaps a spring, linking the two widely spaced RNA-binding domains.

Antonie Van Leeuwenhoek, 1997 May, 71(4), 307 - 11
Digestion of staphylococcal enterotoxin by Bacillus natto; Osawa R et al.; Cooked rice contaminated with staphylococcal enterotoxin A (SEA) was mixed with 'natto', a Bacillus natto fermented soybean food, and the mixture was incubated at 37 degrees C for 1 h . Reversed passive latex agglutination (RPLA) tests performed on the mixture revealed that the RPLA titer against SEA was significantly reduced after incubation . Subsequent analytical tests showed that the SEA protein molecule was fragmented to small peptides by an extracellular protease, subtilisin, produced by B . natto . The proteolytic activity of B . natto was also found to be effective against other types of staphylococcal enterotoxins.

FEMS Immunol Med Microbiol, 1997 May, 18(1), 47 - 53
Characterization of Bacillus thuringiensis isolated from infections in burn wounds; Damgaard PH et al.; Four strains of Bacillus thuringiensis were isolated from infections in burn wounds and from water used in the treatment of burn wounds . The strains produced large parasporal inclusion bodies composed of 141, 83, and 81 kDa protoxins . The four strains were tested for insecticidal activity against larvae of Pieris brassicae and Aedes aegypti but showed no activity; Vero cell assays for the production of enterotoxins were also negative . Attempts to classify the strains according to flagellar H-serotype showed them all to be non-flagellated . Apart from two occupational health accidents that occurred during the handling of highly concentrated B . thuringiensis fluids, this is the first report of B . thuringiensis causing non-gastrointestinal clinical infection in immunosuppressed patients.

Protein Eng, 1997 May, 10(5), 541 - 9
Hyperthermostable mutants of Bacillus licheniformis alpha-amylase: thermodynamic studies and structural interpretation; Declerck N et al.; This paper provides further understanding of the thermodynamic and structural features determining the stability of Bacillus licheniformis alpha-amylase (BLA) at two crucial positions, His133 and Ala209 . Results of protein modelling and saturated site-directed mutagenesis at position 133 and 209 have been reported in a previous paper (Declerck et al., 1995, Prot . Engng, 8, 1029-1037) . In the first part of the present work, evidence is presented supporting the hypothesis that the stabilizing mutations reduce the rate of initial unfolding of the enzyme during the reversible step of the inactivation reaction and do not modify the irreversible processes undergone subsequently by the unfolded molecules . In the second part, we have examined the three-dimensional structure of BLA which has been determined recently by X-ray analysis (Machius et al., 1995, J . Mol . Biol., 246, 545-559) . This analysis showed that our previous predictions made from molecular modelling were partly correct . At position 209, the effect of the stabilizing substitutions can be explained by a groove-filling effect reinforcing the hydrophobic packing between two helices of the central domain, while preserving a well-ordered water structure at the surface . At position 133, the stabilizing substitutions must compensate the loss of the hydrogen bond network in which the original histidine side-chain is involved; this compensation could be achieved through enhanced hydrophobic side-chain interactions within the beta-sheet where residue 133 is located, which correlates with the propensity of the residue to form and maintain a beta-strand conformation of the main chain at this position.

Appl Microbiol Biotechnol, 1997 May, 47(5), 543 - 6
Plasmids for efficient single-copy gene cloning into gdh2 and trpC of Bacillus megaterium DSM319 and QM B1551; Schmiedel D et al.; We constructed integrative plasmids to place xylA-lacZ indicator gene fusions into two different loci of the Bacillus megaterium chromosome, gdh2 and trpC, in lac mutants of strains DSM 319 and QM B1551, which differ markedly . Single-crossover integration was achieved in all cases while double crossovers occurred only in gdh2 of DSM 319 and QM B1551 and in trpC of QM B1551 . Neither of the loci affected regulation of the xylA-lacZ fusions . These results confirm the suitability of the two gene loci for single-copy cloning.

Appl Microbiol Biotechnol, 1997 May, 47(5), 530 - 6
Controlled secretion into the culture medium of a hybrid beta-glucanase by Acetobacter methanolicus mediated by the kil gene of Escherichia coli located on a Tn5-derived transposon; Miksch G et al.; A Tn5-based transposon bearing the kil gene (killing protein), mediating controlled export of periplasmic proteins into the culture medium, was constructed (Tn5-KIL3) . This transposon contained the kil gene of the ColEl plasmid under the growth-phase-dependent promoter of the fic gene (filamentation induced by cAMP) of Escherichia coli, an interposon located upstream of kil, a kanamycin/neomycin-resistance gene, a multiple cloning site and the mob site . The transposition of Tn5-KIL3 to Acetobacter methanolicus showed a moderate transposition frequency (10(-5) -10(-6) . By insertion of a Bacillus hybrid beta-glucanase (bgl) as a model protein into the transposon (Tn5-LF3) it was shown that the secretion function as well as the gene of the target protein had been transferred to and stably integrated into the chromosome of A . methanolicus, and that the transposition of Tn5-LF3 was non-specific . beta-Glucanase was highly overexpressed and secreted into the medium during stationary phase . Total and extra-cellular production of beta-glucanase varied depending on the integration site of the transposon . The viability of the bacterial cells was not affected, and cell lysis did not occur.

J Antibiot (Tokyo), 1997 May, 50(5), 424 - 8
Kinetic isotope effect and reaction mechanism of 2-deoxy-scyllo-inosose synthase derived from butirosin-producing Bacillus circulans; Kudo F et al.; The mechanism of 2-deoxy-scyllo-inosose synthase reaction, a carbocycle formation step from D-glucose-6-phosphate in the biosynthesis of the 2-deoxystreptamine aglycon of clinically important aminocyclitol antibiotics, was investigated with a partially purified enzyme from butirosin-producing Bacillus circulans SANK 72073, Nonlabeled and double-labeled D-{4-2H, 3-15O}glucose-6-phosphate were used for cross-over experiment, and the oxime-TMS ether derivative of the 2-deoxy-scyllo-inosose product was analyzed by GC-MS . The deuterium label at C-4 of the substrate appeared to be retained at C-6 of the inosose product without scrambling of the double-labeled isotopes . Since the transient reduction of NAD+ cofactor was proved to be essential in the 2-deoxy-scyllo-inosose reaction, the hydride abstraction and returning appeared to take place within the same glucose molecule . The observed kinetic isotope effect was estimated to be kH/kD = 2.4 . These results strongly suggest that this carbocycle formation is catalyzed by a single 2-deoxy-scyllo-inosose synthase enzyme with catalytic requirement of NAD+, the mechanism of which appears to be resembled closely to the 2-deoxy-scyllo-inosose synthase in the Streptomyces fradiae.

Br J Dermatol, 1997 May, 136(5), 747 - 51
Molecular diagnosis of deep nodular bacillary angiomatosis and monitoring of therapeutic success; Schlupen EM et al.; A 51-year-old human immunodeficiency virus (HIV)-positive male patient (CDC stage 3C) had had a painful nodule on his external ankle joint for 10 months . A biopsy suggested bacillary angiomatosis, but Kaposi's sarcoma could not be excluded . Rods were detectable in lesional skin by a Warthin-Starry stain . A 298 base pair (bp) gene fragment specific for Bartonella species was amplified from lesional skin and direct nucleotide sequence analysis of the amplification product clearly identified Bartonella quintana . Kaposi's sarcoma-associated herpes virus specific DNA was not amplifiable by polymerase chain reaction (PCR) in our patient, suggesting that the lesion represented bacillary angiomatosis alone, despite clinical and histopathological features which suggested the coexistence of bacillary angiomatosis and Kaposi's sarcoma . The lesion regressed after erythromycin was prescribed . However, 4 and 9 weeks after initiation of therapy, PCR still yielded a positive result in material obtained by a swab . After complete healing, following 12 weeks of antibiotic therapy, PCR became consistently negative . The optimal length of antibiotic treatment in HIV-positive patients with bacillary angiomatosis is not yet known and inadequate therapy may be followed by disseminated disease and a fatal outcome . PCR-based monitoring of the success of treatment is valuable for determining the duration of treatment resulting in a cure.

PDA J Pharm Sci Technol, 1997 May-Jun, 51(3), 111 - 5
Investigation of pulsed light for terminal sterilization of WFI filled blow/fill/seal polyethylene containers; Dunn J et al.; A study was performed to assess the ability of pulsed light to sterilize water for injection in blow/fill/seal polyethylene containers . Pulsed light uses intense, short duration flashes of broad spectrum white light to produce high levels of microbial kill . In a first phase of testing, containers of 0.5, 5, 15, and 120 mL nominal volume were inoculated with Bacillus pumilus endospores, Bacillus subtilus variety niger strain globigii endospores, Bacillus stearothermophilus endospores, and Aspergillus niger conidiospores . Approximately 10(6) colony forming units of each test spore were individually inoculated into 22 replicate containers of each sample volume . Two of these containers served as inoculation recovery controls, and 10 were treated using each of two pulsed light exposure methods: single-sided treatment, or treatment within a reflective cavity . Both treatments employed flashes of intense broad spectrum pulsed light delivered at one flash per second . Cavity treatment used 10 flashes to treat each container within a reflective cavity containing a single lamp . Cavity treatment yielded no recoverable survivors for any of the challenge spores from the contents of any of the 160 total samples . Single-sided treatment used 20 approximately 1-J/cm2 flashes from a single lamp-reflector projecting onto one side of the container . Single-sided treatment yielded no recoverable survivors from the contents of the containers for any of the bacterial endospores tested, but Aspergillus niger survival was detected in 4 of the 40 single-side treated samples . A second phase of tests examined the pulsed light inactivation of Bacillus pumilus spores for a range of inoculation levels . High levels of Bacillus pumilus spore kill were obtained using only a few cavity flashes . The results show that pulsed light can provide high levels of microbial lethality and possesses potential for use as a terminal sterilization method for water for injection in filled, sealed polyethylene containers.

Antonie Van Leeuwenhoek, 1997 May, 71(4), 319 - 23
Laboratory screening of different Bacillus thuringiensis strains against certain lepidopteran pests and subsequent field evaluation on the pod boring pest complex of pigeonpea (Cajanus cajan); Puntambekar US et al.; Laboratory evaluation of different Bacillus thuringiensis subspecies revealed that B . thuringiensis subsp . kurstaki (NCIM 2514) at 10(8) spores/ml concentration caused more than 85% mortality to the neonate larvae of the lepidopteran insects Spodoptera litura (F.) and Phthorimaea operculella (Z.) . This strain at 10(10) spores/ml concentration was effective against the major lepidopteran pests comprising the pod boring complex of pigeonpea (Cajanus cajan), viz . Helicoverpa armigera (H.) and Exelastia atomosa (W.) under the field trials . Total grain yield from this treatment was least 1.5 times more than the untreated control.

Medicine (Baltimore), 1997 May, 76(3), 170 - 84
Whipple disease . Clinical review of 52 cases . The SNFMI Research Group on Whipple Disease . Société Nationale Française de Médecine Interne; Durand DV et al.; Whipple disease is a rare, multiorgan disease with prominent intestinal manifestations . We report a retrospective clinical study of 52 patients recruited in various parts of France from 1967 to 1994 . Seventy-three percent of the patients were male . Clinical manifestations preceding the diagnosis were articular for 35 patients (67%), digestive for 8 patients (15%), general for 7 patients (14%), and neurologic for 2 patients (4%) . At a later stage of the disease, 44 patients (85%) presented diarrhea, weight loss, and malabsorption, while 8 patients (15%) did not show any gastrointestinal symptom throughout the development of the disease . Forty-three patients (83%) presented arthralgia or arthritis, and 11 (21%) had prominent neurologic symptoms . In addition, cardiovascular symptoms were present in 9 patients (17%); mucocutaneous symptoms, in 9 patients (17%); pleuropulmonary symptoms, in 7 patients (13%); and ophthalmologic symptoms, in 5 patients (10%) . All patients but 1 were given a positive diagnosis on histopathologic criteria: jejunal biopsy for 46 patients (90%), lymph node biopsy for 3 patients (6%), brain biopsy for 1 patient (2%), postmortem jejunal and cerebral biopsy for 1 patient (2%) . With treatment, the disease evolved favorably in 47 patients (90%), while 5 patients (10%) had unfavorable outcomes (2 deaths from neurologic involvement, 1 patient with chronic dementia, and 2 patients with digestive symptoms insensitive to antimicrobial therapy) . Of the 41 patients initially treated successfully and whose treatment has been completed, clinical evolution after discontinuation of treatment was favorable in 34 cases (83%) . Clinical relapses occurred in 7 patients . No relapse was observed after treatment by trimethoprim-sulfamethoxazole, alone or following a combination of penicillin and streptomycin, or after the combination of penicillin and streptomycin, whatever the oral follow-up treatment prescribed . The evolution of patients showing a relapse was favorable in all cases after reintroduction of antibiotic therapy . These results are discussed in the light of previously published series and case reports of Whipple disease . The diagnosis of the disease remains difficult at an early phase or when digestive symptoms are absent . It is noteworthy that proximal enteroscopy is sometimes misleading, considered normal on macroscopic examination and nonspecific on pathologic grounds . A normal erythrocyte sedimentation rate represents another pitfall . Histopathology is the key for positive and differential diagnosis, and may require multiple and repeated biopsies . Findings from molecular biology confirm the central role of an uncultured Gram-positive bacillus which was named in 1992 Tropheryma whippelii . A recent report suggests that polymerase chain reaction (PCR) analysis of peripheral blood might allow the diagnosis of Whipple disease in some cases . However, immunologic or cellular parameters such as macrophagic function may play an important, although not clearly elucidated, role in the pathogeny of the disease . Trimethoprim-sulfamethoxazole should be considered the antimicrobial agent of choice in the treatment of Whipple disease, minimizing the risk of cerebral involvement and relapses.

Eur J Biochem, 1997 May 1, 245(3), 797 - 804
Analysis of the large aqueous pores produced by a Bacillus thuringiensis protein insecticide in Manduca sexta midgut-brush-border-membrane vesicles; Carroll J et al.; An osmotic swelling assay utilising carboxyfluorescein self-quenching to measure intravesicular volume changes was adapted to investigate permeability changes induced by the Bacillus thuringiensis Cry1Ac delta-endotoxin in Manduca sexta midgut-brush-border-membrane vesicles (BBMV) . This assay provides a more quantitative analysis of Cry-toxin-induced BBMV permeability changes, extending our previously published protocol which employed a light-scattering signal to monitor delta-endotoxin activity {Carroll, J . & Ellar, D . J . (1993) Eur . J . Biochem . 214, 771-778} . The fluorescence signal changes, supported by electron microscopy of the BBMV, demonstrated that Cry1Ac altered the membrane permeability for large non-electrolyte solutes . With this approach Cry1Ac was observed to induce or form pores freely permeant for raffinose (1.14 nm diameter) and using non-electrolytes of increasing size the pores were estimated to have a limiting diameter of approximately 2.4-2.6 nm under alkaline pH conditions.

Mol Microbiol, 1997 May, 24(3), 545 - 53
Isolation and characterization of the mycobacterial phagosome: segregation from the endosomal/lysosomal pathway; Hasan Z et al.; Mycobacteria have the ability to persist within host phagocytes, and their success as intracellular pathogens is thought to be related to the ability to modify their intracellular environment . After entry into phagocytes, mycobacteria-containing phagosomes acquire markers for the endosomal pathway, but do not fuse with lysosomes . The molecular machinery that is involved in the entry and survival of mycobacteria in host cells is poorly characterized . Here we describe the use of organelle electrophoresis to study the uptake of Mycobacterium bovis bacille Calmette Guerin (BCG) into murine macrophages . We demonstrate that live, but not dead, mycobacteria occupy a phagosome that can be physically separated from endosomal/lysosomal compartments . Biochemical analysis of purified mycobacterial phagosomes revealed the absence of endosomal/lysosomal markers LAMP-1 and beta-hexosaminidase . Combining subcellular fractionation with two-dimensional gel electrophoresis, we found that a set of host proteins was present in phagosomes that were absent from endosomal/lysosomal compartments . The residence of mycobacteria in compartments outside the endosomal/lysosomal system may explain their persistence inside host cells and their sequestration from immune recognition . Furthermore, the approach described here may contribute to an improved understanding of the molecular mechanisms that determine the intracellular fate of mycobacteria during infection.

