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| Biochemistry, 1987 Jun 2, 26(11), 3010 - 6 Catalytic versatility of Bacillus pumilus beta-xylosidase: glycosyl transfer and hydrolysis promoted with alpha- and beta-D-xylosyl fluoride; Kasumi T et al.; Bacillus pumilus beta-xylosidase, an enzyme considered restricted to hydrolyzing a narrow range of beta-D-xylosidic substrates with inversion of configuration, was found to catalyze different stereochemical, essentially irreversible, glycosylation reactions with alpha- and beta-D-xylopyranosyl fluoride . The enzyme promoted the hydrolysis of beta-D-xylopyranosyl fluoride at a high rate, V = 6.25 mumol min-1 mg-1 at 0 degrees C, in a reaction that obeyed Michaelis-Menten kinetics . In contrast, its action upon alpha-D-xylopyranosyl fluoride was slow and characterized by an unusual relation between the rate of fluoride release and the substrate concentration, suggesting the possible need for two substrate molecules to be bound at the active center in order for reaction to occur . Moreover, 1H NMR spectra of a digest of alpha-D-xylosyl fluoride showed the substrate to be specifically converted to alpha-D-xylose by the enzyme . The observed retention of configuration is not consistent with direct hydrolysis by this "inverting" enzyme but is strongly indicative of the occurrence of two successive inverting reactions: xylosyl transfer from alpha-D-xylosyl fluoride to form a beta-D-xylosidic product, followed by hydrolysis of the latter to produce alpha-D-xylose . The transient intermediate product formed enzymically from alpha-D-xylosyl fluoride in the presence of {14C}xylose was isolated and shown by its specific radioactivity and 1H NMR spectrum as well as by methylation and enzymic analyses to be 4-O-beta-D-xylopyranosyl-D-xylopyranose containing one {14C}xylose residue.(ABSTRACT TRUNCATED AT 250 WORDS) Br J Urol, 1987 Jun, 59(6), 554 - 8 Intravesical BCG therapy in the management of multiple superficial bladder carcinoma . Comparison between Glaxo and Pasteur strains; Kaisary AV; Intravesical Bacillus Calmette-Guerin (BCG) therapy in the treatment and prophylaxis of superficial bladder cancer has shown encouraging results in North America but disappointing results have been reported in Great Britain . This was attributed to differences in quality between BCG (Glaxo strain) used in Great Britain and BCG (Pasteur and Tice strains) used in North America . Twenty-one patients with multiple superficial recurrent transitional cell carcinoma of the bladder were entered into a pilot study comparing the efficacy of BCG Glaxo (Evans Medical Ltd, Dunstable, UK) and Pasteur (L'Institut Pasteur, France) strains . Comparable and encouraging results were demonstrated, indicating that this mode of therapy is effective. Ann Cardiol Angeiol (Paris), 1987 Jun, 36(6), 297 - 300 {Streptobacillus moniliformis endocarditis . Apropos of 2 cases}; Rey JL et al.; The authors report two cases of endocarditis secondary to Streptobacillus moniliformis . A 41 year-old man, bitten by a rat, is hospitalized 5 weeks later for an endocarditis demonstrated by echocardiography, with massive aortic escape and hemodynamic failure requiring emergency valve replacement: after a favorable course, the patient dies suddenly 4 months later . A 63 year-old woman is admitted for a septicemic syndrome with sterno-clavicular arthritis which occurred 10 days after a rat bite; followed by a transient ischemic cerebral vascular accident; echocardiogram shows a clubshaped bulge of the distal end of the large mitral valve; the course is uneventful under antibiotherapy . In both cases, blood cultures isolate a Streptobacillus moniliformis . Infections secondary to Streptobacillus moniliformis are rare; this Gram negative bacillus, saprophyte of the rat's rhinopharynx, is transmitted to man, most of the time, by bite, and this causes a septicemia, the evolution of which is usually favorable . Complications, especially endocarditis, are exceptionally rare: only 12 cases are found in the world's literature . The evolution is always fatal in the absence of treatment which must include the association penicillin-aminoside . Prophylaxis of this disease is provided by penicillin antibiotherapy which should be systematic after a rodent's bite. J Dairy Sci, 1987 Jun, 70(6), 1148 - 51 Effect of plate preincubation on Bacillus stearothermophilus disc assay zone diameters; Ryan JJ et al.; Effect of plate preincubation on the Bacillus stearothermophilus disc assay zone size was studied . Each of three raw milk samples were subdivided and treated with five levels of US Pharmacopeial reference standard penicillin G . Freshly prepared, 2.5- and 5-d old seeded disc assay culture plates were preincubated at 64 degrees C for 0 (control), 20, 40, 60, 80, 100, and 120 min prior to placement of discs on the medium surface . Plate age did not have a significant effect on the size of the zone diameter . Following 20 and 40 min of plate preincubation, zone diameters were in some, but not all, cases significantly different from the control . Plate preincubation of 60 min or greater always significantly decreased zone diameter . Routine preincubation of B . stearothermophilus plates is not recommended . In situations where time is a critical factor, plates should be preincubated no longer than 40 min. Appl Environ Microbiol, 1987 Jun, 53(6), 1316 - 21 Purification of the mosquitocidal and cytolytic proteins of Bacillus thuringiensis subsp . israelensis; Hurley JM et al.; Two proteins from parasporal crystals of Bacillus thuringiensis subsp . israelensis were purified to electrophoretic homogeneity by gel filtration and anion-exchange chromatography . The larger of the two proteins (molecular weight, 68,000) was not cytolytic, whereas the smaller protein (molecular weight, 28,000) was highly cytolytic when assayed against rat erythrocytes . When these proteins were assayed against larvae of the yellow fever mosquito, Aedes aegypti, the larger protein was at least 100-fold more toxic than the smaller protein . Although proteolytic activity was not detected in solubilized crystals nor in purified protein preparations, the toxin (molecular weight, 68,000) was readily degraded to smaller, nontoxic molecules, even when maintained at 4 degrees C . Mixtures of the two purified proteins were significantly more toxic to mosquito larvae than was either protein alone . Thus, it is likely that both the mosquitocidal and the cytolytic protein play roles in the overall insecticidal action of the parasporal crystal produced by this bacterium. Int J Lepr Other Mycobact Dis, 1987 Jun, 55(2), 273 - 6 Cold reactive lymphocytotoxic antibodies in patients with tuberculoid and lepromatous leprosy; Naik S et al.; Fifty-seven sera from leprosy patients and 33 sera from age- and sex-matched hospital controls were tested for the presence of cold-reacting lymphocytotoxic antibodies (LCAs) at 15 degrees C against a panel of 30 HLA-typed normal lymphocytes . Eighteen of 57 (31.6%) leprosy sera and 22 of 33 (67%) control sera showed reactivity, but the strength of reactivity of the patients' sera was significantly more than that of the control group (p less than 0.01 by Wilcoxon rank sum test) . Within the leprosy group, there was no significant difference in the reactivity of 30 tuberculoid and 27 lepromatous sera . The occurrence of LCAs was independent of the sex or the HBsAg status of the serum donor . LCA activity was not correlated with treatment status, bacillary load, or reaction state. J Bacteriol, 1987 Jun, 169(6), 2804 - 9 Charge distribution on the S layer of Bacillus stearothermophilus NRS 1536/3c and importance of charged groups for morphogenesis and function; Sara M et al.; The distribution and functional significance of charged groups on the outer and inner faces of the S layer from Bacillus stearothermophilus NRS 1536/3c was investigated . Chemical modification of the exposed amino or carboxyl groups was performed on whole cells, isolated S layers self-assembled in vitro, and cell wall fragments (S layer attached to the peptidoglycan-containing sacculus) . Without chemical modification, S layer self-assembly products could be labeled with polycationic ferritin, while S layers on whole cells could not . Following treatment with glutaraldehyde, whole cells were uniformly labeled with polycationic ferritin . Whole cells treated with glutaraldehyde and glycine methyl ester in the presence of carbodiimide did not bind polycationic ferritin significantly above background . Treatment of cell wall fragments with amino-specific, homobifunctional cross-linkers or with carbodiimide alone rendered the S layer protein nonextractable with sodium dodecyl sulfate . After amidation of the accessible carboxyl groups, the modified, guanidine hydrochloride-extractable S layer protomers did not self-assemble into regularly structured lattices . N-Amidination with ethylacetimidate did not interfere with the self-assembly of the isolated protomers . N-Acetylation resulted in a considerable destabilization of the S layer lattice, as seen by the release of a large amount of modified protomers during the reaction . N-Succinylation led to a complete disintegration of the protein lattice . These results indicated that only the inner face of the S layer carried a net negative charge . On both faces, free amino and carboxyl groups of adjacent protomers were arranged in proximity so as to contribute by electrostatic interactions to the cohesion of the protomers in the two-dimensional array . The native charge of the protomers was required for both the in vitro self-assembly of the isolated subunits and the maintenance of the structural integrity of the S layer lattice . Among other functions, the biological significance of the S layers may be in masking the electronegative charge of the cell wall proper. J Am Mosq Control Assoc, 1987 Jun, 3(2), 201 - 10 Evaluation of larvicides for the control of Simulium damnosum s.l . (Diptera: Simuliidae) in West Africa; Kurtak D et al.; The Onchocerciasis Control Program of the World Health Organization is carrying out an extensive screening program in a search for new larvicides to be used for control of Simulium damnosum s.l . Emphasis has been given to finding a pyrethroid and a carbamate to supplement the organophosphates currently in use . These chemicals with differing modes of action, together with Bacillus thuringiensis H-14, are being used in an attempt to cope with the development and spread of resistance to the organophosphates temephos and chlorphoxim. J Urol, 1987 Jun, 137(6), 1270 - 3 Reduction of bladder cancer growth in mice treated with intravesical Bacillus Calmette Guerin and systemic interleukin 2; Lee KE et al.; The effect of systemic administration of Interleukin 2 (IL2) on intravesical Bacillus Calmette-Guerin (BCG) therapy was studied in an established murine bladder tumor, MBT-2 . BCG (100 micrograms.) was administered intravesically on days 7 and 14 after seeding bladders with MBT-2 cells . IL2 (5,000 U/injection) was given intraperitoneally every eight hours for 10 times (days seven through 10 and 14 through 17) . BCG or IL2 therapy alone failed to reduce incidence of tumor implantation and tumor weight; whereas, combined treatment with BCG and IL2 reduced tumor weight significantly compared to saline or BCG treated mice . Cytotoxicity was assessed in a four-hour 75Semethionine-release assay . Augmentation of natural killer cell activity was only observed in mice treated with BCG plus IL2 . MBT-2 target cells were not lysed by spleen cells from mice treated with BCG or saline . IL2 therapy produced lymphokine-activated killer cell activity, though combining BCG with IL2 suppressed this activity after each course of treatment . The results suggest that combined treatment with IL2 enhances the therapeutic effect of BCG therapy . However, this enhancement of antitumor activity is not clearly explained by augmentation of natural killer or in vivo-generated lymphokine-activated killer cells. Mol Gen Genet, 1987 Jun, 208(1-2), 63 - 9 Chemical synthesis and in vivo hyperexpression of a modular gene coding for Escherichia coli translational initiation factor IF1; Calogero RA et al.; An artificial gene encoding the Escherichia coli translational initiation factor IF1 was synthesized based on the primary structure (71 amino acid residues) of the protein . Codons for individual amino acids were selected on the basis of the preferred codon usage found in the structural genes for the initiation factor IF2 of E . coli and Bacillus stearothermophilus, both of which can be expressed at high levels in E . coli cells . We gave the IF1 gene a modular structure by introducing specific restriction enzyme sites into the sequence, resulting in units of three to ten codons . This was conceived to facilitate site-directed mutagenesis of the gene and thus to obtain IF1 with specific amino acid alterations at desired positions . The IF1 gene was assembled by shot-gun ligation of 9 synthetic oligodeoxyribonucleotides ranging in size from 31 to 65 nucleotides and cloned into an expression vector to place the gene under the control of an inducible promoter . Upon induction, E . coli cells harbouring the artificial gene were found to produce large amounts (greater than or equal to 60 mg/100 g cells) of a protein indistinguishable from natural IF1 in both chemical and biological properties. Appl Environ Microbiol, 1987 Jun, 53(6), 1251 - 6 Cytolytic activity and immunological similarity of the Bacillus thuringiensis subsp . israelensis and Bacillus thuringiensis subsp . morrisoni isolate PG-14 toxins; Gill SS et al.; The parasporal bodies of the mosquitocidal isolates of Bacillus thuringiensis subsp . israelensis and B . thuringiensis subsp . morrisoni isolate PG-14 were compared with regard to their hemolytic and cytolytic activities and the immunological relatedness of the 28- and 65-kilodalton (kDa) proteins that occur in both subspecies . The alkali-solubilized parasporal bodies of B . thuringiensis subsp . israelensis caused 50% lysis of human erythrocytes at 1.14 micrograms/ml, whereas those of B . thuringiensis subsp . morrisoni caused similar lysis at 1.84 micrograms/ml . Preincubation of solubilized parasporal bodies with dioleolyl phosphatidylcholine significantly inhibited the hemolytic activity of both supspecies . In cytolytic assays against Aedes albopictus cells, the toxin concentrations causing 50% lysis for B . thuringiensis subsp . israelensis and B . thuringiensis subsp . morrisoni were 1.87 and 11.98 micrograms/ml, respectively . Polyclonal antibodies raised separately against the 25-kDa protein (a tryptic digest of the 28-kDa protein) or the 65-kDa protein of B . thuringiensis subsp . israelensis cross-reacted, respectively, with the 28- and the 65-kDa proteins of B . thuringiensis subsp . morrisoni . However, neither of these antibodies cross-reacted with the 135-kDa protein of either subspecies . These results indicate that the mosquitocidal and hemolytic properties of B . thuringiensis subsp . israelensis and B . thuringiensis subsp . morrisoni isolate PG-14 are probably due to the biologically related proteins that are present in the parasporal bodies of both subspecies . The lower hemolytic activity of the B . thuringiensis subsp . morrisoni may be due to the presence of lower levels of the 28-kDa protein in that subspecies.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Response Mod, 1987 Jun, 6(3), 313 - 30 Tumoricidal effector mechanisms of murine BCG-activated macrophages . I . Parameters of production and initial characterization of a cytolytic factor serologically related to necrosin; Klostergaard J et al.; Previous studies have shown that peritoneal macrophages from mice chronically infected with Bacillus Calmette-Guerin (BCG) become highly cytotoxic for tumor targets upon further in vitro triggering with a variety of agents . In the current studies, achievement of this activated state was characterized by the production and release of a cytotoxin, herein termed cytolytic factor (CF), which appeared in the fluid phase . Production/release of CF by the macrophage required transcription, translation, glycosylation, and an intact secretory apparatus, as evident from inhibition by treatment with actinomycin-D, cycloheximide, tunicamycin, and monensin, respectively, prior to and during triggering with lipopolysaccharide (LPS) . CF obtained by culture of BCG-activated macrophages appeared rapidly in the supernatant after triggering . Using the actinomycin-D-treated L-929 or EMT-6 targets in a microassay, CF secreted by macrophages cultured in low molecular weight serum components was detected as an approximately 150-kD component on Sephacryl S-200 . CF demonstrated a spectrum of cytotoxic activity against a number of tumor and normal targets in vitro . Its lytic activity appeared equally effective whether the targets were cultured in medium containing fetal calf serum (FCS) or in Neuman-Tytell medium without serum during the assay . CF was moderately sensitive to treatment with TLCK and TAME; however, its activity in serum and apparent molecular weight distinguish it from a moiety obtained from BCG-activated murine macrophages and previously described . A rabbit heteroantiserum raised against highly purified necrosin, a product of the murine macrophage cell line J774.1, was extremely effective in neutralizing the biological activity of CF. DNA, 1987 Jun, 6(3), 255 - 65 Molecular cloning of an amylase gene of Bacillus circulans; Nishizawa M et al.; An amylase gene of Bacillus circulans was cloned in B . subtilis and its nucleotide sequence was determined . The putative proamylase consists of 528 amino acids, which correspond to a molecular weight of 58,776 . Homologous regions with other amylases of Bacillus species were found . A sigma 55-type promoter is located at about 250 bp upstream from the starting codon . This promoter was also functional in Escherichia coli, and able to express beta-galactosidase activity. J Bacteriol, 1987 Jun, 169(6), 2762 - 8 Novel alkaline- and heat-stable serine proteases from alkalophilic Bacillus sp . strain GX6638; Durham DR et al.; An alkalophilic Bacillus sp., strain GX6638 (ATCC 53278), was isolated from soil and shown to produce a minimum of three alkaline proteases . The proteases were purified by ion-exchange chromatography and were distinguishable by their isoelectric point, molecular weight, and electrophoretic mobility . Two of the proteases, AS and HS, which exhibited the greatest alkaline and thermal stability, were characterized further . Protease HS had an apparent molecular weight of 36,000 and an isoelectric point of approximately 4.2, whereas protease AS had a molecular weight of 27,500 and an isoelectric point of 5.2 . Both enzymes had optimal proteolytic activities over a broad pH range (pH 8 to 12) and exhibited temperature optima of 65 degrees C . Proteases HS and AS were further distinguished by their proteolytic activities, esterolytic activities, sensitivity to inhibitors, and their alkaline and thermal stability properties . Protease AS was extremely alkali stable, retaining 88% of initial activity at pH 12 over a 24-h incubation period at 25 degrees C; protease HS exhibited similar alkaline stability properties to pH 11 . In addition, protease HS had exceptional thermal stability properties . At pH 9.5 (0.1 M CAPS buffer, 5 mM EDTA), the enzyme had a half-life of more than 200 min at 50 degrees C and 25 min at 60 degrees C . At pH above 9.5, protease HS readily lost enzymatic activity even in the presence of exogenously supplied Ca2+ . In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C . In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C . The data presented here clearly indicate that these two alkaline proteases from Bacillus sp . strain GX6638 represent novel proteases that differ fundamentally from the proteases previously described for members of the genus Bacillus. DNA, 1987 Jun, 6(3), 273 - 9 Use of lambda exonuclease for efficient oligonucleotide-mediated site-directed deletion and point mutation of double-stranded DNA; Palermo DP et al.; A novel approach to oligonucleotide-mediated, site-directed in vitro mutagenesis is described that allows for the efficient generation of sequence modifications on double-stranded substrates without the need for subcloning into special vectors . Site-directed deletions as well as point mutations were introduced into the genes encoding human tissue plasminogen activator (tPA) and the Bacillus amyloliquefaciens alpha-amylase gene using lambda exonuclease to enzymatically degrade DNA 5' to 3' in order to generate a single-stranded template in the immediate vicinity of the oligonucleotide annealing site . The mutagenizing oligonucleotide, used both to redefine the 5' end of the molecule and to introduce base changes, was annealed to the single-stranded target sequence producing substrates for both the exonucleolytic and polymerizing activities of DNA polymerase Klenow fragment . Resolution of the resultant heteroduplex by Escherichia coli resulted in the generation of the desired deletion point mutation in the tPA sequence with an efficiency of 38% as determined by differential hybridization and 32% as determined by restriction analysis, with final verification by sequence data . As a further test of the method, two point mutations were introduced simultaneously with the desired sequence deletion into the Bacillus amyloliquefaciens alpha-amylase gene, generating a Pst I restriction site at the junction of the DNA encoding the signal peptide and the mature enzyme with an efficiency of 0.3% as determined by sequence data of hybridization-positive/Pst I-positive clones . The lambda exonuclease procedure is designed for use in situations where site-directed deletions must be introduced efficiently alone or with single or double point mutations. Appl Environ Microbiol, 1987 Jun, 53(6), 1333 - 7 Proteolysis in the gut of mosquito larvae results in further activation of the Bacillus sphaericus toxin; Broadwell AH et al.; Gut proteases from the larvae of the mosquito Culex pipiens convert the 43-kilodalton (kDa) toxin from Bacillus sphaericus 2362 to a 40-kDa peptide . The 50% lethal concentration of this peptide for tissue culture-grown cells of Culex quinquefasciatus was 1.0 microgram/ml (as determined by the intracellular ATP assay), 54-fold less than that of the 43-kDa peptide . Gut proteases from Anopheles gambiae and Aedes aegypti, as well as bovine pancreatic trypsin, also converted the 43-kDa protein to a 40-kDa peptide which was indistinguishable from the peptide formed by the proteases from C . pipiens with respect to its toxicity to tissue culture-grown cells of C . quinquefasciatus . Evidence for the in vivo conversion of the 43-kDa protein to the 40-kDa peptide was also obtained from experiments in which larvae of C . pipiens, Anopheles gambiae, and Aedes aegypti were fed crystals from B . sphaericus 2362 . By using the exclusion of trypan blue as an indication of cell viability, it was shown that chitobiose, chitotriose, N-acetylmuramic acid, and N-acetylneuraminic acid decreased the toxicity of the 40-kDa peptide (from 100 to 50% mortality at about 10 mM concentrations of these sugars) . Muramic acid, N-acetylgalactosamine, and N-acetylglucosamine were less effective, while several sugars had no effect, suggesting that the 40-kDa toxin binds to specific receptors on the cell membrane . The 40-kDa protein was less toxic to tissue culture-grown cells of Anopheles gambiae and Aedes dorsalis, and the same sugars which reduced the toxicity for cells of C . quinquefasciatus were also effective in reduction of toxicity for these cell lines.(ABSTRACT TRUNCATED AT 250 WORDS) Biokhimiia, 1987 Jun, 52(6), 918 - 26 {Molecular organization of the N-terminal region of delta-endotoxin of Bacillus thuringiensis subspecies alesti}; Tiurin SA et al.; The tryptic peptide sequences of the N-terminal domain ("true toxin") of delta-endotoxin of Bac . thuringiensis subspecies alesti carrying 282 amino acid residues were determined . A comparison of these sequences with the primary structures of delta-endotoxin of subspecies kurstaki (K-1, K-73) determined by an analysis of corresponding structural genes revealed a conservative region of "true toxin" (residues 29-346) and a hypervariable region (residues 347-617) carrying multiple (not less than 50%) substituents of amino acid residues . It is essential that the amino acid substituents in the variable region are distributed unevenly, being grouped into several highly variable sites carrying 7 to 31 residues . Besides, tryptic peptides of subspecies alesti delta-endotoxin were found to contain peptides having no homologs in the structures of subspecies kurstaki delta-endotoxins . It seems probable that such an uneven distribution of amino acid substituents in the structures of delta-endotoxins of subspecies alesti and kurstaki reflects the functional differences in the two halves of the N-terminal domain ("true toxin"), one of which (i . e., conservative) may be responsible for the toxic effect, while the other one (i . e., variable) seems to participate in toxin interactions with the appropriate receptors of larvae gut. Biochim Biophys Acta, 1987 May 27, 913(1), 72 - 80 A single amino acid substitution deregulates a bacterial lactate dehydrogenase and stabilizes its tetrameric structure; Clarke AR et al.; We have engineered a variant of the lactate dehydrogenase enzyme from Bacillus stearothermophilus in which arginine-173 at the proposed regulatory site has been replaced by glutamine . Like the wild-type enzyme, this mutant undergoes a reversible, protein-concentration-dependent subunit assembly, from dimer to tetramer . However, the mutant tetramer is much more stable (by a factor of 400) than the wild type and is destabilized rather than stabilized by binding the allosteric regulator, fructose 1,6-biphosphate (Fru-1,6-P2) . The mutation has not significantly changed the catalytic properties of the dimer (Kd NADH, Km pyruvate, Ki oxamate and kcat), but has weakened the binding of Fru-1,6-P2 to both the dimeric and tetrameric forms of the enzyme and has almost abolished any stimulatory effect . We conclude that the Arg-173 residue in the wild-type enzyme is directly involved in the binding of Fru-1,6-P2, is important for allosteric communication with the active site, and, in part, regulates the state of quaternary structure through a charge-repulsion mechanism. Biochim Biophys Acta, 1987 May 27, 913(1), 66 - 71 The use of site-directed mutagenesis and time-resolved fluorescence spectroscopy to assign the fluorescence contributions of individual tryptophan residues in Bacillus stearothermophilus lactate dehydrogenase; Waldman AD et al.; Site-directed mutagenesis has been used to generate two mutant Bacillus stearothermophilus lactate dehydrogenases: in one, Trp-150 has been replaced with a tyrosine residue and, in the other, both Trp-150 and -80 are replaced with tyrosines . Both enzymes are fully catalytically active and their affinities for substrates and coenzymes, and thermal stabilities are very similar to those of the native enzyme . Time-resolved fluorescence measurements using a synchrotron source have shown that all three tryptophans in the native enzyme fluoresce . By comparing the mutant and native enzymes it was possible, for the first time, to assign, unambiguously, lifetimes to the individual tryptophans: Trp-203 (7.4 ns), Trp-80 (2.35 ns) and Trp-150 (less than 0.3 ns) . Trp-203 is responsible for 75-80% of the steady-state fluorescence emission, Trp-80 for 20%, and Trp-150 for less than 2%. Science, 1987 May 15, 236(4803), 813 - 6 A tunnel in the large ribosomal subunit revealed by three-dimensional image reconstruction; Yonath A et al.; A better understanding of the molecular mechanism of protein biosynthesis depends on the availability of a reliable model for the ribosome particle . The application of a diffraction technique, namely, three-dimensional image reconstruction from two-dimensional sheets of the large ribosomal subunits of Bacillus stearothermophilus at a resolution of 30 angstroms is described . The resulting three-dimensional model shows at least four projecting arms, arranged radially near the presumed interface with the 30S subunit . The projecting arms are positioned around a cleft, which turns into a tunnel with a length of 100 to 120 angstroms and a diameter of up to 25 angstroms . This tunnel spans the particle and may provide the path taken by the nascent polypeptide chain. Eur J Biochem, 1987 May 15, 165(1), 47 - 53 Biosynthesis of poly(galactosylglycerol phosphate) in Bacillus coagulans; Yokoyama K et al.; The pathway for the de novo synthesis of a teichoic acid, poly(galactosylglycerol phosphate), in Bacillus coagulans AHU 1366 was studied by means of characterization and stepwise conversion of lipid-linked intermediates . Incubation of membranes with UDP-N-acetylglucosamine and UDP-glucose yielded a disaccharide-linked polyprenylpyrophosphate, whose sugar moiety was characterized as glucosyl(beta 1----4)N-acetylglucosamine (Glc-GlcNAc) . By incubation with membranes and CDP-glycerol, Glc-GlcNAc-PP-prenol was converted to a series of glycolipids characterized as (Gro-P)1-6-Glc-GlcNAc-PP-prenol (Gro = glycerol) . Glc-{14C}GlcNAc-PP-prenol was converted to polymer by incubation with membranes, CDP-glycerol and UDP-galactose . Smith degradation of the polymer gave two radioactive fragments corresponding to (Gro-P)3-Glc-GlcNAc and (Gro-P)4-Glc-GlcNAc . These results, together with data on gel chromatography of radioactive polymer synthesized from UDP-{3H}galactose, CDP-glycerol and Glc-{14C}GlcNAc-PP-prenol, led to the conclusion that in this strain poly(galactosylglycerol phosphate) is probably synthesized through the following pathway: GlcNAc-PP-prenol----Glc-GlcNAc-PP-prenol----(Gro-P)3-4 -Glc-GlcNAc-PP-prenol----(Gro-P-Gal)n- (Gro-P)3-4-Glc-GlcNAc-PP-prenol----(Gro-P-Gal)n- (Gro-P)3-4-Glc-GlcNAc-P-peptidoglycan complex. J Biol Chem, 1987 May 15, 262(14), 6683 - 90 Identification and characterization of two functional domains in cytochrome P-450BM-3, a catalytically self-sufficient monooxygenase induced by barbiturates in Bacillus megaterium; Narhi LO et al.; In a previous publication (Narhi, L . O . and Fulco, A . J . (1986) J . Biol . Chem . 