Biosci Biotechnol Biochem, 1997 May, 61(5), 776 - 81
Growth inhibition, morphological change, and ectoenzyme release of LLC-PK1 cells by phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis; Itami C et al.; Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis added to a culture of LLC-PK1 cells inhibited cell growth by 40% . In contrast with normal cells, the cells cultured in the presence of PI-PLC showed needle-like appendages which seemed to have been formed due to portions of the cell remaining adhered to the culture dish as the cell shrank . When LLC-PK1 cells were treated with PI-PLC, significant amounts of alkaline phosphatase and alkaline phosphodiesterase I were released specifically from the apical surface of the LLC-PK1 cells . Furthermore, PI-PLC treatment caused a delay of enzyme production and dome formation . These data indicate that glycosyl-phosphatidylinositol (GPI)-anchored proteins on the surface of LLC-PK1 cells are important in cell growth and differentiation . Also, the combined use of LLC-PK1 cells and PI-PLC of B . thuringiensis is effective for investigating the function of GPI-anchor proteins.

Am J Physiol, 1997 May, 272(5 Pt 1), L989 - 95
SP-A enhances uptake of bacillus Calmette-Guérin by macrophages through a specific SP-A receptor; Weikert LF et al.; Surfactant-associated protein A (SP-A) is a C-type lectin that is involved in surfactant metabolism as well as host defense functions in the lung . We have recently identified a receptor on macrophages {specific 210-kDa SP-A receptor (SPR210)} that binds SP-A . In the current study we have investigated the role of SP-A in mediating uptake of bacillus Calmette-Guerin (BCG) by rat macrophages and human monocytes and have examined the role of the macrophage SPR210 in this process . 125I-labeled SP-A bound BCG in a Ca(2+)-, carbohydrate-, and dose-dependent manner . To examine association of SP-A-BCG complexes with macrophages, BCG were opsonized with SP-A and were incubated with rat bone marrow-derived macrophages (RBMM), rat alveolar macrophages (RAM), or human monocytes at a 1-to-1 ratio for 4 h . The cells were washed, fixed in formalin, and stained with auramine-rhodamine . Cell-associated organisms were enumerated by fluorescent microscopy . The percentage of cells with one or more associated BCG was increased by SP-A from 27% of RBMM with BCG alone to 54% with SP-A-BCG complexes; 1-16% in RAM; and 39-67% in human monocytes . This enhanced uptake was dependent on the dose of SP-A, with maximal increases seen with 10 micrograms/ml . Electron microscopic analysis supported the conclusion that organisms were ingested by and not simply bound to the macrophages . Inclusion of SPR210 antibodies blocked association of SP-A-BCG complexes, suggesting a role for SPR210 in mediating the interaction of SP-A-BCG with the macrophages . This was further supported by the finding that modulation of SPR210 activity resulted in altered SP-A-BCG uptake . These results demonstrate that SP-A binds to BCG and that uptake of these SP-A-BCG complexes is mediated in part by the SPR210 on rat macrophages and human monocytes.

Lett Appl Microbiol, 1997 May, 24(5), 365 - 8
Lactose-induced expression of Bacillus sp . TS-23 amylase gene in Escherichia coli regulated by a T7 promoter; Lin LL et al.; Bacillus sp . TS-23 amylase gene was amplified by polymerase chain reaction and cloned into the pET23(+) transcription vector . Lactose induction of Escherichia coli BL21(DE3) cells harbouring this recombinant plasmid resulted in the extracellular production of gene products . By the addition of 20 mmol l-1 lactose, the amylase activity of fermentation broth had a specific activity of 21 U mg-1 of protein.

Lett Appl Microbiol, 1997 May, 24(5), 340 - 2
A closed system for the filtration of Mycobacterium bovis liquid cultures using disposable capsule filters; Sugden EA et al.; A filtration system was designed to sterilize large volumes of Mycobacterium bovis BCG Tokyo culture safely, needed to purify protein antigens for immunodiagnosis of bovine tuberculosis . A closed system consists of culture bottles connected to three disposable filter capsules of decreasing pore size in series: a depth prefilter over a 1.2 microns filter; 0.8 micron prefilter over a 0.45 micron filter; and a 0.2 micron sterile filter . Low air pressure (3 psi) forces liquid from below the bacillary pellicle . The system features a stainless steel clamp to hold rubber stoppers on the culture bottles, pleated filters to exclude bacillary clumps, a quick disconnector to minimize aerosols, and a closed system with plastic disposable filters that can be autoclaved as a unit without dismantling.

J Invertebr Pathol, 1997 May, 69(3), 197 - 204
Electrophysiological effects of Bacillus sphaericus binary toxin on cultured mosquito cells; Cokmus C et al.; The electrophysiological effects of both trypsinactivated native and 42- and 51-kDa cloned binary toxins of Bacillus sphaericus were investigated on cultured Culex quinquefasciatus cells using the patchclamp technique . Rates of reduction in whole-cell membrane resistance were correlated with increasing native toxin concentration . The 42- or 51-kDa cloned toxin alone at 50 micrograms/ml reduced the resistance . Electrophysiological effects occurred before any changes were visible by phase-contrast microscopy.

J Dairy Res, 1997 May, 64(2), 261 - 70
Temperature characterization of psychrotrophic and mesophilic Bacillus species from milk; Garcia-Armesto MR et al.; A total of 50 isolates of Bacillus spp . and one reference strain were investigated for their growth at 6.5 degrees C for 10 d, 30 degrees C for 3 d and 40 degrees C for 2 d . The results obtained differentiated three physiological groups: one clearly psychrotrophic (able to grow at 6.5 degrees C in 10 d, but not at 40 degrees C in 2 d), one intermediate in psychrotrophy (it grew at both 40 and 6.5 degrees C) and one mesophilic (capable of growth at 30 and 40 degrees C, but not at 6.5 degrees C) . The proportion of strains in the second group was higher among isolates of B . cereus than for other Bacillus spp . However, the proportion of real mesophilic strains was lower for B . cereus isolates . Psychrotrophic B . cereus grew better at both 6.5 and 30 degrees C than other psychrotrophic Bacillus spp . Using eight strains, a correlation between differential growth at mesophilic temperatures (count at 30 degrees C minus count at 40 degrees C) and a standard psychrotrophic count at 6.5 degrees C for 10 d (r = 0.95) was obtained in mixed cultures when the psychrotrophic flora count was < or = 1 log (cfu/ml) lower than the mesophilic count.

J Med Entomol, 1997 May, 34(3), 321 - 7
Resistance to Bacillus sphaericus involves different mechanisms in Culex pipiens (Diptera:Culicidae) larvae; Nielsen-Leroux C et al.; Field Culex pipiens pipiens (L.) mosquitoes that were collected after a control failure with Spherimos in southern France developed high resistance (> 10,000-fold) to Bacillus sphaericus crystal toxin after < 8 generations of laboratory selection . We show that this resistance is encoded by a single major recessive gene on linkage group I at 22.1 recombination units from the sex locus, and that it is not associated with any loss of binding affinity between brush border membrane fractions and the B . sphaericus radiolabeled toxin . Thus, in Southern France, resistance differs from the high B . sphaericus resistance developed after laboratory selection of Californian C . p . quinquefasciatus . This demonstrates that at least 2 different mechanisms may confer high levels of resistance to B . sphaericus crystal toxin in mosquitoes of the C . pipiens complex . These results have important implications for mosquito control strategies.

Urology, 1997 May, 49(5A Suppl), 41 - 7
Mast cells and nerve fibers in interstitial cystitis (IC): an algorithm for histologic diagnosis via quantitative image analysis and morphometry (QIAM); Hofmeister MA et al.; OBJECTIVE: To develop and evaluate a diagnostic algorithm based on the alteration of mast cell and nerve fiber observed in bladder tissue of patients with interstitial cystitis (IC) . MATERIALS AND METHODS: Non-IC samples from 6 control groups (N = 10, 10, 13, 2, 11, and 3, respectively) and nonclassic interstitial cystitis (NC-IC, N = 20) were stained with Giemsa stain in order to calculate the detrusor to mucosa mast cell ratio (DMMCR) using quantitative image analysis and morphometry (QIAM) . Immunohistochemical staining for S-100 protein was also performed to quantify nerve fiber proliferation in the detrusor muscle of the bladder . RESULTS: The average DMMCR of NC-IC was 1.19 . Bacille Calmette-Guerin (BCG) cystitis was 0.84 and microscopically normal bladder tissue from patients with bladder or prostate cancer was 0.45 . No case of IC that we examined had a DMMCR < 0.5 . The number and percentage area of nerve fibers in the detrusor in IC were increased compared to controls and BCG (IC, 2.01%; BCG, 0.95%; control, 1.3%) . CONCLUSION: A diagnostic algorithm is proposed for IC based on the findings that indicate that: 1) if the DMMCR > 0.75, then IC is present; 2) if the DMMCR < 0.5, then IC is negative; and 3) if the DMMCR is between 0.5 and 0.75, a quantitative S-100 protein staining analysis can be employed to evaluate nerve fiber proliferation to detect those marginal cases of NC-IC . The findings of the study also suggest that a neuroimmune process or mediation may be involved in the pathogenesis of IC.

Urology, 1997 May, 49(5), 687 - 90; discussion 690-1
Additional bacillus Calmette-Guérin therapy for recurrent transitional cell carcinoma after an initial complete response; Bui TT et al.; OBJECTIVES: To determine the success of additional bacillus Calmette-Guerin (BCG) therapy for transitional cell carcinoma recurring after a complete response (CR) to the initial treatment course of BCG . METHODS: All patients treated with BCG with a minimum follow-up of 5 years were reviewed to identify complete responders who subsequently recurred and received additional BCG therapy . The duration of initial response and the incidence and duration of a second CR were recorded . RESULTS: Of 11 patients with an initial CR to a 6-week course of BCG, 9 (82%) achieved a second CR and 5 of the 9 (42%) maintained tumor-free status beyond 5 years of follow-up (median 87 months, range 64 to 110) . Patients who again recurred after the second CR did not benefit from further BCG therapy . CONCLUSIONS: A repeat course of BCG is a reasonable option of therapy for transitional cell carcinoma that has recurred after a CR to a prior course of BCG . Careful monitoring by cytology, cystoscopy, and biopsy is mandatory to direct nonresponders to prompt alternative therapy and to ensure continued disease-free status among responders.

Neuroscience, 1997 May, 78(2), 549 - 60
Ultrastructural studies of an immune-mediated inflammatory response in the CNS parenchyma directed against a non-CNS antigen; Matyszak MK et al.; We have shown previously that heat-killed bacillus Calmette-Guerin injected into the brain parenchyma becomes sequestered behind the blood brain barrier for months undetected by the immune system . However, independent peripheral sensitization of the immune system to bacillus Calmette-Guerin results in recognition of bacillus Calmette-Guerin in the brain and the induction of focal chronic lesions {Matyszak M . K . and Perry V . H . (1995) Neuroscience 64, 967 977} . We carried out ultrastructural studies of these lesions . Prior to subcutaneous challenge we used immunohistochemistry to detect bacillus Calmette-Guerin which was found in cells with the morphology of macrophages/microglia and in perivascular macrophages . Eight to 14 days after subcutaneous challenge there was a conspicuous leucocyte infiltration at the site of bacillus Calmette-Guerin deposits within the brain parenchyma . The majority of these cells were macrophages and lymphocytes, with some lymphocytes showing characteristic blast morphology . Dendritic cells in close contact with lymphocytes were prominent . Inflammatory cells were found in perivascular cuffs and within the brain parenchyma . The tissue was oedematous and some axons were undergoing Wallerian degeneration with associated myelin degeneration . Throughout the lesions, but more commonly at the edges, we detected macrophages containing myelin in their cytoplasm close to intact axons and axons with evidence of remyelinating sheaths, suggestive of primary demyelination . In older lesions, two to three months after the peripheral challenge, the oedema was less pronounced and there was little evidence of Wallerian degeneration . There were still many macrophages . lymphocytes and dendritic cells, although the number of these cells was lower than in earlier lesions . Late lesions also contained many plasma cells which were not present in early lesions . In these late lesions there were bundles of axons with no myelin or a few axons with thin myelin sheaths, suggestive of persistent or ongoing demyelination or remyelination . These observations show that, during a delayed-type hypersensitivity lesion in the CNS, the leucocyte populations change with time, and suggest that the mechanisms and type of tissue damage are different in the early and late stages of the lesion.

Appl Environ Microbiol, 1997 May, 63(5), 1953 - 8
Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy; Beuchat LR et al.; The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern . A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B . cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin . A portion of unheated vegetative cells from 24-h BHI broth cultures was sensitive to nisin as evidenced by an inability to form colonies on BHI agar containing 10 micrograms of nisin/ml . Heat-stressed cells exhibited increased sensitivity to nisin . At concentrations as low as 1 microgram/ml, nisin was lethal to B . cereus, the effect being more pronounced in BHI broth than in beef gravy . The inhibitory effect of nisin (1 microgram/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8 degrees C than at 15 degrees C . Nisin, at a concentration of 5 or 50 micrograms/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15 degrees C, respectively, for 14 days . Growth of vegetative cells and spores of B . cereus after an initial period of inhibition is attributed to loss of activity of nisin . One of two test strains produced diarrheal enterotoxin in gravy stored at 8 or 15 degrees C within 9 or 3 days, respectively . Enterotoxin production was inhibited in gravy supplemented with 1 microgram of nisin/ml and stored at 8 degrees C for 14 days; 5 micrograms of nisin/ml was required for inhibition at 15 degrees C . Enterotoxin was not detected in gravy in which less than 5.85 log10 CFU of B . cereus/ml had grown . Results indicate that as little as 1 microgram of nisin/ml may be effective in inhibiting or retarding growth of and diarrheal enterotoxin production by vegetative cells and spores of psychrotrophic B . cereus in beef gravy at 8 degrees C, a temperature exceeding that recommended for storage or for most unpasteurized, ready-to-serve meat products.

Appl Environ Microbiol, 1997 May, 63(5), 1679 - 84
Effect of Bacillus thuringiensis toxins on the membrane potential of lepidopteran insect midgut cells; Peyronnet O et al.; To test whether the ability of Bacillus thuringiensis toxins to form pores in the midgut epithelial cell membrane of susceptible insects correlates with their in vivo toxicity, we measured the effects of different toxins on the electrical potential of the apical membrane of freshly isolated midguts from gypsy moth (Lymantria dispar) and silkworm (Bombyx mori) larvae . In the absence of toxin, the membrane potential, measured with a conventional glass microelectrode, was stable for up to 30 min . It was sensitive to the K+ concentration and the oxygenation of the external medium . Addition of toxins to which L . dispar is highly {CryIA(a) and CryIA(b)} or only slightly {CryIA(c) and CryIC} sensitive caused a rapid, irreversible, and dose-dependent depolarization of the membrane . CryIF, whose toxicity towards L . dispar is unknown, and CryIE, which is at best poorly active in vivo, were also active in vitro . In contrast, CryIB and CryIIIA, a coleopteran-specific toxin, had no significant effect . The basolateral-membrane potential was unaffected by CryIA(a) or CryIC when the toxin was applied to the basal side of the epithelium . In B . mori midguts, the apical-membrane potential was abolished by CryIA(a), to which silkworm larvae are susceptible, but CryIA(b) and CryIA(c); to which they are resistant, had no detectable effect . Although the technique discriminated between active and inactive toxins, the concentration required to produce a given effect varied much less extensively than the sensitivity of gypsy moth larvae, suggesting that additional factors influence the toxins' level of toxicity in vivo.

Appl Environ Microbiol, 1997 May, 63(5), 1643 - 6
Immunomagnetic detection of Bacillus stearothermophilus spores in food and environmental samples; Blake MR et al.; There are currently no methods for the rapid and sensitive detection of bacterial spores that could be used to direct raw materials containing high spore loads away from products that pose a food safety risk . Existing methods require an overnight incubation, cannot detect spores below 10(5) CFU/ml, or are not specific to particular species . This work describes a method to specifically detect < 10(4) CFU of bacterial spores per ml within 2 h . Polyclonal antibodies to Bacillus stearothermophilus spores were attached to 2.8-micron-diameter magnetic polystyrene beads by using a polythreonine cross-linker via the antibody carbohydrate moiety . A biotin-avidin-amplified sandwich enzyme-linked immunosorbent assay coupled to a fluorescent substrate was used to quantitate captured spores . The concentration of B . stearothermophilus spores in samples was linearly correlated to fluorescent activity (r2 = 0.99) with a lower detection limit of 8 x 10(3) CFU/ml and an upper detection limit of 8 x 10(5) CFU/ml . The detection limits are not fixed and can be changed by varying the immunomagnetic bead concentration . Several food and environmental samples were tested to demonstrate the versatility of the assay.

Clin Infect Dis, 1997 May, 24(5), 860 - 6
Kingella kingae: an emerging cause of invasive infections in young children; Yagupsky P et al.; Kingella kingae, a fastidious hemolytic gram-negative bacillus once considered to be an exceptional cause of disease, has emerged in recent years as an important invasive pathogen in children . When synovial fluid and other exudates were inoculated into blood culture bottles, enhanced recovery of the organism was observed, and an annual incidence of invasive K . kingae infections of 27.4 per 100,000 children younger than age 24 months was demonstrated in southern Israel . Skeletal infections are the most common clinical presentation of K . kingae, and studies conducted in that region have shown that this organism is the most common etiology of septic arthritis in children below the age of 24 months . Other invasive diseases caused by K . kingae include bacteremia, endocarditis, and infections involving the lower respiratory tract, the eyes, or the central nervous system . Recent studies have demonstrated that K . kingae is part of the normal oropharyngeal flora of young children . Clinical data suggest that the organism may gain access to the bloodstream in the course of an upper respiratory infection or stomatitis . The organism is susceptible to a wide range of antimicrobial drugs, and with the exception of some cases of endocarditis, K . kingae infections in children usually run a benign clinical course.

Diabetes Care, 1997 May, 20(5), 767 - 72
Bacille Calmette-Guérin vaccination and incidence of IDDM in Montreal, Canada; Parent ME et al.; OBJECTIVE: To describe the association between Bacille Calmette-Guerin (BCG) vaccination and IDDM development in two different case-control series (A and B) in Montreal . RESEARCH DESIGN AND METHODS: Case-control series A comprised 93 IDDM cases and 2,903 control subjects who participated in a community-based tuberculin reactivity survey and who belonged to the same birth cohorts and areas of residence as the IDDM cases, Case-control series B comprised 249 IDDM cases and 431 age- and sex-matched friends and neighborhood control subjects . RESULTS: In series A, the BCG vaccination prevalence among cases and control subjects was 21.5% (95% CI 13.2-29.8%) and 22.3% (95% CI 20.8-23.8%), respectively . The odds ratio (OR) for IDDM associated with BCG vaccination was 1.09 (95% CI 0.62-1.91), after adjusting for the birth cohorts and areas of residence . The vaccination prevalence in series B was 17.7% (95% CI 13.0-22.4%) among cases and 15.1% (95% CI 11.7-18.5%) among control subjects . The OR for IDDM due to BCG vaccination was 1.26 (95% CI 0.79-2.02), taking into account the matched sets . Only one case (3.3%) from series B who had been vaccinated at birth was diagnosed by age 5, compared with 52 cases (24.5%) who had not been vaccinated (P < 0.01) . CONCLUSIONS: The lower proportion of birth-vaccinated IDDM cases diagnosed at a very young age, compared with nonvaccinated cases, possibly reflects a temporary boost of the immune functions after vaccination . However, as a whole, results from these analyses fail to support a protective role of BCG vaccination against juvenile-onset IDDM.

J Urol, 1997 May, 157(5), 1655 - 9
Long-term efficacy of intravesical bacillus Calmette-Guerin for carcinoma in situ: relationship of progression to histological response and p53 nuclear accumulation; Ovesen H et al.; PURPOSE: We assessed the influence of the histological response to intravesical bacillus Calmette-Guerin (BCG) and the prevalence of p53 nuclear accumulation on the clinical behavior of patients with carcinoma in situ . MATERIALS AND METHODS: Of 60 patients with Bergquist grade 3 carcinoma in situ 13 had primary and 47 had secondary carcinoma in situ . Patients received 6 weekly instillations and nonresponders received an additional 6 instillations at 2-week intervals . No maintenance was administered . Median followup was 48 months . The p53 nuclear accumulation was detected by immunohistochemical analysis with antibody PAb 1801 . RESULTS: The complete histological response rate to BCG therapy was 64%, which decreased to 52% at 4 years . BCG was more effective for treatment of primary than secondary carcinoma in situ (complete response rate 85 versus 57%, respectively) . The 45% progression rate was related to the initial histological response occurring in 26% of patients with a complete versus 77% with a partial and no response . Consequently, the progression rate was only 8% for primary versus 57% for secondary carcinoma in situ . Of the patients receiving only 1 course of BCG 40% had progression compared to 62% of those who received 2 courses . Patients in whom both courses failed had a progression rate of 89% . Intravesical BCG converted the p53 nuclear immunoreactivity from positive to negative in 73% of the 26 patients expressing reactivity before treatment, of whom 68% also had a complete response . The progression rate was related to the prevalence of p53 nuclear reactivity after but not before treatment (90% of patients with versus 37% without p53 nuclear accumulation had progression) . All 3 complete responders with p53 nuclear reactivity after BCG had progression, which suggests that molecular genetic change may precede histological change . Complete responders without p53 nuclear accumulation after BCG treatment experienced the lowest progression rate (21%) . CONCLUSIONS: Our results suggest that patients with a persistent complete histological response and without p53 nuclear accumulation after BCG treatment can be followed conservatively . Cystectomy should be considered in all other patients.

Nucleic Acids Res, 1997 May 1, 25(9), 1720 - 6
Leader peptides of inducible chloramphenicol resistance genes from gram-positive and gram-negative bacteria bind to yeast and Archaea large subunit rRNA; Harrod R et al.; catA86 is the second gene in a constitutively transcribed, two-gene operon cloned from Bacillus pumilus . The region that intervenes between the upstream gene, termed the leader, and the catA86 coding sequence contains a pair of inverted repeat sequences which cause sequestration of the catA86 ribosome binding site in mRNA secondary structure . As a consequence, the catA86 coding sequence is untranslatable in the absence of inducer . Translation of the catA86 coding sequence is induced by chloramphenicol in Gram-positives and induction requires a function of the leader coding sequence . The leader-encoded peptide has been proposed to instruct its translating ribosome to pause at leader codon 6, enabling chloramphenicol to stall the ribosome at that site . Ribosome stalling causes destabilization of the RNA secondary structure, exposing the catA86 ribosome binding site, allowing activation of its translation . A comparable mechanism of induction by chloramphenicol has been proposed for the regulated cmlA gene from Gram-negative bacteria . The catA86 and cmlA leader-encoded peptides are in vitro inhibitors of peptidyl transferase, which is thought to be the basis for selection of the site of ribosome stalling . Both leader-encoded peptides have been shown to alter the secondary structure of Escherichia coli 23S rRNA in vitro . All peptide-induced changes in rRNA conformation are within domains IV and V, which contains the peptidyl transferase center . Here we demonstrate that the leader peptides alter the conformation of domains IV and V of large subunit rRNA from yeast and a representative of the Archaea . The rRNA target for binding the leader peptides is therefore conserved across kingdoms.

Mol Cells, 1997 Apr 30, 7(2), 251 - 8
Crystal structure of thermostable alpha-amylase from Bacillus licheniformis refined at 1.7 A resolution; Hwang KY et al.; alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolase, E.C.3.2.1.1) catalyze the cleavage of alpha-1, 4-glucosidic linkages of starch components, glycogen, and various oligosaccharides . Thermostable alpha-amylases from Bacillus species are of great industrial importance in the production of corn syrup or dextrose . Thermostable alpha-amylase from Bacillus licheniformis, a monomeric enzyme with molecular mass of 55,200 Da (483 amino acid residues), shows a remarkable heat stability . This enzyme provides an attractive model for investigating the structural basis for thermostability of proteins . The three-dimensional structure of thermostable alpha-amylase from Bacillus licheniformis has been determined by the multiple isomorphous replacement method of X-ray crystallography . The structure has been refined to a crystallographic R-factor of 19.9% for 58,601 independent reflections with F0 > 2 sigma F0 between 8.0 and 1.7 A resolution, with root mean square deviations of 0.013 A from ideal bond lengths and 1.72 degrees from ideal bond angles . The final model consists of 469 amino acid residues and 294 water molecules . Missing from the model are the N- and C-termini and the segment between Trp182 and Asn192 . Like other alpha-amylases, the polypeptide chain folds into three distinct domains . The first domain (domain A), consisting of 291 residues (from residue 3 to 103 and 207 to 396), forms a (beta/alpha)8-barrel structure . The second domain (domain B), consisting of residues 104 to 206, is inserted between the third beta-strand and the third alpha-helix of domain A . The third C-terminal domain (domain C), consisting of residues 397 to 482, folds into an eight-stranded antiparallel beta-barrel . Neither calcium ion nor chloride ion is located near the active site . This study reveals the architecture of the thermostable alpha-amylase from Bacillus licheniformis . By homology with other alpha-amylases, important active site residues can be identified as Asp231, Glu261, and Asp328, which are all located at the C-terminal end of the central (beta/alpha)8-barrel . Since many of the stabilizing and destabilizing mutations obtained so far fall in domain B or at its border, this region of the enzyme appears to be important for thermostability . The factors responsible for the remarkable thermostability of this enzyme may be increased ionic interactions, reduced surface area, and increased packing interactions in the interior.

J Biol Chem, 1997 Apr 25, 272(17), 11557 - 65
Reversion of Ras- and phosphatidylcholine-hydrolyzing phospholipase C-mediated transformation of NIH 3T3 cells by a dominant interfering mutant of protein kinase C lambda is accompanied by the loss of constitutive nuclear mitogen-activated protein kinase/extracellular signal-regulated kinase activity; Bjorkoy G et al.; The transformed phenotype of v-Ras- or Bacillus cereus phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC)-expressing NIH 3T3 cells is reverted by expressing a kinase-defective mutant of protein kinase C lambda (lambdaPKC) . We report here that extracellular signal-regulated kinase (ERK)-1 and -2 are constitutively activated in v-Ras- and PC-PLC-transformed cells in the absence of added growth factors . Interestingly, the activated ERKs were exclusively localized to the cell nucleus . Consistently, the transactivating potential of the C-terminal domain of Elk-1, which is activated upon ERK-mediated phosphorylation, was strongly induced in serum-starved cells expressing v-Ras or PC-PLC . Reversion of v-Ras- or PC-PLC-induced transformation by expression of dominant negative lambdaPKC abolished the nuclear ERK activation suggesting lambdaPKC as a novel, direct or indirect, activator of mitogen-activated protein kinase/ERK kinase in response to activated Ras or elevated levels of phosphatidylcholine-derived diacylglycerol . Transient transfection experiments confirmed that lambdaPKC acts downstream of Ras but upstream of mitogen-activated protein kinase/ERK kinase . We found both the v-Ras- and PC-PLC-transformed cells to be insensitive to stimulation with platelet-derived growth factor (PDGF) . No detectable receptor level, autophosphorylation, or superinduction of DNA synthesis could be observed in response to treatment with PDGF . Reversion of the transformed cell lines by expression of dominant negative lambdaPKC restored the receptor level and the ability to respond to PDGF in terms of receptor autophosphorylation, ERK activation, and induction of DNA synthesis.

J Biol Chem, 1997 Apr 25, 272(17), 11152 - 6
Extreme stabilization of a thermolysin-like protease by an engineered disulfide bond; Mansfeld J et al.; The thermal inactivation of broad specificity proteases such as thermolysin and subtilisin is initiated by partial unfolding processes that render the enzyme susceptible to autolysis . Previous studies have revealed that a surface-located region in the N-terminal domain of the thermolysin-like protease produced by Bacillus stearothermophilus is crucial for thermal stability . In this region a disulfide bridge between residues 8 and 60 was designed by molecular modelling, and the corresponding single and double cysteine mutants were constructed . The disulfide bridge was spontaneously formed in vivo and resulted in a drastic stabilization of the enzyme . This stabilization presents one of the very few examples of successful stabilization of a broad specificity protease by a designed disulfide bond . We propose that the success of the present stabilization strategy is the result of the localization and mutation of an area of the molecule involved in the partial unfolding processes that determine thermal stability.

J Biol Chem, 1997 Apr 25, 272(17), 11011 - 6
Involvement of phosphatidylcholine-specific phospholipase C in platelet-derived growth factor-induced activation of the mitogen-activated protein kinase pathway in Rat-1 fibroblasts; van Dijk MC et al.; The role of phosphatidylcholine (PC) hydrolysis in activation of the mitogen-activated protein kinase (MAPK) pathway by platelet-derived growth factor (PDGF) was studied in Rat-1 fibroblasts . PDGF induced the transient formation of phosphatidic acid, choline, diacylglycerol (DG), and phosphocholine, the respective products of phospholipase D (PLD) and phospholipase C (PC-PLC) activity, with peak levels at 5-10 min . PLD-catalyzed transphosphatidylation (with n-butyl alcohol) diminished DG formation at 5 min but not at later stages of PDGF stimulation . Phorbol ester-induced down-regulation of protein kinase C (PKC) completely blocked PLD activation but not the formation of DG and phosphocholine at 10 min of PDGF stimulation . Collectively, these data indicate that PDGF activates both PLD and PC-PLC . In contrast, epidermal growth factor did not activate PC-PLC in these cells, and it activated PLD only weakly . DG formation by itself, through Bacillus cereus PC-PLC treatment of cells, was sufficient to mimic PDGF in activation of MAPK independent of phorbol ester-sensitive PKC . Since PKC down-regulation blocked PDGF-induced PLD but not MAPK activation, we conclude that PLD is not involved in MAPK signaling . In contrast, MAPK activation by exogenous (bacterial) PLD was not affected by PKC down-regulation, indicating that signals evoked by exogenous PLD differ from endogenous PLD . D609 (2-10 microg/ml), an inhibitor of PC-PLC, blocked PDGF- but not epidermal growth factor-induced MAPK activation . However, D609 should be used with caution since it also affects PLD activity . The results suggest that PC-PLC rather than PLD plays a critical role in the PDGF-activated MAPK pathway.

Biochemistry, 1997 Apr 22, 36(16), 4969 - 78
Transbilayer movement of fluorescent phospholipids in Bacillus megaterium membrane vesicles; Hrafnsdottir S et al.; We investigated the transbilayer movement or flip-flop of phospholipids in vesicles derived from the cytoplasmic membrane of Bacillus megaterium . Since common assay techniques were found to be inapplicable to the Bacillus system, we exploited and elaborated a newly described method in which fluorescent phospholipids (1-myristoyl-2-C6-NBD phospholipids) are used as tracers to monitor flip-flop . These lipids were introduced into Bacillus vesicles from synthetic donor vesicles containing a fluorescence quencher . Transport was measured by monitoring the increase in fluorescence as the tracers departed the quenched environment of the donor vesicle and entered first the outer membrane leaflet and subsequently the inner leaflet of Bacillus vesicles . Independent experiments involving cobalt quenching of NBD fluorescence provided results consistent with the existence of pools of fluorescent phospholipid in the outer and inner leaflets of Bacillus vesicles at the completion of transport . Using the assay we show that phospholipid flip-flop in Bacillus vesicles occurs rapidly (half-time approximately 30 s at 37 degrees C) with no preference for a particular phospholipid headgroup and that it is sensitive to proteolysis . We also establish that flip-flop does not occur in synthetic phospholipid vesicles or vesicles made from Bacillus phospholipids . We conclude that Bacillus vesicles possess the ability to promote rapid transbilayer movement of phospholipids, and that the transport is probably protein (flippase)-mediated.