261, 7160-7169) we described the characterization of a soluble 119,000-dalton P-450 cytochrome (P-450BM-3) that was induced by barbiturates in Bacillus megaterium . This single polypeptide contained 1 mol each of FAD and FMN/mol of heme and, in the presence of NADPH and O2, catalyzed the oxygenation of long-chain fatty acids without the aid of any other protein . We have now utilized limited trypsin proteolysis in the presence of substrate to cleave P-450BM-3 into two polypeptides (domains) of about 66,000 and 55,000 daltons . The 66-kDa domain contains both FAD and FMN but no heme, reduces cytochrome c in the presence of NADPH, and is derived from the C-terminal portion of P-450BM-3 . The 55-kDa domain is actually a mixture of three discrete peptides (T-I, T-II, and T-III) separable by high performance liquid chromatography . All three contain heme and show a P-450 absorption peak in the presence of CO and dithionite . The major component, T-I (Mr = 55 kDa), binds fatty acid substrate and has an N-terminal amino acid sequence identical to that of intact P-450BM-3, an indication that this domain constitutes the N-terminal portion of the 119-kDa protein . T-II (54 kDa) is the same as T-I except that it is missing the first nine N-terminal amino acids and does not bind substrate . T-III (Mr = 53.5 kDa) has lost the first 15 N-terminal residues and does not bind substrate . Since trypsin digestion of P-450BM-3 carried out in the absence of substrate yields T-II and T-III but no T-I, it appears that 1 or more residues of the first nine N-terminal amino acids of this protein are intimately involved in substrate binding . Although both the heme- and flavin-containing tryptic peptides retain their original half-reactions, fatty acid monooxygenase activity cannot be reconstituted after proteolysis, and the two domains, once separated, show no affinity for each other . In most respects, the reductase domain of P-450BM-3 more closely resembles the mammalian microsomal P-450 reductases than it does any known bacterial protein. J Biol Chem, 1987 May 15, 262(14), 6676 - 82 Cloning of the gene encoding a catalytically self-sufficient cytochrome P-450 fatty acid monooxygenase induced by barbiturates in Bacillus megaterium and its functional expression and regulation in heterologous (Escherichia coli) and homologous (Bacillus megaterium) hosts; Wen LP et al.; In a previous publication (Narhi, L . O., and Fulco, A . J . (1986) J . Biol . Chem . 261, 7160-7169) we described the characterization of a 119,000-dalton P-450 cytochrome that is strongly induced by barbiturates in Bacillus megaterium . In the presence of NADPH and O2, this single polypeptide can catalyze the hydroxylation of long-chain fatty acids without the aid of any other protein . The gene encoding this unique monooxygenase (cytochrome P-450BM-3) has now been cloned by an immunochemical screening technique . The Escherichia coli clone harboring the recombinant plasmid produces a 119,000-dalton protein that appears to be electrophoretically and immunochemically identical to the B . megaterium enzyme and contains the same N-terminal amino acid sequence . The recombinant DNA product also exhibits the characteristic cytochrome P-450 spectrum and is fully functional as a fatty acid monooxygenase . In E . coli, the synthesis of P-450BM-3 is directed by its own promoter included in the DNA insert and proceeds constitutively at a very high rate but is not stimulated by pentobarbital . However, when the cloned P-450BM-3 gene, either intact or in a truncated form, is introduced back into B . megaterium via an E . coli/Bacillus subtilis shuttle vector, its expression is constitutively repressed but is induced by pentobarbital . This finding demonstrates that the regulatory region of the P-450BM-3 gene that responds to barbiturates is included in the cloned DNA . The evidence also indicates that pentobarbital cannot directly act on the gene to cause induction but presumably interacts with another component such as a repressor molecule that is present in B . megaterium but is absent in the E . coli clone. Arch Biochem Biophys, 1987 May 15, 255(1), 127 - 35 The action of sphingomyelinase from Bacillus cereus on ATP-depleted bovine erythrocyte membranes and different lipid composition of liposomes; Tomita M et al.; The presence of cholesterol or phosphatidylethanolamine in sphingomyelin liposomes enhanced 2- to 10-fold the breakdown of sphingomyelin by sphingomyelinase from Bacillus cereus . On the other hand, the presence of phosphatidylcholine was either without effect or slightly stimulative at a higher molar ratio of phosphatidylcholine to sphingomyelin (3/1) . In the bovine erythrocytes and their ghosts, the increase by 40-50% or the decrease by 10-23% in membranous cholesterol brought about acceleration or deceleration of enzymatic degradation of sphingomyelin by 50 or 40-50%, respectively . The depletion of ATP (less than 0.9 mg ATP/100 ml packed erythrocytes) enhanced K+ leakage from, and hot hemolysis (lysis without cold shock) of, bovine erythrocytes but decelerated the breakdown of sphingomyelin and hot-cold hemolysis (lysis induced by ice-cold shock to sphingomyelinase-treated erythrocytes), either in the presence of 1 mM MgCl2 alone or in the presence of 1 mM MgCl2 and 1 mM CaCl2 . Also, ATP depletion enhanced the adsorption of sphingomyelinase onto bovine erythrocyte membranes in the presence of 1 mM CaCl2 up to 81% of total activity, without appreciable K+ leakage and hot or hot-cold hemolysis . These results suggest that the presence of cholesterol or phosphatidylethanolamine in biomembranes makes the membranes more susceptible to the attack of sphingomyelinase from B . cereus and that the segregation of lipids and proteins in the erythrocyte membranes by ATP depletion causes the deceleration of sphingomyelin hydrolysis despite the enhanced enzyme adsorption onto the erythrocyte membranes. Eur J Biochem, 1987 May 15, 165(1), 223 - 7 The GTP pool in Bacillus brevis and its significance for sporulation; Federn H et al.; The GTP pool of Bacillus brevis as well as that of other nucleotides is highly sensitive to all kinds of environmental changes like the cell transfer procedures or nutrient depletion of the cells . In growing cultures, as well as in cells transferred from rich to nitrogen-deficient medium, the nucleotide pool decreases significantly . This decrease is followed by the onset of sporulation only when cells are allowed to produce the peptide antibiotic tyrocidine or if tyrocidine is added to the culture . However, exogenous tyrocidine is active in triggering sporulation only when it is added within a short period of time immediately after shift down, that is when the nucleotide pool is observed to decrease. Biochemistry, 1987 May 5, 26(9), 2480 - 6 The valyl-tRNA synthetase from Bacillus stearothermophilus has considerable sequence homology with the isoleucyl-tRNA synthetase from Escherichia coli; Borgford TJ et al.; We report the DNA sequence of the valS gene from Bacillus stearothermophilus and the predicted amino acid sequence of the valyl-tRNA synthetase encoded by the gene . The predicted primary structure is for a protein of 880 amino acids with a molecular mass of 102,036 . The molecular mass and amino acid composition of the expressed enzyme are in close agreement with those values deduced from the DNA sequence . Comparison of the predicted protein sequence with known protein sequences revealed a considerable homology with the isoleucyl-tRNA synthetase of Escherichia coli . The two enzymes are identical in some 20-25% of their amino acid residues, and the homology is distributed approximately evenly from N-terminus to C-terminus . There are several regions which are highly conservative between the valyl- and isoleucyl-tRNA synthetases . In one of these regions, 15 of 20 amino acids are identical, and in another, 10 of 14 are identical . The valyl-tRNA synthetase also contains a region HLGH (His-Leu-Gly-His) near its N-terminus equivalent to the consensus HIGH (His-Ile-Gly-His) sequence known to participate in the binding of ATP in the tyrosyl-tRNA synthetase . This is the first example of extensive homology found between two different aminoacyl-tRNA synthetases. Nature, 1987 May 28-Jun 3, 327(6120), 341 - 3 Reconstitution of a phospholipid flippase from rat liver microsomes; Backer JM et al.; The endoplasmic reticulum is the principal site of synthesis and initial incorporation of membrane lipids in eukaryotic cells; the enzymes of glycerolipid biosynthesis are exclusively located on its cytoplasmic surface . To maintain a phospholipid bilayer in this organelle, newly synthesized phospholipids must be translocated to the lumenal surface . Consistent with this are measurements indicating that movement of phospholipids across microsomal membranes is rapid, with a half-time less than 5 min (refs 3 and 4) . Rapid movement of phospholipids has also been detected across the plasma membrane of Bacillus megaterium, another site of de novo lipid biosynthesis . The rapid transmembrane movement of phosphatidylcholine has not been detected, however, in vesicles prepared from microsomal lipids . These latter data suggest involvement in the endoplasmic reticulum of a phospholipid-translocating protein, as was first proposed by Bretscher who called it 'flippase' . Here we report reconstitution of a phospholipid flippase from rat liver microsomes into lipid vesicles. Sci Sin {B}, 1987 May, 30(5), 503 - 6 Heat-stable DNA polymerase I large fragment resolves hairpin structure in DNA sequencing; Ye SY et al.; A heat-stable large fragment was obtained by subtilisin digestion of DNA polymerase, prepared from Bacillus stearothermophilus . The dideoxy sequencing method, combined with the use of M13 vector has proved to be the most powerful one for obtaining the sequences of large genomes . However, the hairpin structure formed along the single-stranded DNA template often prevents the DNA polymerase from moving on, with the result that no sequence information can be obtained . The heat-stable large fragment that we have obtained has proved to be the most active at 65 degrees C . When the sequencing reaction was carried out at this temperature, the hairpin structure was resolved and the sequencing gels obtained were satisfactory. Biochem J, 1987 May 1, 243(3), 701 - 7 The isolation of a peptide from the catalytic domain of Bacillus stearothermophilus tryptophyl-tRNA synthetase . The interaction of Brown MX-5BR with tyrosyl-tRNA synthetase; McArdell JE et al.; Tryptophyl-tRNA synthetase is irreversibly inactivated by Procion Brown MX-5BR with an apparent dissociation constant (KD) of 8.8 microM and maximum rate of inactivation k3 0.192 s-1 . The specificity of the interaction is supported by two previously reported observations . Firstly, Brown MX-5BR inactivation of tryptophyl-tRNA synthetase is inhibited by substrates, and secondly, the animated derivative of Brown MX-5BR is a competitive inhibitor of tryptophyl-tRNA synthetase with a Ki of 2 X 10(-4) M with respect to both tryptophan and ATP . Tryptic digestion of the dye-affinity-labelled enzyme and subsequent resolution of the peptides by h.p.l.c . yielded one major dye-peptide peak . Amino acid sequence analysis resulted in the identification of the dye-binding domain centred on lysine-178 . Tyrosyl-tRNA synthetase is also inactivated by Procion Brown MX-5BR, and this inactivation is prevented by ATP but not by tyrosine . The interaction of tyrosyl-tRNA synthetase with hydroxylated Brown MX-5BR exhibited non-competitive kinetics with respect to the amino acid-binding site and competitive kinetics against ATP with a Ki of 6 X 10(-6) M. J Gen Microbiol, 1987 May, 133 ( Pt 5), 1389 - 95 Isolation and characterization of the facultative methylotroph Mycobacterium ID-Y; Reed WM et al.; A facultatively methylotrophic Mycobacterium was isolated from Cleveland Harbor, Ohio, USA . The isolate, designated ID-Y, used a wide range of carbon and energy sources including methane and several other hydrocarbons . It displayed a growth cycle from rod-shaped exponential-phase cells, with many cell pairs exhibiting V-formation, to cocco-bacillary stationary-phase cells . A fixation technique involving glutaraldehyde/alcian blue resulted in the observation of a three-layered cell wall . Isolate ID-Y has an ultrastructure similar to that of other mycobacteria, particularly Mycobacterium phlei and Mycobacterium flavum, which is presently classified as a Xanthobacter species. Pediatr Infect Dis J, 1987 May, 6(5), 451 - 4 Prospective study of the magnitude and duration of changes in tuberculin reactivity during uncomplicated and complicated measles; Tamashiro VG et al.; The suppression of cellular immune responses during measles is thought to contribute to the development of secondary infections which often complicate this disease . To determine whether there was a difference in the altered cellular immune responses of children with and without complications we performed a prospective study of purified protein derivative skin test reactivity in children with natural measles virus infections who had received Bacillus Calmette-Guerin as infants . Twenty-five tuberculin-positive children who developed measles (13 uncomplicated and 12 complicated) were skin-tested weekly beginning 1 to 2 weeks before and ending 2 to 7 weeks after the onset of the rash . All children became anergic during the acute phase of measles . Children with complications remained unreactive for a significantly longer period of time after the rash (mean, 4 weeks) than did children without complications (mean, 2.3 weeks, P less than 0.001). Mutat Res, 1987 May, 183(3), 225 - 9 Presence of inducible DNA repair in Bacillus thuringiensis; Auffray Y et al.; Weigle reactivation of ultraviolet-irradiated luminal diameter 8 bacteriophage was observed after ultraviolet treatment of Bacillus thuringiensis cells . A slight increased frequency of clear plaque mutants was detected among the survivors . The kinetics of induction of the phage reactivation and phage mutagenesis have been determined . The presence of chloramphenicol before and after irradiation abolished the induction of repair and mutagenesis . These experiments suggest that, in spite of the relatively small mutagenic response in bacteriophage progeny, B . thuringiensis has an inducible repair system responsible to the significant Weigle reactivation of irradiated phage. Infect Immun, 1987 May, 55(5), 1300 - 8 Cell membrane interaction of Bacillus thuringiensis subsp . israelensis cytolytic toxins; Gill SS et al.; Two toxic polypeptides of 24 and 25 kilodaltons (kDa) were purified from parasporal proteinaceous crystals of Bacillus thuringiensis subsp . israelensis . Both of these polypeptides, which are antigenically similar and have identical N terminals, lysed human erythrocytes and cultured mosquito cells . Although the 24-kDa peptide was more toxic than the 25-kDa peptide, both were less toxic than the crude alkali-solubilized crystal toxin . However, a 1:1 mixture of these 24- and 25-kDa proteins was more toxic than either of these polypeptides individually, indicating a possible interaction between these proteins at the cell membrane . Both the 24- and the 25-kDa proteins were inactivated by aqueous suspensions of dioleolylphosphatidylcholine, indicating the involvement of phospholipids in the cytotoxic action of these toxins . Thus the role of cell membrane phospholipids in mediating the toxin action was studied by using phospholipases as probes . Treatment of erythrocytes with high levels of phospholipase D increased their susceptibility to the toxin; however, phospholipase A2-treated erythrocytes were less susceptible to the toxin . These erythrocytes also bound less 125I-labeled 25-kDa toxin . These results support the role of fatty acyl residues at the syn-2 position of membrane phospholipids in toxin action . The cytolytic toxin of B . thuringiensis subsp . israelensis is thought to damage cell membranes in a detergentlike manner . However, there was a difference between the cytolytic action of this toxin and that of a nonionic detergent such as Triton X-100 because phospholipase A2-treated erythrocytes were more susceptible to Triton X-100, whereas such erythrocytes were less sensitive to the toxin . Thus, the cytolytic toxin apparently did not act as a nonspecific detergent, but rather interacted with phospholipid receptors on the cell membrane . Such an interaction of the toxin with phospholipid receptors probably results in the increased cell permeability, thereby causing cell lysis. Mol Cell Biochem, 1987 May, 75(1), 15 - 21 Synthesis of hybrid bisnucleoside 5',5"'-P1,P4-tetraphosphates by aminoacyl-tRNA synthetases; Traut TW; Aminoacyl tRNA synthetases, by means of a back reaction, are able to synthesize certain 5', 5"'-P1, P4-bisnucleoside tetraphosphates of biological importance, such as Ap4A . Here it is shown that HisRS and TrpRS (Bacillus stearothermophilus) and AlaRS (E . coli) also synthesize the hybrid compounds Ap4G, Ap4C, and Ap4U . GlnRS (E . coli) is unable to synthesize any of the above compounds . AlaRS synthesizes Ap4U very poorly, and Ap4C and Ap4G almost as effectively as Ap4A . HisRS and TrpRS synthesize Ap4G, Ap4U and Ap3U quite effectively, and Ap4C very poorly . The fact that hybrid bisnucleoside tetraphosphates can be made by the same enzymes, and at rates comparable to Ap4A, suggests that these compounds may also occur in vivo. J Gen Microbiol, 1987 May, 133 ( Pt 5), 1257 - 63 Oxygen profiles in, and in the agar beneath, colonies of Bacillus cereus, Staphylococcus albus and Escherichia coli; Peters AC et al.; The paper reports the use of microelectrodes to measure O2 penetration in different aged colonies of Bacillus cereus, Escherichia coli and Staphylococcus albus . In young (18 h) colonies of B . cereus and E . coli O2 disappeared at depths of 25-30 micron and 35-40 micron respectively . In young S . albus colonies, O2 reached a minimum but was never completely absent . As colonies aged (24-168 h) the depth to which O2 penetrated increased. Zentralbl Bakteriol Mikrobiol Hyg {B}, 1987 May, 184(2), 108 - 21 {Dependence of microbiologic test results of formaldehyde gas sterilization methods on the nature of the test material}; Spicher G et al.; The efficiency of a formaldehyde gas sterilization procedure was evaluated with the aid of test pieces consisting of various materials . Both rigid and flexible tubes served as test pieces . The tubes were 75 cm long with an inner diameter of 1 mm and were sealed at one end . The bioindicators were placed inside the tubes close to the sealed end . Dried spores of Bacillus stearothermophilus adhering to linen threads served as test organisms . The test results varied according to the material of the test pieces and the thickness of their walls (see Table 1) . In flexible tubes made of silicon rubber, all bioindicators became sterile, in tubes of stainless steel, all bioindicators exhibited test organisms that had survived . The findings for materials such as polyvinyl chloride, polyethylene, polyamide and polytetrafluorethylene ranged between these two extremes; the frequencies of bioindicators containing viable germs were 10, 55, 68 and 85%, respectively . Rigid and flexible tubes which had been sealed at both ends served to demonstrate that silicon rubber and polyvinyl chloride were highly permeable for formaldehyde and water vapour . Also the other plastic materials tested were permeable for formaldehyde and water vapour but longer exposure periods were needed to create conditions in the interior of the tubes that would result in a killing of the test organisms (see Fig 2) . In this respect, polyamide exhibited a peculiar behaviour . The number of viable spores remained at the initial level for a long period before a decline took place . From the results of testing, it is concluded that test pieces must conform to the objects to be sterilized not only in their dimensions (length, inner diameter) but also in the characteristics of their material . The walls of the test pieces should not have a higher permeability for formaldehyde and water vapour than the material to be sterilized . The highest demands on the efficiency of formaldehyde gas sterilization procedures are those created by mental tubes and thick-walled flexible polytetrafluorethylene . Instruments and devices to be sterilized by a formaldehyde gas procedure should be preferentially made of materials which are sufficiently permeable for formaldehyde and water vapour as e.g . silicon rubber . Such gas-permeable components may considerably facilitate the sterilization of cavities which have a small lumen and are difficult to reach. Appl Environ Microbiol, 1987 May, 53(5), 1142 - 6 Stimulation by Hyphopichia burtonii and Bacillus amyloliquefaciens of aflatoxin production by Aspergillus flavus in irradiated maize and rice grains; Cuero RG et al.; Aspergillus flavus was grown on maize and rice extract agars and on irradiated viable cracked maize and rice grains, either in pure culture or in dual culture with wild strains of either Hyphopichia burtonii or Bacillus amyloliquefaciens . Aflatoxin production by A . flavus and its growth and interactions with the other microorganisms were studied at three water activities (aw) (0.98, 0.95, and 0.90) and two temperatures (25 and 16 degrees C) . Both H . burtonii and B . amyloliquefaciens markedly stimulated growth and aflatoxin production by A . flavus on cracked maize, especially at 25 degrees C and 0.95 and 0.98 aw . No aflatoxin was detected in pure cultures of A . flavus on cracked rice after 12 days of incubation at 25 degrees C, but some was produced by mixed cultures at 16 degrees C and 0.98 aw . The morphological interactions among A . flavus, H . burtonii, and B . amyloliquefaciens were also examined on maize and rice extract agars under similar controlled conditions. Medicine (Baltimore), 1987 May, 66(3), 218 - 23 Serious infections caused by Bacillus species; Sliman R et al.; Thirty-eight patients with serious infections caused by organisms belonging to the genus Bacillus are described . Our experience, and that reported in the literature, indicates that, in most cases, isolated Bacillus bacteremia is not a particularly serious disease . Therefore, under most circumstances, empiric antibiotic therapy designed specifically for treatment of Bacillus is probably not necessary . Endocarditis can occur, but apparently follows bacteremia only infrequently . When these bacteria cause localized infection such as pneumonia, pan-ophthalmitis, visceral abscess, or musculoskeletal infections, tissue necrosis and profound morbidity are the rule . The frequency of these complications following bacteremia appears to be low but cannot be estimated from our experience or that reported in the literature reviewed . The role of intravascular devices and trauma as predisposing factors is emphasized . Immunocompromised hosts and intravenous drug abusers appear predisposed, but intravascular devices in the former group may play an important role in the pathogenesis of Bacillus infections . Antibiotics which appear especially useful in the treatment of Bacillus infections are clindamycin and vancomycin, to which the vast majority of strains are susceptible in vitro . Beta-lactam antibiotics, including the new cephalosporins and penicillins, are of little value in this setting. J Urol, 1987 May, 137(5), 871 - 3 Bacillus Calmette-Guerin for treatment of superficial transitional cell carcinoma of the bladder in patients who have failed thiotepa and/or mitomycin C; Soloway MS et al.; Thirty patients with stage Ta carcinoma in situ or T1 superficial bladder cancer received 6 weeks of intravesical bacillus Calmette-Guerin . All patients had persistent or recurrent tumor despite thiotepa and/or mitomycin C . Response was determined by the results of endoscopy, bladder wash cytology and biopsy performed 4 weeks after the last dose of bacillus Calmette-Guerin . Of the 30 patients 15 (50 per cent) had a complete response . The likelihood of a complete response was better for those with initial Ta lesions (62 per cent) and carcinoma in situ (56 per cent) than for patients with an initial T1 lesion (25 per cent) . Although the longest followup is only 36 months (mean 16 months) patients with a complete response have a much better prognosis in terms of subsequent tumor, need for cystectomy and death of bladder cancer. J Bacteriol, 1987 May, 169(5), 2165 - 70 Use of bacterial luciferase to establish a promoter probe vehicle capable of nondestructive real-time analysis of gene expression in Bacillus spp; Carmi OA et al.; We report the construction and use of a new promoter probe vehicle capable of allowing extremely sensitive measurements of transcriptional activity promoted from random, chromosomal DNA fragment inserts . Coupled with the advantage of sensitivity, the detection system is noninvasive, nondestructive, and provides real-time reportage of expression potential . These latter aspects make it an especially valuable system for a continuing analysis of the complex transcriptional regulation patterns now recognized as a dominant control feature during the differentiation and morphogenesis characteristic of the sporulation cycle in Bacillus species . In this respect we describe the isolation of DNA fragments from B . megaterium and B . subtilis capable of initiating transcription in both the respective parent organisms and, in certain instances, also in Escherichia coli . Detailed luminescence studies showed that several promoter regions which are entirely or substantially developmentally controlled were isolated. Biochim Biophys Acta, 1987 Apr 23, 898(3), 293 - 8 Effect of K+ on the membrane functions of an alkalophilic Bacillus; Koyama N et al.; We have examined the involvement of K+ in the membrane functions of a facultatively alkalophilic Bacillus at neutral and alkaline pH . The effects of K+ on membrane functions, such as maintenance of the membrane potential, leucine uptake and respiratory activity, were dependent on the external pH . K+ uptake, which induced alkalinization of the cytoplasm, is suggested to be electrogenic at neutral pH and 'electroneutral' at alkaline pH, resulting in a similar level of net accumulation . We suggest that the bacterial membrane is highly permeable to K+ at neutral pH, compared to alkaline pH, which results in a pH-dependent effect of K+ on the above membrane functions. FEBS Lett, 1987 Apr 20, 214(2), 305 - 7 BliI, a restriction endonuclease from Bacillus licheniformis; Manachini PL et al.; From Bacillus licheniformis a site-specific restriction endonuclease, named BliI, has been purified and characterized . BliI was able to digest lambda DNA at pH 9.1 over a wide temperature range (25-65 degrees C) . Digestion of lambda and psi X174 DNAs with BliI produced banding patterns identical to those seen with HaeIII . Therefore, BliI and HaeIII endonculeases are isoschizomers. Cancer Res, 1987 Apr 15, 47(8), 2067 - 72 Tumor cytokinetics in the presence of normal, alloimmune, or Bacillus Calmette-Guérin-activated host cells simultaneously assayed in vivo and in vitro; Normann SJ et al.; Rates of tumor cell loss, replication, and growth were determined simultaneously for P-815 mastocytoma cells placed in culture or transplanted as a peritoneal ascites tumor . Cytokinetic parameters were concordant in vitro to in vivo for P-815 cells growing in the presence of syngeneic DBA/2 resident or proteose peptone-elicited macrophages and under allogeneic C57BL/6 nonimmune conditions . Under alloimmune conditions, measured parameters differed in vitro from in vivo but conclusions were consistent in that alloimmune host cells were cytolytic and cytostatic and caused tumor regression . In contrast, syngeneic Bacillus Calmette-Guerin-activated macrophages were cytolytic and cytostatic in vitro but not in vivo despite equivalent or greater effector to target ratios, presence or absence of endotoxin in the tumor inoculi, or changes in the injection schedule of Bacillus Calmette-Guerin . Similarly, Bacillus Calmette-Guerin-activated macrophages were cytolytic in vitro but not in vivo when admixed with tumor cells prior to injection into the leg . This study is the first simultaneously conducted cytokinetic analysis of a common pool of labeled tumor cells growing in vitro and in vivo using randomly selected mice as donors of host effector cells or as recipients of tumor transplantation . It demonstrates that activated macrophages which are cytolytic and cytostatic in vitro for P-815 cells may not function analogously in vivo in controlling tumor growth. Cancer Res, 1987 Apr 15, 47(8), 2014 - 9 Tumoricidal effector mechanisms of murine Bacillus Calmette-Guérin-activated macrophages: mediation of cytolysis, mitochondrial respiration inhibition, and release of intracellular iron by distinct mechanisms; Klostergaard J et al.; Murine Bacillus Calmette-Guerin-activated macrophages mediate discrete cytotoxic effects in cocultured tumor target cells in vitro . These effects include: the loss of intracellular iron, in part associated with reversible inhibition of the Kreb's cycle enzyme, aconitase; cytostasis, associated with reversible lesions inflicted in the electron transport chain (ETC) of the mitochondria resulting in reversible loss of proliferative capacity; and cytolysis, manifested by eventual gross perturbation of the integrity of the plasma membrane . We demonstrate that these manifestations of cytotoxicity are the result of three independent mechanisms employing apparently distinct macromolecules for their commission . Analysis of target cells that are highly susceptible (L-929), highly resistant (L-1210), or have incomplete resistance (EMT-6) to the cytolytic effects of cocultured activated macrophages indicates that there is no consistent relationship between the release of intracellular 59Fe and 51Cr . Thus, perturbation of intracellular iron pools did not appear to be an obligatory step on the pathway to cytolysis . Further evidence for this dissociation was obtained by employing a specific heteroantiserum reactive with cytolytic molecule(s) . This antiserum could block the cytolytic response (51Cr release of cocultured L-929 and EMT-6 targets) but had no effect on the extent of iron release from viable EMT-6 or L-1210 targets . Furthermore, the cytolytic factor itself was incapable of mediating effects on the ETC or in causing release of intracellular iron . Two lines of evidence suggested that effects on the ETC are not linked with loss of intracellular iron . First, the monokine respiration inhibitory factor was incapable of causing release of intracellular iron from target cells in which the mitochondria were strongly suppressed . Second, the kinetics of release of respiration inhibitory factor from endotoxin-triggered Bacillus Calmette-Guerin-activated macrophages indicate a retarded appearance compared to the time at which a factor mediating release of intracellular iron was detectable . Our results strongly suggest that these three distinct cytotoxic reactions are under differential control by the effector cell. Experientia, 1987 Apr 15, 43(4), 464 - 5 3-Amino-3-deoxy-D-glucose: an antibiotic produced by a deep-sea bacterium; Fusetani N et al.; Gram-positive bacteria isolated from deep-sea sediments of the Pacific basin showed considerable antibacterial activity . A Bacillus strain, isolated from a sediment sample collected at a depth of 4310 m, was shown to produce 3-amino-3-deoxy-D-glucose, a known antibiotic. J Immunol, 1987 Apr 15, 138(8), 2739 - 44 Expression of a 120,000 dalton protein during tumoricidal activation in murine peritoneal macrophages; Johnston PA et al.; Modulation of protein expression during interferon-gamma (IFN-gamma)-lipopolysaccharide (LPS)-mediated macrophage tumoricidal activation has been examined by metabolic radiolabeling of various murine peritoneal macrophage populations with {35S}methionine followed by SDS-PAGE analysis . Although both IFN-gamma and LPS are capable of stimulating the expression of several proteins when used independently, combined treatment induced the enhanced or de novo expression of a 120,000 dalton polypeptide . The expression of this protein was synergistically regulated by both IFN-gamma and LPS in a manner strongly reminiscent of the functional synergism that these two agents exhibit with respect to induction of tumoricidal activity . p120 expression could be seen first at approximately 3 hr after the addition of both agents, reached optimal expression by 6 hr, and maintained elevated synthesis for up to 24 hr . This time course corresponds closely to that seen for the acquisition of tumoricidal competence . Macrophages elicited in the primed state of activity in vivo with methyl vinyl ether co-polymer II (MVE-II) did not express p120, but could be induced to do so when treated with low doses of LPS . Under similar conditions, MVE-II-elicited cells also acquire tumoricidal activity . Macrophages obtained from mice chronically infected with bacillus Calmette-Guerin constitutively expressed both p120 and cytolytic activity . If such macrophages were cultured for 24 hr, the expression of both events decayed and was lost, but could be restored by treatment with low doses of LPS . Thus the data support a strong correlation between the expression by macrophages of a novel 120,000 dalton protein and the expression of tumor cytotoxicity. Biochem Biophys Res Commun, 1987 Apr 14, 144(1), 203 - 9 Membranous phosphoglyceride-linked biosynthesis of pentadecapeptide, linear gramicidin, by Bacillus brevis ATCC 8185; Kubota K; A peptide antibiotic, linear gramicidin A, from Bacillus brevis ATCC 8185 was biosynthesized with a cell-free preparation . An ethanolamine donor required for masking of a carboxyl terminal in this linear peptide was detected . Phosphatidylethanolamine, one of phosphoglycerides and a major structural element of membranes in bacterial cells, was verified to be the primary donor of terminal ethanolamine in the total synthesis of the peptide . This paper suggests that one of the non-ribosomal peptidyl products undergoes tight linkage to a component of cellular membranes. Biochemistry, 1987 Apr 7, 26(7), 1969 - 73 Inhibition of thiaminase I from Bacillus thiaminolyticus . Evidence supporting a covalent 1,6-dihydropyrimidinyl-enzyme intermediate; Hutter JA et al.; Thiaminase I from Bacillus thiaminolyticus strain Matsukawa et Misawa is completely and irreversibly inhibited by treatment with 4-amino-6-chloro-2-methylpyrimidine . Inhibition is a time-dependent first-order process, exhibiting a half-time of 4 h at an inhibitor concentration of 5 mM . A specific active-site-directed inactivation is supported by protection of the enzymatic activity in the presence of the substrates thiamin and quinoline as well as by the observation that a stoichiometric amount of inorganic chloride is released during inactivation . 