Brain Res, 1997 Apr 18, 754(1-2), 142 - 6
Barbiturate-induced expression of neuronal nitric oxide synthase in the rat cerebellum; Thompson CM et al.; Neuronal nitric oxide synthase (nNOS) is known to share some structural and functional similarities with the cytochrome P450 mixed function oxidase system . Unlike P450, it does not require a second enzyme, reductase, to transfer electrons . This characteristic is similar to P450(BM-3) of Bacillus megaterium . P450(BM-3) and certain mammalian subfamilies of P450, such as P4502B, are known to be induced by phenobarbital (PB), and these P450s share a consensus sequence called the Barbie box . Because of the similarities nNOS shares with P450(BM-3) and other mammalian P450s, we have examined whether nNOS also responds to PB treatment . We have used semi-quantitative PCR, Western blot analysis, a functional assay, and immunohistochemistry in order to answer this question . These data show a threefold increase in nNOS mRNA expression, more modest nNOS protein and activity induction, and no discernible changes in localization of nNOS within the cerebellum following PB treatment.

N Engl J Med, 1997 Apr 17, 336(16), 1142 - 8
Fulminant liver failure in association with the emetic toxin of Bacillus cereus; Mahler H et al.; BACKGROUND: A 17-year-old boy and his father had acute gastroenteritis after eating spaghetti and pesto that had been prepared four days earlier . Within two days, fulminant liver failure and rhabdomyolysis developed in the boy and he died . The father had hyperbilirubinemia and rhabdomyolysis but recovered . We investigated the cause of these illnesses . METHODS: Bacteria were isolated and characterized by conventional methods, and bacterial toxins were quantified by immunoassays and cell-culture techniques . The effect of the isolated toxin on the rates of oxidation of various substrates was analyzed in rat-liver mitochondria . RESULTS: Autopsy of the boy's liver revealed diffuse microvesicular steatosis and midzonal necrosis that suggested impaired beta-oxidation of liver mitochondria due to a mitochondrial toxin . There was no evidence of ingestion of heavy metals, halogenated compounds, hepatotoxic drugs, or staphylococcal enterotoxin . However, high concentrations of Bacillus cereus emetic toxin were found in both the residue from the pan used to reheat the food and the boy's liver and bile . B . cereus was cultured from the intestinal contents and the pan residue . The emetic toxin isolated from the B . cereus cultures was found to be a mitochondrial toxin . CONCLUSIONS: Fulminant liver failure developed after the ingestion of food contaminated with the B . cereus emetic toxin . The toxin inhibits hepatic mitochondrial fatty-acid oxidation, indicating that it caused liver failure in this patient.

Arch Biochem Biophys, 1997 Apr 15, 340(2), 231 - 8
Reconstitution of the fatty acid hydroxylase activity of cytochrome P450BM-3 utilizing its functional domains; Sevrioukova I et al.; Cytochrome P450BM-3, a catalytically self-sufficient fatty acid monooxygenase from Bacillus megaterium, is a multidomain protein containing heme, FAD, and FMN . Previous attempts to reconstitute the fatty acid monooxygenase activity of intact P450BM-3 utilizing equimolar concentrations of the separate heme (BMP) and reductase (BMR) domains, have been unsuccessful because two-electron reduced FMN, which rapidly accumulates, is incapable of electron transfer to the heme iron . The present study of the reconstitution of the monooxygenase activity of P450BM-3 utilized combinations of the different functional domains of P450BM-3 . For this purpose, the FAD/NADPH- and FMN-binding domains of P450BM-3 as well as the combination of the heme- and FMN-binding domains (BMP/FMN) have been expressed and purified . The reconstitution systems, consisting of either BMP/FMN and FAD domains or BMP, FMN, and FAD domains, were still less effective than the holoenzyme, P450BM-3, but were much more effective than a system consisting of BMP and BMR . The maximal rate of oxidation of palmitic acid by the newly developed reconstitution systems is still only approximately 5% of the activity of the holoenzyme . The reconstitution systems produced omega-1, omega-2, and omega-3 monohydroxy palmitic acid, but not the secondary products of palmitic acid hydroxylation observed with the holoenzyme . The physical cause of the inability to reconstitute fully the maximal activity of the holoenzyme as well as the lack of secondary product formation is not presently understood.

FEMS Microbiol Lett, 1997 Apr 15, 149(2), 245 - 8
Bacillus cereus may produce two or more diarrheal enterotoxins; Ombui JN et al.; Bacillus cereus strains were tested for production of diarrheal enterotoxin by the reverse passive latex agglutination test and for presence of B . cereus enterotoxin gene (bceT) by polymerase chain reaction . About 50% of 56 B . cereus strains reacted positive in broth culture in the reverse passive latex agglutination test, while the bceT gene was detected in 41.1% . Sixteen percent of the strains were positive for both diarrheal enterotoxin and bceT gene . A 741 bp probe prepared from the polymerase chain reaction product detected bceT gene in all strains that were positive with the polymerase chain reaction . This study indicates a likelihood of two or more enterotoxins being produced by B . cereus which may be involved in causing diarrheal type food poisoning.

FEMS Microbiol Lett, 1997 Apr 15, 149(2), 221 - 6
Regulation of cyclodextrin glucanotransferase synthesis in Bacillus ohbensis; Nishida T et al.; The production of cyclodextrin glucanotransferase (CGTase) by Bacillus ohbensis is dependent on the presence of starch and inhibited by glucose in the medium . Northern blot analysis revealed that the CGTase gene (cgt) was transcribed to almost the same level irrespective of the presence or absence of starch, but glucose completely repressed the transcription . Furthermore, a relatively high amount of CGTase protein was detected on Western blotting only in the medium with starch, showing the lack of posttranslational control of the CGTase activity . These findings suggest some starch induction mechanism for the cgt gene, possibly at the posttranscriptional level, besides negative transcriptional regulation by glucose.

Structure, 1997 Apr 15, 5(4), 521 - 32
The solution structure of serine protease PB92 from Bacillus alcalophilus presents a rigid fold with a flexible substrate-binding site; Martin JR et al.; BACKGROUND: Research on high-alkaline proteases, such as serine protease PB92, has been largely inspired by their industrial application as protein-degrading components of washing powders . Serine protease PB92 is a member of the subtilase family of enzymes, which has been extensively studied . These studies have included exhaustive protein engineering investigations and X-ray crystallography, in order to provide insight into the mechanism and specificity of enzyme catalysis . Distortions have been observed in the substrate-binding region of subtilisin crystal structures, due to crystal contacts . In addition, the structural variability in the substrate-binding region of subtilisins is often attributed to flexibility . It was hoped that the solution structure of this enzyme would provide further details about the conformation of this key region and give new insights into the functional properties of these enzymes . RESULTS: The three-dimensional solution structure of the 269-residue (27 kDa) serine protease PB92 has been determined using distance and dihedral angle constraints derived from triple-resonance NMR data . The solution structure is represented by a family of 18 conformers which overlay onto the average structure with backbone and all-heavy-atom root mean square deviations (for the main body of the molecule) of 0.88 and 1.21 A, respectively . The family of structures contains a number of regions of relatively high conformational heterogeneity, including various segments that are involved in the formation of the substrate-binding site . The presence of flexibility within these segments has been established from NMR relaxation parameters and measurements of amide proton exchange rates . CONCLUSIONS: The solution structure of the serine protease PB92 presents a well defined global fold which is rigid with the exception of a restricted number of sites . Among the limited number of residues involved in significant internal mobility are those of two pockets, termed S1 and S4, within the substrate-binding site . The presence of flexibility within the binding site supports the proposed induced fit mechanism of substrate binding.

J Immunol, 1997 Apr 15, 158(8), 3796 - 9
Nitric oxide and TNF-alpha production by murine peritoneal macrophages activated with a novel 20-kDa protein isolated from Bacillus thuringiensis var . thuringiensis parasporal bodies; Gomez-Flores R et al.; The effects of a novel 20-kDa protein isolated from Bacillus thuringiensis var . thuringiensis (BTp20) parasporal bodies on macrophage activation were investigated and compared with the activity of LPS . Murine resident or IFN-gamma-primed peritoneal macrophages (50 U/ml of IFN-gamma for 16 h) were treated with 50 microg/ml of BTp20, alone or in combination with LPS (1 to 100 ng/ml) . BTp20 was not toxic for macrophages as determined by the MTT (3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide) reduction assay, however, 16% reduction of viability was observed when macrophages were treated with a combination of IFN-gamma, BTp20, and LPS at 100 ng/ml . BTp20 was able to stimulate significant cellular adherence and spreading, and significant production of nitrite and TNF-alpha by resident macrophages after 1.5 h of treatment; nitrite release, however, was induced with only 10 min of macrophage exposure to BTp20 . BTp20 activities were significantly potentiated by IFN-gamma pretreatment . It was also observed that nitrite release by IFN-gamma-primed macrophages activated with LPS (1-100 ng/ml) was 20 times lower than that induced by IFN-gamma + BTp20 . BTp20 and LPS independently stimulated significant TNF-alpha production by IFN-gamma-primed macrophages, the effect of BTp20 + LPS (1 and 10 ng/ml) combination was additive . In summary, this study demonstrated that BTp20 activates macrophage functions in the absence of IFN-gamma or LPS, and that IFN-gamma enhances BTp20 activities.

J Biol Chem, 1997 Apr 11, 272(15), 9609 - 12
Expression of neutral sphingomyelinase identifies a distinct pool of sphingomyelin involved in apoptosis; Zhang P et al.; The activation of sphingomyelinase and generation of ceramide have been implicated as important regulatory pathways in cell growth and apoptosis . Bacterial sphingomyelinase has been used in many cell systems to mimic the activation of endogenous sphingomyelinase . These studies, however, have been complicated by the inability of exogenously applied bacterial sphingomyelinase to perform many of the effects of short chain cell permeable ceramides, indicating that there may be a distinct signal transducing pool of sphingomyelin not accessible to exogenous sphingomyelinase or that endogenous ceramide is not sufficient to induce these changes . We cloned the Bacillus cereus sphingomyelinase gene by polymerase chain reaction and subcloned it into a mammalian expression vector under the control of an inducible promoter . Upon stable transfection and induction of B . cereus sphingomyelinase, there were increases in neutral sphingomyelinase activity, cellular ceramide levels, cleavage of the death substrate poly(ADP-ribosyl)polymerase, and cell death . In contrast, exogenously applied B . cereus sphingomyelinase, despite causing higher elevations in ceramide levels, was unable to induce poly(ADP-ribosyl)polymerase cleavage or cell death . These results support the existence of a signal transducing pool of sphingomyelin that is distinct from the pool accessible to exogenous sphingomyelinase.

Yi Chuan Xue Bao, 1997 Apr, 24(2), 178 - 82
{Study on transposition behavior of IS5376 in Bacillus stearothermophilus}; Xu K et al.; IS5376 and IS5377 are two transposable elements discovered in Bacillus stearothermophilus . Analysis of random samples revealed that the frequency of transposition of IS5376 from CU21 chromosome to plasmids pFDC5 and pFDC12 was much higher at 65 degrees C than that at 48 degrees C while that of IS5377 was very low at both 48 degrees C and 65 degrees C . The exact nature of the temperature effect is obscure at present from evidences obtained so far it is concluded that this is a consequence of the innate property of IS5376 . Furthermore, it was found that a certain degree of site specificity in transposition was evident and that a direct repeat of 4 or 5 bp of the target DNA appeared at the site of transposition.

Tierarztl Prax, 1997 Apr, 25(2), 94 - 9
{Cat-scratch disease: historical, clinical, phylogenetic and taxonomic aspects}; Muller HE; The cat-scratch disease (CSD) is known as a nosological entity since 1950 . It was diagnosed by the clinical symptoms, epidemiologic data, and the intracutaneous test of Hanger and Rose . The aetiologic agent is Bartonella (formerly Rochalimaea) henselae occurring in thirty to fifty percent of healthy cats . The gramnegative alpha-2-proteobacteria cause the CSD but also fever in healthy humans . Patients suffering from AIDS show bacillary angiomatosis, bacillary peliosis hepatis, endocarditis, and septicemia . There is an open question for other aetiologic agents causing CSD as cofactors . For example, Afipia felis is found to a certain extent from patients suffering from CSD . Furthermore, Rothia dentocariosa was isolated in lymphnodes of CSD patients, and also other grampositive rods may play an important role together with B . henselae in CSD.

Vaccine, 1997 Apr-May, 15(6-7), 689 - 99
Immunobiological properties of a 30 kDa secretory protein of Mycobacterium tuberculosis H37Ra; Sinha RK et al.; Six different secretory proteins of molecular weights (15, 26, 30, 41, 55 and 70 kDa) were isolated from 8-day-old culture filtrate of Mycobacterium tuberculosis H37Ra using different column chromatography techniques . These proteins were further examined for their ability to induce cell mediated (T-cell proliferation assay) and humoral immune response (ELISA) in mice immunized with total culture filtrate proteins . Out of six proteins, three proteins showed good reactivity . However, the activity was at a maximum with 30 kDa antigen . The immune response induced by 30 kDa antigen emulsified in Freund's incomplete adjuvant (FIA) was investigated and was found to be dose dependent . The T-cell response induced by this protein was skewed towards T-helper (Th1) cells as determined by the pronounced secretion of interleukin-2 (IL-2) and gamma-interferon (IFN-gamma) . The protective activity of the 30 kDa protein was also evaluated and compared with reference to Bacillus Calmette Guerin (BCG) vaccine in the mice challenged with virulent M . tuberculosis H37Rv . The degree of protection afforded by the 30 kDa antigen on the basis of mortality and the significant decrease in c.f.u.'s recovered from different organs (lung, liver, spleen) after 30 days of challenge with LD50 of M . tuberculosis H37Rv was significantly higher in comparison to BCG vaccinated animals . However, the degree of immunity induced by this antigen decreased with time (when challenged 8 and 12 weeks post-immunization) but it was still comparable with BCG . These findings suggest that 30 kDa secretory protein of M . tuberculosis is the key immunoprotective antigen and may be a suitable candidate for the development of an alternative subunit vaccine against tuberculosis.

Eur J Clin Microbiol Infect Dis, 1997 Apr, 16(4), 308 - 11
Cervical lymphadenitis due to an unusual mycobacterium; Tortoli E et al.; A scotochromogenic acid-fast bacillus was isolated from a lymph node of a 2-year-old female . On the basis of conventional testing, the mycobacterium appeared to be Mycobacterium scrofulaceum . Its mycolic acid profile, however, was not identical to that of Mycobacterium scrofulaceum but was similar to that of Mycobacterium interjectum . Direct sequencing of the 16S rRNA gene revealed a unique nucleic acid sequence, suggesting that the isolate represents a previously undescribed pathogenic species.

Appl Microbiol Biotechnol, 1997 Apr, 47(4), 379 - 84
Cloning and expression of the Bacillus sphaericus 2362 mosquitocidal genes in a non-toxic unicellular cyanobacterium, Synechococcus PCC6301; Sangthongpitag K et al.; Genes encoding the mosquitocidal binary toxin of Bacillus sphaericus 2362 were introduced into Synechococcus PCC6301, a cyanobacterium that can tolerate a number of potential variations in the mosquito breeding environment, and can serve as a food source for mosquito larvae . The toxin genes, preceded by a Synechococcus rbcL promoter, were located on a mobilizable Escherichia coli Synechococcus shuttle vector, which was introduced into Synechococcus PCC6301 at frequencies of 10(-5)-10(-7) exconjugants/recipient, depending on the selective conditions used . Recombinant Synechococcus exhibited significant toxicity against 2-day-old and 6-day-old Culex quinquefasciatus larvae, the concentration required to kill 50% of larvae (LC50) being 2.1 x 10(5) and 1.3 x 10(5) cells/ml respectively . Mosquitocidal activity decreased tenfold after 20 generations of non-selective growth.

J Clin Microbiol, 1997 Apr, 35(4), 1030 - 1
The diagnostic yield of acid-fast-bacillus smear-positive sputum specimens; Stone BL et al.; The yield of mycobacterial culture from acid-fast-bacillus smear-positive sputum specimens was 387 or 439 (88.2%) . Forty-nine of 52 culture-negative specimens came from patients on treatment . We conclude that the yield of culture from smear-positive sputum specimens is very high and that only two acid-fast-bacillus smear-positive specimens are needed for the initial evaluation of pulmonary mycobacteriosis.

In Vitro Cell Dev Biol Anim, 1997 Apr, 33(4), 315 - 23
Cytolytic activity of Bacillus thuringiensis CryIC and CryIAc toxins to Spodoptera sp . midgut epithelial cells in vitro; Wang SW et al.; A sensitive lactate dehydrogenase (LDH) assay was modified to determine the cytolytic activity of Bacillus thuringiensis CryIC and CryIAc delta endotoxins to viable collagenase-dissociated midgut epithelial cells (MEC) from larvae of Spodoptera frugiperda and Spodoptera exigua . The MEC preparations from these Spodoptera sp . consisted predominantly of columnar cells (65-75%) and goblet cells (25-35%) . Time course microscopy experiments indicated that only the columnar cells became swollen during CryIC toxin incubation . Also, comparative cytotoxicity studies were run with cell lines of nonmidgut origin established from S . frugiperda (SF21AE) and S . exigua (SEUCR1A) . Optimum conditions for the cytotoxicity assay were similar for MEC and cell lines of both species, and were met in an assay in which 0.1-ml cell concentrations (8.5 +/- 0.5 x 10(4) cells) were incubated with toxin dilutions (0.01-20 micrograms) for 1 h at 24 degrees C at a final pH of 7.8 . The Spodoptera sp . MEC were twofold more sensitive to CryIC (68% lysis) than CryIAc (32% lysis) at optimum toxin levels (2.5-5 micrograms) . Also, the SEUCR1A cells were more sensitive (2.3-fold) to CryIC (70% lysis) than CryIAc (30% lysis) at optimum toxin levels of 5-10 micrograms . The SF21AE cells, however, were twofold less sensitive to CryIC (30% lysis) than SEUCR1A cells and response to CryIAc and CryIC was similar . Immunoblot analysis of either Spodoptera sp . MEC or brush border membrane vesicles (BBMV) identified seven CryIC binding proteins with molecular mass of 137, 120, 115, 68, 65, 63, and 45 kDa . Occasionally, a 148-kDa protein band was observed . The CryIAc toxin bound to two proteins on MEC and BBMV with molecular mass of 137 and 120 kDa.

Clin Infect Dis, 1997 Apr, 24(4), 562 - 4
Bacillary angiomatosis associated with myositis in a patient infected with human immunodeficiency virus; Whitfeld MJ et al.; A man with AIDS presented with a deep soft-tissue mass involving the right thigh . Biopsy of a skin lesion on the back and culture of a specimen from this lesion showed bacillary angiomatosis due to Bartonella (formerly Rochalimaea) quintana . Magnetic resonance imaging revealed a large heterogeneous mass involving the vastus medialis and intermedius muscles . Therapy with erythromycin caused rapid resolution of both the cutaneous lesion and the muscle lesion . Bartonella infection is proposed as an additional cause of bacterial myositis and expands the spectrum of presentation of bacillary angiomatosis.

Biosci Biotechnol Biochem, 1997 Apr, 61(4), 670 - 4
Secretory production of erythropoietin and the extracellular domain of the erythropoietin receptor by Bacillus brevis: affinity purification and characterization; Nagao M et al.; Bacillus brevis secretes a large amount of cell wall proteins into the culture medium . For construction of Bacillus brevis expression-secretion vectors of human erythropoietin (EPO) and the extracellular domain of mouse erythropoietin receptor (sEPOR), cDNA for each mature form was inserted into a plasmid containing the promoter region and the signal-peptide encoding region of a cell wall protein . Culture supernatants of transformants were affinity purified using a monoclonal antibody-fixed gel for EPO and and EPO-fixed gel for sEPOR . The affinity purification efficiently removed unwanted proteins, giving samples with sufficiently high purity to analyze amino acid sequences of N-terminal regions and biological activities . Combination of this secretory production and affinity purification may facilitate isolation of a large amount of pure EPO and sEPOR, and is useful for further understanding the molecular mechanism of interaction between EPO and EPOR.

Chem Pharm Bull (Tokyo), 1997 Apr, 45(4), 733 - 6
Synthesis and biological evaluation of monoindolyl and indolocarbazolyl oxazolones and imidazolones; Pereira ER et al.; Eight compounds structurally related to protein kinase C inhibitor MDL 27032 and substituted with indole moieties were synthesized . Their activities towards protein kinase C (PKC) and protein kinase A (PKA) were determined . Their effect on PKC-mediated contraction of rat tracheal smooth muscle, their antiproliferative activity on two murine tumor cell lines, melanoma B16 and leukemia P388 and their antimicrobial activity on a gram-positive bacterium Bacillus cereus were also examined . The mammalian and bacterial cell antiproliferative activity, as well as vasorelaxant effect, observed for some of them could not be correlated to PKC or PKA inhibition . Only bulky bis-indolyl compounds exhibited biological activity in these experiments . Rigid indolocarbazoles had the strongest antiproliferative activity.

J Econ Entomol, 1997 Apr, 90(2), 361 - 9
Parameters influencing potency of Bacillus thuringiensis var . israelensis products; Skovmand O et al.; Bioassays of products based on Bacillus thuringiensis var . israelensis have been carried out according to standard protocols . These analyses revealed that the slopes of the log-probit transformed concentration mortality curves of various products were different from that of the international standards (IPS82 for B . thuringiensis israelensis) . For statistical reasons, this invalidates the tests . Products giving various slopes of the concentration mortality curves will obtain different potencies when estimated at a LC90 level than when estimated at LC50 level, as normally done . The LC90 level is probably more relevant for the field effect . Changing the median particle size of a product in a non destructive way results in change of slope and LC50 and thereby potency . Therefore, potency of a product as measured in these bioassays is not just a measure of the quantity of B . thuringiensis israelensis crystal protein present, but a function of product parameters like median particle size . Biochemical methods for quantification of toxin can therefore not relate simply to potency of the products obtained with this method . It is suggested that standard protocols for bioassay may be changed to assure equal particle size of products and samples to obtain parallel dose response.

J Econ Entomol, 1997 Apr, 90(2), 299 - 303
Reversal of low-level resistance to Bacillus sphaericus in a field population of the southern house mosquito (Diptera:Culicidae) from an urban area of Recife, Brazil; Silva-Filha MH et al.; A larval population of Culex quinquefasciatus Say from an urban area (Coque) of Recife, Brazil, submitted to selection with Bacillus sphaericus Neide for a 26-mo trial, was 10 times less susceptible to B . sphaericus and slightly more susceptible to Bacillus thuringiensis israelensis, compared with untreated populations . The low-level resistance to B . sphaericus was unstable in the absence of selection pressure . The LC50 of B . sphaericus to the Coque population declined gradually and attained a susceptibility level similar to that observed in a laboratory control colony 16 mo after the treatment period was interrupted . In parallel to the recovery of B . sphaericus susceptibility . Coque larvae also were as susceptible as laboratory larvae to B . thuringiensis israelensis.

Microbiology, 1997 Apr, 143 ( Pt 4), 1221 - 33
A carboxy-terminal processing protease gene is located immediately upstream of the invasion-associated locus from Bartonella bacilliformis; Mitchell SJ et al.; A gene with homology to those encoding an unusual class of C-terminal processing proteases that flanks the invasion-associated locus ialAB of Bartonella bacilliformis has been identified . The 1302 bp gene, termed ctpA, is located immediately upstream of the ialA gene and encodes a predicted nascent product of 434 amino acids, producing a mature protein of 411 amino acid residues . The Bartonella CtpA appears to undergo autolysis in vitro, producing multiple products of 43-46 kDa, and a second group of products of 36-37 kDa . Production of CtpA in vivo gives a single product of 41.8 kDa . In addition to a computer-predicted N-terminal secretory signal sequence, the molecular mass difference in vivo versus in vitro indicates that CtpA is likely to be secreted and post-translationally modified . The full-length CtpA protein shows 30% identify to the CtpA protein of Synechocystis sp . 6803 (69% overall sequence similarity) . The mature CtpA protein also has significant homology to the tail-specific protease (Tsp) of Escherichia coli, with 22% identify and 62% similarity to an internal region of the 660 amino acid Tsp . The CtpA protein does not appear to exhibit haemolysin, collagenase, or caseinase activity . The ctpA gene is conserved in all Bartonella species examined, as determined by hybridization analyses, but it was not found in Brucella abortus or E . coli . The ctpA gene does not directly affect the erythrocyte-invasion phenotype conferred by ialAB, but its homology to other stress-response processing proteases implies an important role in survival of this intracellular pathogen.

Am J Ophthalmol, 1997 Apr, 123(4), 560 - 2
Endogenous Ochrobactrum anthropi endophthalmitis; Berman AJ et al.; PURPOSE: To describe bilateral endogenous endophthalmitis caused by Ochrobactrum anthropi in a partially immunosuppressed patient who had undergone central venous access for hyperalimentation and home intravenous therapy . METHODS: Case report . RESULTS: Blood cultures were positive for O anthropi . Vitreous cultures grew a gram-variable bacillus . The patient's ocular and systemic condition markedly improved after intravitreal antibiotics and systemic ciprofloxacin . CONCLUSIONS: Ochrobactrum anthropi may cause endogenous endophthalmitis in patients with a history of indwelling catheters for venous access or other permanent medical devices.

J Urol, 1997 Apr, 157(4), 1254 - 8; discussion 1258-9
Deoxyribonucleic acid ploidy and the clinical pattern of grade 2 superficial bladder cancer; Neulander E et al.; PURPOSE: We attempted to find an objective and quantitative parameter that would enable us to differentiate between aggressive and nonaggressive grade 2 superficial transitional cell carcinoma of the bladder . This type of tumor belongs to a heterogeneous group, 30% of which behave aggressively by invading lamina propria and muscle tissue in the course of the evolution . The remaining 70% of tumors have less aggressive qualities, are nonprogressive and have a low recurrence rate . To date to our knowledge there is no way to differentiate between these 2 subpopulations of tumors . MATERIALS AND METHODS: We performed a retrospective flow cytometric analysis of deoxyribonucleic acid (DNA) ploidy and cell cycle phases on primary and solitary formalin fixed and paraffin embedded tumor tissue . RESULTS: Of the 41 specimens studied 16 were aneuploid and 25 were diploid . Aneuploidy was associated with progressive disease and high mortality rates, particularly in patients who did not receive postoperative adjuvant intravesical instillation . DNA diploidy was equated with lack of mortality and a low progression rate . The use of intravesical bacillus Calmette-Guerin as an adjuvant postoperative treatment was associated with low recurrence rates . In this group elevated G2M percent and S phase fractions correlated with higher recurrence rates . CONCLUSIONS: DNA ploidy was a prognostic factor in stage Ta grade 2 bladder transitional cell carcinoma and should be considered as a complementary test to histopathological analysis . Adjuvant instillation with bacillus Calmette-Guerin is advised after transurethral resection of primary solitary aneuploid stage Ta grade 2 bladder transitional cell carcinomas and of primary solitary diploid tumors with elevated G2M percent and S phase fraction.

J Urol, 1997 Apr, 157(4), 1246 - 9
Does isoniazid reduce side effects of intravesical bacillus Calmette-Guerin therapy in superficial bladder cancer? Interim results of European Organization for Research and Treatment of Cancer Protocol 30911; Vegt PD et al.; PURPOSE: We analyzed the influence of the tuberculostatic agent isoniazid on the incidence and severity of adverse effects of intravesical bacillus Calmette-Guerin (BCG) therapy in patients with superficial bladder cancer . MATERIALS AND METHODS: In a prospective randomized multicenter study the side effects of intravesical instillations with Tice strain BCG with and without isoniazid were compared in patients with stages pTa and pT1 bladder tumors . Isoniazid was given orally at a dose of 300 mg . daily at every instillation in an attempt to decrease the side effects of BCG . RESULTS: No differences in local or systemic adverse reactions after intravesical immune therapy with BCG could be observed between patients treated with or without prophylactic isoniazid therapy . However, analysis of liver function tests after BCG with isoniazid showed slightly more liver toxicity compared to BCG alone . CONCLUSIONS: Prophylactic administration of isoniazid during BCG instillations provides no decrease in any known side effect of BCG . In contrast, transient liver function disturbances are encountered slightly more frequently when isoniazid is administered . The use of prophylactic isoniazid in patients treated with BCG is not recommended.

Pediatr Infect Dis J, 1997 Apr, 16(4), 354 - 8
Mycobacterial lung disease in cystic fibrosis: a prospective study; Fauroux B et al.; BACKGROUND: Patients with cystic fibrosis (CF) may be predisposed to airway infections with unusual organisms, such as mycobacteria . The aim of the study was to determine the incidence and clinical picture of mycobacterial infection in CF children . METHODS: At least 2 acid-fast bacillus (AFB) smears and mycobacterial cultures were performed on a prospective basis on 682 sputum specimens from 106 patients during a 1-year period . RESULTS: Thirty-three percent of the cultures were contaminated with other bacteria . Seven children had at least one sputum culture positive for one mycobacterium . Five children had only one positive AFB culture . Their clinical status and lung function remained stable during follow-up . Two teenagers with severe lung disease had several positive AFB smears and cultures for Mycobacterium chelonae and Mycobacterium abscessus . The isolation of M . chelonae and M . abscessus was associated with a clinical and functional decline . Clarithromycin treatment resulted in temporary improvement with the disappearance of the mycobacteria after 6 months of treatment . This prospective study shows an incidence of 2.3% for positive cultures . The prevalence was 6.6% for mycobacterial colonization but only 1.9% for mycobacterial lung disease in our pediatric population . CONCLUSIONS: We recommend performing AFB smears and cultures in CF children with severe lung disease and/or during a lung exacerbation . In these patients persistence of M . chelonae or M . abscessus in sputum should lead to consideration of treatment with clarithromycin.

Immunol Cell Biol, 1997 Apr, 75(2), 161 - 6
Sequential activation of alveolar macrophages by IFN-gamma and LPS is required for enhanced growth inhibition of virulent Mycobacterium bovis but not M . bovis BCG; Aldwell FE et al.; Alveolar macrophages (AM) form the first line of defence against most respiratory pathogens and, unlike tissue macrophages, are constantly exposed to a wide variety of antigenic stimuli . In this study we investigated the in vitro effects of IFN-gamma and LPS on growth of virulent Mycobacterium bovis and M . bovis bacille Calmette-Guerin (BCG) in bovine AM . Bovine AM were purified from bronchial lavage fluid and cultured in serum-free medium . Pretreatment of bovine AM with IFN-gamma resulted in growth inhibition of M . bovis BCG but only partially inhibited growth of virulent M.bovis . Enhanced inhibition of virulent M.bovis by bovine AM required sequential stimulation with IFN-gamma and LPS and was associated with increased induction of nitric oxide (NO) and IL-12 mRNa . Growth inhibition of M . bovis was not affected by treatment of macrophages with the L-arginine analogue, NG-monomethyl-L-arginine although this treatment decreased NO production . These results suggest that a second activation signal in the form of TNF-alpha or LPS may be required to induce bacteriostasis of virulent M . bovis by bovine AM in vivo . The ability of bovine AM to respond to activation stimuli in vitro suggests that these cells may play an important role in preventing establishment of intracellular bacterial infections in the lung.