4-Amino-5-(anilinomethyl)-6-chloro-2-methylpyrimidine, which resembles the structure of the product of base exchange of thiamin with aniline, inactivates thiaminase approximately 2 orders of magnitude faster . Inactivation is again complete and irreversible and is a time-dependent first-order process, in this case exhibiting saturation at low inhibitor concentrations (KI = 96 microM) . Enzyme inactivation can be explained as the result of displacement of chloride from the chloropyrimidine by a nucleophile at the enzyme active site . The inactivation suggests that the Zoltewicz-Kauffman model of bisulfite-catalyzed thiamin cleavage {Zoltewicz, J . A., & Kauffman, G . M . (1977) J . Am . Chem . Soc . 99, 3134-3142}, which calls for the reversible nucleophilic addition of catalyst across the 1,6 double bond of thiamin's pyrimidine ring, may be applicable to thiaminase as well. Cancer Res, 1987 Apr 1, 47(7), 1785 - 92 Induction of a tumor necrosis factor-like activity by Nocardia rubra cell wall skeleton; Izumi S et al.; Nocardia rubra cell wall skeleton (N-CWS) stimulated adherent cells harvested from the peritoneal cavities of thioglycollate-treated mice to produce cytotoxic activity . Depletion of macrophages from the adherent cells by 2-chloroadenosine or silica abrogated the production of this cytotoxic activity, whereas treatment of the adherent cells with anti-Thy-1.2 antibody and complement did not . This suggested that macrophages were the producer cells of the activity . Cytotoxic activity became detectable as early as 2 h after N-CWS treatment and reached peak activity at 9 h, then declined to a lower level, indicating rapid onset without persistent effects . N-CWS-induced cytotoxic factors have a fairly narrow temperature range, pH optimum for storage, and are sensitive to pronase and trypsin . By using column chromatography, N-CWS-induced cytotoxic factors were compared in detail with tumor necrosis serum obtained from Bacillus Calmette-Guerin endotoxin-treated mice . Both toxins were found to be nearly identical with respect to their behavior in ion-exchange, gel filtration, and concanavalin A affinity columns . N-CWS also induced human peripheral blood lymphocytes to release cytotoxic activity . Monocytes predominantly participated in production of this activity as confirmed by treatment with monoclonal antibody and complement . The cytotoxic activity was completely neutralized by anti-human tumor necrosis factor antiserum, but not by anti-human lymphotoxin antiserum . The fact that human peripheral blood lymphocytes release tumor necrosis-like factors after stimulation with N-CWS might account for the antitumor effects of this agent. Lab Anim Sci, 1987 Apr, 37(2), 176 - 9 An enzyme-linked immunosorbent assay for detection of anti-Bacillus piliformis serum antibody in rabbits; Waggie KS et al.; An enzyme-linked immunosorbent assay (ELISA) is described for the detection of rabbit serum antibody directed against the causative agent of Tyzzer's disease, Bacillus piliformis . Ninety-four percent agreement was found between the ELISA and an indirect fluorescent antibody test . The sensitivity of the ELISA was 95% and its specificity was 92% as compared to the indirect fluorescent antibody test (IFAT) . The rabbit origin B . piliformis isolate used in this ELISA was found to be cross-reactive by ELISA and IFAT to B . piliformis isolates of rat, gerbil and horse origin . This suggests that a single B . piliformis isolate may be used as antigen for an ELISA utilizable for multiple species. Lab Anim, 1987 Apr, 21(2), 155 - 60 Lesions of experimentally induced Tyzzer's disease in Syrian hamsters, guineapigs, mice and rats; Waggie KS et al.; The relative susceptibilities of C57BL/6NCR and BALB/cANNCR mice, F344/NCR rats, 2/NCR guineapigs and CR:RGH Syrian hamsters to Bacillus piliformis infection were determined by orally inoculating 20 weanling females from each species with suspensions of B . piliformis spores . Animals from each group were sequentially necropsied over 2 week periods to document the lesions produced . No significant gross or microscopic lesions were observed in the BALB mice or the Fischer rats . Gross and microscopic lesions were observed in the livers and intestines of many guineapigs and hamsters killed 3-14 days after inoculation . A large lesion was observed in the left cardiac ventricle of one C57BL mouse 10 days after inoculation . Warthin-Starry silver-stained tissue sections revealed clusters of B . piliformis within the cytoplasm of intestinal epithelial cells, smooth muscle cells, hepatocytes and myocytes bordering foci of necrosis in the intestines, liver and heart. J Clin Microbiol, 1987 Apr, 25(4), 672 - 4 Clinical features and therapeutic interventions in 17 cases of Bacillus bacteremia in an immunosuppressed patient population; Cotton DJ et al.; We retrospectively examined episodes of Bacillus bacteremia at a hospital with a large proportion of immunosuppressed patients . Seventeen episodes in 9.5 years met our case definition: two of two bottles of one blood culture or one of two bottles of two or more separately obtained blood cultures drawn on the same date . During the same period, there were 59 additional episodes in which a single blood culture had only one of two bottles positive for Bacillus species . Only 2 of 59 such episodes resulted in recurrent bacteremia (3%), as compared with 5 of 17 episodes meeting our case definition (29%) (P = 0.004) . In four of five episodes complicated by recurrent bacteremia and in which appropriate antibiotics were used, a Hickman-Broviac catheter was in place and was not removed . We suggest that our case definition permits the differentiation of infection from contamination based on outcome and that patients with Bacillus bacteremia have chronic venous catheters removed as well as receive antibiotic treatment. Cancer Res, 1987 Apr 1, 47(7), 1762 - 6 Intravesical Bacillus Calmette-Guérin therapy for murine bladder tumors: initiation of the response by fibronectin-mediated attachment of Bacillus Calmette-Guérin; Ratliff TL et al.; Intravesical Bacillus Calmette-Guerin (BCG) is considered to be one of the most effective treatments for superficial bladder cancer . Although the mechanisms by which BCG inhibits tumor growth are not known, previous studies have shown that systemic immunization to BCG and the local expression of the immune response in the bladder are associated with a favorable response to BCG therapy . We have investigated the conditions required for the initiation of an immunological response after the intravesical instillation of BCG . Initial histological studies showed that BCG attached to the bladder wall only in areas where the urothelium was damaged by electrocautery and suggested that attachment was associated with the fibrin clot . Quantitative studies verified the histological observations . Minimal BCG attachment (mean less than 10(2) colony forming units) was observed in normal bladders in contrast with a mean of 1.42 X 10(4) colony forming units/bladder in bladders damaged by electrocautery (10 separate experiments) . BCG attachment to the bladder wall was durable since organisms were observed in bladders 48 h after instillation . To investigate the proteins to which BCG attached, we tested the binding of BCG to extracellular matrix and inflammatory proteins which comprise a significant portion of the fibrin clot . BCG bound in vitro to coverslips coated in vivo with extracellular matrix proteins but did not bind to control albumin-coated coverslips . BCG also bound to coverslips coated with purified plasma fibronectin but not to coverslips coated with other purified extracellular matrix proteins including laminin, fibrinogen, and type IV collagen . BCG attachment to coverslips coated with either extracellular matrix proteins or purified fibronectin was inhibited by antibodies specific for fibronectin . Moreover, BCG attachment to cauterized bladders in vivo was inhibited by antifibronectin antibodies . These results demonstrate that fibronectin mediates the attachment of BCG to surfaces and suggest that it is the primary component mediating attachment within the bladder . Moreover, the data suggest that the BCG-fibronectin interaction may be a requisite first step for the initiation of the antitumor activity in intravesical BCG for bladder cancer. Pharm Res, 1987 Apr, 4(2), 154 - 7 A novel method for the synthesis of kyotorphin, Tyr-Arg, and 3H-Tyr-Arg, catalyzed by tyrosyl-tRNA synthetase from Bacillus stearothermophilus; Kitabatake S et al.; A novel method of dipeptide synthesis is described that can be carried out in aqueous solution and does not require complicated protecting and deprotecting procedures . An analgesic neuropeptide named kyotorphin, H-Tyr-Arg-OH, was synthesized from unprotected tyrosine and arginine in a new enzymatic reaction catalyzed by immobilized tyrosyl-tRNA synthetase from Bacillus stearothermophilus . The reaction could be a useful tool in the syntheses of radioisotope-labeled oligopeptides to be used in receptor binding assays . 3H-Kyotorphin was prepared by this method at a yield of 72% and could be used in receptor binding assays after a single chromatographic separation. Ophthalmology, 1987 Apr, 94(4), 407 - 13 Microbial endophthalmitis resulting from ocular trauma; Affeldt JC et al.; Twenty-seven cases of culture-positive endophthalmitis that developed after ocular trauma were reviewed . The intraocular culture specimens showed a virulent microbiologic spectrum with Bacillus sp as the most common isolate (8 eyes) . The visual prognosis was poor, with only 22% of patients retaining 20/400 or better vision . This level of vision was achieved in 2 of 22 (9%) bacterial cases and in four of five (80%) fungal cases . Retinal detachment (5 cases) or retinal breaks (2 cases) at the time of the initial injury had a uniformly poor visual prognosis . Postoperative retinal detachment not associated with phthisis bulbi occurred in five eyes, three of which had successful retinal reattachment surgery . Delayed onset retinal detachment after successful initial management of traumatic endophthalmitis had a greater frequency of successful retinal reattachment surgery. Biochem Int, 1987 Apr, 14(4), 597 - 603 Order of binding of substrate to valyl-tRNA synthetase from Bacillus stearothermophilus in amino acid activation reaction; Kakitani M et al.; Amino acid activation reaction with valyl-tRNA synthetase (EC 6.1.1.9) from Bacillus stearothermophilus was studied kinetically by measuring ATP-PPi exchange to find the order of the binding of substrate to the enzyme . The effects of the concentration of the substrates (L-valine and ATP) and two dead-end inhibitors (L-valinol and adenosine) on the reaction rate were analyzed . The results indicate that L-valine and ATP are bound to the enzyme in a random sequence . This conclusion is consistent with the one previously suggested by static binding experiments. Mikrobiyol Bul, 1987 Apr, 21(2), 131 - 7 {Effect of physiological factors on biosynthesis of urease in Bacillus spp.}; Aksoz N; In this study, urea was shown to be the inducer of the urease enzyme of the soil isolate Bacillus spp . The extracellular urease enzyme production was repressed in UGT cultures containing ammonia, ammonium chloride or tryptophane and in nutrient broth cultures . The optimal urease production culture conditions were determined as pH: 7.0, 30 degrees C and 150 rpm. Antibiot Med Biotekhnol, 1987 Apr, 32(4), 279 - 82 {Solid-phase immunoenzyme analysis of insulin using Bacillus licheniformis 749/c beta-lactamase and horseradish peroxidase as markers}; Zhvirblene AA et al.; Two modifications of solid phase enzyme immunoassay (EIAA) for insulin with the use of beta-lactamase from B . licheniformis 749/c and horse radish peroxidase as the markers were developed . beta-Lactamase and peroxidase conjugates with insulin were prepared with the glutaric aldehyde and periodate methods respectively . Both the conjugates were stable and preserved high immunospecific and enzymatic activity . Sensitivity of EIAA with the use of the beta-lactamase or peroxidase as the markers was the same and amounted to 15 ng/ml of insulin . However, with the use of the beta-lactamase in EIAA not only spectrophotometric recording but also visual registration of the results was possible. J Parasitol, 1987 Apr, 73(2), 295 - 9 Alteration of Trichostrongylus colubriformis egg permeability by Bacillus thuringiensis israelensis toxin; Bone LW et al.; A toxin from the bacterium Bacillus thuringiensis israelensis is lethal to nematode eggs . Exposure of eggs of the ruminant nematode Trichostrongylus colubriformis to the toxin significantly increased the eggs' permeability to radiolabeled phenylalanine within 2 hr . Calcium chloride inhibited the toxin-induced change in egg permeability . Iodine staining of eggs that were exposed to the microbial toxin revealed that egg permeability was altered within 5 min and was dependent on the dose of toxin . Addition of 34 mM sucrose, 17 mM sodium chloride, or 17 mM potassium chloride to the eggs' medium increased the toxin's lethality . Exopeptidase activity in eggs of T . colubriformis was reduced significantly after exposure to the B . t . israelensis toxin . Tetrodotoxin, tetraethylammonium chloride, ouabain, 4-acetamido-4'-isothiocyano-stilbene-2,2'disulfonic acid (SITS), 4,4'-diisothiocyano-2,2'disulfonic acid stilbene (DIDS), valinomycin, and sodium vanadate, which affect membrane transport, had no significant effect on the activity of B . t . israelensis toxin for eggs . Likewise, a series of nucleotides and their derivatives had no effect on the toxin's activity . Ovicidal activity of the microbial toxin was increased by 4-aminopyridine (4.4 X), but was decreased by furosemide (97 X), nigericin (263 X), or monensin (125 X) . Microscopic measurement of T . colubriformis eggs after treatment with the microbial toxin revealed no significant size change. J Biomol Struct Dyn, 1987 Apr, 4(5), 885 - 93 Distribution of charges in Bacillus intermedius 7P ribonuclease determines the number of cooperatively melting regions of the globule; Protasevich II et al.; A correlation between the distribution of charged side groups in the globule of Bacillus intermedius 7P ribonuclease (binase) and the process of heat denaturation was studied at different pH values in order to estimate a relation between charge distribution in globular proteins and the character of cooperative thermodynamic transitions . As was shown by comparing the results of scanning microcalorimetric analysis of heat denaturation with the three-dimensional structure of binase, at optimal pH the molecule exists as a single cooperative system stabilized by hydrogen bonds, Van der Waals' contacts, and electrostatic interactions like salt bridges . At pH lower than 4.0 (below the physiological optimum) the cooperativity type of the system was found to change due to a reversible cooperative transition in the ternary structure of the protein globule . It has been concluded that the molecular architecture and the arrangement of atoms do not change considerably in different environments; thus the thermodynamic properties of the globule vary due to the alteration of charge distribution and the consequent changes in the size and number of cooperative regions of the globule . Thus, structural and energetic domains may be non-coincident in proteins. Pathology, 1987 Apr, 19(2), 115 - 9 On the "cause" of tuberculosis; Stehbens WE; In epidemiology, tuberculosis is currently considered to have multiple causes and "cause" is used for all secondary, aggravating and conditional factors in its pathogenesis . The tubercle bacillus is the cause of tuberculosis, and without it tuberculosis cannot occur . The failure to differentiate the cause from noncausative associated factors has wide and serious implications . It is retrogressive, scientifically inaccurate and to be avoided for the sake of logic and precision in scientific communication . Associated noncausative factors need to be classified according to their roles in the pathogenesis of the disease. Can J Microbiol, 1987 Apr, 33(4), 304 - 13 Microincineration and elemental X-ray microanalysis of single Bacillus cereus T spores; Scherrer R et al.; Single whole spores of bacillus cereus T were analyzed by scanning electron microscopy and electron microprobe X-ray microanalysis before and after high-temperature (600 degrees C) ashing in air . High-temperature ashing consisted of a centripetal oxidation of the spore surface combined with pyrolysis of the spore's interior . Ashing of single spores produced a compact central ash particle, mimicking the much larger unashed spore body in outline but containing craterlike microregions, and a peripheral thin ash film . Ashing mostly eliminated the spore's organic matrix; however, ash residues still gave residual carbon-characteristic X-ray counts . Ashing of single spores produced a two-, five-, and six-fold increase of potassium, magnesium, and calcium X-ray intensities, respectively . Iron, although low in actual counts, became detectable after ashing . Phosphorus characteristic X-rays were decreased by 41% after ashing, while volatilization lowered sodium and manganese X-ray intensities by over 80% . High-temperature ashing enhanced element-characteristic X-ray intensities of the non-volatilizable mineral(ized) elements of spores by compacting them into ash residues, more so than by simply abolishing their organic matrix . Microincineration appears a generally useful preconcentration technique for elemental detection and localization in X-ray microanalysis. J Nutr Sci Vitaminol (Tokyo), 1987 Apr, 33(2), 113 - 27 Hydrolysis and synthesis of thiamin triphosphate in bacteria; Nishimune T et al.; Thiamin triphosphate (ThTP) in early stationary phase cells of Escherichia coli grown in nutrient broth with 0.1% yeast extract was found to constitute approximately 5-7% of cellular thiamin diphosphate (ThDP) or around 5 nmol/g cell . Nearly the same level of ThTP was obtained in a Bacillus strain . When E . coli was loaded with an excess of ThTP or ThDP, cellular ThTP was found to be controlled in the course of the long term to maintain its ratio to the amount of cellular ThDP . The ThTP vs . ThDP ratio in E . coli cells after short-term ThDP uptake was found to be a function of the cellular growth phase . The ratio in early exponential phase E . coli cells was found to be approximately 4% and it became lower (less than 3%) when cell growth proceeded to the late exponential stage . Two phosphatases specific for ThTP (ThTPase) among thiamin phosphates were detected in E . coli . One required Mg2+ and was found mainly in the soluble fraction, while the other was Mg2+-independent and originated from the membrane . The two ThTPases were similar to their rat tissue counterparts. Mol Gen Mikrobiol Virusol, 1987 Apr, (4), 19 - 22 {Purification and substrate specificity of BcmI restriction endonuclease}; Tsvetkova NV et al.; A strain producing the new specific restriction endonuclease BcmI has been found in the Bacillus generum . The enzyme has been purified by chromatography on the blue sepharose, phosphocellulose PII, heparin sepharose . The analogous purification has been obtained when the blue sepharose has been substituted for the orange sepharose, the home produced sorbent . The BcmI enzyme has been shown by the substrate specificity definition to be an isoschizomer of the restriction endonuclease ClaI. Arch Biochem Biophys, 1987 Apr, 254(1), 376 - 9 The c-type cytochromes of the gram-positive bacterium Bacillus licheniformis; Woolley KJ; The release of soluble c-type cytochromes from cells of the gram-positive bacterium Bacillus licheniformis was effected by treatment with lysolytic buffer . After further purification three different c-type cytochromes designated c-551, c-552, and c-554 were isolated . Oxidized and reduced spectra, molecular weight, isoelectric point, and amino acid compositions are reported for each of these monoheme proteins. Med Vet Entomol, 1987 Apr, 1(2), 157 - 62 Efficacy of Bacillus sphaericus 2362 against larvae of Anopheles gambiae under laboratory and field conditions in West Africa; Nicolas L et al.; A flowable concentrate of Bacillus sphaericus (Neide) strain 2362 was applied against Anopheles gambiae Giles s.l . mosquito larve in small plot field trials in Bobo-Dioulasso area . Burkina-Faso . Third and fourth instar larvae were controlled for 10-15 days with a dosage of 10 g/m2, 3-10 days with 1 or 0.1 mg/m2, and 2 days with 0.01 g/m2 . Complete elimination of larval populations required 1 x 10(2) to 2 x 10(3) viable spores/ml in the larval feeding zone . After treatment, the total numbers of viable spores decreased in the ponds, due to ingestion of spores by non-target as well as target organisms and/or loss of viability of some spores by sunlight . This formulation was less effective against An . gambiae than against Culex quinquefasciatus Say larvae, both in laboratory bioassays and under field conditions. Med Vet Entomol, 1987 Apr, 1(2), 137 - 46 Management of insecticide resistance in control of the Simulium damnosum complex by the Onchocerciasis Control Programme, West Africa: potential use of negative correlation between organophosphate resistance and pyrethroid susceptibility; Kurtak D et al.; 1 . Resistance of some populations of the Simulium damnosum complex to temephos (100-fold at the LC50 level), with degrees of cross-resistance to chlorphoxim (14-fold) and other organophosphate insecticides, follows intensive larvicidal control of S . damnosum s.l . in West African river systems since 1975 by the WHO Onchocerciasis Control Programme . 2 . Larvae of at least three sibling species of the S . damnosum complex have become organophosphate-resistant: these are the forest species S . sanctipauli Vajime & Dunbar and the savanna species S . sirbanum V . & D . and S . damnosum Theobald sensu stricto . 3 . Organophosphate-resistant S . damnosum s.l . larvae show increased susceptibility to some organochlorine and pyrethroid insecticides, especially to permethrin (up to 11-fold) and OMS 3002 (up to 17-fold), as compared with organophosphate-susceptible populations . 4 . This differential susceptibility is reflected by increased pyrethroid efficacy in operational use for river treatments against organophosphate-resistant field populations of S . damnosum s.l . larvae . Treatment of 100 km of the lower Bandama River in 1985 showed that permethrin at the highly selective dosage of 10 min exposure to 0.01 mg/l caused reversion towards organophosphate-susceptibility of the target population of S . sanctipauli . This effect was less pronounced when the Comoe River was treated at the lower dosage of 0.005 mg/l for 10 min . 5 . To overcome temephos-resistance, it is proposed that the most rational usage of currently available larvicides would involve the following annual sequence of treatments: Bacillus thuringiensis serotype H-14 when river discharge is below 75 m3/s; chlorphoxim for about eight weekly treatment cycles after river discharge rises; permethrin (or alternative pyrethroid) for up to six treatment cycles--this should eliminate any incipient selection for chlorphoxim-resistance; resume chlorphoxim (or perhaps carbosulfan) treatments until river discharge falls below 75 m3/s permitting resumed use of B.t . H-14. J Biochem (Tokyo), 1987 Apr, 101(4), 871 - 8 Purification and properties of the aromatic amino acid aminotransferase from gramicidin S-producing Bacillus brevis; Kanda M et al.; The aromatic amino acid aminotransferase was purified to a homogenous state from a gramicidin S-producing strain of Bacillus brevis . The enzyme shows a molecular weight of about 71,000 on gel-filtration . The subunit molecular weight is about 35,000 as determined by sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is a dimer . The enzyme exhibits absorption maxima near 425 and 330 nm at neutral pH . One mole of pyridoxal phosphate is bound per subunit . The enzyme has amino donor specificity for aromatic amino acids, L-phenylalanine, L-tyrosine, and L-tryptophan, and utilizes 2-oxoglutarate as the amino acceptor . This enzyme activity was separated from both the aspartate aminotransferase activity and the branched chain amino acid aminotransferase activity by chromatography on DEAE-Sephadex. J Bacteriol, 1987 Apr, 169(4), 1564 - 70 Cloning and nucleotide sequence of the gene coding for enzymatically active fragments of the Bacillus polymyxa beta-amylase; Kawazu T et al.; The gene encoding beta-amylase was cloned from Bacillus polymyxa 72 into Escherichia coli HB101 by inserting HindIII-generated DNA fragments into the HindIII site of pBR322 . The 4.8-kilobase insert was shown to direct the synthesis of beta-amylase . A 1.8-kilobase AccI-AccI fragment of the donor strain DNA was sufficient for the beta-amylase synthesis . Homologous DNA was found by Southern blot analysis to be present only in B . polymyxa 72 and not in other bacteria such as E . coli or B . subtilis . B . polymyxa, as well as E . coli harboring the cloned DNA, was found to produce enzymatically active fragments of beta-amylases (70,000, 56,000, or 58,000, and 42,000 daltons), which were detected in situ by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Nucleotide sequence analysis of the cloned 3.1-kilobase DNA revealed that it contains one open reading frame of 2,808 nucleotides without a translational stop codon . The deduced amino acid sequence for these 2,808 nucleotides encoding a secretory precursor of the beta-amylase protein is 936 amino acids including a signal peptide of 33 or 35 residues at its amino-terminal end . The existence of a beta-amylase of larger than 100,000 daltons, which was predicted on the basis of the results of nucleotide sequence analysis of the gene, was confirmed by examining culture supernatants after various cultivation periods . It existed only transiently during cultivation, but the multiform beta-amylases described above existed for a long time . The large beta-amylase (approximately 160,000 daltons) existed for longer in the presence of a protease inhibitor such as chymostatin, suggesting that proteolytic cleavage is the cause of the formation of multiform beta-amylases. Biochem Biophys Res Commun, 1987 Mar 30, 143(3), 901 - 7 Purification and characterization of the larvicidal toxin of Bacillus sphaericus 1593M; Sgarrella F et al.; We present here a procedure for purifying the larvicidal toxin from sporulating cells of Bacillus sphaericus 1593M and describe some of the biochemical and biophysical properties of this toxin . The procedure involves solubilization of the cell-wall/membrane bound toxin by sonication of cells followed by repeated rounds of freezing and thawing at 50 degrees C . Further purification involved Sephadex G-100 and DEAE Sephacel chromatography . We show by Sephadex G-100 chromatography that at pH 7.5 the smallest active form of the toxin has an Mr of 38,000 and that this toxin can reversibly aggregate to molecular forms of a size higher than 2 X 10(5) Mr . By shifting the pH from 7.5 to 8.5 only the aggregated forms can be observed. J Biol Chem, 1987 Mar 25, 262(9), 4280 - 3 Crystallization and preliminary x-ray diffraction studies of subtilisin GX from Bacillus sp . GX6644; Gilliland GL et al.; Subtilisin GX, a serine protease from Bacillus species GX6644, has been crystallized by the vapor diffusion method using ammonium sulfate as the precipitant . The space group is P212121 with a = 38.4 A, b = 70.3 A, c = 73.5 A, and one molecule in the asymmetric unit . The crystals diffract to beyond 2.0-A resolution and are suitable for a high resolution three-dimensional structure determination . All x-ray data used in the preliminary crystallographic study were collected with an electronic area detector. J Mol Biol, 1987 Mar 20, 194(2), 287 - 97 Crystal structure of a deletion mutant of a tyrosyl-tRNA synthetase complexed with tyrosine; Brick P et al.; The crystal structure of a deletion mutant of tyrosyl-tRNA synthetase from Bacillus stearothermophilus