Clin Microbiol Rev, 1997 Apr, 10(2), 203 - 19
Bartonella spp . as emerging human pathogens; Anderson BE et al.; Members of the genus Bartonella (formerly Rochalimaea) were virtually unknown to modern-day clinicians and microbiologists until they were associated with opportunistic infections in AIDS patients about 6 years ago . Since that time, Bartonella species have been associated with cat scratch disease, bacillary angiomatosis, and a variety of other disease syndromes . Clinical presentation of infection with Bartonella ranges from a relatively mild lymphadenopathy with few other symptoms, seen in cat scratch disease, to life-threatening systemic disease in the immunocompromised patient . In some individuals, infection manifests as lesions that exhibit proliferation of endothelial cells and neovascularization, a pathogenic process unique to this genus of bacteria . As the spectrum of disease attributed to Bartonella is further defined, the need for reliable laboratory methods to diagnose infections caused by these unique organisms also increases . A brief summary of the clinical presentations associated with Bartonella infections is presented, and the current status of laboratory diagnosis and identification of these organisms is reviewed.

Am Fam Physician, 1997 Apr, 55(5), 1783 - 9, 1793-4
Cat-scratch disease and related clinical syndromes; Smith DL; Bartonella (Rochalimaea) henselae is a common cause of cat-scratch disease . This newly identified bacterium is also the cause of several other clinical syndromes, including bacillary angiomatosis, bacillary peliosis hepatitis and splenitis, and acute and relapsing bacteremia . A high percentage of young cats carry B . henselae . Fortunately, serious complications of B . henselae infections are rare in immunocompetent patients . Cat-scratch disease is usually a self-limited illness that does not necessarily require antibiotic therapy . Severe or persistent cases respond well to several antibiotics, including erythromycin and doxycycline . Cat-scratch disease should be included in the differential diagnosis of serious neurologic disease, particularly when regional lymphadenopathy develops suddenly in a previously healthy patient who owns a cat . Treatment of uncomplicated central nervous system disease is generally supportive . Antibiotic therapy is reserved for patients with atypical or severe involvement, including encephalopathy and retinitis . Other internal and cutaneous manifestations of B . henselae infection have recently been described . These potentially life-threatening infections respond well to antibiotic therapy, even in immunocompromised patients.

J Rheumatol, 1997 Apr, 24(4), 752 - 8
Synovial membrane cytokine profiles in reactive arthritis secondary to intravesical bacillus Calmette-Guérin therapy; Smith MD et al.; We describe the cellular infiltrate and cytokine profile in sequential synovial membrane biopsies from a patient with acute followed by chronic synovitis after intravesical bacillus Calmette-Guerin (BCG) therapy for an in situ transitional cell carcinoma of the bladder . Histological and immunohistochemical analysis of 3 synovial biopsies were done sequentially over a 9 month period . The patient was HLA-B27 positive, but HLA-DR4 negative, and did not have the "shared epitope." Unlike other cases, this patient's arthritis did not respond initially to nonsteroidal antiinflammatory drugs and was exacerbated by corticosteroid therapy . The synovitis took a neutrophilic form, with marked synovial membrane content of interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) . It subsequently developed into chronic lymphoplasmacytoid synovitis, similar to rheumatoid arthritis (RA), with decreased IL-8 but continuing IL-1 and TNF-alpha production in the synovial membrane . The synovitis resolved to a fibrotic synovium with residual thickening of the synovial lining layer and continued production of TNF-alpha . Thus, during the evolution of this arthritis, the synovial layer and continued production of TNF-alpha . Thus, during the evolution of this arthritis, the synovial membrane yielded a cellular infiltrate and cytokine content that had marked similarities with that seen in RA; however, the arthritis eventually remitted spontaneously.

J Bacteriol, 1997 Apr, 179(8), 2551 - 6
Molecular cloning and characterization of the genes encoding the L1 and L2 components of hemolysin BL from Bacillus cereus; Ryan PA et al.; Hemolysin BL, which is composed of a binding component, B, and two lytic components, L1 and L2, is the enterotoxin responsible for the diarrheal food poisoning syndrome caused by strains of Bacillus cereus . To further characterize the toxin, we sought to clone and sequence the genes encoding the L1 and L2 proteins . A genomic library was screened with polyclonal antibody to the L1 and L2 proteins to identify recombinant clones containing the genes . Five clones reacted with the antibody to L2, but none reacted with the antibody to L1 . Southern hybridization analysis with oligonucleotide probes designed from the N-terminal amino acid sequences of the L1 and L2 proteins, in conjunction with immunoblot and nucleotide sequence analysis, revealed that the recombinant plasmid from one of the clones contained two genes, hblC and hblD, which encode L2 and L1, respectively . The two genes are arranged in tandem and are separated by only 37 bases . The gene which encodes the B component of hemolysin BL (hblA) is located immediately downstream from the gene encoding the L1 protein . Northern blot analysis of B . cereus RNA showed a 5.5-kb transcript which hybridized with DNA fragments internal to, or including a portion of, the coding sequences of the B, L1, and L2 genes, suggesting that the clustered genes which encode the components of hemolysin BL are cotranscribed and constitute an operon.

Appl Environ Microbiol, 1997 Apr, 63(4), 1406 - 20
Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph; Cue D et al.; We report the fist genetic transformation system, shuttle vectors, and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus . By using a polyethylene glycol-mediated transformation procedure, we have successfully transformed B . methanolicus with both integrative and multicopy plasmids . For plasmids with a single BmeTI recognition site, dam methylation of plasmid DNA (in vivo or in vitro) was found to enhance transformation efficiency from 7- to 11-fold . Two low-copy-number Escherichia coli-B, methanolicus shuttle plasmids, pDQ507 and pDQ508, are described . pDQ508 caries the replication origin cloned from a 17-kb endogenous B . methanolicus plasmid, pBM1 . pDQ507 carries a cloned B . methanolicus DNA fragment, pmr-1, possibly of chromosomal origin, that supports maintenance of pDQ507 as a circular, extrachromosomal DNA molecule . Deletion analysis of pDQ507 indicated two regions required for replication, i.e., a 90-bp AT-rich segment containing a 46-bp imperfect, inverted repeat sequence and a second region 65% homologous to the B . subtilis dpp operon . We also evaluated two E . coli-B . subtilis vectors, pEN1 and pHP13, for use as E . coli-B . methanolicus shuttle vectors . The plasmids pHP13, pDQ507, and pDQ508 were segregationally and structurally stable in B . methanolicus for greater than 60 generations of growth under nonselective conditions; pEN1 was segregationally unstable . Single-stranded plasmid DNA was detected in B . methanolicus transformants carrying either pEN1, pHP13, or pDQ508, suggesting that pDQ508, like the B . subtilis plasmids, is replicated by a rolling-circle mechanism . These studies provide the basic tools for the genetic manipulation of B . methanolicus.

Appl Environ Microbiol, 1997 Apr, 63(4), 1195 - 8
Distribution and characterization of mosquitocidal toxin genes in some strains of Bacillus sphaericus; Priest FG et al.; The binary toxin of Bacillus sphaericus strains forms a crystal in sporulating cells, while the mosquitocidal toxin is located in the cytoplasm of vegetative cells . The distribution of binary toxin (btx) and mosquitocidal toxin (mtx) genes in 53 strains of B . sphaericus was determined by hybridization of specific gene probes to chromosomal DNA in Southern blots . btx genes were found in all strains of serotype 5a5b examined and in some strains of serotypes 1a, 3, 6, 25, and 48, while mtx genes were detected in strains of serotypes 1a, 2a2b, 5a5b, 6, 9a9c, 25, and 48 . Serotype 26a26b strains lacked both toxin genes, as did some strains of serotypes 2a2b, 3, 6, and 48 . Partial DNA sequences of btx genes from five strains, together with published sequences, revealed four types of toxin among mosquitocidal B . sphaericus strains . most of the 42-kDa toxin gene of btx was identical in strains from serotypes 1a, 3, 6, and 48, and the gene is here classified as a type 1 btx gene . A serotype 3 strain isolated in Singapore possessed a unique 42-kDa toxin gene, here designated type 4; while the btx genes from strains of serotypes 5a5b and 25 are referred to as types 2 and 3, respectively.

Antimicrob Agents Chemother, 1997 Apr, 41(4), 866 - 8
Sensitivity of Aeromonas hydrophila carbapenemase to delta3-cephems: comparative study with other metallo-beta-lactamases; Felici A et al.; Ceftriaxone and ceftriaxone S-oxide behaved as inactivators against the metallo-beta-lactamase of Aeromonas hydrophila AE036 and as substrates for the zinc beta-lactamase produced by Bacillus cereus (569/H/9) and Stenotrophomonas maltophilia ULA 511 . Moreover, RO 09-1428, a catechol-cephalosporin, was not recognized by the A . hydrophila enzyme . Panipenem, cephalosporin C, cephalosporin C-gamma-lactone, and loracarbef were substrates for the three studied beta-lactamases.

Curr Microbiol, 1997 Apr, 34(4), 199 - 204
Gramicidin S production by Bacillus brevis in simulated microgravity; Fang A et al.; In a continuing study of microbial secondary metabolism in simulated microgravity, we have examined gramicidin S (GS) production by Bacillus brevis strain Nagano in NASA High Aspect Rotating Vessels (HARVs), which are designed to simulate some aspects of microgravity . Growth and GS production were found to occur under simulated microgravity . When performance under simulated microgravity was compared with that under normal gravity conditions in the bioreactors, GS production was found to be unaffected by simulated microgravity . The repressive effect of glycerol in flask fermentations was not observed in the HARV . Thus the negative effect of glycerol on specific GS formation is dependent on shear and/or vessel geometry, not gravity.

Yeast, 1997 Mar 30, 13(4), 337 - 51
Enhancement of protein secretion in Saccharomyces cerevisiae by overproduction of Sso protein, a late-acting component of the secretory machinery; Ruohonen L et al.; Increased production of secreted proteins in Saccharomyces cerevisiae was achieved by overexpressing the yeast syntaxins . Sso1 or Sso2 protein, the t-SNAREs functioning at the targeting/fusion of the Golgi-derived secretory vesicles to the plasma membrane . Up to four- or six-fold yields of a heterologous secreted protein, Bacillus alpha-amylase, or an endogenous secreted protein, invertase, were obtained respectively when expressing either one of the SSO genes, SSO1 or SSO2, from the ADH1 promoter on a multicopy plasmid . Direct correlation between the Sso protein level and the amount of secreted alpha-amylase was demonstrated by modulating the expression level of the SSO2 gene . Quantitation of the alpha-amylase activity in the culture medium, periplasmic space and cytoplasm suggests that secretion into the periplasmic space is the primary stage at which the SSO genes exert the secretion-enhancing function . Pulse-chase data also support enhanced secretion efficiently obtained by SSO overexpression . Our data suggest that the Sso proteins may be rate-limiting components of the protein secretion machinery at the exocytosis step in yeast.

Gene, 1997 Mar 25, 188(1), 35 - 9
Overexpression of BsoBI restriction endonuclease in E . coli, purification of the recombinant BsoBI, and identification of catalytic residues of BsoBI by random mutagenesis; Ruan H et al.; BsoBI is a type II restriction enzyme found in Bacillus stearothermophilus JN209 that recognizes the symmetric sequence 5'-CYCGRG-3' (Y=C or T; R=A or G) and cleaves between the first and second base to generate a four-base 5' extension . The cloning and sequencing of BsoBI restriction-modification system has been described by Ruan et al . {Mol . Gen . Genet . 252 (1996) 695-699} . Here we report the overexpression of BsoBI restriction endonuclease gene in E . coli by insertion of the endonuclease gene into an expression vector pRRS . The recombinant BsoBI was purified to homogeneity and its N-terminus sequence was determined . It has the same N-terminal aa sequence as the native enzyme . The constitutive expression of BsoBI from pRRS is lethal to E . coli in the absence of the cognate methylase . The bsoBIR gene was mutagenized with either hydroxylamine or by error-prone polymerase chain reaction in vitro and transferred into E . coli via plasmid vectors in the absence of the cognate methylase . Surviving transformants were selected that carry BsoBI variants which lost endonuclease activity . DNA sequencing of the mutant alleles revealed that G123, D124, D212, D246, E252 and H253 are important residues for enzymatic activity . An electrophoretic mobility shift assay was used to identify binding-proficient and cleavage-deficient variants . Seven variants I95M&D124Y, G123R, D212N, K207R&D212V, D246N, D246G and E252K can still bind DNA despite the loss of cleavage activity . Thus, residues D124, D212, D246 and E252 may be located near or within the catalytic center, and are likely involved in metal ion binding.

FEBS Lett, 1997 Mar 24, 405(2), 241 - 4
Engineering thermolysin-like proteases whose stability is largely independent of calcium; Veltman OR et al.; Thermal stability of the thermolysin-like protease produced by Bacillus stearothermophilus (TLP-ste) is highly dependent on calcium at concentrations in the millimolar range . We describe the rational design and production of a fully active TLP-ste variant whose stability is only slightly dependent on calcium concentration.

Pediatr Surg Int, 1997 Mar 21, 12(2/3), 220 - 3
Is BCG vaccine innocent?
Karnak I I, Senocak ME, Buyukpamukcu N, Gocmen A.
Four patients admitted to the Hacettepe University Department of Pediatric Surgery between 1987 and 1995, two with Bacille Calmette-Guerin (BCG) lymphadenitis and two with multisystem postvaccination tuberculosis (MPT), are presented . The hospital records and records of the Ministery of Health Tuberculosis Control Department were evaluated to determine the complications of BCG vaccine . The most common complication was lymphadenitis with or without suppuration (0.3 per thousand - 3 per thousand) . Surgical intervention was required in two BCG lymphadenitis cases and two cases of MPT . Involved lymph nodes were excised in two lymphadenitis cases . Colostomy and percutaneous nephrostomy was performed in the first case of MPT in addition to triple antituberculous drug therapy . Although BCG lymphadenitis is self limited, chronically discharging nodes and tumor-like lymphadenopathy masses need to be excised . On the other hand, MPT has a silent nature with resistance to antituberculous drug therapy . Surgical intervention may be required, directed to the involved systems.

Bull Acad Natl Med, 1997 Mar 18, 181(3), 441 - 50; discussion 451-4
{Cat scratch disease and associated infections}; Chomel BB et al.; Cat scratch disease (CSD) was first described in France by Debre et al . in 1950, yet the causative bacterial agent of CSD remained obscure until 1992, when Bartonella (formerly Rochalimaea) henselae was implicated in CSD by serological and microbiologic studies . B . henselae had been linked initially to bacillary angiomatosis (BA), but also bacillary peliosis, relapsing bacteremia and endocarditis . Cats are healthy carriers of B . henselae and B . clarridgeiae, and can be bacteremic for months to years . Cat to cat transmission of the organism involves the cat flea in absence of direct contact transmission . Present knowledge on the etiology, clinical features and epidemiological characteristics of cat scratch disease/bacillary angiomatosis are presented.

Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2111 - 6
Transgenic elite indica rice plants expressing CryIAc delta-endotoxin of Bacillus thuringiensis are resistant against yellow stem borer (Scirpophaga incertulas); Nayak P et al.; Generation of insect-resistant, transgenic crop plants by expression of the insecticidal crystal protein (ICP) gene of Bacillus thuringiensis (Bt) is a standard crop improvement approach . In such cases, adequate expression of the most appropriate ICP against the target insect pest of the crop species is desirable . It is also considered advantageous to generate Bt-transgenics with multiple toxin systems to control rapid development of pest resistance to the ICP . Larvae of yellow stem borer (YSB), Scirpophaga incertulas, a major lepidopteran insect pest of rice, cause massive losses of rice yield . Studies on insect feeding and on the binding properties of ICP to brush border membrane receptors in the midgut of YSB larvae revealed that cryIAb and cryIAc are two individually suitable candidate genes for developing YSB-resistant rice . Programs were undertaken to develop Bt-transgenic rice with these ICP genes independently in a single cultivar . A cryIAc gene was reconstructed and placed under control of the maize ubiquitin 1 promoter, along with the first intron of the maize ubiquitin 1 gene, and the nos terminator . The gene construct was delivered to embryogenic calli of IR64, an elite indica rice cultivar, using the particle bombardment method . Six highly expressive independent transgenic ICP lines were identified . Molecular analyses and insect-feeding assays of two such lines revealed that the transferred synthetic cryIAc gene was expressed stably in the T2 generation of these lines and that the transgenic rice plants were highly toxic to YSB larvae and lessened the damage caused by their feeding.