has been determined at 2.5 A resolution using molecular replacement techniques . The genetically engineered molecule catalyses the activation of tyrosine with kinetic properties similar to those of the wild-type enzyme but no longer binds tRNATyr . It contains 319 residues corresponding to the region of the polypeptide chain for which interpretable electron density is present in crystals of the wild-type enzyme . The partly refined model of the wild-type enzyme was used as a starting point in determining the structure of the truncated mutant . The new crystals are of space group P2(1) and contain the molecular dimer within the asymmetric unit . The refined model has a crystallographic R-factor of 18.7% for all reflections between 8 and 2.5 A . Each subunit contains two structural domains: the alpha/beta domain (residues 1 to 220) containing a six-stranded beta-sheet and the alpha-helical domain (residues 248 to 319) containing five helices . The alpha/beta domains are related by a non-crystallographic dyad while the alpha-helical domains are in slightly different orientations in the two subunits . The tyrosine substrate binds in a slot at the bottom of a deep active site cleft in the middle of the alpha/beta domain . It is surrounded by polar side-chains and water molecules that are involved in an intricate hydrogen bonding network . Both the alpha-amino and hydroxyl groups of the substrate make good hydrogen bonds with the protein . The amino group forms hydrogen bonds with Tyr169-OH, Asp78-OD1 and Gln173-OE1 . The phenolic hydroxyl group forms hydrogen bonds with Asp76-OD1 and Tyr34-OH . In contrast, the substrate carboxyl group makes no direct interactions with the enzyme . The results of both substrate inhibition studies and site-directed mutagenesis experiments have been examined in the light of the refined structure. Biochim Biophys Acta, 1987 Mar 19, 923(3), 381 - 8 Isolation and partial characterization of biologically active Fc receptor of chicken red cells; Manghi MA et al.; It has previously been demonstrated that chicken red cells have a receptor with the capacity to bind aggregated IgG, IgM 7 S or antigen-complex IgG . This receptor was isolated from Nonidet P-40 soluble extracts of chicken red cells by immunoadsorption with either immobilized aggregated IgG or monomeric IgM (IgM 7 S) and further gel filtration through a Sephacryl S-300 column . The Fc binding material was characterized as a glycoprotein with a molecular weight of 30,000 which retained its Fc receptor activity after the isolation procedure . This was demonstrated by its capacity to inhibit the binding of 125I-IgM 7 S or 125I-labelled aggregated IgG to chicken red cells . After Bacillus cereus phospholipase C treatment the Fc receptor activity remained unchanged, but the molecular weight (15,000) did not, suggesting that the phospholipids cleaved by this treatment were not essential for the interactions of the receptor with specific ligands . However, this Fc-binding component was shown to have a molecular weight of 13,000 and a diminished Fc receptor activity after reduction with dithiothreitol, suggesting the presence of at least one disulphide bridge, necessary to maintain the total ligand-binding activity. Am Rev Respir Dis, 1987 Mar, 135(3), 763 - 5 Pulmonary complications of intravesical Bacille Calmette-Guérin immunotherapy; Israel-Biet D et al.; Lungs have rarely been reported to be affected by any side effects of BCG therapy . Interstitial pneumonitis, though, is known to occur under such circumstances, but its pathogenesis is still debated between an infectious and a hypersensitivity mechanism . We report here 3 cases of pulmonary complications of BCG therapy evaluated by bronchoalveolar lavage (BAL) . Cellular data obtained from all 3 patients were characterized by a markedly increased alveolar lymphocytosis . The T4/T8 ratio was elevated compared with that in normal subjects and with the T4/T8 ratio of circulating lymphocytes . Furthermore, alveolar lymphocytes were highly sensitized to PPD, as evaluated by their proliferation and their production of interleukin 2 in the presence of PPD . Mycobacteria were not found in the 3 patients . We conclude that interstitial pneumonitis occurring during BCG therapy could be explained by a hypersensitivity phenomenon, leading to an intense immune and lymphocyte-mediated response within involved organs. J Lab Clin Med, 1987 Mar, 109(3), 300 - 7 Assessment of tamoxifen as adjuvant therapy in stage II breast cancer: a long-term follow-up; Marshall JS et al.; Results of a prospective, randomized clinical trial of three treatment regimens--cyclophosphamide, methotrexate, and 5-fluorouracil (C); C plus the antiestrogen, tamoxifen citrate (CT); and CT plus bacillus Calmette-Guerin (CTBCG)--in 311 women with stage II breast cancer are reported . The data were analyzed by univariate (product limit and log rank) analysis and by multivariate analysis . Estrogen receptors were measured in all primary tumors . The mean follow-up period was 78.2 months . The regimens containing tamoxifen citrate significantly decreased the risk of recurrence in patients with positive estrogen receptors . The addition of tamoxifen does not, however, appear to provide an advantage in overall survival . No benefit in disease-free or overall survival was observed resulting from the addition of BCG to the treatment regimen . The design of the study did not permit an evaluation of the efficacy of the chemotherapy used inasmuch as all patients received it. J Am Geriatr Soc, 1987 Mar, 35(3), 213 - 8 Gram-negative bacillary bacteremia in the elderly: incidence, ecology, etiology, and mortality; McCue JD; The incidence, ecology, and mortality of gram-negative bacillary bacteremia in elderly patients were studied in an analysis of 334 episodes over a four-year-period in a 489-bed North Carolina community teaching hospital, 135 (40.4%) of which occurred in patients 70 years of age or older . The bacteremia rate per 1000 hospital admissions increased sharply with increasing age . The ecology and in vitro antimicrobial susceptibilities of the bacterial isolates were strongly influenced by community v hospital acquisition, but not by age . Urosepsis was significantly more likely to be the underlying source of hospital-acquired bacteremia in patients 70 years or older (P less than 0.01) . Total bacteremia-related mortality did not increase with increasing age; in the group of patients aged 70 years or older with nonfatal/ultimately fatal underlying diseases (NF/UFUD), however, mortality was 9.1% compared to 2.9% in the younger age group (P less than 0.001) . Significantly increased bacteremia-related mortality was also noted in the older patients with NF/UFUD admitted from nursing homes (P less than 0.05) and those not treated with an appropriate antimicrobial agent within 24 hours (P less than 0.01) . Overall, the older patients with hospital-acquired bacteremia, neutropenia-associated infection, those bacteremic from a nonurinary source of infection, and those treated with multiple-drug regimens had higher mortality (P less than 0.05) . Gram-negative bacteremia is much more common in patients 70 years of age or older and compared with younger patients mortality appears to be significantly increased for the important subgroup of older patients with nonfatal or ultimately fatal underlying diseases. J Bacteriol, 1987 Mar, 169(3), 1147 - 52 Transformation of vegetative cells of Bacillus thuringiensis by plasmid DNA; Heierson A et al.; Plasmid DNA-mediated transformation of vegetative cells of Bacillus thuringiensis was studied with the following two plasmids: pBC16 coding for tetracycline resistance and pC194 expressing chloramphenicol resistance . A key step was the induction of competence by treatment of the bacteria with 50 mM Tris hydrochloride buffer (pH 8.9) containing 30% sucrose . Transformation frequency was strongly influenced by culture density during the uptake of DNA and required the presence of polyethylene glycol . Growth in a minimal medium supplemented with Casamino Acids gave 35 times more transformants than growth in a rich medium . The highest frequencies were obtained with covalently closed circular DNA . With all parameters optimized, the frequency was 10(-3) transformants per viable cell or 10(4) transformants per microgram of DNA . Cells previously frozen were also used as recipients in transformation experiments; such cells gave frequencies similar to those obtained with freshly grown cells . The procedure was optimized for B . thuringiensis subsp . gelechiae, but B . thuringiensis subsp . kurstaki, B . thuringiensis subsp . galleriae, B . thuringiensis subsp . thuringiensis, and B . thuringiensis subsp . israelensis were also transformed . Compared with protoplast transformation, our method is much faster and 3 orders of magnitude more efficient per microgram of added DNA. Am Rev Respir Dis, 1987 Mar, 135(3), 671 - 5 Increased salivary elastase precedes gram-negative bacillary colonization in postoperative patients; Dal Nogare AR et al.; The upper airway epithelium is coated with fibronectin, a glycoprotein that covers receptor sites for gram-negative bacteria and prevents them from colonizing the oropharynx . We investigated the identity of salivary proteolytic enzymes capable of degrading fibronectin in a group of 16 patients who had elective cardiac surgery . Six patients became colonized with gram-negative bacteria (Group C) and 10 did not (Group NC) . Salivary elastase activity was low in both groups preoperatively . Twenty-four hours after surgery, salivary elastase activity increased in Group C, and it remained elevated at 48 and at 72 h . Fibronectin digestive activity of the saliva of patients in Group C was also increased within 24 h of surgery, and salivary elastase and fibronectin digestive activity were highly correlated (r = 0.86, p less than 0.001) . Enzyme inhibition experiments showed that most of the fibronectin digestive activity was due to elastase from polymorphonuclear cells (PMN), and the molecular weight of the salivary enzyme digesting fibronectin was 30,000 daltons (similar to the molecular weight of elastase) . Levels of antileukoprotease, the major elastase inhibitor in saliva, were normal in patients with increased elastase activity . We conclude that salivary elastase is of PMN origin, increases prior to gram-negative bacillary colonization of the pharynx, and is responsible for most of the fibronectin digestive activity of the saliva. Mikrobiologiia, 1987 Mar-Apr, 56(2), 243 - 8 {Comparative characteristics of extrachromosomal DNA in cultures of Bacillus thuringiensis serotype H-14}; Ambartsumian NS et al.; The extrachromosomal DNAs from different strains of Bacillus thuringiensis subsp . israelensis were comparatively analysed . Within the serotype studied, two groups of strains have been revealed . These are characterised by a certain plasmid composition as well as by specific physiological, biochemical and insecticide properties. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1987 Mar, 263(4), 509 - 24 Examination of 20 Bacillus species by crossed immunoelectrophoresis under taxonomic aspects; Hartung M et al.; In order to examine the efficiency of crossed immunoelectrophoresis (CIE) for the differentiation of species and for the determination of taxonomic relationships within the genus Bacillus, an investigation including strains of 20 species was performed . Ultrasonic extracts (USE) of sporefree-grown vegetative cells were used . With rabbit antisera against USE of type strains, the CIE was accomplished in homologous and heterologous combinations . USE's of additional strains were included into this investigation . In order to evaluate the immunoelectropherograms, the number of precipitates was counted . A mean of 103 precipitates was found in homologous reactions . With few exceptions, heterologous combinations showed less similarity in the number of antigens . Because of their high cross-reactions, the following species could not be differentiated: B . subtilis from B . amyloliquefaciens and strains of B . coagulans, furthermore, B . cereus from B . thuringiensis . The different species revealed lower numbers of precipitates with decreasing taxonomic relationships in heterologous combinations . This observation was used to classify the investigated species by a 'position-frequency analysis' (PFA) . After sorting the matrix of precipitate numbers in the ascertained optimal sequence of species, a cluster analysis was carried out . The phenogram showed 6 (respectively 8) group clusters . The members of the morphologic group I (Smith et al., 1952) was found only in group cluster 2 . The phenogram was partly in agreement with phenograms based on other characteristics, e.g . DNA hybridization. Pediatr Infect Dis J, 1987 Mar, 6(3), 272 - 80 Tuberculosis in Bacillus Calmette-Guérin-immunized and unimmunized children in Sweden: a ten-year evaluation following the cessation of general Bacillus Calmette-Guérin immunization of the newborn in 1975; Romanus V; An analysis was made of childhood tuberculosis in Sweden between 1969 and 1984 which included the 6.25 years before and the 9.75 years after the cessation of general Bacillus Calmette-Guerin (BCG) immunization of the new-born on April 1, 1975 . The annual incidence of tuberculosis per 100,000 children ages 0 to 4 years increased from an average of 1.1 cases in the period 1970 to 1974 to 1.3 cases in the period 1975 to 1979 and to 2.1 cases in the period 1980 to 1984, including both children born in Sweden and those born abroad . Among children born in Sweden after April 1, 1975, tuberculosis occurred in 58 (57 unimmunized and one BCG-immunized), or 1.3 cases per 100,000 person years up to and including 1984 . Eighteen of the 58 children were asymptomatic . Minor symptoms were reported in 13 and clinical illness in 27 children, 2 of whom developed meningitis and 1 of whom died of miliary infection . The relative increase of tuberculosis in the mainly unimmunized cohorts born in Sweden after April 1, 1975, compared with the mainly BCG-immunized cohorts born in Sweden in the period 1969 to 1974 was, by the end of 1984, estimated at 6.0 (95% confidence interval, 2.3, 16.1) . Tuberculosis was about 10 times more common in non-BCG-immunized children born in Sweden of foreign parents than in those born of Swedish parents.(ABSTRACT TRUNCATED AT 250 WORDS) J Clin Oncol, 1987 Mar, 5(3), 441 - 9 A prospective randomized trial of maintenance versus nonmaintenance intravesical bacillus Calmette-Guérin therapy of superficial bladder cancer; Badalament RA et al.; Between August 1981 and July 1984, 93 patients with polychronotopic superficial papillary carcinoma (Ta and/or T1), flat carcinoma in situ (Tis), or concomitant superficial papillary and in situ bladder carcinoma were entered into a prospective randomized trial of maintenance v nonmaintenance intravesical bacillus Calmette-Guerin (BCG) therapy . Forty-six patients who received BCG weekly for 6 weeks were compared with 47 patients receiving the six-weekly doses of BCG plus monthly BCG for 2 years . Both groups were evaluated every 3 months by cytology, cystoscopy, and biopsy . A significant reduction in the number of recurrent tumors per patient-month was demonstrated for both groups (P less than .0001); however, the difference in reduction of tumors between the two groups was not significant . Additionally, patients receiving maintenance and nonmaintenance therapy had similar tumor recurrence and progression rates . These results indicate that monthly maintenance BCG does not prevent, delay, or reduce tumor recurrence or progression observed with the 6-week regimen . Maintenance BCG was associated with increased local toxicity, primarily dysuria, frequency, and urgency . Dosage reduction was required in 22 of 47 patients (46.8%) . When the data were subjected to multivariate analysis, the presence or absence of tumor following induction BCG and PPD skin test results were found to be significant variables . Controlling for either the presence or absence of tumor following induction BCG, tumor recurrence and progression rates were not significantly different for the two treatment groups . However, the absence of tumor after induction BCG was associated with a longer disease-free duration (P = .00001) and time to progression (P = .095) . Patients with a reactive tuberculin skin test before and after induction BCG had significantly less tumor recurrences than patients with different PPD skin tests results (P = .02) . Tumor progression was not related to tuberculin skin testing. J Bacteriol, 1987 Mar, 169(3), 1017 - 23 Expression in Escherichia coli of a cloned crystal protein gene of Bacillus thuringiensis subsp . israelensis; McLean KM et al.; A ca . 10-kilobase (kb) HindIII fragment of plasmid DNA from Bacillus thuringiensis subsp . israelensis was cloned into plasmid pUC9 and transformed into Escherichia coli . Extracts of the recombinant strain contained a 27-kilodalton (kDa) peptide that reacted with antibodies to a 27-kDa peptide isolated from crystals produced by B . thuringiensis subsp . israelensis . Extracts of the recombinant strain were hemolytic and toxic to Aedes aegypti larvae . Full expression of the 27-kDa peptide required the presence of a ca . 0.8-kb region of DNA located 4 kb upstream from the structural gene; the 0.8-kb region could be present in cis or trans relative to the gene and apparently acted post-transcriptionally . Analysis of maxicells showed that the 10-kb insert also coded for peptides of 67, 20, and 16 kDa; data obtained with different subclones suggest that the 20-kDa peptide is encoded in the 0.8-kb DNA region. J Am Mosq Control Assoc, 1987 Mar, 3(1), 20 - 5 Laboratory and field efficacy of Bacillus thuringiensis var . Israelensis and Bacillus sphaericus against Anopheles gambiae s.l . and Culex quinquefasciatus in Ouagadougou, Burkina Faso; Majori G et al.; Two wettable powders (Bactimos and Vectobac) and one flowable concentrate (Teknar) of Bacillus thuringiensis var . israelensis (B.t.i.) and primary powders of Bacillus sphaericus isolates 1593 and 2362 were evaluated (laboratory) against field-collected larvae of Anopheles gambiae s.l . and Culex quinquefasciatus in Ouagadougou, Burkina Faso . Bactimos, Vectobac and a Corn-cob B.t.i . formulation (ABG-6138G) were field tested against Cx . quinquefasciatus and An . gambiae s.l . The isolates of B . sphaericus were also tested against An . gambiae s.l . in artificial ponds . Both wettable powders of B.t.i . showed superior activity than the flowable concentrate formulation against An . gambiae s.l . in the laboratory . Culex quinquefasciatus was more susceptible (3-4X) to B.t.i . (Bactimos) than An . gambiae s.l . The isolates of B . sphaericus were more effective (2-3X) against both mosquito species than Bactimos . In a ditch and two channels, Bactimos, Vectobac and ABG-6138G at 0.65, 1.5 and 5.6 kg/ha, respectively, gave 91-100% control of Cx . quinquefasciatus within 3 days of treatment . The same formulations at rates ranging from 0.25 to 5.6 kg/ha, produced 82-97% control of An . gambiae s.l . in rainwater pools 24 h after treatment . Isolates 1593 and 2362 at 0.12 and 0.24 kg/ha gave excellent control of An . gambiae s.l . in artificial ponds. Mutagenesis, 1987 Mar, 2(2), 107 - 9 U.v.-induced and N-methyl-N'-nitro-N-nitrosoguanidine-induced mutagenesis in Bacillus thuringiensis; Auffray Y et al.; The lethal and mutagenic effects of u.v . light and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on Bacillus thuringiensis were investigated . Lethality studies demonstrated that B . thuringiensis was relatively sensitive to these agents . This bacterium was mutated at the rifampicin resistance marker by u.v . light and to a lesser extent by the direct acting alkylating agent MNNG . One mutant selected for its greater sensitivity to u.v . light expressed a higher frequency of mutagenesis after u.v . light treatment and appeared to be defective in an excision repair pathway . However, this mutant was only slightly mutable by MNNG in comparison with the wild-type strain . This unusual phenotype does not yet have a parallel among the radiation sensitive mutants described in other bacterial species. J Gen Microbiol, 1987 Mar, 133 ( Pt 3), 527 - 34 Morphology, growth and reversion in a stable L-form of Escherichia coli K12; Onoda T et al.; An L-form isolated from Escherichia coli K12 by sequential treatment with N-methyl-N'-nitro-N-nitrosoguanidine and lysozyme was adapted to grow in hyperosmolar liquid cultures . It was stable in the absence of antibiotic when cultured in brain heart infusion (BHI) broth containing NaCl and CaCl2, the optimal concentrations being 0.34 M and 1 mM, respectively . No growth of the L-form was observed when CaCl2 was not added to BHI medium containing 0.34 M-NaCl . On the other hand, when KCl replaced NaCl as the osmotic stabilizer, growth of the L-form was repressed in the presence of CaCl2 . Electron microscopy of the L-form confirmed the absence of a cell wall . A revertant strain derived from the L-form grew as a stable bacillary form in BHI medium without osmotic stabilizer . The growth characteristics of the revertant strain resembled those of the parent strain . The revertant strain produced L-forms in the presence of NaCl. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1987 Mar, 263(4), 525 - 9 Represent the batches 2 of strain NCTC 3991 and NCTC 3992 B . coagulans? Hartung M, Hellmann E. In a previous publication (M . Hartung and E . Hellmann: Examination of 20 Bacillus Species by Crossed Immunoelectrophoresis under Taxonomic Aspects, Zbl . Bakt . Hyg . A 263 (1987) 509-524) we came to the conclusion that the species B . subtilis and B . coagulans were closely related . This statement has to be revised: The antigenic relationship is low . Further investigations have shown that all three B . coagulans-strains, although originating from different sources, were overgrown by B . subtilis . The same fact may have caused errors in other publications (e.g . Parry et al.: A Colour Atlas of Bacillus Species, Wolfe Medical Publications Ltd., London, 1983. Biochem J, 1987 Mar 1, 242(2), 573 - 9 Purification and properties of a germination-specific cortex-lytic enzyme from spores of Bacillus megaterium KM; Foster SJ et al.; Two peptidoglycan-lytic enzyme activities were isolated from spores of Bacillus megaterium KM . Surface-bound lytic enzyme was extracted from dormant spores and hydrolysed a variety of peptidoglycan substrates including isolated spore cortex, but did not cause refractility changes in permeabilized spores . Germination-specific lytic enzyme activity appeared early in germination and had minimal activity on isolated peptidoglycan substrates, but caused refractility changes in permeabilized spores of several Bacillus isolated peptidoglycan substrates, but caused refractility changes in permeabilized spores of several Bacillus species . The germination-specific lytic enzyme was shown to be a heat-sensitive 29 kDa protein with maximal activity at pH 6.5 . It catalysed post-commitment muramic acid delta-lactam synthesis and displayed an inhibitor profile similar to that for post-commitment A600 loss . The relationship of the germination-specific enzyme to a recently proposed model of spore germination is discussed. Arch Ophthalmol, 1987 Mar, 105(3), 342 - 4 Posttraumatic Bacillus cereus endophthalmitis; Schemmer GB et al.; We encountered a patient who developed Bacillus cereus endophthalmitis following trauma . Early therapy, which included intravitreal clindamycin phosphate and gentamicin sulfate, resulted in a visual acuity of 20/60 . A five-year retrospective review of all cases of endophthalmitis following trauma reported at our institution revealed Bacillus as the infecting organism in six (46%) of 13 culture-positive cases . The high frequency of virulent Bacillus infections in the setting of trauma necessitates the use of antibiotics that are active against this organism in posttraumatic endophthalmitis . The combination of clindamycin and gentamicin can be effective therapy in Bacillus species infections if used early in the course of the disease. J Bacteriol, 1987 Mar, 169(3), 1338 - 40 Occurrence in Bacillus megaterium of uridine 5'-diphospho-N-acetylgalactosamine and uridine 5'-diphosphogalactosamine, intermediates in the biosynthesis of galactosamine-6-phosphate polymer; Nishikawa J et al.; We isolated two galactosamine derivatives from Bacillus megaterium sporulating cells by lectin affinity chromatography followed by DEAE-Sephadex A-25 chromatography . From chemical analyses and measurements of these compounds, it was determined that one was uridine 5'-diphospho-N-acetylgalactosamine and that the other was uridine 5'-diphosphogalactosamine . They appeared in the middle stage of sporulation and disappeared during the period when galactosamine-6-phosphate is deposited on the forespore surface . These results suggest that uridine 5'-diphospho-N-acetylgalactosamine and uridine 5'-diphosphogalactosamine are intermediates in the biosynthesis of the galactosamine-6-phosphate polymer, a backbone structure of the exosporium. FEBS Lett, 1987 Feb 23, 212(2), 193 - 8 An integrated prediction of secondary, tertiary and quaternary structure of glucose dehydrogenase; Hones J et al.; Based on homology of partial sequences, on physico-chemical evidence and on studies using chemical modification, we came to the tentative conclusion that tetrameric glucose dehydrogenases from Bacillus megaterium and B . subtilis should have a structure closely related to that of lactate dehydrogenase . The overall homology of primary structures was found to be very low, however, and independent predictions of secondary structure produced a clearly different pattern of beta-strands and alpha-helices . We nevertheless tried a manual prediction based on the hydrophobic nature of internal beta-sheet and on the amphiphilic character of external helices . This treatment led to the identification of analogues of all the beta-strands present in lactate dehydrogenase with the exception of beta C . Six amphiphilic helices were identified corresponding to alpha B, alpha C, alpha D, alpha 1F, alpha 2F and alpha 3G in lactate dehydrogenase . Conserved functional residues were found at analogous positions . The Q and R intersubunit contacts could be identified and partial proteolysis was found to occur on the outer surface of the tetramer . The structure was found to explain the better binding of NADP as compared to NAD+ and offered a rationalization of the role of the essential lysine at position 201. FEBS Lett, 1987 Feb 23, 212(2), 289 - 91 Tetrameric alkaline phosphatase from human liver is converted to dimers by phosphatidylinositol phospholipase C; Hawrylak K et al.; Membrane-bound human liver alkaline phosphatase solubilized by a non-ionic detergent, Nonidet P-40 (NP-40), has the molecular mass of a tetramer . It can be converted to a dimeric form by treatment with a phosphatidylinositol phospholipase C (PI-PLC) obtained from Bacillus cereus . When human liver plasma membranes were directly treated with PI-PLC, the released alkaline phosphatase was dimeric . Thus, phosphatidylinositol may help maintain the tetrameric quaternary structure of alkaline phosphatase and aid its binding to human liver plasma membranes. Eur J Biochem, 1987 Feb 16, 163(1), 105 - 12 Purification and characterization of cystathionine gamma-synthase type II from Bacillus sphaericus; Kanzaki H et al.; Cystathionine gamma-synthase type II, which catalyzes L-cystathionine synthesis from O-acetyl-L-homoserine and L-cysteine was purified from Bacillus sphaericus (IFO 3536) in seven steps . The purified enzyme appeared to be homogeneous by the results of polyacrylamide electrophoresis and ampholyte electrofocusing . The enzyme is a typical pyridoxal-P dependent enzyme, has a molecular mass of 165 kDa and consists of four subunits identical in molecular mass . The enzyme catalyzed the gamma-replacement reaction and the elimination reaction was hardly detected even when a large amount of enzyme was added . In the replacement reaction, O-acetyl-L-homoserine and the following thiol compounds: L and D-cysteine, L and D-homocysteine, sodium sulfide, various alkyl and aryl mercaptans, acted as the most suitable substrate to produce L-cystathionine and the corresponding S-substituted L-homocysteine derivatives. Cell Immunol, 1987 Feb, 104(2), 334 - 42 Regulation of bone marrow cell survival in short-term cultures: a new macrophage function; Blasi E et al.; The involvement of macrophages (M phi) in the regulation of bone marrow (BM) cell survival in short-term cultures was studied . We developed a system to measure the survival of fresh BM cells in vitro, by evaluating 111indium (111In) release from prelabeled BM cells . 