Biochemistry, 1997 Mar 18, 36(11), 3170 - 8
Translational initiation factor IF2 from Bacillus stearothermophilus: a spectroscopic and microcalorimetric study of the C-domain; Misselwitz R et al.; Conformation and stability of the C-terminal domain of initiation factor IF2 from Bacillus stearothermophilus were analyzed by circular dichroism, fluorescence and Raman spectroscopy, and microcalorimetry under different solvent conditions . From circular dichroism and Raman measurements, IF2C at neutral pH can be classified as an alpha + beta protein . Solvent perturbation and Raman spectroscopy indicate a high accessibility of the tyrosine residues in the native protein . The Gdn/HCl-induced unfolding of IF2C was monitored by circular dichroism . IF2C unfolding at neutral pH proceeds in two discrete steps . The midpoints (c(m)) and the free energy of unfolding (deltaG(u)H2O) of the first and second transition are 2.05 M and 6.2 kcal x mol(-1) and 4.1 M and 12.9 kcal x mol(-1), respectively . ANS does not bind to the stable intermediate formed at 3 M Gdn/HCl . It seems likely that IF2C is composed of two subdomains which unfold in a stepwise process . Melting experiments at pH 7.0 are impaired by irreversible aggregation at higher temperatures . However, in Gdn/HCl containing buffer at denaturant concentrations up to 1.5 M the melting becomes a reversible process and can be analyzed by differential scanning calorimetry . At Gdn/HCl concentrations between 1.0 and 1.5 M, IF2C seems to be composed of two folding units with Tm values of about 60 and 78 degrees C and folding enthalpy values (deltaHm) of about 37 and 58 kcal x mol(-1) . At pH values below pH 3.0, IF2C can adopt a new acid-induced conformation, which is characterized by a high secondary structure content and a strong ANS binding . The Gdn/HCl-induced unfolding of IF2C at pH 2.6 takes place only in one discrete step with a midpoint c(m) of 3.3 M and a deltaG(AUa)H2O of 11.9 kcal x mol(-1).

Gene, 1997 Mar 18, 187(2), 217 - 9
Primary structure and strand specificity of BstF5I-1 DNA methyltransferase which recognizes 5'-GGATG-3'; Degtyarev SKh et al.; The gene for BstF5I-1 DNA-methyltransferase (MTase) from Bacillus stearothermophilus F5 (a FokI isoschizomer, recognizing 5'-GGATG-3') was cloned and its nucleotide (nt) sequence was determined . Analysis of deduced amino acid (aa) sequence shows that M x BstF5I-1 belongs to D21 class of MTases and has a little homology with M x FokI . M x BstF5I-1 modifies only the upper strand of recognition sequence (5'-GGATG-3').

FEBS Lett, 1997 Mar 17, 405(1), 47 - 54
Mechanical stability of compact modules of barnase; Takahashi K et al.; Globular proteins are composed of structural elements such as secondary structures and modules . Modules are compact segments consisting of 10-40 contiguous amino acid residues and are often encoded by exons . Therefore, the view that the modular organization of proteins is a result of exon-shuffling or -fusion is given support . Secondary structures such as alpha-helix and beta-sheet are stabilized by hydrogen bonds and are thus considered to be stable, structural elements of a globular domain . Since module boundaries are often located on alpha-helices or beta-sheets, it is not obvious whether the modules are mechanically stable . We carried out molecular dynamics simulations on modules of barnase, a bacterial RNase from Bacillus amyloliquefaciens, for 1 ns in vacuo and 150 ps in water . Five of six modules (M1, 1-24; M2, 25-52; M3, 53-73; M4, 74-88; M5, 89-98) retained native-like conformations during these simulations . Only the C-terminal module (M6, 99-110) was deformed; it is less compact than the other modules . As the modules are mechanically stable they are suitable as parts combined into proteins . Together with RNase activity of the three isolated modules of barnase, M2, M3 and M6, our study supports the view that modules were indeed original building blocks of proteins.

FEBS Lett, 1997 Mar 10, 404(2-3), 241 - 4
Glutamyl endopeptidase of Bacillus intermedius, strain 3-19; Leshchinskaya IB et al.; A homogeneous glutamyl endopeptidase splitting peptide bonds of glutamic, rarely of aspartic acid residues in peptides and proteins, was isolated from Bacillus intermedius 3-19 culture filtrate using chromatography on CM cellulose and Mono S . The enzyme molecular mass is equal to 29 kDa, pI 8.4 . The protease is inhibited by diisopropylfluorophosphate . The enzyme, like other glutamyl endopeptidases, reveals two pH optima at pH 7.5 and 9.0 for casein and one at pH 8.0 for Z-Glu-pNA hydrolysis . A 6 mM K(m) is found for hydrolysis of the latter substrate . The enzyme activity optimum lies at 55 degrees C, and it is stable at pH 6.5-11.0 . Its N-terminal sequence shows 56% coinciding residues when compared with that of Bacillus licheniformis glutamyl endopeptidase.

FEBS Lett, 1997 Mar 10, 404(2-3), 148 - 52
A novel delta-endotoxin gene cryIM from Bacillus thuringiensis ssp . wuhanensis; Shevelev AB et al.; A new cryI-related sequence designated cryIM was cloned using an immunoscreening technique from ssp . wuhanensis of Bacillus thuringiensis (BT), previously reported to produce multiple Cry proteins {Chestukhina et al . (1994) Can . J . Microbiol . 240, 1026-1034} . Analysis of the cryIM nucleotide sequence revealed an ORF, BTII-type promoter-like sequence, peculiar for such genes, a translation initiation element and a putative transcription terminator . Nevertheless, its product was not previously found in the crystals of the host strain {Chestukhina et al . (1994) Can . J . Microbiol . 240, 1026-1034} which shows its weak or absent natural expression . The amino acid sequence of 1151 residues encoded by the continuous reading frame of cryIM is not identical but is essentially similar to the other delta-endotoxins of the CryI class . An IS231-like sequence was found 400 bp downstream of the cryIM stop codon and a fragment of the cryIAb gene was located 3 kb upstream of its initiator codon in the same orientation . Artificial expression of the cloned gene in E . coli under the control of the lacZ promoter allowed us to obtain its hypothetical protein product.

Gene, 1997 Mar 10, 187(1), 19 - 27
Cloning and characterization of the Bg/II restriction-modification system reveals a possible evolutionary footprint; Anton BP et al.; Bg/II, a type II restriction-modification (R-M) system from Bacillus globigii, recognizes the sequence 5'-AGATCT-3' . The system has been cloned into E . coli in multiple steps: first the methyltransferase (MTase) gene, bglIIM, was cloned from B . globigii RUB561, a variant containing an inactivated endonuclease (ENase) gene (bglIIR) . Next the ENase protein (R.BglII) was purified to homogeneity from RUB562, a strain expressing the complete R-M system . Oligonucleotide probes specific for the 5' end of the gene were then synthesized and used to locate bglIIR, and the gene was isolated and cloned in a subsequent step . The nucleotide sequence of the system has been determined, and several interesting features have been found . The genes are tandemly arranged, with bglIIR preceding bglIIM . The amino acid sequence of M.BglII is compared to those of other known MTases . A third gene encoding a protein with sequence similarity to known C elements of other R-M systems is found upstream of bglIIR . This is the first instance of a C gene being associated with an R-M system where the R and M genes are collinear . In addition, open reading frames (ORFs) resembling genes involved with DNA mobility are found in close association with BglII . These may shed light on the evolution of the R-M system.

J Chromatogr B Biomed Sci Appl, 1997 Mar 7, 690(1-2), 338 - 42
Analysis of dipalmitoyl phosphatidylcholine in amniotic fluid by high-performance liquid chromatography; Alvarez JG et al.; A novel method for the determination of dipalmitoyl phosphatidylcholine (DPPC) in amniotic fluid by high-performance liquid chromatography (HPLC) is described . Aliquots of 50 microliters of amniotic fluid were hydrolyzed with phospholipase C from Bacillus cereus and the resulting dipalmitoylglycerol analyzed by HPLC . Run-to-run and day-to-day precision for DPPC analysis were 4.2 and 6.1%, respectively, and analysis time was 10 min . Recoveries for DPPC ranged between 92 and 98% . In summarizing, this method provides a high precision and fast turnaround time means for the analysis of DPPC in amniotic fluid.

Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 1640 - 4
One gene in diamondback moth confers resistance to four Bacillus thuringiensis toxins; Tabashnik BE et al.; Environmentally benign insecticides derived from the soil bacterium Bacillus thuringiensis (Bt) are the most widely used biopesticides, but their success will be short-lived if pests quickly adapt to them . The risk of evolution of resistance by pests has increased, because transgenic crops producing insecticidal proteins from Bt are being grown commercially . Efforts to delay resistance with two or more Bt toxins assume that independent mutations are required to counter each toxin . Moreover, it generally is assumed that resistance alleles are rare in susceptible populations . We tested these assumptions by conducting single-pair crosses with diamondback moth (Plutella xylostella), the first insect known to have evolved resistance to Bt in open field populations . An autosomal recessive gene conferred extremely high resistance to four Bt toxins (Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F) . The finding that 21% of the individuals from a susceptible strain were heterozygous for the multiple-toxin resistance gene implies that the resistance allele frequency was 10 times higher than the most widely cited estimate of the upper limit for the initial frequency of resistance alleles in susceptible populations . These findings suggest that pests may evolve resistance to some groups of toxins much faster than previously expected.

Int J Food Microbiol, 1997 Mar 3, 34(3), 307 - 18
Isolation and characterisation of Bacillus cereus from pasteurised milk in household refrigerators in The Netherlands; Te Giffel MC et al.; The incidence and some characteristics (carbohydrate metabolism, growth profiles, haemolysin production and enterotoxin production) of Bacillus cereus, in pasteurised, low-fat (1.5%) milk, in household refrigerators in the Netherlands was investigated . In 247 (74%) of the 334 milk samples analyzed, the mesophilic aerobic counts were between 50 and 5000 per millilitre . B . cereus could be isolated from 133 (40%) of the samples . In general the B . cereus counts were low; numbers of less than five per millilitre were observed in 258 (77%) of the samples . As expected, both the mesophilic aerobic counts and levels of B . cereus increased with increasing storage temperatures in the refrigerator and prolonged storage times . In total, 143 presumptive B . cereus colonies were isolated . According to the ISO confirmation tests and the carbohydrate patterns (API 50 CHB) 134 (94%) of these isolates were confirmed to be B . cereus . Of these 134 isolates 20% fermented lactose and 53% of the 106 strains tested were able to grow at 7 degrees C . These percentages are much higher than expected for strains isolated from non-dairy products, suggesting that strains can adapt to environmental conditions in milk . All 106 strains tested, produced haemolysin, 27% showed the discontinuous haemolytic pattern characteristic for haemolysin BL, possibly a virulence factor . Of the 37 B . cereus isolates tested for enterotoxin production 27 (73%), 28 (76%) and 26 (70%) were found to be enterotoxigenic (as determined by the Western immunoblot technique, polymerase chain reaction (PCR) and Vero cell assays, respectively) . Isolates unable to ferment lactose, produced less enterotoxin in comparison with those able to utilize lactose . Although only a few outbreaks of food poisoning caused by B . cereus in milk (products) have been reported, most strains isolated from these products are able to produce enterotoxins and may represent a health hazard.

Rev Clin Esp, 1997 Mar, 197(3), 152 - 7
{Tuberculosis outbreak at a public school}; Navarro Gracia JF et al.; BACKGROUND: At school there are special circumstances of living together and a particular susceptibility, which favour the emergence of tuberculosis microepidemics . We report here the microepidemic occurred at a school among 9-year old children . METHODS: After ruling out a possible familiar source in a child with pulmonary tuberculosis, we detected a case with high bacillar shedding in a female teacher and conducted a tuberculin search among children and teachers, initially outlining the theoretical groups at risk . Tuberculin positive children underwent chest-X-ray and when abnormalities were found, children were derived to the pediatrician for chemotherapy . All converters received secondary chemoprophylaxis and all non-respondents primary chemoprophylaxis . RESULTS: The classroom where the teacher spent most of het time had a higher rate of converters (70%) than other classroom, where the index teacher spent only a partial time (40%; RR: 1.75; CI: 1.06-2.88) or the collective of teachers (45.4%; RR: 1.45; CI: 0.94-2.23) . Three additional cases of secondary disease were detected, all of them children . The initial compliance with chemoprophylaxis was greater among (for) children (97.0%) than among teachers (41.6%) . Among children there was one case of tuberculin conversion compared with three cases among teachers . No additional cases were detected; also, an abnormal rate of reactors outside the initially studied groups was also not detected . CONCLUSIONS: Our results somehow agree with those reported from other school outbreaks . To note the anergy and lack of symptoms in the index case and the suggestion to delineate the degree of spending hours together to identify groups with a higher theoretical risk of being infected . Thus, an unnecessary expense of resources and a social alarm would be avoided.

Genetika, 1997 Mar, 33(3), 308 - 13
{Extrachromosomal DNA spectrum of Bacillus thuringiensis Berliner and Bacillus sphaericus Meyer et Neide}; Chichian VG et al.; Analysis of the pattern of extrachromosomal DNA in different cultures of Bacillus thuringiensis and Bacillus sphaericus demonstrated a higher content of extrachromosomal DNA in B . thuringiensis than in B . sphaericus . The quantity and approximate molecular weights of the plasmids were determined . The assumption that the plasmid DNA content in B . thuringiensis strains is higher than in the other representatives of the genus Bacillus was confirmed.

Mikrobiologiia, 1997 Mar-Apr, 66(2), 247 - 53
{Restriction analysis of DNA from phages isolated from type strains of Bacillus thuringiensis}; Azizbekian KR et al.; Comparative analysis of the DNA of 11 phages isolated from type strains of Bacillus thuringiensis was performed by means of digestion with restriction nucleases . According to the results obtained, the studied phages were classified into five groups . Within each group, complete identity was revealed in both the restriction fragment number and the molecular mass of individual fragments . No homology was observed between genome structures of phages assigned to different groups.

Mikrobiologiia, 1997 Mar-Apr, 66(2), 242 - 6
{Morphologic characterization and protein analysis of phages isolated from type strains of Bacillus thuringiensis}; Azizbekian KR et al.; The lysogeny of eleven type strains of Bacillus thuringiensis was studied . Eight new phages were isolated from the variants B . thuringiensis var . tochigiensis, yunnanensis, colmeri, shandongiensis, neoleonensis, silo, mexicanensis, and toguchini belonging to the B1 morphotype . The phages that were isolated from B . thuringiensis var . tochigiensis, yunnanensis, shandongiensis, and mexicanensis possessed transverse disks at their tails and were classified into one morphological group . The protein patterns of the isolated phages were determined.

Int J Urol, 1997 Mar, 4(2), 139 - 43
Recurrence of primary superficial bladder cancer treated with prophylactic intravesical Tokyo 172 bacillus Calmette-Guérin: a long-term follow-up; Shinka T et al.; BACKGROUND: Long-term results after transurethral resection (TUR) and prophylactic intravesical Tokyo 172 bacillus Calmette-Guerin (BCG) therapy for primary superficial bladder cancer were analyzed by multivariate analysis, and factors affecting the recurrence of bladder tumors after this therapy were examined . METHODS: One-hundred and forty-one consecutive patients with primary superficial bladder cancer who consulted the Department of Urology at Wakayama Medical College and affiliated hospitals between May 1985 and May 1990 were studied . Tokyo strain BCG was given intravesically (80 mg in 40 ml saline) weekly for 6 weeks . RESULTS: The 5-year cumulative recurrence-free rate by the Kaplan-Meier method was 0.702 in 141 patients with primary superficial bladder cancer . The 5-year recurrence-free function using the proportional hazard model was 0.743 . Using the Cox proportional hazard model, variables that significantly contributed to recurrence after intravesical BCG included female sex, tumor size less than 1 cm in diameter, and T1 tumor stage . Patient age, tumor type, multiplicity, tumor grade, and concomitant carcinoma in situ did not contribute to recurrence . CONCLUSION: Long-term results showed that prophylactic intravesical Tokyo strain BCG after TUR for primary superficial bladder cancer is also effective in preventing the recurrence of bladder cancer, and the biologic behavior of superficial bladder cancer other than stage T1 tumor may be altered after intravesical BCG.

Biol Chem, 1997 Mar-Apr, 378(3-4), 327 - 9
Crystallographic analysis of phosphoglycerate kinase from the hyperthermophilic bacterium Thermotoga maritima; Auerbach G et al.; Phosphoglycerate kinase from the hyperthermophilic bacterium Thermotoga maritima has been co-crystallized with its substrate 3-phosphoglycerate and the ATP analogue AMP-PNP using the vapour diffusion method . Crystals were obtained from a solution containing polyethylene glycol (MW 3000/8000) as precipitating agent . A complete diffraction data set from orthorhombic crystals was collected up to 2.0 A resolution . The TmPGK crystallizes in the space group P2(1)2(1)2 (cell dimensions: a = 62.0 A, b = 76.9 A, c = 87.5 A) with one molecule in the asymmetric unit . The structure was solved by Patterson search methods using Bacillus stearothermophilus PGK as a search model and was refined to a crystallographic R factor of 22.0% . Compared to the enzyme from B . stearothermophilus, horse, pig and yeast, the Thermotoga enzyme exhibits a drastically reduced interdomain angle, similar to the one reported for PGK from Trypanosoma brucei . Here we present crystallographic data of the first high-resolution structure of a PGK in largely closed conformation, complexed with the two products of the catalyzed reaction, and, at the same time, the first PGK structure from a hyperthermophilic organism.

Protein Eng, 1997 Mar, 10(3), 273 - 8
RNA cleavage without hydrolysis . Splitting the catalytic activities of binase with Asn101 and Thr101 mutations; Okorokov AL et al.; Members of the microbial guanyl-specific ribonuclease family catalyse the endonucleolytic cleavage of single-stranded RNA in a two-step reaction involving transesterification to form a 2',3'-cyclic phosphate and its subsequent hydrolysis to yield the respective 3'-phosphate . The extracellular ribonuclease from Bacillus intermedius (binase, RNase Bi) shares a common mechanism for RNA hydrolysis with mammalian RNases . Two catalytic residues in the active site of binase, Glu72 and His101, are thought to be involved in general acid-general base catalysis of RNA cleavage . Using site-directed mutagenesis, binase mutants were produced containing amino acid substitutions H101N and H101T and their catalytic properties towards RNA, poly(I), poly(A), GpC and guanosine 2',3'-cyclic phosphate (cGMP) substrates were studied . The engineered mutant proteins are active in the transesterification step which produces the 2',3'-cyclic phosphate species but they have lost the ability to catalyse hydrolysis of the cyclic phosphate to give the 3' monophosphate product.

J Am Mosq Control Assoc, 1997 Mar, 13(1), 87 - 9
Use of Bactimos briquets (B.t.i . formulation) combined with the backswimmer Notonecta irrorata (Hemiptera:Notonectidae) for control of mosquito larvae; Neri-Barbosa JF et al.; The efficacies of Bacillus thuringiensis var . israelensis (Bactimos briquets) and the backswimmer Notonecta irrorata were evaluated both individually and in combination to control mosquito larvae in plastic containers in Monterrey, Mexico . The combined strategy proved to be the most effective one.

Intern Med, 1997 Mar, 36(3), 221 - 6
Fulminant septicemic syndrome of Bacillus cereus in a leukemic patient; Akiyama N et al.; We report a rapidly fatal Bacillus cereus septicemia in a leukemic patient receiving remission-induction therapy . Symptoms resembling food poisoning and fever preceded coma accompanied by neurologic abnormalities . Autopsy revealed necrotizing leptomeningitis with subarachnoid hemorrhage and coagulation necrosis of the liver with bacterial infiltration . These clinicopathologic findings were closely similar to those of reported cases . Because of a rapidly fatal clinical course, suspicion of this syndrome early in the course is important to determine an appropriate treatment . Therefore, we propose that this type of septicemia should be termed as fulminant septicemic syndrome of Bacillus cereus.

Jpn J Cancer Res, 1997 Mar, 88(3), 281 - 8
Bacillus Calmette-Guérin enhances production and secretion of type IV collagenases in peripheral blood mononuclear cells; Kageyama Y et al.; Intravesical administration of bacillus Calmette-Guerin (BCG) is an effective and widely accepted treatment for superficial bladder cancer . Rapid progression of the disease after BCG therapy, however, has been reported in some cases refractory to the treatment . We examined whether BCG treatment and coexistence of peripheral blood mononuclear cells (PBMCs) alter the invasive potential of bladder cancer cells . Production and secretion of two type IV collagenases, matrix metalloproteinase (MMP) 2 and MMP 9, by PBMCs from five healthy donors or bladder cancer cells (T24, JTC 30, and JTC 32) were evaluated by gelatin zymography, western blot analysis, and northern blot analysis . Invasion of bladder cancer cells was also examined using reconstituted basement membrane (Matrigel) . BCG (5, 50, and 500 micrograms/ml) had no effect on secretion of MMP 2 and MMP 9 by bladder cancer cells, but increased the production and secretion of MMP 9 by PBMCs in a dose-dependent manner . The coexistence of PBMCs increased invasion of T24 cells and BCG further enhanced the invasion . Thus, BCG promotes invasion of bladder cancer cells under certain conditions . An increase in the secretion of MMP 9 by PBMCs may account in part for the effect.

J Antibiot (Tokyo), 1997 Mar, 50(3), 220 - 8
Fusaricidins B, C and D, new depsipeptide antibiotics produced by Bacillus polymyxa KT-8: isolation, structure elucidation and biological activity; Kajimura Y et al.; Fusaricidins B, C and D, new depsipeptide antibiotics, have been isolated as minor components from the culture broth of Bacillus polymyxa KT-8 which was obtained from the rhizosphere of garlic suffering from the basal rot caused by Fusarium oxysporum . The structure of fusaricidin B has been elucidated mainly by various NMR experiments coupled with amino acid analysis in relation to fusaricidin A, the main component of the complex, whose structure was reported previously . The fraction consisting of fusaricidins C and D was unsuccessfully separated, giving roughly a 4:1 mixture of the two, respectively . The structures of fusaricidins C and D have been determined within the mixture by detailed analyses of the 2D NMR spectra . Fusaricidins B, C and D are active against fungi and Gram-positive bacteria almost as well as fusaricidin A.

Eur J Biochem, 1997 Mar 1, 244(2), 426 - 33
Properties of an NAD(H)-containing methanol dehydrogenase and its activator protein from Bacillus methanolicus; Arfman N et al.; Oxidation of C1-C4 primary alcohols in thermotolerant Bacillus methanolicus strains is catalyzed by an NAD-dependent methanol dehydrogenase (MDH), composed of ten identical 43,000-Mr subunits . Each MDH subunit contains a tightly, but non-covalently, bound NAD(H) molecule, in addition to 1 Zn2+ and 1-2 Mg2+ ions . The NAD(H) cofactor is oxidized and reduced by formaldehyde and methanol, respectively, while it remains bound to the enzyme . Incubation of MDH with methanol and exogenous NAD (coenzyme) results in reduction of this NAD coenzyme . Both NAD species are not exchanged during catalysis . NAD thus plays two different and important roles in the MDH-catalyzed reaction, with the bound NAD cofactor acting as primary electron acceptor and the NAD coenzyme being responsible for reoxidation of the reduced cofactor . MDH obeys a ping-pong type reaction mechanism, which is consistent with such a temporary parking of reducing equivalents at the MDH-bound cofactor . Spectral studies show that, in the presence of exogenous NAD and Mg2+ ions, MDH interacts with a previously identified 50,000-Mr activator protein . The activator protein appears to facilitate the oxidation of the reduced NADH cofactor of MDH, which results in a strongly increased turnover rate of MDH.

Eur J Biochem, 1997 Mar 1, 244(2), 361 - 70
Steady-state and picosecond-time-resolved fluorescence studies on the recombinant heme domain of Bacillus megaterium cytochrome P-450; Khan KK et al.; The conformational changes associated with the interaction of sodium laurate with the recombinant heme domain for cytochrome P-450BM3 have been investigated by steady-state and picosecond-time-resolved fluorescence spectroscopy . The steady-state quenching experiments show that while all the five tryptophan residues are accessible to acrylamide in the free enzyme as well as the enzyme x substrate complex, the number of tryptophan residues accessible to ionic quenchers decreases on interaction of the substrate with the enzyme . This indicates that some of the tryptophan residues move towards the core of the protein on interaction with the substrate . The number of tryptophan residues accessible to the solvent as determined by the calculation of the solvent-accessible area for the free enzyme agrees with the values obtained by the quenching experiments . The time-resolved fluorescence studies carried out by means of the time-correlated single-photon-counting technique show that the fluorescence-decay curve is best fitted to a three-exponential model (0.2, 1.0 and 5.4 ns) . Lifetime distributions, as recovered by the maximum-entropy method, agree with the discrete exponential model . The binding of the substrate does not lead to any significant change in the lifetime components of the enzyme, indicating that the tryptophan residues are possibly away from the substrate-binding domain . The decay-associated emission spectra and the magnitudes of amplitude of different lifetimes indicate that the shortest lifetime component (tau1) originates from the three tryptophan residues that are completely or partially accessible to the solvent, and tau2 originates from the tryptophan residues that are buried in the core of the enzyme and not accessible to the solvent . X-ray crystallographic data and solvent-acessible-area calculations have been used to identify these residues.

Br J Urol, 1997 Mar, 79(3), 373 - 7
Nuclear accumulation of mutant p53 protein: a possible predictor of failure of intravesical therapy in bladder cancer; Caliskan M et al.; OBJECTIVE: To investigate the expression and importance of the nuclear accumulation of p53 in superficial transitional cell carcinoma (TCC) of the bladder and its role as a predictor of response to treatment . PATIENTS AND METHODS: Tumour samples from 30 patients (two women and 28 men, mean age 60.1 years, range 44-75) with pTa/pT1 tumours were assessed immunohistochemically using the Pab1801 monoclonal antibody and standard avidin-biotin peroxidase staining for p53 protein . RESULTS: Tumours from six patients (20%) showed nuclear accumulation of p53; five of these patients failed intravesical therapy with bacille Calmette-Guerin (BCG) and progressed to muscle invasive and/or metastatic disease, in contrast to six of 24 patients with no detectable nuclear oncoprotein . CONCLUSIONS: The nuclear accumulation of p53 appears to be a prognostic indicator of tumour unresponsive to intravesical treatment, even with the most potent agent (BCG) . Therefore, early radical treatment modalities must be seriously considered in this group of patients.

Appl Microbiol Biotechnol, 1997 Mar, 47(3), 262 - 6
Cloning of the Bacillus pumilus beta-xylosidase gene (xynB) and its expression in Saccharomyces cerevisiae; La Grange DC et al.; A genomic DNA library of the bacterium Bacillus pumilus PLS was constructed and the beta-xylosidase gene (xynB) was amplified from a 3-kb genomic DNA fragment with the aid of the polymerase chain reaction technique . The amplified xynB gene was inserted between the yeast alcohol dehydrogenase II gene promoter (ADH2P) and terminator (ADH2T) sequences on a multicopy episomal plasmid (pDLG11) . The xynB gene was also fused in-frame to the secretion signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1S) before insertion between the ADH2P and ADH2T sequences on a similar multicopy episomal plasmid (pDLG12) . The resulting construct ADH2P-MF alpha 1S-xynB-ADH2T was designated XLO1 . Both plasmids pDLG11 and PDLG12 were introduced into Saccharomyces cerevisiae but only the expression of the XLO1 gene yielded biologically functional beta-xylosidase . The total beta-xylosidase activity remained cell-associated with a maximum activity of 0.09 nkat/ml obtained when the recombinant S . cerevisiae strain was grown for 143 h in synthetic medium . The temperature and pH optima of the recombinant Xlo1 enzyme were 45-50 degrees C and pH 6.6 respectively . The enzyme was thermostable at 45 degrees C; however, at 60 degrees C most of the Xlo1 was inactive after 5 min.

Appl Microbiol Biotechnol, 1997 Mar, 47(3), 255 - 61
Large-scale production and characterization of Bacillus thuringiensis subsp . tenebrionis insecticidal protein from Escherichia coli; Gustafson ME et al.; Bacillus thuringiensis subsp . tenebrionis insecticidal protein was produced in recombinant Escherichia coli and purified to near homogeneity to provide quantities of protein for safety-assessment studies associated with the registration of transgenic potato plants . The 68-kDa protein is produced naturally by Bacillus thuringiensis subsp . tenebrionis by translation initiation at an internal initiation site in the native DNA sequence . The gene sequence specific for this truncated protein was expressed in E . coli strain JM 101 and fermented at the 1000-1 scale . The protein accumulated as insoluble inclusion bodies, and was purified by extraction at pH 10.8 with carbonate buffer, selective precipitation at pH 9.0, and differential centrifugation . No chromatography steps were required to produce over 50 g purified protein as a lyophilized powder with a purity greater than 95% and demonstrating full insecticidal activity against Colorado potato beetle larvae . The protein was further characterized to assure identity and suitability for use in safety-assessment studies.

Cancer Immunol Immunother, 1997 Mar, 44(1), 35 - 40
Effects of colony-stimulating factors on cellular cytotoxicity; Durek C et al.; Colony-stimulating factors (CSF) are used clinically in the treatment of chemotherapy-induced myelosuppression and in support of bone marrow transplantation . As CSF are known to have pleiotropic functions, their effects on cellular cytotoxicity were analysed in vitro against bladder carcinoma cell lines . By means of an L-{3H}methionine-release assay, the cytotoxicity of peripheral blood mononuclear cells against the natural-killer(NK)-cell-resistant bladder carcinoma cell lines BT-A and SBC-7 was measured using different effector/target-cell ratios . Costimulatory effects of granulocyte-colony-stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and stem cell factor (SCF) on the generation of lymphokine-activated killer (LAK), bacillus Calmette-Guerin-activated killer (BAK) and natural killer (NK) cell cytotoxicity were investigated in this assay . Furthermore, the effect of CSF on proliferation of urothelial tumor cells in vitro was determined by a {3H}thymidine DNA-labelling technique . GM-CSF, but not G-CSF, IL-3 or SCF, was able to increase NK, BAK and LAK cytotoxicity in a dose-dependent manner . No acceleration of carcinoma cell proliferation was evident under the conditions of our assay . These data indicate the costimulatory effect of GM-CSF on cellular cytotoxicity, which might be used for immunotherapeutic purposes.

Biosci Biotechnol Biochem, 1997 Mar, 61(3), 520 - 4
Structural study of an exocellular polysaccharide of Bacillus circulans; Isobe Y et al.; Bacillus circulans, a soil bacterium, produced an exocellular polysaccharide of high viscosity . On the basis of the results of methylation analysis, mild acid hydrolysis, and 1D and 2D 1H-NMR spectroscopy, it was concluded that the polysaccharide has a basic repeating unit composed of beta-L-rhamnopyranose, alpha-D-mannopyranose, alpha-D-galactopyranose, and alpha-D-glucopyranuronic acid with the following structure . {symbol: see text}

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