111In release was proportional to cell death and inversely related to the number of trypan blue excluding cells . Upon 24 hr of culture in conventional medium, more than 50% of BM cells died . In order to investigate whether BM cell death could be reduced by coculture with other cell types, 111In-labeled BM cells were incubated for 24 hr with peritoneal M phi, thymocytes (THY), or polymorphonuclear cells (PMN) and then assayed for their survival . We found that coculture of BM cells with M phi dramatically increased BM survival, whereas THY or PMN consistently failed to enhance BM survival . The ability to promote BM cell survival, here designated nurse activity, represented a novel function of M phi and was further characterized . The stage of activation of M phi did not influence their nurse activity, since M phi elicited in vivo by proteose-peptone, thioglycollate, or Bacillus Calmette-Guerin, as well as resident M phi unstimulated or activated in vitro with lipopolysaccharide, equally sustained survival of BM cells . BM-derived M phi (adherent cells from BM cultures maintained in 20% L-cell-conditioned medium for 14 days) were equally effective in exerting nurse activity . Moreover, nurse activity was also exerted across the histocompatibility barriers . Supernatants from M phi cultures or killed M phi were ineffective . We propose that the nurse effect of M phi on BM is a primitive function that may play an important role in the development of the hemopoietic system. J Urol, 1987 Feb, 137(2), 220 - 4 Risks and benefits of repeated courses of intravesical bacillus Calmette-Guerin therapy for superficial bladder cancer; Catalona WJ et al.; An actuarial analysis of the risks and benefits of repeated courses of intravesical bacillus Calmette-Guerin therapy for superficial bladder cancer was performed for 100 consecutive patients treated for carcinoma in situ (29), prophylaxis against recurrent tumor (51) or residual superficial papillary tumor (21) . The risk-to-benefit ratio at entry into bacillus Calmette-Guerin therapy (7 per cent risk of invasive cancer developing, 5 per cent risk of metastases and 77 per cent prospect for status free of tumor) and in patients who had failed only 1 course of bacillus Calmette-Guerin therapy (11 per cent invasive cancer, 14 per cent metastases and 58 per cent free of tumor) were highly favorable . However, among patients who had failed 2 or more courses of bacillus Calmette-Guerin therapy the risks of invasive (30 per cent) or metastatic (50 per cent) cancer developing exceeded the prospects for eradicating the superficial tumor present (20 per cent) with further therapy . The results suggest that patients who have failed 2 courses of bacillus Calmette-Guerin therapy (as given in our treatment protocol) should be considered for alternative treatment. J Bacteriol, 1987 Feb, 169(2), 796 - 801 Amino sugars in the glycoprotein toxin from Bacillus thuringiensis subsp . israelensis; Pfannenstiel MA et al.; The carbohydrate content of purified Bacillus thuriniensis subsp . israelensis crystal toxin was determined by six biochemical tests, column chromatography on an amino acid analyzer, and the binding of 11 fluorescent lectins . The crystals contained approximately 1.0% neutral sugars and 1.7% amino sugars . The amino sugars consisted of 70% glucosamine and 30% galactosamine . No N-acetylneuraminic acid (sialic acid) was detected . The presence of amino sugars was confirmed by the strong binding of fluorescent wheat germ agglutinin and the weak binding of fluorescent soybean agglutinin . These lectins recognize N-acetyl-D-glucosamine and N-acetyl-D-galactosamine, respectively . The lectin-binding sites appeared evenly distributed among the protein subunits of the crystal . The sugars were covalently attached to the crystal toxin because wheat germ agglutinin still bound alkali-solubilized toxin which had been boiled in sodium dodecyl sulfate, separate by polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes . This study demonstrates the covalent attachment of amino sugars and indicates that the B . thuringiensis subsp . israelensis protein toxins should be viewed as glycoprotein toxins . The crystals used in the present study were purified on sodium bromide density gradients . Studies employing crystals purified on Renografin density gradients can give artificially high values for the anthrone test for neutral sugars. J Biochem (Tokyo), 1987 Feb, 101(2), 477 - 84 Fluorometric study on the interaction of amino acids and ATP with valyl-tRNA synthetase from Bacillus stearothermophilus; Kakitani M et al.; Interactions of several amino acids and nucleotides with valyl-tRNA synthetase {EC 6.1.1.9} (VRS) from Bacillus stearothermophilus were investigated using as a probe the ligand-induced quenching of protein fluorescence (lambda ex = 295 nm, lambda em = 340 nm) of VRS . L-Valine, L-threonine, L-isoleucine, L-glutamic acid, L-leucine, and D-valine caused fluorescence quenching . Among them, L-threonine had a Kd value comparable to that for the cognate substrate, L-valine, but the other amino acids were bound more weakly as estimated by the fluorescence titration method . L-Alanine, L-histidine, and L-serine did not cause any fluorescence change . Among the nucleotides tested (ATP, ADP, AMP, GTP, ITP, CTP, and UTP), only ATP caused the fluorescence change . In the presence of an excess amount of ATP, only L-valine and L-threonine, among the tested amino acids, induced the fluorescence quenching, and the binding of L-valine was greatly favored under this condition . This is consistent with the results of the ATP-PP1 exchange reaction by VRS, in which only L-valine and L-threonine, of these 9 amino acids tested, could serve as substrates, and the Km value for L-valine was much smaller than that for L-threonine . Thus the binding of ATP to VRS enhances the substrate specificity of VRS towards amino acids.(ABSTRACT TRUNCATED AT 250 WORDS) Appl Environ Microbiol, 1987 Feb, 53(2), 365 - 70 Thermal inactivation and injury of Bacillus stearothermophilus spores; Feeherry FE et al.; Aqueous spore suspensions of Bacillus stearothermophilus ATCC 12980 were heated at different temperatures for various time intervals in a resistometer, spread plated on antibiotic assay medium supplemented with 0.1% soluble starch without (AAMS) or with (AAMS-S) 0.9% NaCl, and incubated at 55 degrees C unless otherwise indicated . Uninjured spores formed colonies on AAMS and AAMS-S; injured spores formed colonies only on AAMS . Values of D, the decimal reduction time (time required at a given temperature for destruction of 90% of the cells), when survivors were recovered on AAMS were 62.04, 18.00, 8.00, 3.33, and 1.05 min at 112.8, 115.6, 118.3, 121.1, and 123.9 degrees C, respectively . Recovery on AAMS-S resulted in reduced decimal reduction time . The computed z value (the temperature change which will alter the D value by a factor of 10) for spores recovered on AAMS was 8.3 degrees C; for spores recovered on AAMS-S, it was 7.6 degrees C . The rates of inactivation and injury were similar . Injury (judged by salt sensitivity) was a linear function of the heating temperature . At a heating temperature of less than or equal to 118.3 degrees C, spore injury was indicated by the curvilinear portion of the survival curve (judged by salt sensitivity), showing that injury occurred early in the thermal treatment as well as during logarithmic inactivation (reduced decimal reduction time) . Heat-injured spores showed an increased sensitivity not only to 0.9% NaCl but also to other postprocessing environmental factors such as incubation temperatures, a pH of 6.6 for the medium, and anaerobiosis during incubation. J Biol Response Mod, 1987 Feb, 6(1), 88 - 95 Endogenous production of TNF-like cytotoxic factor in BCG-primed mice by heterologous fibrinogen; Kajikawa T et al.; The triggering activities of heterologous fibrinogen and fibrin on endogenous production of tumor necrosis factor (TNF)-like cytotoxic factor in vivo were examined . The triggering activities of fibrinogen or fibrin from four species injected into the peritoneal cavity of C3H/He mice infected i.p . with bacillus Calmette-Guerin (BCG) were tested . Heterologous, but not homologous, fibrinogen and fibrin showed triggering activity . The route of triggering by heterologous fibrinogen to elicit TNF-like activity systemically was studied . Injection of heterologous fibrinogen i.v . into mice infected i.v . with BCG resulted in a 40-fold higher serum TNF-like activity level than after its i.p . injection . The serum TNF-like activity level was maximal 1 h after i.v . injection of heterologous fibrinogen . When heterologous fibrinogen was injected several times i.v . into mice bearing solid-tumors, TNF-like activity was also released into the serum after every injection, although the activity decreased progressively on second and third injections to 10 and 1%, respectively, of that after the first injection . We used heterologous fibrinogens derived from different species for triggering every week to avoid this gradual decrease of TNF-like activity . In this way TNF-like activity was induced as highly as the primary induction . These results showed that TNF-like cytotoxic factor could be produced in vivo locally or systemically by heterologous fibrinogen or fibrin . Thus both agents should be useful as nontoxic triggering agents. J Biol Response Mod, 1987 Feb, 6(1), 1 - 19 Observations on the combined systemic administration of mixed bacterial vaccine, bacillus Calmette-Guerin, transfer factor, and lymphoblastoid lymphocytes to patients with cancer, 1974-1985; Waisbren BA Sr; Herein are reported the results of treating 139 cancer patients with combined immunomodulation that consisted of bacillus Calmette-Guerin, transfer factor, and mixed bacterial vaccine . In addition 28 patients were given infusions of lymphoblastoid lymphocytes . Patients were admitted to this treatment program who either had failed to respond to other modalities, had elected to add immunomodulation to usual therapy, or had refused chemotherapy and/or radiation therapy . The results suggested that combined immunomodulation therapy is well tolerated and safe and that this approach on a prima facie basis had a salutary effect on the courses of a number of the patients treated . The results also illustrate alternative pathways that can be taken by patients and physicians who are not comfortable with protocolized double-blind methods of approaching patients with poor prognosis cancer. Fundam Appl Toxicol, 1987 Feb, 8(2), 236 - 42 Five-month oral (diet) toxicity/infectivity study of Bacillus thuringiensis insecticides in sheep; Hadley WM et al.; Bacillus thuringiensis insecticides (Bt) {Dipel (test substance D or Thuricide-HP (test substance T)} were administered in the diet for 5 months to castrated mixed rambouillet/merino sheep (24-34 kg at the beginning of the study) at a dose of 500 mg/kg/day (approximately 10(12) spores per day) . No treatment-related effect was seen on weight gain or clinical chemistry parameters nor were significant gross clinical changes observed . Several blood and tissue samples taken just prior to the time the animals were killed or at necropsy were found to be positive for Bt when cultured . Detailed gross and microscopic pathologic examination of the sheep revealed several incidental lesions . However, the only lesion that may have been associated with the treatment was lymphocytic hyperplasia in Peyer's patches seen in the cecum of three sheep and it was not considered to be clinically significant. J Urol, 1987 Feb, 137(2), 216 - 9 Intraluminal interleukin 2 and bacillus Calmette-Guerin for treatment of bladder cancer: a preliminary report; Merguerian PA et al.; The authors treated 13 patients who had undergone resection of transitional cell carcinoma of the bladder with a combination of 3,500 units of interleukin 2 and half the recommended dose of bacillus Calmette-Guerin . Of the patients 11 (85 per cent) remained free of tumor for a mean of 13 months (range 6 to 24 months) . These results are comparable to those with bacillus Calmette-Guerin therapy alone . However, a longer followup with a large number of patients is needed to assess the efficacy of this modality compared to conventional bacillus Calmette-Guerin therapy . Side effects after treatment were minor, self limiting (fever, hematuria and bladder irritability) and lasted for 24 hours. Proc Natl Acad Sci U S A, 1987 Feb, 84(4), 955 - 8 Cloning and purification of a unique lysozyme produced by Bacillus phage phi 29; Saedi MS et al.; A DNA fragment of the bacteriophage phi 29 chromosome, encoding the entire sequence of phi 29 gene 15, has been cloned into the Escherichia coli expression vector pPLc245 under the control of the phage lambda major leftward promoter, PL . Upon heat induction, a protein with an apparent molecular mass of 26 kDa was overproduced . The molecular mass of this protein corresponds to the 28 kDa predicted for the product of gene 15 from its nucleotide sequence . The overproduced protein has been purified to near homogeneity and confirmed to be the product of gene 15 by amino acid sequence analysis of its N terminus . The purified product of gene 15 has a lysozyme activity similar to other phage-type lysozymes: products of phage T4 gene e and of phage P22 gene 19 . However, to our knowledge phi 29 lysozyme is structurally unique among the phage-type lysozymes. Appl Environ Microbiol, 1987 Feb, 53(2), 390 - 6 Microorganisms capable of metabolizing the herbicide metolachlor; Saxena A et al.; We screened several strains of microorganisms and microbial populations for their ability to mineralize or transform the herbicide metolachlor {2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)-acetami de} because such cultures would potentially be useful in the cleanup of contaminated sites . Although we used various inocula and enrichment culture techniques, we were not able to isolate microorganisms that could mineralize metolachlor . However, strains of Bacillus circulans, Bacillus megaterium, Fusarium sp., Mucor racemosus, and an actinomycete were found to transform metolachlor . Several metabolites could be determined with high-performance liquid chromatography . The tolerance of the strains to high concentrations of metolachlor was also evaluated for the usefulness of the strains for decontamination . Tolerance of the actinomycete to metolachlor concentrations over 200 ppm (200 micrograms/ml) was low and could not be increased by doubling the sucrose concentration in the growth medium or by using a large biomass as inoculum . However, a Fusarium sp . could grow and transform metolachlor up to a concentration of 300 ppm. Ann Ophthalmol, 1987 Feb, 19(2), 65 - 8 Endogenous Bacillus cereus panophthalmitis; Cowan CL Jr et al.; Over the past seven years we have treated three cases of drug abusers in whom endogenous Bacillus cereus endophthalmitis rapidly progressed to panophthalmitis . Ocular features of infection with this organism include severe pain, chemosis, proptosis, corneal infiltration and ring abscess, subretinal exudation, retinal hemorrhages, and perivasculitis . The process becomes fulminant in an explosive manner and may be accompanied by fever and leukocytosis . Ophthalmologists should be cognizant of the apparent susceptibility of drug abusers to Bacillus cereus infections and should consider this organism in any severe, rapidly evolving intraocular infectious process. J Bacteriol, 1987 Feb, 169(2), 579 - 86 Cloning and sequencing of the blaZ gene encoding beta-lactamase III, a lipoprotein of Bacillus cereus 569/H; Hussain M et al.; It has not been clear whether the membrane-bound beta-lactamase III of Bacillus cereus 569 is a separate enzyme or a modified form of the secreted beta-lactamase I . The membrane enzyme is an acyl-glyceride thioether-linked lipoprotein (J . B . K . Nielsen and J . O . Lampen, Biochemistry 22:4652-4656, 1983) and thus is probably a separate entity . We cloned the beta-lactamase III gene (blaZ) on a 4.9-kilobase-pair ClaI fragment from mutant strain 569/H (constitutive for high-level production of beta-lactamases I, II, and III), and the nucleotide sequence was determined . The structural gene was flanked by typical promoter, transcription termination, and translation initiation sequences . Expression of the cloned gene in Escherichia coli was low in exponential-phase cultures and increased only as the cultures reached the stationary phase . The deduced amino acid sequence indicates a pre-beta-lactamase III of 316 amino acid residues (35,021 daltons), with a 29-residue signal peptide and a mature lipoprotein form of approximately 32,500 daltons . The 12 NH2-terminal residues of a 21-kilodalton tryptic peptide from the B . cereus membrane enzyme were in agreement with the sequence deduced from the cloned gene . The amino acid sequence was highly homologous to the class A beta-lactamases, especially that of Bacillus licheniformis 749 . beta-Lactamase III is a distinct class A enzyme and the product of a separate gene (blaZ). J Biol Response Mod, 1987 Feb, 6(1), 28 - 34 Effect of forphenicinol on the production of lipopolysaccharide-induced interferon and tumor necrosis factor and on the growth of Meth A fibrosarcoma in mice; Okura A et al.; Oral administration of forphenicinol stimulated the production of lipopolysaccharide (LPS)-induced interferon (IFN) in mice up to 21-fold over control when given more than 10 days before the elicitation . The IFN production in mice sensitized with bacillus Calmette-Guerin (BCG) 14 days before LPS injection was further augmented by forphenicinol given after BCG . In addition to the IFN production, forphenicinol increased production of LPS-induced tumor necrosis factor (TNF) in mice sensitized with BCG, and the dose of LPS required to induce TNF was reduced by the drug . Furthermore, forphenicinol augmented the antitumor effect of BCG and LPS on Meth A fibrosarcoma. Biochem Biophys Res Commun, 1987 Jan 30, 142(2), 475 - 82 Independent bindings of Mn2+ and Mg2+ to the active site of B . cereus glutamine synthetase; Nakano Y et al.; Glutamine synthetase purified from Bacillus cereus IFO 3131 was modified by iodoacetamide and the ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA) . Only Mg2+-dependent activity was inactivated by iodoacetamide, whereas only Mn2+-dependent activity was inactivated by FSBA . When iodoacetamide-treated enzyme was reacted with FSBA, Mn2+-dependent activity was also inactivated . Mg2+ plus Mn2+-dependent activity was inactivated in any case . The results suggested that the binding sites of Mn2+ and Mg2+ are separate from each other in the active site of B . cereus glutamine synthetase and that bindings of Mg2+ and Mn2+ to each site are required for normal activity in vivo. Biochim Biophys Acta, 1987 Jan 20, 923(1), 150 - 5 Lysosomal and cytosolic sialidases in rabbit alveolar macrophages: demonstration of increased lysosomal activity after in vivo activation with bacillus Calmette-Guerin; Pilatte Y et al.; Sialidase activity was assayed in homogenized rabbit alveolar macrophages using a fluorogenic substrate: sodium 4-methylumbelliferyl-alpha-D-neuraminate . After differential centrifugation one acid-active enzyme (optimum pH 4.2) was detected in the 16,000 X g pellet that contained lysosomes, mitochondria and peroxisomes . A second activity, with an optimum pH of 5.4, was found in the cytosolic fraction . The acid-active sialidase accounted for more than 95% of the total sialidase activity in crude homogenate . When alveolar macrophages were collected from rabbits stimulated with bacillus Calmette-Guerin (BCG), the acid-active sialidase specific activity was increased 2.5-fold whereas other lysosomal enzymes such as N-acetylglucosaminidase and beta-galactosidase were stable . The cytosolic sialidase activity did not change. J Mol Biol, 1987 Jan 5, 193(1), 237 - 8 Crystallization and preliminary X-ray studies of Bacillus pumilus IPO xylanase; Moriyama H et al.; The xylan-degrading enzyme xylanase, from Bacillus pumilus IPO, has been crystallized . The crystals are monoclinic, space group P21 with a = 40.8 A, b = 66.8 A, c = 34.7 A and beta = 103.0 degrees . The asymmetric unit contains one molecule of Mr 22,500 . The crystals diffract to at least 2.5 A resolution, and they are suitable for X-ray crystal structure analysis at high resolution. J Mol Biol, 1987 Jan 5, 193(1), 171 - 87 Structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus at 1.8 A resolution; Skarzynski T et al.; The structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus has been crystallographically refined at 1.8 A resolution using restrained least-squares refinement methods . The final crystallographic R-factor for 93,120 reflexions with F greater than 3 sigma (F) is 0.177 . The asymmetric unit of the crystal contains a complete tetramer, the final model of which incorporates a total of 10,272 unique protein and coenzyme atoms together with 677 bound solvent molecules . The structure has been analysed with respect to molecular symmetry, intersubunit contacts, coenzyme binding and active site geometry . The refined model shows the four independent subunits to be remarkable similar apart from local deviations due to intermolecular contacts within the crystal lattice . A number of features are revealed that had previously been misinterpreted from an earlier 2.7 A electron density map . Arginine at position 195 (previously thought to be a glycine) contributes to the formation of the anion binding sites in the active site pocket, which are involved in binding of the substrate and inorganic phosphates during catalysis . This residue seems to be structurally equivalent to the conserved Arg194 in the enzyme from other sources . In the crystal both of the anion binding sites are occupied by sulphate ions . The ND atom of the catalytically important His176 is hydrogen-bonded to the main-chain carbonyl oxygen of Ser177, thus fixing the plane of the histidine imidazole ring and preventing rotation . The analysis has revealed the presence of several internal salt-bridges stabilizing the tertiary and quaternary structure . A significant number of buried water molecules have been found that play an important role in the structural integrity of the molecule. J Occup Med, 1987 Jan, 29(1), 29 - 31 Unusually aggressive transmission of tuberculosis in a factory; Mosher CB et al.; A case of pulmonary tuberculosis in a factory worker was studied to identify infected contacts and to gauge the degree of transmission . Initial findings suggested far more extensive transmission than anticipated, and the study was expanded . Of all factory workers tested, 33% were infected with the tubercule bacillus in spite of massive air volume-to-contact ratio . Deliberate stagnation of the air to prevent occupational exposure to lead and asbestos was believed to be a contributing variable. Ukr Biokhim Zh, 1987 Jan-Feb, 59(1), 70 - 5 {Interaction of the glycoprotein from the Bacillus pumilis cell wall with liposomes}; Karamushka VI et al.; The methods of centrifugation and gel-filtration on Sephadexes G-50 and G-150 were used to study the interaction of Bacillus pumilis cell wall glycoprotein component having the molecular weight of 50 kDa (GP-50) with lyposomes from bacterial lipids . GP-50 is shown to sorb on such liposomes and disturb their barrier properties inducing yield of low-molecular label . GP-50 exerts no effect on properties of liposomes from egg lecithin . Electrostatic forces are supposed to play a decisive role in initial acts of GP-50 interactions with lipid phase of microbial envelopes. Diagn Microbiol Infect Dis, 1987 Jan, 6(1), 53 - 8 Ovine pulmonary transit of tetracycline and minocycline; Cohen SH et al.; Following cannulation of the right external jugular vein and the efferent duct of the right caudal mediastinal lymph node (the caudal end of this node was ligated to cut off the inflow of systemic lymph, i.e., 90%-95% of the efferent lymph was of pulmonary origin), sheep were given either tetracycline or minocycline as single doses of 5 mg/kg body weight infused intravenously over 30 min . Venous blood plasma and pulmonary lymph collected contemporaneously before infusion and from 5 min to 24 hr postinfusion were assayed by a well-agar diffusion method using Bacillus cereus . Peak concentrations of both drugs were observed in both plasma and lymph at 5 min postinfusion . Tetracycline penetrated into the lymph better than minocycline (percent penetration 67.3% of cf . 38.2%) . The concentration of tetracycline was significantly higher in lymph during and 5 min postinfusion (p less than 0.01), a factor that may be of importance when selecting a tetracycline for the treatment of a pulmonary infection. Am Rev Respir Dis, 1987 Jan, 135(1), 3 - 9 Tuberculosis in physicians . Compliance with preventive measures; Geiseler PJ et al.; Compliance with public health recommendations for tuberculosis control was evaluated by a survey of 4,417 physicians who had not contracted tuberculosis during medical school nor during the 4 decades after graduation through 1981 . Thirty-one percent of the cohort had been vaccinated with bacille Calmette-Guerin (BCG) and 47% considered themselves tuberculin-positive . Thirty-two percent of 1,088 physicians who graduated after 1974 had been exposed to 3 or more patients with infectious tuberculosis in the previous year . Fifty-eight percent of 738 unimmunized, tuberculin-negative physicians who had been exposed to one or more patients with infectious tuberculosis in the previous year had tuberculin tests every 1 to 2 yr . Forty-nine percent of 597 unimmunized tuberculin reactors with similar occupational exposure had chest roentgenograms every 1 to 2 yr . The BCG-vaccinated physicians were less likely to have frequent tuberculin tests but no different frequency of chest roentgenograms . Eight percent of 1,460 unimmunized tuberculin reactors received isoniazid chemoprophylaxis, including 39% of 128 tuberculin reactors who graduated after 1974 (the majority of the latter were younger than 35 yr of age) . Of 66 physicians who had active tuberculosis during medical school or after graduation, 20 (30%) had not received any antituberculosis chemotherapy, whereas 2 of 46 who did, received chemotherapy only after a second episode of tuberculosis . In summary, our study documents poor compliance by physicians with recommended policies for the prevention of tuberculosis in health care workers. Urology, 1987 Jan, 29(1), 26 - 30 Use of Nd:YAG laser in treatment of bladder cancer; Shanberg AM et al.; A total of 53 patients were treated with neodymium:yttrium-aluminum garnet (Nd:YAG) laser and followed up for two years . Three patients had carcinoma in situ (CIS); 4 had Ta lesions; and 28 had TINxMo, 8 had T2NxMo, and 10 had T3NxMo lesions . The patients with CIS received a combination of Nd:YAG laser and intravesical bacillus Calmette Guerin (BCG) instillation . The remainder of the patients received no supplementary chemotherapy . No tumor recurrence was noted in those with Ta lesions . Tumor recurred in 18 per cent of patients with T1 lesions, and 33 per cent of those with T2 lesions; 80 per cent of patients with T3 lesions had resistant or residual tumors . In patients with CIS, there was no tumor recurrence . Nd:YAG laser is effective in Ta, T1, and T2 bladder cancers, with low recurrence rate and minimal complications . In very select, high-risk patients with small lesions, and in those who refuse cystectomy, there may be a place for laser therapy for either palliation or definitive treatment of T3 invasive tumors of the bladder. Oncology, 1987, 44(5), 279 - 82 Induction of cutaneous plasma cell tumor as a complication of adjuvant immunotherapy with methanol extraction residue of bacillus Calmette-Guérin; Suster SM et al.; Methanol extraction residue (MER), a cell wall fraction of bacillus Calmette-Guerin, has been reported to exhibit immunomodulating properties which permit its successful use as an adjuvant of immunotherapy in cancer patients . Its beneficial immunostimulatory effects are generally thought to be due to nonspecific stimulation of cell-mediated immunity . Little attention has been focused on its humoral stimulating properties . A case is presented of a patient undergoing treatment with adjuvant immunotherapy with MER for malignant lymphoma who developed a cutaneous plasma cell tumor at the injection site of the drug . This complication may represent the result of enhanced humoral immunity induced by the MER treatment . The development of a cutaneous plasma cell tumor must be added to the list of known MER toxicities, and may constitute an indication for the discontinuation of treatment with this agent. Int Arch Allergy Appl Immunol, 1987, 84(3), 306 - 10 Immunogenicity of hapten-carrier conjugates associated with liposomes; Neveu PJ; Immunogenicity of liposome-entrapped hapten-carrier conjugates was studied in guinea pigs . Animals immunized intravenously with a subimmunogenic dose of a hapten-carrier complex entrapped in liposomes exhibited cutaneous anaphylactic reactions to the hapten, even when the liposomes were treated with trypsin, but not delayed hypersensitivity (DH) reactions to the carrier . Animals immunized with a mixture of empty liposomes and antigen showed anaphylactic reactions to the hapten that appeared to be elicited by antigen adsorbed on the outer membrane of liposomes . The induction of anaphylaxis to the hapten by liposome-associated antigen was not due to an adjuvant property of liposomes . DH reactions to the carrier after injection of a liposome-entrapped subimmunogenic dose of a hapten-carrier conjugate could be observed only after activation of macrophages by Bacillus Calmette-Guerin. Indian J Lepr, 1987 Jan-Mar, 59(1), 50 - 3 Follow-up study of pauci-bacillary leprosy on-multi-drug regimen; Ramanan R et al.; One hundred and twenty nine newly registered cases of pauci-bacillary leprosy were put on Dapsone daily and Rifampicin once a month and were followed up for one year . Out of 129 cases, 108 (83.7%) were found to be clinically active at the end of one year of multidrug treatment (MDT) . In 25 out of these 108 cases, skin biopsy was done and well defined granulomas were seen after therapy in 11 patients (44%). Indian J Lepr, 1987 Jan-Mar, 59(1), 36 - 43 Follow up of BL/LL patients on a slightly modified WHO regimen of multidrug therapy; Katoch K et al.; 56 lepromatous leprosy patients with an initial average BI of 4.45 were administered once a month 600 mg of Rifampicin, 100 mg of Clofazimine on alternate days and 100 mg of Dapsone daily . None of these patients became smear negative in 2 years, and the same regimen was continued further . Two patients have become negative in 3 years and treatment has been stopped in them . The study indicates that highly bacilliferous LL/BL patients are likely to need 3 years or more of MDT for achieving bacterial negativity. Indian J Lepr, 1987 Jan-Mar, 59(1), 20 - 5 Eighth nerve evaluation in leprosy; Mann SB et al.; 25 cases of bacillary positive leprosy patients and 25 age and sex matched controls were investigated for assessment of cochleo-vestibular status . Impaired hearing was complained of by 4 patients . None had tinnitus, dizziness or vertigo . On testing 44% patients were found to have unilateral or bilateral perceptive deafness . Specialised tests of hearing indicated that the deafness was of cochlear type . The vestibular functions were not affected . Leprosy seems to selectively involve the cochlea. Exp Biol, 1987, 46(3), 149 - 53 Sub-toxic effects of Bacillus sphaericus 1593 M on feeding growth and reproduction of Laccotrephes griseus (Hemiptera: Nepidae); Mathavan S et al.; Long-term effects of sub-lethal doses of biocide (25, 50, 100 and 200 micrograms/ml) from B . sphaericus 1593 M on feeding, growth and reproduction of L . griseus were studied . The deleterous effects of the biocide include: reduced feeding, retarded growth, delayed maturity, reduction in fecundity, and decrease in protein content of the ovary . The effects are convincingly dependent on biocide dose and exposure duration . Extensive use of the biocide may bring a population depletion of non-target species even at sub-lethal concentrations . The possible mode of action of biocide is discussed. Eur Urol, 1987, 13(1-2), 1 - 6 Bladder tumors: when to do and what to expect from random mucosal biopsies with reference to blood group cell surface antigens; Boccon-Gibod L et al.; Routine random biopsies of normal looking bladder mucosa in the evaluation of bladder tumors demonstrated a high occurrence of anomalies ranging from dysplasia to carcinoma in situ (CIS) . 75 patients with a urothelial bladder tumor were submitted to 165 endoscopic procedures under anesthesia including transurethral resection of any bladder tumor and random mucosal biopsies in the 4 quadrants of the bladder . The frequency and severity of mucosal anomalies rise with the tumor grade and stage: in stage 0.Ta, the percentage of anomalies rises from 15% in grade-1 lesions to 53% in grade-3 tumors . 80% of the stage A (T1) B and C (T2, T3) tumors are associated with anomalies of the normal looking bladder mucosa characterized by CIS in 50% of the cases . These lesions which may persist in the absence of any visible tumor respond dramatically to endovesical bacillus Calmette-Guerin therapy . Simultaneous determinations of blood group antigens fail to demonstrate a clear correlation between the antigenic status of the tumor and that of random biopsies . These results may help to clarify the indications of random mucosal biopsies which should be reserved to the treatment and surveillance of grade 3-tumors, irrespective of their stage. Pediatr Radiol, 1987, 17(2), 166 - 7 Osteomyelitis deriving from BCG-vaccination; Arias FG et al.; Secondary complications after BCG-vaccination are unusual . There is an almost invariably lethal generalized infection affecting the immunodepressed . Late dissemination of BCG to bone is characterized by lytic lesions, it is not normally associated with immunologic changes and has a good prognosis . The histology of BCG-osteomyelitis resembles that produced by Koch bacillus . We present a rare case of a lytic lesion in the femur improving after specific treatment. Vet Clin North Am Small Anim Pract, 1987 Jan, 17(1), 91 - 103 Cat scratch disease . A therapeutic dilemma; Margileth AM; Cat scratch disease, a relatively common infectious disease, is caused by a small gram-negative pleomorphic bacillus . The course of CSD is usually benign and self-limiting and is characterized by tender regional chronic (3 weeks or longer) lymphadenopathy and frequently preceded by a primary skin lesion following cat contact or scratches . Persistence of adenopathy for several months in a generally healthy patient with gradual spontaneous resolution of the enlarged node is the natural course. FEBS Lett, 1987 Jan 1, 210(1), 91 - 6 The complete amino acid sequence of ribosomal protein S12 from Bacillus stearothermophilus; Kimura M et al.; The amino acid sequence of ribosomal protein S12 from Bacillus stearothermophilus has been completely determined . The sequence data were mainly obtained by manual sequencing of peptides derived from digestion with trypsin, Staphylococcus aureas protease and pepsin . A few overlaps of tryptic peptides were established by DNA sequence analysis of a chromosomal fragment containing the rpsL gene coding for ribosomal protein S12 . The protein contains 138 amino acid residues and has an Mr of 15,208 . Comparison of this sequence with the sequences of the ribosomal S12 proteins from E . coli as well as from Euglena, tobacco and liverwort chloroplasts shows that 75% of the amino acid residues are identical within the S12 proteins of all four species . Therefore, S12 is the most strongly conserved ribosomal protein known so far. Mem Inst Oswaldo Cruz, 1987, 82 Suppl 3, 29 - 34 The potassium impermeable apical membrane of insect epithelia: a target for development of safe pesticides; Harvey WR et al.; Columnar cell apical membranes (CCAM) in series with goblet cell apical membranes (GCAM) form an electroosmotic barrier separating the midgut lumen from epithelial cell cytoplasm . A unique K+ ATPase in GCAM generates three gradients across this barrier . A greater than 180 mV electrical gradient (lumen positive) drives amino acid uptake through voltage-dependent K+ symports . A greater than 1000-fold {H+} gradient (lumen alkaline) and a greater than 10-fold {K+} gradient (lumen concentrated) are adaptations to the high tannin and high K+ content, respectively, in dietary plant material . Agents which act on the apical membrane and disrupt the PD, H+, or K+ gradients are potential insecticides . Insect sensory epithelia and mammalian stria vascularis maintain similar PD and K+ gradients but would not be exposed to ingested anti-apical membrane insecticides . Following the demonstration by Sacchi et al . that Bacillus thuringiensis delta-endotoxin (Bt) induces specifically a K+ conductance increase in CCAM vesicles, we find that the K+ channel blocking agent, Ba2+, completely reverses Bt inhibition of the K+-carried short circuit current in the isolated midgut of Manduca sexta . Progress in characterizing the apical membrane includes finding that fluorosulfonylbenzoyladenosine binds specifically to certain GCAM polypeptides and that CCAM vesicles can be mass produced by Ca2+ or Mg2+ precipitation from Manduca sexta midgut. Free Radic Res Commun, 1987, 4(2), 105 - 8 Effects of oxygen free radical scavengers on the membrane myoinositol dehydrogenase of Bacillus pumilus strain 5; Eze MO et al.; Micromolar amounts of superoxide dismutase (SOD) or parabenzoquinone (PBQ) inhibit the membrane-bound myoinositol dehydrogenase of Bacillus pumilus strain 5 in the mode of this enzyme transferring electrons to 2,6-dichlorophenol indophenol (DCPIP) . The inhibition trends are similar to those reported earlier by us for the inhibition by mannitol and benzoate . We postulate that the transfer of electrons from the enzyme to DCPIP involves in its rate-limiting step, a catalytic intermediate in the nature of superoxide (O2-) and/or hydroyl free radical (OH.) . Scavenging of any one or both of these radicals, therefore, inhibits the electron transfer reaction . PBQ serves as an electron sink in the reaction preventing the reduction of DCPIP. Biomed Pharmacother, 1987, 41(5), 227 - 32 Spergualin: a new antitumour antibiotic; Umezawa K et al.; Spergualin was isolated from the culture filtrate of Bacillus laterosporus as an antitumour substance . It had a unique structure and was shown to have chemotherapeutic effects on mouse transplantable leukaemias such as L-1210, P-388, P-815, C-1489, EL-4 and RL male 1 . It was especially effective to L-1210 leukaemia and the leukaemia-bearing mice were even curable by the optimal dose of this drug . When the spergualin-treated cured mice were inoculated again by L-1210 cells, those leukaemic cells did not grow in the animals suggesting that specific immunity to L-1210 had been induced . In this induction of immunity cytotoxic T lymphocytes were suggested to be involved . Cytostatic effect of spergualin in cell culture was dependent on the content of amine oxidase in serum . In the study of structure-activity relationship, the 15-hydroxy group was found to be not necessary, while the spermidine moiety was essential for antitumour activity . 15-Deoxy derivative of spergualin was found to be more potent in antitumour activity. Cell Immunol, 1987 Jan, 104(1), 12 - 23 Interleukin 1 and prostaglandin production by cells of the mononuclear phagocyte system isolated from mycobacterial granulomas; Montreewasuwat N et al.; A study has been made of the activity of interleukin 1 (IL-1) and prostaglandins (PGs) in the culture supernatants from unstimulated and lipopolysaccharide (LPS)-stimulated mycobacteria-induced granuloma cells . Both epithelioid cells from bacillus Calmette-Guerin (BCG)-induced granulomas and macrophages from Mycobacterium leprae-induced granulomas, separated on a fluorescence-activated cell sorter using monoclonal antibody specific to guinea pig macrophages, spontaneously secreted low levels of IL-1 (assayed by thymocyte comitogenic and fibroblast mitogenic activities) into culture supernatants . However, culture supernatants from LPS-stimulated epithelioid cells showed significantly higher IL-1 activity than those from unstimulated cells . In contrast, LPS stimulation of M . leprae granuloma macrophages failed to enhance IL-1 production . Nevertheless, IL-1 activity in the culture supernatants from stimulated mycobacterial granuloma cells of both types was much lower than that from LPS-stimulated peritoneal exudate macrophage culture supernatants . There was no detectable amount of prostaglandin E2 (PGE2) in the culture supernatants from both unstimulated and LPS-stimulated BCG- and M . leprae-induced granuloma cells in comparison to much higher levels of PGE2 produced by unstimulated (0.28-6.2 ng/ml) or LPS-stimulated (greater than 15 ng/ml) peritoneal exudate macrophages . However, BCG granuloma cells either secreted prostaglandin F2 alpha (PGF2 alpha) spontaneously or produced comparable levels of PGF2 alpha to those from peritoneal exudate macrophages on stimulation, while M . leprae granuloma macrophages produced much lower levels of PGF2 alpha. J Urol, 1987 Jan, 137(1), 155 - 8 Requirement of a thymus dependent immune response for BCG-mediated antitumor activity; Ratliff TL et al.; Surgical adjuvant intravesical bacille Calmette-Guerin (BCG) therapy is an effective method of treating superficial transitional cell carcinoma of the bladder . The role of the immune response in the antitumor activity of intravesical BCG is not known . We investigated the requirement of a thymus-dependent immune response for the inhibition of the growth of the intravesically implanted mouse bladder tumor, MBT-2 . Intravesical BCG had no antitumor activity when administered to athymic nude mice bearing MBT-2 tumors . In two experiments tumor outgrowth in control and BCG-treated mice was identical . Adoptive transfer of BCG sensitized splenocytes (one spleen equivalent per mouse injected intravenously immediately prior to the first BCG treatment) syngeneic to the MBT-2 tumor transferred delayed hypersensitivity reactivity to BCG antigens and restored the antitumor activity of intravesical BCG . In two separate experiments mice receiving splenocytes plus BCG had 0 and 20% tumor outgrowth compared with 100% in control mice (p less than .02 and p less than .05, respectively) . These results demonstrate that the antitumor activity of intravesical BCG therapy requires a thymus-dependent immune response. Gene, 1987, 54(2-3), 267 - 73 The effects of deletions in the leader sequence of cat-86, a chloramphenicol-resistance gene isolated from Bacillus pumilus; Harwood CR et al.; The cat-86 gene of Bacillus pumilus, specifying a Cm-inducible CAT enzyme, was cloned previously into B . subtilis on plasmid pUB110 . Various lines of evidence suggest that control of expression of this gene is at the level of translation and involves inverted complementary repeat sequences 5' to the initiation codon . A series of deletions have been generated in this region and their effects on the induction of cat-86 observed in B . subtilis, Escherichia coli and a number of ribosomal mutant strains of B . subtilis . The results indicate that the inverted complementary repeat sequences, which are capable of forming a stable stem-loop structure in the mRNA (delta G = -24.4 kcal/mol), form a barrier to translation in E . coli and B . subtilis. J Exp Pathol, 1987 Summer, 3(4), 579 - 86 Immunotherapy of bladder cancer with intralesional injection with BCG; Calsini P et al.; Immunotherapy of bladder cancer with non specific immunostimulants, such as bacillus Calmette Guerin (BCG), has been shown to significantly reduce tumor recurrences, and, when applied through intravesical BCG instillation to induce the regression of some established tumors, including carcinomas "in situ" and even some invasive tumors . Since in other tumors, namely melanomas, BCG has been proved to be most successful when infected directly into the tumor, intralesional therapy with BCG (1.5 x 10 BCG weekly for four weeks) has been applied in 20 patients affected by bladder cancer (T1 - T2) . The local reaction has induced substantial reduction of the tumor mass and, in some cases, (four) total disappearance of the tumor . On the sixteen patients without total disappearance of the tumor, TUR was applied . The successive follow-up of these patients offer at present the following figures: 3 out of the four patients with remission after BCG alone are disease-free after 26-30 months, whereas one has developed after 18 months a papilloma surgically removed; 15 out of the sixteen patients treated with BCG followed by TUR are disease-free after 26-30 months . These data, compared with literature findings, support the idea that intratumoral BCG instillation of bladder cancer permits a longer disease-free period than other therapeutical approaches. Vet Med Nauki, 1987, 24(9), 78 - 84 {Histological characteristics of the intraorbital glands of pheasants with tuberculosis}; Dimitrov DS et al.; For the first time in the country are investigated histologically lacrimal and Harderian glands of pheasants, affected by tuberculosis, raised in pens . By histological analysis were reported the results of the cell reactivity in the affected by the tubercular process tissues and organs . Under the influence of the mycobacteria and their disintegration products the tissues react specifically . In pheasants, affected by tuberculosis are established destructive processes in the intraorbital glands . Mainly in the connective tissue of the glands are observed tubercular knots, which morphologically differ from these in the hen . In the interlobular tissue, in the interacinar and peritubular connective tissue exist strongly expressed infiltration with cells of the lymphoid series, phagocytes and heterophils, as a result of the affected-tissue reactivity, on account of the local and diffusional spreading of the tubercular bacillus in the glands. Mikrobiyol Bul, 1987 Jan, 21(1), 34 - 41 {Effects of some physiologic factors on Bacillus sp . urease activity}; Aksoz N; In this study, culture supernatant of Bacillus sp . has been used as crude enzyme source . It has been determined that 1% urea is the optimal substrate concentration for maximal urease activity . On the other hand, Bacillus sp . urease showed its maximal activity at pH:7.0-8.0, 30 degrees C . 50 minutes of incubation has been found to be the most suitable incubation period . The activity of the enzyme showed a 36% decrease when stored at +4 degrees C for a month. Eur Biophys J, 1987, 15(4), 225 - 30 The active site of manganese-containing superoxide dismutase from Bacillus stearothermophilus studied by 1H and 19F magnetic relaxation; Viglino P et al.; The dependence of the magnetic relaxation rates of 1H and 19F- on temperature, frequency, pH and N-3 concentration, were measured in solutions of Manganese-containing superoxide dismutase of Bacillus stearothermophilus, and were compared to activity measurements, in order to obtain some information on the structure and dynamics at Mn(III) present in the active site of the enzyme . The experimental data lead us to hypothesize the presence of two binding sites in the coordination sphere of the enzyme bound Mn(III), which are accessible to water and anions and have different chemical and spectroscopic properties . NMR measurements carried out in the presence of competitive inhibitors and the pH dependence of both NMR relaxation rates suggest that F-, N-3 and OH- ions bind to one site, while a water molecule binds to the other one . The stability constant values of the complexes between these anions and the enzyme are reported . The influence of the anions on activity and the pH dependence of NMR parameters are discussed. Crit Rev Biotechnol, 1987, 6(2), 163 - 232 The biotechnology of Bacillus thuringiensis; Andrews RE Jr et al.; One of the challenges in the application of biotechnology to pest control is the identification of agents found in nature which can be used effectively . Biotechnology offers the potential of developing pesticides based on such agents which will provide environmentally sound and economically feasible insect control alternatives . Such an agent, the insect pathogen Bacillus thuringiensis, is the subject of intense investigations in several laboratories . Insecticides which use the entomocidal properties of B . thuringiensis are currently produced and sold worldwide; new products are currently in the development stage . Herein, the biology and genetics of B . thuringiensis and the problems associated with current products are critically reviewed with respect to biotechnology . Moreover, the economic and regulatory implications of technologically advanced products are evaluated. Gene, 1987, 61(2), 165 - 76 Extracellular production of cloned alpha-amylase by Escherichia coli; Suominen I et al.; Overexpression of Bacillus stearothermophilus gene coding for thermostable alpha-amylase in Escherichia coli was shown to cause outer-membrane damage leading to extracellular location of periplasmic proteins . Prolonged high expression of the alpha-amylase gene under lacZpo control eventually also lysed cells . Surprisingly, expression controlled by the pL promoter of phage lambda allowed specific release of periplasmic proteins into the growth medium without total cell lysis . Accumulation of alpha-amylase in the growth medium continued for at least 24 h under lambda pL control, whereas beta-lactamase activity ceased to increase beyond the exponential growth phase . The extent of outer membrane damage caused by alpha-amylase expression was monitored by following growth kinetics in the presence of lysozyme and by electron microscopy of the cells . Supplementing growth medium with Mg2+ restored the normal growth kinetics . It is suggested that periplasmic protein release caused by alpha-amylase overexpression is a stress response of the cell . A role for induced autolytic activity of the cell as a final effector of protein release is also proposed. Med Cutan Ibero Lat Am, 1987, 15(6), 504 - 10 {Delayed cutaneous response to different antigens in household contacts of leprosy patients and controls}; Bottasso O et al.; The delayed type hypersensitivity (DTH) response to PPD, candidin and histoplasmin, and Mitsuda reaction (MR) in household contacts (HC) of multibacillary (HCM) and paucibacillary (HCP) leprosy patients, and non exposed controls was studied . Intradermal tests with 0.1 ml . of each antigen were performed and read after 48 hours . Late nodular reaction to ML (MR) was evaluated at the 21st day post-challenge . As we had formerly demonstrated, MR was depressed in HCM . Interestingly, this reaction was also depressed in HCP to a lesser magnitude . HCM had a lower DTH response to histoplasmin in relation to PPD and candidin when persons were simultaneously challenged with the three antigens . According to these results we conclude that: a), HCM have a depression in late lepromin reaction; b), in HCP the depression in MR is of a lower magnitude . We hypothesize that in healthy contacts of bacilliferous patients an active mechanism of immunosuppression to mycobacterial antigens may be produced . Alterations in the skin response to histoplasmin is discussed. J Gen Microbiol, 1987 Jan, 133 ( Pt 1), 193 - 9 Exochelin-mediated iron acquisition by the leprosy bacillus, Mycobacterium leprae; Hall RM et al.; Exochelins, water-soluble siderophores of mycobacteria, were isolated and partially purified from culture filtrates of iron-deficiently grown cultures of Mycobacterium neoaurum NCTC 10439 and an armadillo-derived Mycobacterium (ADM 8563) . Two biologically active fractions mediating iron uptake were isolated from each bacterium which not only were able to transport iron into the producing organism but also into suspensions of Mycobacterium leprae isolated from armadillo liver . The rate of exochelin-mediated iron uptake into M . leprae was about 1.5% of the rate observed into the producing organisms . The process of iron uptake appears to be by facilitated diffusion as it was not inhibited by HgCl2, NaN3, KCN, dinitrophenol or carbonyl cyanide m-chlorophenylhydrazone . Since no uptake of iron occurred into iron-sufficient ADM cells, this may indicate that M . leprae, as recovered from an animal tissue, had been growing iron-deficiently in order for iron uptake to have been demonstrated in vitro. Gene, 1987, 54(2-3), 155 - 65 Tolerated variations in a genome: the case of closely related Bacillus phages PZA, phi 29 and phi 15--a review; Paces V et al.; Restriction-site analysis was used to estimate the relationship of bacteriophages PZA, phi 29 and phi 15 . Complete nucleotide sequences of PZA and luminal diameter 29 genomes were compared and tolerated variations were assessed . Most of the base-pair changes are silent nucleotide substitutions in the third position of codons but amino acid changing substitutions are also observed . The terminal portions of the phage genomes diverged faster than their central parts . Gene mutations in phage PZA were induced by hydroxylamine and their frequency was compared with the evolutionary mutability. Biomed Biochim Acta, 1987, 46(4), 293 - 6 Purification of trypsin and bacterial proteinases by column chromatography on coffee grain particles; Safarik I; Trypsin and extracellular proteinases produced by Bacillus sp . were purified by column chromatography on coffee grain particles . The ballast proteins were eluted with water, while the adsorbed proteinases were eluted with 1 M sodium chloride solution . The capacity is approximately 2 mg of trypsin per ml of the packed sorbent. Gene, 1987, 53(1), 113 - 9 Nucleotide sequence of the insecticidal protein gene of Bacillus thuringiensis strain aizawai IPL7 and its high-level expression in Escherichia coli; Oeda K et al.; A DNA fragment carrying the insecticidal protein gene of Bacillus thuringiensis subsp . aizawai IPL7 was cloned from a 78-kb plasmid . The nucleotide sequence revealed that the cloned DNA fragment contained a 3465-bp protein-coding region with 156-bp 5'-flanking, and 168-bp 3'-flanking regions . The open reading frame encoded a 130,690 Da protein consisting of 1155 amino acid residues . Nucleotide sequence comparison of the aizawai gene with the published berliner 1715 gene showed only 8 nt changes in the coding regions . It was found that 72 bp of the 5'-flanking sequence of the cloned aizawai gene was responsible for constitutive expression of the 130-kDa protein gene in Escherichia coli . The expression was greatly enhanced by introducing the tac promoter upstream from the 72-bp 5'-flanking region of the aizawai gene . Under optimal conditions, the 130-kDa insecticidal protein amounted to 38% of the total cellular protein. Gene, 1987, 53(1), 11 - 9 Expression of Bacillus amyloliquefaciens extracellular ribonuclease (barnase) in Escherichia coli following an inactivating mutation; Paddon CJ et al.; An inactivated gene for Bacillus amyloliquefaciens extracellular ribonuclease (barnase) has previously been cloned and sequenced following transposon mutagenesis . The intact gene could not be assembled in Escherichia coli and is presumed to be lethal . Therefore, we introduced specific mutations into the barnase gene to prevent its lethal effect . A Gln-73 mutant gene was stable in E . coli but only produced low amounts of barnase antigen . Mutants containing Asp, Gln or Arg, instead of His-102, at the active site were identified by immunological screening for barnase antigen . None of the mutant proteins with alterations at aa residue 102 possessed RNase activity . The level of barnase (Asp-102) was higher in E . coli than in B . subtilis but the protein was not processed to the correct size in E . coli . To obtain correct processing, the barnase (Asp-102) structural gene was fused to the E . coli alkaline phosphatase promoter and signal sequence (phoA) . Cells containing this construct secreted correctly processed barnase (Asp-102) into the periplasmic space and culture supernatant at a level of 20 mg/l . Barnase (Asp-102) was purified and found to have an identical N-terminus and a thermal unfolding curve that was nearly identical to that of active barnase (His-102) . The cloning and expression of barnase in E . coli will allow detailed analysis of barnase protein folding by molecular genetic approaches. Pediatr Hematol Oncol, 1987, 4(2), 137 - 43 Central venous catheter with subcutaneous injection port (Port-A-Cath): clinical experience with children; Wesenberg F et al.; Long-term intermittent venous access was established in 26 children by means of a central venous catheter (CVC) with a subcutaneous injection port (Port-A-Cath) (PAC) . As of December, 1985, PACs had been in place for 20-750 days (cumulative 10,890 days) with 647 entries into the system . The PACs were used for blood sampling and administration of chemotherapy, antibiotics, fluids, total parenteral nutrition (TPN), and blood products . One patient with sever neutropenia (absolute neutrophil granulocyte count {ANC} less than 0.1 x 10(9)/L) at the time of the PAC implant developed an infection around the port after 2 days, with subsequent septicemia (Bacillus cereus) necessitating removal of the PAC . Otherwise, no definite PAC-related infections occurred, including 258 days of neutropenia (ANC less than 0.5 x 10(9)/L) . Two PACs were found occluded with greyish deposits of fat and organic material after long-term (45 and 61 days) continuous TPN and were removed . Malposition of catheter, extravasation, thrombosis, and other potential technical or psychological complications were not observed . The children continued normal activities, and the easy venous access decreased emotional stress during treatment . Local doctors were trained to use the PACs, with which they administered maintenance chemotherapy . We conclude that the use of PACs in children is safe, even in the first year of life, and has many advantages when compared with other CVCs currently in use . Strict indications, meticulous implantation technique, and adequate handling are, however, mandatory. Ophthal Plast Reconstr Surg, 1987, 3(4), 231 - 5 Ocular adnexal injury and complications in orbital dog bites; Gonnering RS; Orbital dog bites, though statistically uncommon, occur most frequently in children and are associated with severe ocular adnexal injury . Of 16 victims, two-thirds were under 10 and over half under 5 years of age . The wounds consisted of numerous periorbital punctures, and in most cases, full-thickness lid lacerations involving the tear system . There were no serious injuries to the globe . Reversible amblyopia occurred in two children under 3 years of age with damage to the levator muscle . One child suffered a naso-orbital fracture . Because of the obvious nature of the injury, most patients present early and can be managed well with meticulous wound care and primary surgical repair . The use of prophylactic antibiotics, though controversial, appears prudent in such cases . Ophthalmologists treating these injuries must be aware of serious potential complications including occult facial fracture or intracranial penetration in young children, septicemia caused by bacillus DF-2 in patients with prior splenectomy, tetanus, and rabies. Microbiol Immunol, 1987, 31(10), 967 - 74 A highly sensitive method for detection of 32P incorporation into acid-soluble compounds and its application to evaluate ATP-forming ability of Bacillus megaterium QM B1551 spores at the very early stage of germination; Otani M et al.; A method for specific removal of {32P}orthophosphate (Pi) as phosphomolybdate-triethylamine complex was slightly modified by repeating the Pi precipitation procedures in the presence of unlabeled Pi, which resulted in a complete removal of 32Pi (greater than 99.98%) . Using this modified method, we determined 32P incorporation into acid-soluble compounds in order to evaluate the ATP-forming ability of Bacillus megaterium spores at the very early stage of germination . Addition of fructose as a substrate started the 32P incorporation later than a few min after triggering germination . This delay of a few min was well coincident with the onset of 3-phosphoglycerate (3PGA) breakdown, indicating that fructose metabolism and the accompanying aerobic ATP formation were initiated only after fructose phosphorylation by the ATP derived from anaerobic breakdown of endogenous 3PGA . In contrast, addition of glucose started incorporation of 32P into acid-soluble compounds immediately after germination . In the latter case, NADH generated by glucose oxidation to gluconate (catalyzed by glucose dehydrogenase) might serve as an initial ATP source without depending on 3PGA breakdown and glucose metabolism via the Embden-Meyerhof pathway. Ter Arkh, 1987, 59(7), 80 - 2 {Effectiveness of the combined therapy of pulmonary mycobacterial infections depending on the species classification of the causative agent}; Kochetov BI et al.; The purpose of the study was to define the effect of specific tubercle bacillus on a course of pulmonary tuberculosis and the role of atypical mycobacteria in the development of "nontuberculous mycobacterioses" . A total of 1896 mycobacterial cultures isolated from 1285 patients were identified . Mycobacterial cultures from 86 patients were identified as M . bovis; mycobacteria of the "atypical" group from 12 patients were agents of nontuberculous mycobacterioses . Cultures in 7 patients were identified as M . avium . in 2 patients as M . xenopi, and in 3 as M . fortuitum . Therapeutic results showed an unfavorable course of tuberculosis caused by M . bovis (disintegration cavity closing in 40.7% only and bacillus elimination in 52.3% of the cases) . A tendency to a torpid course and progression of nontuberculous mycobacterioses was noted . The best results were obtained with the use of a rapid method of i.v . injection of isoniazid and with the use of rifampicin. Microbiol Immunol, 1987, 31(6), 597 - 601 17K-spore coat protein antigen in sporulating cells of Bacillus megaterium ATCC 19213; el-Belbasi HI et al.; Using immunological techniques, we studied the behavior of spore coat protein during sporulation of Bacillus megaterium ATCC 19213 . Antibody specific to the main coat protein of 17,000 daltons was prepared and used to demonstrate that the spore coat protein was synthesized and deposited at a later stage during sporulation. Microbiol Immunol, 1987, 31(2), 101 - 11 Isolation and characterization of forespores from Bacillus megaterium; Watabe K et al.; A procedure for isolation of intact forespores from sporulating Bacillus megaterium cells was developed . The cells were digested with lysozyme and made to release free forespores from the protoplasts by disruption of the cytoplasmic membrane with sonication in phosphate buffer containing 10% glycerol . The suitability of the procedure was confirmed by recovery of dipicolinic acid in the isolated forespores and an electron microscopic observation . The fine structure of the forespores prepared at 6 hr (t6) after initiation of sporulation was similar to that of mature spores, except that the cortex layer and primordial cell wall were thinner and the core was larger . The density, determined by density gradient centrifugation, of the forespores isolated at t6, t10, t12, and mature spores was estimated to be 1.2783, 1.2875, 1.2861, and 1.2858, respectively . The isolated forespores at t6 and t8 were extremely heat labile (D80 of 9.5 and 21.5 min, respectively) relative to mature spores (D80 of 277.8 min) . These forespores were also less resistant to organic solvents . Germination of the forespores as well as mature spores was induced by KNO3, D-glucose, and L-leucine . Forespores at t6 were more sensitive to KNO3-induced germination than those at t10, t12, and mature spores when measured by reduction in the optical density of cell suspension. Dev Comp Immunol, 1987 Winter, 11(1), 125 - 37 Ultrastructural characterization of leucocytes in the pronephros of carp (Cyprinus carpio, L.); Temmink JH et al.; In the pronephros of carp (Cyprinus carpio, L.) the following cells were found and ultrastructurally characterized: erythrocytes; lymphocytes and plasma cells; thrombocytes; neutrophilic, eosinophilic and basophilic granulocytes; phagocytic reticular cells and monocytes and non-phagocytic reticular cells . The cells could be separated on a Percoll continuous density gradient and relatively pure fractions could be obtained . In vitro phagocytosis of bacteria (Bacillus megaterium) was found in monocytes and neutrophilic granulocytes, while basophilic and eosinophilic granulocytes engulfed bacterial cells without actual endocytotic uptake. Microbiol Immunol, 1987, 31(1), 27 - 34 Comparative antigenicity of spore coat proteins from Bacillus species using antibody to spore coat proteins of Bacillus megaterium; el-Belbasi HI et al.; Spore coat proteins obtained by extraction with sodium dodecylsulfate/dithiothreitol from six Bacillus spores were compared by immunoblot analysis using antibodies to spore coat proteins from two strains of B . megaterium . Although the extract from spores of each strain had heterogenous proteins with various molecular weights, there were some bands which cross-reacted with specific antibodies from B . megaterium spores . Specific antibody to 48K protein from B . megaterium ATCC 12872 cross-reacted with 17K protein from B . megaterium ATCC 19213, 13K protein from B . cereus and 50K protein from B . subtilis 60015 and B . subtilis NRRL B558 . Also, specific antibody to 22K protein from the same strain cross-reacted with 22K and 17K proteins from B . megaterium ATCC 19213 and 13K protein from B . cereus T . Specific antibody to 17K protein from B . megaterium ATCC 19213 reacted with 22K and 19K proteins in addition to 17K protein of own strain, and it was cross-reactive with 16K protein from B . megaterium ATCC 12872, 19K and 27K proteins from B . thiaminolyticus, 13K protein from B . cereus. Folia Microbiol (Praha), 1987, 32(2), 89 - 95 Factors influencing the activity of cellular alkaline phosphatase during growth and sporulation of Bacillus cereus; Vinter V et al.; Alkaline phosphatase (EC 3.1.3.1) is synthesized in media with a low phosphate concentration (0.37 mM of total and 19 microM of inorganic phosphate, respectively) already during the exponential phase of growth of Bacillus cereus . The enzyme is repressed by higher phosphate concentrations (3.7 mM) during the whole growth period; during sporogenesis the enzyme activity in cells slightly increases even under these conditions . During growth the enzyme is not secreted into the medium, a minor amount being released after cessation of growth . The enzyme activity can be increased by adding Zn2+ ions (10 microM) . When during growth without phosphate the pH of the medium decreases below 5.0, the enzyme activity temporarily decreases and growth is slowed down, followed by a subsequent increase of the enzyme activity . In this case the onset of sporulation is also delayed. Biochem Int, 1987 Jan, 14(1), 15 - 26 On the formation of a dicloxacillin-p-hydroxymercuribenzoate suicide complex mediated by beta-lactamase I from Bacillus cereus; Amicosante G et al.; p-Hydroxymercuribenzoate is a non-competitive inhibitor of beta-lactamase I from Bacillus cereus and also, after preliminary preincubation, an inactivator of the enzyme . Submitted to the simultaneous action of PCMB plus dicloxacillin, the enzyme completely loses its activity . Extensive dialysis can restore the enzymatic activity only if preincubation had been carried out with either PCMB or dicloxacillin but not if both inhibitors had been simultaneously present . Mercaptoethanol protects the enzyme from the action of PCMB, but not from the severe inactivation caused by dicloxacillin-PCMB mixtures . All these data suggest the formation of a complex between PCMB and the acyl-enzyme intermediate generated upon hydrolysis of the beta-lactam bond of dicloxacillin. Appl Environ Microbiol, 1987 Jan, 53(1), 79 - 82 Modification of membrane sulfhydryl groups in bacteriostatic action of nitrite; Buchman GW 3rd et al.; The mechanism by which nitrite inhibits outgrowing spores of Bacillus cereus T was examined by using techniques developed earlier for nitrite analogs . The morphological stage of inhibition, cooperativity effects, effect of pH on inhibition, kinetics of protection against iodoacetate incorporation into membrane sulfhydryl groups, and protection against the bacteriocidal effect of carboxymethylation by iodoacetate indicate that nitrite acts as a membrane-directed sulfhydryl agent . The mechanism by which nitrite modifies the chemical reactivity of the sulfhydryl group could be either direct covalent modification or inactivation through communication with another modified membrane component . Profiles of pH effects suggest that the active agent is the protonated form of nitrite . The nitrite concentrations which modify membrane sulfhydryl activity coincide with those which have a bacteriostatic effect . These results are consistent with membrane sulfhydryl modification as a component of the mechanism of nitrite-induced bacteriostasis in this aerobic sporeformer. Appl Environ Microbiol, 1987 Jan, 53(1), 47 - 52 Involvement of the spore coat in germination of Bacillus cereus T spores; Kutima PM et al.; Bacillus cereus T spores were prepared on fortified nutrient agar, and the spore coat and outer membrane were extracted by 0.5% sodium dodecyl sulfate-100 mM dithiothreitol in 0.1 M sodium chloride (SDS-DTT) at pH 10.5 (coat-defective spores) . Coat-defective spores in L-alanine plus adenosine germinated slowly and to a lesser extent than spores not treated with SDS-DTT, as determined by decrease in absorbance and release of dipicolinic acid and Ca2+ . Spores germinated in calcium dipicolinate only after treatment with SDS-DTT . Biphasic and triphasic germination kinetics were observed with normal and coat-defective spores, respectively, in an environment with temperature increasing from 20 to 65 degrees C at a rate of 1 degree C/min . Therefore, the physical and biochemical processes involved in germination are modified by coat removal . The data suggest that a portion of the germination apparatus located interior to the coat may be protected by the coat and outer membrane or that the coat and outer membrane otherwise enhance germination in L-alanine plus adenosine . When coat-defective spores were heat activated with the dialyzed (12,000-Mr cutoff) components extracted from the spores, germination of the SDS-DTT-treated spores was enhanced; thus, one or more components located in the spore coat or outer membrane with a molecular weight greater than 12,000 were essential for fast germination. Rev Infect Dis, 1987 Jan-Feb, 9(1), 110 - 23 Posttraumatic endophthalmitis: the emerging role of Bacillus cereus infection; Davey RT Jr et al.; Endophthalmitis resulting from nonsurgical penetrating trauma to the eye is a relatively uncommon infection in the United States . Data are limited, but most recently published series have attributed the highest incidence of infection to gram-positive organisms, in particular Staphylococcus epidermidis . Fungal causes have been reported far less frequently . Bacillus species are being recognized increasingly as major causes of posttraumatic ocular disease, with rates of infection often making them the second most commonly isolated organisms . Bacillus cereus, an especially virulent pathogen, causes a fulminant endophthalmitis characterized by rapid destruction of intravitreal contents and a uniformly poor visual outcome . Certain toxins elaborated by the organism may contribute to its particular virulence . The currently recommended approach to suspected posttraumatic infection involves early use of diagnostic vitrectomy and intraocular culture, use of intravitreal antibiotics, and combination treatment with systemic and periocular antibiotics. Can J Surg, 1987 Jan, 30(1), 28 - 9 Bacillus cereus endophthalmitis; Le Saux N et al.; The authors present a case of a young man with post-traumatic endophthalmitis caused by Bacillus cereus . The clinical course was typical of the panophthalmitis caused by this toxin-producing organism: rapid onset of signs of systemic infection, a corneal ring abscess and eventual loss of the globe requiring enucleation . Studies of experimental rabbit models of the infection have indicated that the most efficacious regimen consists of systemic and topical clindamycin and gentamicin . Successful therapy depends upon immediate systemic administration of these agents when the clinical setting suggests Bacillus cereus infection. Int J Radiat Biol Relat Stud Phys Chem Med, 1987 Jan, 51(1), 7 - 18 Involvement of calcium and dipicolinic acid in the resistance of Bacillus cereus BIS-59 spores to u.v . and gamma radiations; Kamat AS et al.; The role of dipicolinic acid (DPA) in determining the resistance of Bacillus cereus spores to u.v . and gamma radiation was investigated . B . cereus BIS-59 spores containing varying amounts of DPA were prepared by appropriate compositional adjustments in the secondary media . Compared with spores containing 6 per cent DPA (dry weight) those containing 0.8 per cent DPA were far more sensitive to u.v . radiation . Similar u.v . radiation sensitivity was also found in respect of a DPA-less mutant of B . cereus T 6A 1 . Pre-treatment of DPA deficient spores (of wild type or mutant B . cereus) with DPA or the presence of DPA during irradiation resulted in increased resistance of these spores to u.v . radiation . In the range 0.2 to 1 per cent DPA content of spores of B . cereus BIS-59, a striking inverse relationship could be discerned between the DPA content and the number of spore photo-products (5-thymidyl, 5,6-dihydrothymine) formed in DNA and spore viability . The resistance of B . cereus spores to gamma radiation did not seem to be influenced by their DPA content. Toxicon, 1987, 25(5), 555 - 64 Effects of phospholipases C from bacteria on binding of enkephalin to rat brain membranes; Nakajima M et al.; The effects of phospholipases C on the equilibrium constants and maximal binding capacities of tritiated {D-Ala2,-D-Leu5} enkephalin to rat brain membranes were investigated using phosphatidylcholine-hydrolyzing phospholipase C and sphingomyelinase C of Bacillus cereus and, phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis . When 72% of the phosphatidylinositol in the rat brain membranes was hydrolyzed by phosphatidylinositol-specific phospholipase C, the affinity of opiate receptor for {D-Ala2,-D-Leu5} enkephalin was almost doubled and maximal binding of {D-Ala2,-D-Leu5} enkephalin was decreased to 87% of control . Although specific {D-Ala2,-D-Leu5} enkephalin binding was decreased with phosphatidylinositol hydrolysis when measured at higher concentration (30 nM) of {D-Ala2,-D-Leu5} enkephalin, the specific binding was increased with the hydrolysis of phosphatidylinositol when measured at lower concentration (6 nM) of the ligand . On treatment of membranes with phosphatidylcholine-hydrolyzing phospholipase C, specific {D-Ala2,-D-Leu5} enkephalin binding was drastically decreased with the progressive hydrolysis of phosphatidylcholine in the rat brain membranes, and specific binding was completely lost after 81% hydrolysis of phosphatidylcholine . However, the affinity of opiate receptor for {D-Ala2,-D-Leu5} enkephalin was not influenced, and maximal binding was decreased to 32% of the control when 61% of phosphatidylcholine was hydrolyzed . Treatment with sphingomyelinase C did not cause any appreciable reduction of specific {D-Ala2,-D-Leu5} enkephalin binding . From these results, it is concluded that the binding of {D-Ala2,-D-Leu5} enkephalin to opiate receptor is influenced by changes in the phospholipid environment of the rat brain membranes, and that phosphatidylinositol may be a modulator for the function of the receptor. Gene, 1987, 52(2-3), 285 - 90 Cloning and heterologous expression of an insecticidal delta-endotoxin gene from Bacillus thuringiensis var . aizawai IC1 toxic to both lepidoptera and diptera; Haider MZ et al.; Bacillus thuringiensis var . aizawai IC1 synthesises an insecticidal protein delta-endotoxin (130-135 kDa) that is toxic to both lepidopteran and dipteran larvae, and cross-reacts immunologically with certain monospecific lepidopteran toxins . A 166-kb plasmid from this bacterium was found to hybridise with an intragenic probe derived from the clone B . thuringiensis var . sotto lepidopteran-specific delta-endotoxin gene . A strongly hybridising 5.2-kb SstI fragment from var . aizawai plasmid DNA was cloned in pUC18 . After subcloning of this DNA in Escherichia coli, recombinants were obtained that synthesised large amounts of a 130-135-kDa protein . The protein was deposited in the cytoplasm as microscopically visible inclusion bodies and lysates of these cells were found to be toxic to both lepidopteran and dipteran larvae by comparison with controls . The structural basis for the dual specificity of this var . aizawai toxin is now amenable to further study. Gene, 1987, 51(2-3), 187 - 96 Cloning and nucleotide sequence of different iso-IS231 elements and their structural association with the Tn4430 transposon in Bacillus thuringiensis; Mahillon J et al.; A family of five repetitive sequences (RS) has been isolated from a plasmid DNA library of Bacillus thuringiensis strain berliner 1715 . In a previous paper {Mahillon et al., EMBO J . 4(1985)3895-3899} one of these was shown to harbor all the features of an IS element (IS231) . Further nucleotide sequence analysis revealed that two other RS, flanking the delta-endotoxin gene, are actually variants of IS231 . Comparison of the nucleotide sequences surrounding the iso-IS231 elements showed a unique structural association between some of these elements and the transposon Tn4430 . Although these IS231 elements have transposed into Tn4430, both these IS231 s and the transposon Tn4430 remain structurally intact. J Bacteriol, 1987 Jan, 169(1), 340 - 50 Cloning and analysis of ermG, a new macrolide-lincosamide-streptogramin B resistance element from Bacillus sphaericus; Monod M et al.; To analyze the regulation of a newly discovered macrolide-lincosamide-streptogramin B resistance element (ermG) found in a soil isolate of Bacillus sphaericus, we cloned this determinant and obtained its DNA sequence . Minicell analysis revealed that ermG specifies a 29,000-dalton protein, the synthesis of which is induced by erythromycin . S1 nuclease mapping was used to identify the transcriptional start site . These experiments demonstrated the presence on the ermG mRNA of a 197 to 198-base leader . Within the leader are two small open reading frames (ORFs) capable of encoding 11- and 19-amino-acid peptides . Each ORF is preceded by a suitably spaced Shine-Dalgarno sequence . The ermG protein is encoded by a large ORF that encodes a 244-amino-acid protein, in agreement with the minicell results . This protein and the 19-amino-acid peptide are highly homologous to the equivalent products of ermC and ermA . We conclude, on the basis of this homology, that ermG encodes an rRNA transmethylase . The leader of ermG can be folded into a structure that sequesters the Shine-Dalgarno sequence and start codon for the large ORF (SD3) . On the basis of these data and on the observed greater responsiveness of the ermG system than of the ermC system to low concentrations of erythromycin, we propose a model for the regulation of this gene in which the stalling of a ribosome under the influence of an inducer, while reading either peptide, suffices to uncover SD3 and allow translation of the rRNA transmethylase . The evolution of ermG is discussed. Med Vet Entomol, 1987 Jan, 1(1), 23 - 7 Differential effects of Bacillus sphaericus strain 2362 on Culex quinquefasciatus and its competitor Culex cinereus in West Africa; Nicolas L et al.; The larval susceptibility to Bacillus sphaericus strain 2362 of the non-man-biting mosquito Culex cinereus and the urban filariasis vector Culex quinquefasciatus, two competitor mosquitoes in polluted habitats, was compared . In the laboratory, both species ingested a similar amount of B . sphaericus spores when fed c . 2 x 10(5) spores per ml for 30 min . However, in the same experiment, third-instar larvae of Cx quinquefasciatus were reduced by 98% at 24 h exposure while Cx cinereus larvae were only reduced by 6% at 72 h . In the field, preimaginal populations of Cx cinereus ingested, within a week, more than 99% of the applied spores, but showed no significant decrease through 14 days in cesspools treated at 10 g/m2 of a flowable concentrate of B . sphaericus 2362, containing 2 x 10(10) spores/g . It is proposed that specific biological control of Cx quinquefasciatus could result from appropriate treatment of breeding-sites with larvicidal B . sphaericus and competitive displacement by Cx cinereus or other mosquitoes with larvae that are more tolerant of B . sphaericus. Gene, 1987, 59(2-3), 279 - 83 High-level expression of Bacillus stearothermophilus 6-phosphofructo-1-kinase in Escherichia coli; French BA et al.; The 6-phosphofructo-1-kinase (PFK) gene from Bacillus stearothermophilus has been expressed at high levels in Escherichia coli . This expression has been demonstrated by complementation studies, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and PFK assays of cell extracts . A level of B . stearothermophilus PFK expression corresponding to 20% of the total extracted protein was calculated from densitometric scans of an SDS-polyacrylamide gel . The high level of recombinant gene expression will enable this laboratory to determine structure-function relationships in B . stearothermophilus PFK by the method of oligodeoxynucleotide-directed mutagenesis. Cancer Immunol Immunother, 1987, 25(3), 180 - 4 The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives . II . Establishment of tumor-specific immunotherapy models utilizing MDP hapten-reactive helper T cell activity; Sano H et al.; Preinduction of potent haptenic muramyl dipeptide (MDP)-reactive helper T cell activity and subsequent immunization with MDP hapten-coupled syngeneic tumor cells resulted in enhanced induction of tumor-specific immunity through T-T cell collaboration between anti-MDP hapten helper T cells and tumor-specific effector T cells . The present study establishes two types of tumor-specific immunotherapy protocols utilizing helper T cells against MDP hapten cross-reactive with Bacillus Calmette Guerin (BCG) . In the first model, naive normal C3H/He mice or mice in which MDP hapten-reactive helper T cells had been generated by BCG-sensitization were inoculated i.d . with syngeneic X5563 tumor cells . When both groups of mice were allowed to generate MDP hapten-modified tumor cells in the tumor mass in situ by intratumoral injection of MDP hapten, an appreciable number of growing tumors in the BCG-presensitized but not in the unsensitized group were observed to regress . In the second model, a growing X5563 tumor mass was removed by the surgical resection 9 days after the tumor implantation . Approximately 90% of C3H/He mice receiving such treatment died from tumor metastasis by about 30 days after the tumor resection . However, immunization of mice with MDP hapten-coupled X5563 tumor cells subsequent to the tumor resection resulted in an increased survival rate . Such protection from the tumor metastasis was appreciably stronger when compared to the protection obtained by immunization with MDP hapten-uncoupled tumor cells . The mice surviving in both models were also demonstrated to retain X5563 tumor-specific immunity . These results indicate that the presentation of MDP hapten-modified tumor cells to BCG-sensitized recipients results in potent tumor-specific immunity which contributes to the regression of the primary tumor or inhibition of metastatic tumor growth. J Hyg Epidemiol Microbiol Immunol, 1987, 31(1), 53 - 63 Efficacy of Czechoslovak and Soviet Bacillus thuringiensis (serotype H-14) formulations against mosquito larvae; Rettich F; Laboratory and field comparisons were made with two wettable powder formulations of Bacillus thuringiensis serotype H-14 (B . t . H-14) prepared in Czechoslovakia ("Moskitur") and the USSR ("Baktokulicid") . Expressed in the international Aedes aegypti toxic units (TU X mg-1) the potency of these two test formulations was greater than that of the Institute Pasteur Standard IPS-78 (= 1,000 TU X mg-1), i.e . Moskitur had a potency of about 1,500 TU X mg-1 and the Soviet Baktokulicid 2,000 TU X mg-1 . The Baktokulicid and Moskitur LC 90 values for laboratory-reared Aedes aegypti larvae were, respectively, 0.11 and 0.16 mg X l-1 . The range of LC 90 values for the Czechoslovak wild-caught mosquito species of the genera Aedes and Culex was 0.14-0.31 mg X liter-1 with Moskitur, 0.11-0.41 mg X l-1 with Baktokulicid, and 0.16-0.48 mg X l-1 with IPS-78 . The susceptibility of laboratory Anopheles stephensi larvae was close to that of Aedes aegypti, larvae of An . messeae required many times as much Baktokulicid (1.6 mg X l-1) and Moskitur (more than 6.4 mg X l-1) for 90% mortality as did other mosquito species . The aim of outdoor assays was to establish the minimum Moskitur and Baktokulicid rates giving a 100% control of mosquito larvae . For Ae . cantans breeding habitat in flood plain forest areas these rates ranged between 0.1-0.5 mg X l-1 (0.2-1.0 kg X ha-1), for Ae . vexans control on artificially irrigated meadows between 0.8-2.0 mg X l-1 (1.2-3.0 kg X ha-1) . Consistently with laboratory bioassays, Baktokulicid gave 100% control of An . messeae 4th instar larvae at a dose as high as 3.2 mg X l-1, Moskitur gave 23.1% kill at 6.4 mg X l-1 . The effect of Moskitur and Baktokulicid formulations was immediate, larvae that hatched 7-14 days posttreatment survived . The efficacy of B . t . H-14 outdoor treatments tended to markedly decrease with the larval densities exceeding 100 larvae per 1 dm2 . Species of nontarget aquatic organisms, including the Diptera Chaoborus crystallinus, Mychlonyx sp . and Dixidae, were not noticeably affected by treatments with B . t . H-14 formulations used. J Bacteriol, 1987 Jan, 169(1), 72 - 9 Characterization of the surface protein layers of the mosquito-pathogenic strains of Bacillus sphaericus; Lewis LO et al.; The protein surface layers on the cell walls of mosquito-pathogenic and nonpathogenic Bacillus sphaericus strains were studied by structural, biochemical, and serological methods . The surface structure of two representative insect-pathogenic strains had the form of a delicate linear array with a repeat interval of 5 nm . This was distinctly different from the tetragonal array of the P-1 strain in spacing and arrangement . The surface layers were composed of acidic glycoproteins with molecular weights ranging from approximately 133,000 to 155,000 . Peptide mapping and serological analysis of the surface proteins revealed eight distinct groups among the pathogens . These groups were very similar to the groupings determined by flagellar-antigen serotyping and bacteriophage typing. Gene, 1987, 59(2-3), 161 - 70 Efficient secretion of Bacillus amyloliquefaciens alpha-amylase by {corrected} its own signal peptide from Saccharomyces cerevisiae host cells {corrected}; Ruohonen L et al.; The expression and secretion of Bacillus amyloliquefaciens alpha-amylase was studied in yeast Saccharomyces cerevisiae . The Bacillus promoter was removed by BAL 31 digestion and three forms of the alpha-amylase gene were constructed: the Bacillus signal sequence was either complete (YEp alpha a1), partial (YEp alpha a2) or missing (YEp alpha a3) . Secretion of alpha-amylase into the culture medium was obtained with the complete signal sequence only . The secreted alpha-amylase was glycosylated and its signal peptide was apparently processed . The glycosylated alpha-amylase remained active . The enzyme produced by the other constructions was not glycosylated and thus probably remained in the cytoplasm. Gene, 1987, 57(1), 37 - 46 Nucleotide sequence and deduced amino acid sequence of a coleopteran-active delta-endotoxin gene from Bacillus thuringiensis subsp . san diego; Herrnstadt C et al.; A gene from Bacillus thuringiensis subsp . san diego that is responsible for a delta-endotoxin active against Colorado potato beetle and some other Coleoptera was sequenced and shown to have surprising regional homology with both lepidopteran and dipteran active delta-endotoxins from other strains of B . thuringiensis . Unlike the lepidopteran active toxins from B . thuringiensis subsp . kurstaki that exist as approx . 130-kDa protoxins and form bipyramidal crystalline inclusions, the coleopteran toxic protein forms a square-shaped crystal composed of an approx . 65-kDa protein . Comparisons of the gene sequences encoding the active portions of these protoxins indicate conservation of N-terminal hydrophilic and hydrophobic regions, and suggest a distant ancestral origin for these insecticidal proteins. Gene, 1987, 54(2-3), 197 - 202 Construction of an excretion vector and extracellular production of human growth hormone from Escherichia coli; Kato C et al.; A new excretion vector, pEAP8, was constructed to develop an excretion system for Escherichia coli . This plasmid, derived from pEAP37, carried the weakly activated kil gene of plasmid pMB9 {Kobayashi et al., J . Bacteriol . 166 (1986) 728-732} and the penicillinase promoter and signal region of an alkalophilic Bacillus sp . to excrete foreign gene products . A gene for human growth hormone (hGH) was joined to this signal sequence through the HindIII site . The recombinant plasmid p8hGH1 thus constructed, was introduced into E . coli . The hybrid protein which was produced in E . coli carrying p8hGH1 was processed during transport through the inner membrane, with the mature hGH being excreted into the medium through the outer membrane which was made permeable by the action of the kil gene . The N-terminal amino acid sequence and the biological activity of the extracellular hGH were consistent with those of the authentic hGH. Eur Urol, 1987, 13(4), 246 - 50 Experience with bacillus Calmette-Guérin in patients with superficial bladder tumors . Value of monocyte activation in intravesical bacillus Calmette-Guérin therapy; Nissenkorn I et al.; Twenty-four patients with transitional cell carcinoma of the bladder, who had failed thiotepa therapy, were subjected to immunotherapy with intravesical bacillus Calmette-Guerin (BCG) by a modification of the Morales protocol . The results show a marked decline in the number of recurrent tumors following BCG immunotherapy . From a cumulative recurrence of 1 in 8.3 months, recurrences declined to 1 in 82 months . In parallel with the clinical follow-up, the level of the in vivo activation of peripheral blood monocytes of the treated patients, as a result of immunostimulation with BCG, was monitored by the quantitation of oxidative burst products released by the cells . The oxidative burst capacity of peripheral blood monocytes were found to undergo an increase of 80-140% in 5 of 10 patients tested . The patients with a high level of activation of peripheral blood monocytes did not have tumor recurrence while 3 of the 5 patients who did not show an increase in the oxidative burst had recurrent tumors . The possibilities for utilizing the oxidative burst test as an early indicator for distinction between patients who will respond to BCG immunotherapy and nonresponders is discussed. Mikrobiologiia, 1987 Jan-Feb, 56(1), 152 - 4 {Electron microscope study of the serological relations of Bacillus thuringiensis phages}; Kochkina ZM; The kinetics of neutralization cross-reactions was studied with Bacillus thuringiensis subsp . galleriae 1-97 phages, and serological complexes were investigated using immunoelectron microscopy . The results of these studies have made it possible to locate both common and specific antigenic components of the phages. Biochem Biophys Res Commun, 1986 Dec 30, 141(3), 937 - 41 Effect of removal of the cytolytic factor of Bacillus thuringiensis subsp . israelensis on mosquito toxicity; Held GA et al.; Solubilized crystal protein of Bacillus thuringiensis subsp . israelensis was fractionated by affinity chromatography using a monoclonal antibody directed against the crystal's 28 kDa peptide . The 28 kDa peptide was found to be relatively nontoxic to mosquito larvae although it does contain the hemolytic activity of the crystals . The crystal protein fraction depleted of the 28 kDa peptide was found to be nonhemolytic and to retain nearly full toxicity to mosquito larvae . These results suggest that the 28 kDa peptide is not required for the toxicity of solubilized crystal protein. Biochem Biophys Res Commun, 1986 Dec 15, 141(2), 756 - 61 Purification of larvicidal protein from Bacillus sphaericus 1593; Narasu ML et al.; Coat proteins from the spores of Bacillus sphaericus 1593 were separated by preparative polyacrylamide gel electrophoresis . Neutralising antibodies were raised against a single protein band exhibiting toxicity to mosquito larvae . IgG was purified and coupled to CNBr-activated Sepharose 4B to be used as an immunoaffinity matrix . The larvicidal protein was purified to electrophoretic homogeneity using this immunoaffinity column . The single protein species resolved into four peptides of molecular weights 42.6, 44.1, 50.7 and 51.3 KDa on polyacrylamide gel electrophoresis under denaturing conditions . This protein contained 12% carbohydrates . The purified protein exhibited an LC50 value of 8.3 +/- 1.6 ng/ml when tested against early third instar larvae of the mosquito Culex pipiens var quinquefasciatus. Biochim Biophys Acta, 1986 Dec 10, 884(3), 490 - 6 Purification, stability and kinetic properties of highly purified adenosine deaminase from Bacillus cereus NCIB 8122; Gabellieri E et al.; Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) from Bacillus cereus NCIB 8122 has been purified to electrophoretic homogeneity by ammonium sulfate precipitation, gel filtration through Sephadex G-100, DEAE-Sephadex A-50 chromatography and ion-exchange HPLC on DEAE-Polyol . The enzyme activity is stabilized (at temperatures from 0 degrees C to 40 degrees C) by 50 mM NH4+ or K+, while it is irreversibly lost in the absence of these or a few other monovalent cations . Glycerol (24% by volume) helps the cation in stabilizing the enzyme activity above 40 degrees C, but also exerts per se a noticeable protecting effect at room temperature . B . cereus adenosine deaminase displays the following properties: Mr on Sephadex G-200, 68,000; Mr in SDS-polyacrylamide gel electrophoresis, 53,700; optimal pH-stability (in the presence of 50 mM KCl) over the range 8-11 at 4 degrees C, and maximal catalytic activity at 30 degrees C between pH 7 and 10; Km for adenosine around 50 microM over the same pH range and Km for 2'-deoxyadenosine around 400 microM. S Afr Med J, 1986 Dec 6, 70(12), 761 - 2 Epstein-Barr virus infection in a patient with active pulmonary tuberculosis . A case report; Gledhill RF et al.; Active pulmonary tuberculosis is highly prevalent among the black population but factors predisposing to a defective cellular immune response to the tubercle bacillus are not readily apparent in many . Serological evidence of a recent reactivation of Epstein-Barr virus (EBV) infection in such a patient could therefore be a relevant finding, since EBV has the potential to suppress cellular immune responses. J Vet Pharmacol Ther, 1986 Dec, 9(4), 402 - 11 Oral treatment of polioencephalomalacia and subclinical thiamine deficiency with thiamine propyl disulphide and thiamine hydrochloride; Thomas KW; Thiaminase type I production by Bacillus thiaminolyticus and activity in vitro were repressed by the primary substate thiamine and by thiamine monophosphate and thiamine propyl disulphide . At thiamine concentrations of 300-3000 mumol/l production of active enzyme by B . thiaminolyticus, and activity of purified enzyme, were totally repressed . Growth of B . thiaminolyticus was inhibited by thiamine propyl disulphide at 3000 mumol/l . Activity of purified thiaminase was lost when incubated with ruminal fluid from healthy thiaminase free sheep . Enzyme activity was also lost when exposed to mildly alkaline conditions but was stimulated and stabilized against heat denaturation if treated with dithiothreitol . Thiaminase activities in the ruminal fluids of cases of ovine polioencephalomalacia were repressed within 2 h of oral administration of thiamine propyl disulphide . Blood pyruvate levels and transketolase activities were restored to normal following treatment . Treated animals recovered clinically and were returned to pasture. FEBS Lett, 1986 Dec 1, 209(1), 104 - 6 The growth of ordered two-dimensional sheets of 70 S ribosomes from Bacillus stearothermophilus; Piefke J et al.; Well ordered two-dimensional sheets of intact 70 S ribosomes from Bacillus stearothermophilus have been obtained in vitro using salt-alcohol mixtures . These sheets consist of relatively small unit cells with dimensions of 200 +/- 20 A and 400 +/- 30 A . Diffraction patterns of electron micrographs of these sheets stained with uranyl acetate contain features to 42 A resolution. J Steroid Biochem, 1986 Dec, 25(6), 995 - 9 Metabolism of 11-deoxycortisol by a Bacillus species; Mahato SB et al.; Microbial transformation by a Bacillus species was employed for the preparation of potentially important derivatives of 11-deoxycortisol . Each microbial metabolite was characterised by the application of various spectroscopic methods . The five metabolites of 11-deoxycortisol were characterised as 4-androstene-3,17-dione (2), 14-hydroxy-4-androstene-3,17-dione (3), 14,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (4), 6 beta,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (5) and 15 alpha,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (6) . The availability of the metabolites enabled complete elucidation of their {13C}NMR spectra. Appl Environ Microbiol, 1986 Dec, 52(6), 1293 - 8 Cadmium- and mercury-resistant Bacillus strains from a salt marsh and from Boston Harbor; Mahler I et al.; Bacteria resistant to cadmium or mercury or both were isolated from the Great Sippewissett Marsh (Cape Cod, Mass.) and from Boston Harbor . Many of these metal-resistant isolates were gram-positive aerobic sporeformers, although not necessarily isolated as spores . Although several of the isolated strains bore plasmids, cadmium and mercury resistances appeared to be, for the most part, chromosomally encoded . DNA sequence homology of the gram-positive cadmium- and mercury-resistant isolates was not demonstrable with metal resistance genes from plasmids of either gram-positive (pI258) or gram-negative (pDB7) origin . Cadmium resistance of all the marsh isolates tested resulted from reduced Cd2+ transport . On the other hand, three cadmium-resistant harbor isolates displayed considerable influx but no efflux of Cd2+ . Hg-resistant strains detoxified mercury by transforming Hg2+ to volatile Hg0 via mercuric reductase. South Med J, 1986 Dec, 79(12), 1499 - 502 Gram-negative bacillary meningitis in the adult: review of 39 cases; Gower DJ et al.; From 1976 through 1984, the period covered in this report, we reviewed our total experience with gram-negative meningitis in adult patients, looking especially at how treatment and mortality had changed . Thirty-nine adults had 45 episodes of gram-negative meningitis . Twenty-five patients had had a dura-arachnoid disruption, 12 a septic episode, and two a bacterial mastoiditis . The overall mortality was 35.9% . Thirteen patients were treated with a full course of intrathecal antibiotics (five or more days) and eight patients with an abbreviated course (one or two doses) . The use of chloramphenicol was associated with poor patient outcome, a finding consistent with both experimental and clinical findings of others. J Epidemiol Community Health, 1986 Dec, 40(4), 295 - 300 The nature of mycobacterial disease in south east England, 1977-84; Yates MD et al.; The nature and incidence of bacteriologically confirmed mycobacterial disease in south east England over the eight year period 1977-84 has been determined by a study of cultures received by the PHLS Regional Centre for Tuberculosis Bacteriology at Dulwich . The number of cases of tuberculosis in the ethnic European population has shown a decline, more so among males than females, but there has not been a significant decline in cases among ethnic Asians . Most tuberculosis is due to the classical human tubercle bacillus but cases due to the Asian human type, the bovine type (M . bovis), and the African types (M . africanum) also occur . The number of cases of disease due to 'atypical' mycobacteria has doubled over the eight year period, and these now account for about 5% of bacteriologically diagnosed mycobacterial disease in this region . The continuing role of reference facilities for the surveillance of tuberculosis and the diagnosis and management of the growing numbers of other mycobacterial infections is stressed. J Rheumatol, 1986 Dec, 13(6), 1076 - 80 Routine analysis of synovial fluid cells is of value in the differential diagnosis of arthritis in children; Kunnamo I et al.; Results of synovianalysis are reported in 129 children, 91 with juvenile rheumatoid arthritis (JRA), 13 with septic arthritis, 12 with enteroarthritis, 12 with acute transient arthritis and 1 with bacillus Calmette-Guerin arthritis . Mononuclear cells were dominant in the patients with oligoarticular JRA (mean 64%, median 74%), and among these, significantly lower proportions of polymorphonuclear (PMN) cells were found in patients with early onset disease with antinuclear antibodies or chronic uveitis than in those with late onset with HLA-B27 . PMN cells were dominant in polyarticular and systemic onset JRA, septic arthritis and enteroarthritis . The sensitivity and specificity of a white cell count above 40,000/mm3 exceeded 90% in differentiating septic arthritis from the other kinds of arthritis . Measurement of total protein, glucose and lactate in synovial fluid was of limited diagnostic value. Antibiot Med Biotekhnol, 1986 Dec, 31(12), 886 - 9 {Sporulation in Bacillus licheniformis strains that do not synthesize bacitracin and serine exo- and endoproteases}; Lukin AA et al.; Sporulation and synthesis of bacitracin, a peptide antibiotic and serine exoproteases and endoproteases were studied in 6 strains of Bacillus licheniformis at various periods of cultivation in sporulation media . Thermostable spores in all the strains were shown to form in the absence of both the exogenic and endogenic antibiotic and proteases . It was suggested that bacitracin and the serine proteases were not specific regulators of sporogenesis in B . licheniformis and their biological function under the natural conditions included, respectively, the antibiotic action and hydrolysis of proteins and peptides of the environment and cells. Int J Lepr Other Mycobact Dis, 1986 Dec, 54(4), 556 - 9 Evaluation of monitoring antibodies to PGL-I in armadillos experimentally infected with M . leprae; Truman RW et al.; An enzyme-linked immunosorbent assay (ELISA) for IgM antibodies to the phenolic glycolipid-I antigen of Mycobacterium leprae was evaluated for efficacy in monitoring armadillos experimentally infected with the bacillus . IgM antibodies were detected in armadillos from 186 days after experimental infection until the animals were sacrificed . The ELISA demonstrated the establishment of infection earlier and more reliably than the histologic methods previously applied . Satisfactory yields of M . leprae from individual armadillos could be predicted 97% of the time, and the technique may be useful in identifying appropriate harvest times or resistance among armadillos . The ELISA seems to be a valuable adjunct for managing experimental infections of M . leprae in armadillos. J Clin Microbiol, 1986 Dec, 24(6), 917 - 21 Serodiagnosis of leprosy: relationships between antibodies to Mycobacterium leprae phenolic glycolipid I and protein antigens; Levis WR et al.; Sera from leprosy patients and controls were assayed for immunoglobulin M (IgM) and IgG antibodies to the Mycobacterium leprae-specific phenolic glycolipid I antigen (PG) by enzyme-linked immunosorbent assay, for IgG antibodies to M . leprae protein antigens by Western immunoblot, and for antibodies to a 65-kilodalton (kDa) protein antigen of M . leprae by a competition antibody binding assay . Elevated levels of anti-PG IgM were seen in lepromatous and borderline lepromatous patients, and elevated levels of anti-PG IgG were seen in borderline lepromatous patients . There was a significant correlation between the bacillary index (BI) and anti-PG IgM whether all leprosy patients or only multibacillary patients were analyzed . A significant correlation was seen between anti-PG IgG and BI when all leprosy patients were used for analysis, but not when only multibacillary patients were used . IgG antibodies to protein antigens of M . leprae, as detected by Western immunoblot, were more prevalent in lepromatous and borderline lepromatous patients than in borderline tuberculoid patients, while one of eight controls showed one weak band . There were significant correlations between the number of M . leprae protein antigens detected by the sera of patients and both BI and the level of anti-PG IgM . The 65-kDa competition antibody binding assay detected active multibacillary leprosy . Patients positive for antibody to the 65-kDa antigen had a significantly higher BI and levels of anti-PG IgM and anti-PG IgG than did patients that were negative . In addition, the level of antibody to the 65-kDa antigen correlated with both the BI and anti-PG IgM . We conclude that testing for antibodies to protein antigens of M . leprae may provide a useful adjunct to testing for antibodies to PG. J Am Mosq Control Assoc, 1986 Dec, 2(4), 548 - 51 Beecomist-applied Bacillus sphaericus for the control of riceland mosquitoes; Lacey LA et al.; An aerially applied flowable concentrate (FC) of Bacillus sphaericus Neide (isolate 2362) was evaluated against riceland mosquitoes using the Beecomist spray head . Application of the FC at 0.58 and 1.17 liter/ha in maturing rice fields resulted in a 48 hr mean posttreatment reduction of Anopheles quadrimaculatus of 71 and 82%, respectively . A significant reduction in larval populations one week posttreatment was also observed . Treatment rates of 0.29, 0.44, and 0.58 liter/ha in reflooded second crop rice fields resulted in 48 hr posttreatment reduction of Psorophora columbiae larvae of 50, 76 and 98% respectively. J Am Mosq Control Assoc, 1986 Dec, 2(4), 461 - 8 Swath width determination for Beecomist-applied Bacillus thuringiensis (H-14) against Anopheles quadrimaculatus larvae in rice fields; Sandoski CA et al.; Optimum flight path interval of Beecomist-applied Bacillus thuringiensis var . israelensis (serotype H-14) (Bti) against Anopheles quadrimaculatus larvae was determined by assessing the effective swath width in rice fields . Droplet sensitive cards, laboratory-reared and naturally occurring populations of larvae were used to monitor aerial treatments 1 day posttreatment . Overlapping swaths were necessary to provide high levels of larval reduction . Based on tests where flight path intervals were 18.3, 36.6 and 73.2 m, optimal flight path interval was estimated to be approximately 67 m downwind of the extreme downwind flight path when flow rate was 1.44 liter/min. Proteins, 1986 Dec, 1(4), 326 - 34 Proteases of enhanced stability: characterization of a thermostable variant of subtilisin; Bryan PN et al.; A procedure has been developed for the isolation and identification of mutants in the bacterial serine protease subtilisin that exhibit enhanced thermal stability . The cloned subtilisin BPN' gene from Bacillus amyloliquefaciens was treated with bisulfite, a chemical mutagen that deaminates cytosine to uracil in single-stranded DNA . Strains containing the cloned, mutagenized subtilisin gene which produced subtilisin with enhanced thermal stability were selected by a simple plate assay procedure which screens for esterase activity on nitrocellulose filters after preincubation at elevated temperatures . One thermostable subtilisin variant, designated 7150, has been fully characterized and found to differ from wild-type subtilisin by a single substitution of Ser for Asn at position 218 . The 7150 enzyme was found to undergo thermal inactivation at one-fourth the rate of the wild-type enzyme when incubated at elevated temperatures . Moreover, the mid-point in the thermally induced transition from the folded to unfolded state was found to be 2.4-3.9 degrees C higher for 7150 as determined by differential scanning calorimetry under a variety of conditions . The refined, 1.8-A crystal structures of the wild-type and 7150 subtilisin have been compared in detail, leading to the conclusion that slight improvements in hydrogen bond parameters in the vicinity of position 218 result in the enhanced thermal stability of 7150. Jpn J Exp Med, 1986 Dec, 56(6), 297 - 307 Action of the bacterial neutral protease, dispase, on cultured cells and its application to fluid suspension culture with a review on biomedical application of this protease; Nagata M et al.; The bacterial neutral protease from Bacillus polymyxa, dispase, has been characterized using 21 mammalian cell lines in its cell-dispersing capability, cytotoxicity, and effects on cell growth . The previous observation that fibroblast-like cells are detached by this protease from culture substrate, as well as dissociated into a monodisperse cell suspension, while that epithelial-like cells are detached but not completely dissociated, was generally confirmed, although exceptions to these general rules were noted . Efforts were made here by the use of dispase to develop techniques for suspension culture and single cell plating: Successful conditions of suspension culture for 6 lines, and single cell plating for 3 lines were respectively described . Key factors in these techniques were choice of a low concentration of dispase in growth media to avoid its cytotoxic effect, and choice of appropriate serum concentration in the medium to allow this protease to remain active . Recent literatures on the application of dispase in biomedical science were reviewed. Arch Microbiol, 1986 Dec, 146(3), 275 - 9 Acetyl-coenzyme A: arylamine N-acetyltransferases in microorganisms: screening and isolation of an enzyme from Bacillus cereus; Hasmann MJ et al.; The raw extracts of a series of microorganisms were screened for the presence of acetyl-coenzyme A: arylamine N-acetyltransferase (AAAT) using a radioactive assay with 3H-acetyl-coenzyme A and aniline as substrates . Enzyme activities were primarily detected in the soluble fractions of Bacillus and Nocardia species, and in some further soil organisms . Only strains of Bacillus cereus were able to acetylate 4-nitroaniline and 3,5-dimethyl-4-nitroaniline . The fermentation conditions for the production of the enzyme were optimized . The AAAT from one strain of Bacillus cereus was purified 24-fold and characterized. Mol Gen Genet, 1986 Dec, 205(3), 530 - 4 Physical mapping of LP51 and LP52 prophages of lysogenic strains of Bacillus licheniformis; Doskocil J et al.; The physical maps of the LP51 and LP52 prophages in lysogenic strains of Bacillus licheniformis were constructed on the basis of data obtained by hybridization of phage DNA probes with Southern blots of restricted DNA of the lysogens . The data were compatible with the Campbell model for chromosomal integration; the attP site was mapped at 58.7-61.8 map units of the genomes of both phages . Identification of prophage-host DNA junction fragments indicated the presence of a unique attB site on the bacterial chromosome; the set of junction fragments in the strain B . licheniformis ATCC 10716 was identical to that of ATCC 11946, but different from ATCC 8187 . Both the LP51 and LP52 phages used the same integration sites . Upon reinfection with either phage, the cured strains UM12 and UM18 (i.e . 10716 and 11946 cured of LP52 or LP51, respectively) turned out to be integration deficient . In surface cultures the reinfected bacteria could be maintained in the lysogenic state without, however, integrating the phage genome; when these bacteria were passaged in submerged cultures, several modes of anomalous integration were observed, and the phage segregated into a variety of forms, discernible by virulence and plaque morphology . In liquid cultures of UM12(LP51) or UM12(LP52) lytic forms finally predominated, while most lysogenized UM18 were converted into defective lysogens which contained a defective prophage in a stably integrated form. Mol Gen Genet, 1986 Dec, 205(3), 390 - 7 Characterization of the genes encoding the haemolytic toxin and the mosquitocidal delta-endotoxin of Bacillus thuringiensis israelensis; Bourgouin C et al.; The crystalline parasporal inclusions (crystals) of Bacillus thuringiensis israelensis (Bti), which are specifically toxic to mosquito and black fly larvae, contain three main polypeptides of 28 kDa, 68 kDa and 130 kDa . The genes encoding the 28 kDa protein and the 130 kDa protein have been cloned from a large plasmid of Bti . Escherichia-coli recombinant clones containing the 130 kDa protein gene were highly active against larvae of Aedes aegypti and Culex pipiens, while B . subtilis recombinant cells containing the 28 kDa protein gene were haemolytic for sheep red blood cells . A fragment of the Bti plasmid which is partially homologous to the 130 kDa protein gene was also isolated; it probably corresponds to part of a second type of mosquitocidal toxin gene . Furthermore, restriction enzyme analysis suggested that the 130 kDa protein gene is located on the same Bti EcoRI fragment as another kind of Bti mosquitocidal protein gene cloned by Thorne et al . (1986) . Hybridization experiments conducted with the 28 kDa protein gene and the 130 kDa protein gene showed that these two Bti genes are probably present in the plasmid DNA of B . thuringiensis subsp . morrisoni (PG14), which is also highly active against mosquito larvae. Int J Radiat Biol Relat Stud Phys Chem Med, 1986 Dec, 50(6), 983 - 93 Purine and its analogues and radiation damage in Bacillus megaterium spores; Powers EL; As an extension of results obtained from radiation studies on caffeine both in other laboratories and more recently in this laboratory using the bacterial spore as the test system, six compounds with chemical structures closely resembling that of caffeine were tested as radiation modifiers . Of these compounds, purine, adenine and hypoxanthine resembled caffeine in sensitizing spores to radiation, while theobromine, xanthine and theophylline did not . These responses are discussed in relation to the electron sequestration hypothesis of cellular sensitization to high-energy radiation. Eur J Biochem, 1986 Dec 1, 161(2), 273 - 80 Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715; Hofte H et al.; A plasmid-encoded crystal protein gene (bt2) has been cloned from Bacillus thuringiensis berliner 1715 . In Escherichia coli, it directs the synthesis of the 130-kDa protein (Bt2) which is toxic to larvae of Pieris brassicae and Manduca sexta . Comparison of the deduced amino acid sequence of this Bt2 protein with the B . thuringiensis kurstaki HD1 Dipel, B . thuringiensis kurstaki HD73 and B . thuringiensis sotto crystal protein sequences suggests that homologous recombination between the different genes has occurred during evolution . Treatment of the Bt2 protein with trypsin or chymotrypsin yields a 60-kDa protease-resistant and fully toxic polypeptide . The minimal portion of the Bt2 protein required for toxicity has been determined by analysing the polypeptides produced by deletion derivatives of the bt2 gene . It coincides with the 60-kDa protease-resistant Bt2 fragment and it starts between amino acids 29 and 35 at the N-terminus and terminates between positions 599 and 607 at the C-terminus. J Biochem (Tokyo), 1986 Dec, 100(6), 1507 - 21 Studies on glucosyltransferase and endogenous glucosyl acceptor in Bacillus cereus AHU 1030 membranes; Shimada A et al.; A glucosyltransferase, extracted from the membranes of Bacillus cereus AHU 1030 with Tris-HCl buffer containing 0.1% Triton X-100 at pH 9.5, was separated from an endogenous glucosyl acceptor by chromatography on DEAE-Sepharose CL-6B subsequent to chromatography on Sepharose 6B . Structural analysis data showed that the glucosyl acceptor was a glycerol phosphate polymer linked to beta-gentiobiosyl diglyceride . The enzyme catalyzed the transfer of glucosyl residues from UDP-glucose to C-2 of the glycerol residues of repeating units of the acceptor . On the other hand, a lipoteichoic acid which contained 0.3 D-alanine residue per phosphorus was isolated from the cells by phenol treatment at pH 4.6 . Except for the presence of D-alanine, this lipoteichoic acid had the same structure as the glucosyl acceptor . The rate of glucosylation observed with the D-alanine-containing lipoteichoic acid as the substrate was less than 40% of that observed with the D-alanine-free lipoteichoic acid, indicating that the ester-linked D-alanine in the lipoteichoic acid interferes with the action of the glucosyltransferase . The enzyme also catalyzed glucosylation of poly(glycerol phosphate) which was synthesized in the reaction of a separate enzyme fraction with CDP-glycerol . Thus, it is likely that the glucosyltransferase functions in the synthesis of cell wall teichoic acid. Cancer, 1986 Dec 1, 58(11), 2411 - 6 Immunostimulation with intrapleural BCG as adjuvant therapy in resected non-small cell lung cancer . The Ludwig Lung Cancer Study Group (LLCSG); Purification and characterization of the active fragment from Bacillus thuringiensis delta-toxin; Limited tryptic hydrolysis of a partially purified delta-toxin (Mr = 100,000) from Bacillus thuringiensis, has produced a polypeptide fragment of Mr = 60,000 containing the full biological activity . The fragment was the only polypeptide observed in the polyacrylamide-gel electrophoresis of the delta-toxin after treatment with trypsin and could be purified by DEAE-cellulose chromatography . Amino acid and partial sequence analyses indicate that the 60,000 Mr fragment has been derived from the mid-section of the holotoxin peptide; over 80% of Lys, 65% of Pro and 50% of His residues in the holotoxin have been lost in the active fragment . This section must contain the active site since its specific insecticidal activity is approximately twice that of the holotoxin . The active fragment shows complete cross-reactivity with the antiserum raised against the native toxin, and appeared to possess higher thermal stability than the mother protein . It provides a powerful tool for studies of the structure involved in the insecticidal activity. Eur J Biochem, 1986 Nov 17, 161(1), 51 - 9 Biosynthesis of the wall neutral polysaccharide in Bacillus cereus AHU 1356; Murazumi N et al.; The pathway for the biosynthesis of a cell wall polysaccharide, composed of glucosamine, mannosamine, galactosamine and glucose in a molar ratio of 4:1:1:1, was studied with a membrane system from Bacillus cereus AHU 1356 . In this system a glycolipid characterized as GalNAc(alpha 1----4)ManNAc(beta 1----4)GlcNAc-PP-undecaprenol was formed from GlcNAc-PP-undecaprenol by sequential transfer of N-acetylmannosamine and N-acetylgalactosamine residues from UDP-ManNAc and UDP-GalNAc respectively . An additional N-acetylglucosamine residue and a glucose residue were individually transferred from their UDP derivatives to the trisaccharide-linked lipid with the formation of tetrasaccharide-linked lipids, which seem to serve as intermediates in the polysaccharide synthesis . Incubation of membranes with the trisaccharide-linked lipid even in the absence of sugar-linked nucleotides led to the formation of polysaccharide . These results, together with the data on Smith degradation of the synthesized polysaccharide, indicate that the repeating trisaccharide units of the main chain of the polysaccharide arise from the GalNAc-ManNAc-GlcNAc moiety of the glycolipid intermediates and that the sugar residues in the lateral branches of the polymer are at least partly introduced through oligosaccharide-linked lipid intermediates . In addition, the structure of native polysaccharide was re-examined, and the presence of the disaccharide sequence ManNAc(beta 1----4)GlcNAc in the polysaccharide chain was confirmed. J Mol Biol, 1986 Nov 5, 192(1), 161 - 2 Characterization of single crystals of the large ribosomal particles from a mutant of Bacillus stearothermophilus; Yonath A et al.; Single, three-dimensional crystals of 50 S ribosomal subunits, from a mutant of Bacillus stearothermophilus that lacks the protein L11, have been characterized using a synchrotron X-ray source . The crystals of the mutated particles grow under the same conditions and are isomorphous to those of the wild type of the same bacteria . They are orthorhombic, contain at least one 2-fold screw axis, and have unit cell dimensions of a = 350(+/- 10) A, b = 670(+/- 10) A, and c = 910(+/- 10) A . They diffract to 15 to 18 A resolution at 4 degrees C and are stable in the synchrotron beam for several hours. Biochemistry, 1986 Nov 4, 25(22), 7208 - 15 Changes in the coordination geometry of the active-site metal during catalysis of benzylpenicillin hydrolysis by Bacillus cereus beta-lactamase II; Bicknell R et al.; Rapid-scanning stopped-flow spectroscopy (425-700 nm) has been used to study spectral changes in cobalt(II)-substituted Bacillus cereus beta-lactamase II during the binding and hydrolysis of benzylpenicillin . The experiments were carried out in aqueous solution over a temperature range of 3-20 degrees C . Three metallointermediates have been characterized by their visible absorption spectra . Two of them have visible absorption spectra identical with the intermediates ES1 and ES2 previously observed at subzero temperatures in a mixed aqueous/organic solvent {Bicknell, R., & Waley, S.G . (1985) Biochemistry 24, 6876-6887} . In addition, the branched kinetic pathway observed with the zinc(II) and cobalt(II) beta-lactamase II at subzero temperatures has been shown to occur with the cobalt(II)-substituted enzyme in aqueous solution at above-zero temperatures; thus, at pH 6.0 and 3 degrees C, the rate and equilibrium constants are readily determined for the reaction scheme: (Formula: see text) . A third transient intermediate (called ES*) was found to precede ES1 in the pre-steady-state time period . The identity of the intermediates formed in aqueous solution with those previously observed in the cryostudy confirms that the mechanism is not changed either by the presence of an organic cosolvent or by subzero temperatures . Further characterization of ES1 and the steady-state intermediate ES2 at subzero temperatures, where their lifetime may be extended for up to several hours, has involved circular and magnetic circular dichroic studies . The magnetic circular dichroic spectra identify changes in the coordination sphere of the active-site metal during catalysis.(ABSTRACT TRUNCATED AT 250 WORDS) Eur J Biochem, 1986 Nov 3, 160(3), 587 - 91 DNA-supercoiling is affected in vitro by the peptide antibiotics tyrocidine and gramicidin; Bohg A et al.; Tyrocidine, a peptide antibiotic produced by Bacillus brevis (ATCC 8185), relaxes superhelical DNA in a biphasic manner and induces 'packaging' of the DNA at higher concentrations . This was concluded from studies using the sensitive 4,5',8-trimethylpsoralen photobinding technique {Sinden, R . R., Carlson, J . O . & Pettijohn, D.-E . (1980) Cell 21, 773-783} . Relaxed DNA is not affected by tyrocidine whereas linearized molecules become packaged . The linear gramicidin synthesized by the same strain reverses the tyrocidine-induced relaxation as well as the packaging, an observation which might be of biological relevance. Mikrobiologiia, 1986 Nov-Dec, 55(6), 1005 - 8 {Structural characteristics of DNA of Bacillus thuringiensis phages}; Kochkina ZM; The spectral characteristics of DNA from two phages of the polylysogenic Bacillus thuringiensis subsp . galleriae culture 1-97 were studied . The typical parameters of melting and the negative reaction with formaldehyde are indicative of the double-helical structure of these DNAs . The phage DNAs differ in the molar content of nitrogen bases (32 and 38 mole% of GC) and in their distribution along the molecule . This distribution is uniform in the DNA of one phage whereas the other phage DNA is composed of heterological segments with a different nucleotide composition. J Comp Pathol, 1986 Nov, 96(6), 671 - 84 Chronic inflammatory and lymphoproliferative lesions of the equine small intestine; Platt H; A retrospective study was made of 20 horses with severe and extensive chronic disease of the small intestine . Many of the animals had clinical evidence of malabsorption, with progressive loss of weight, hypoalbuminaemia and sometimes anaemia . All but two of the horses were Thoroughbreds . The pathology was diverse . Nine of the cases were alimentary lymphomas (Platt, 1986) and five had lymphocytic and eosinophilic infiltrations in the bowel wall which were considered to be probable reactions to parasitic invasion . One had acute thrombosis associated with partial occlusion of the anterior mesenteric artery by a verminous thrombus, superimposed on granulomatous lesions resulting from earlier ischaemic episodes . Two animals, from one stud, had dense mononuclear infiltration of the intestinal mucosa with villous atropy accompanying an unidentified acid-fast bacillary infection in the mesenteric lymph nodes and other sites . Three horses had granulomatous or lymphogranulomatous infiltration of the small intestine accompanied by marked mucosal and villous atrophy . One of these had multiple abscessation in part of the affected bowel . Only the three latter cases had lesions resembling those of equine granulomatous enteritis and the results of this study indicate the rarity of this condition in Thoroughbreds in Britain . The different types of lesion were only distinguishable by histological examination, since their clinical effects and gross pathology were not characteristic. J Bacteriol, 1986 Nov, 168(2), 479 - 85 Nucleotide sequences of two cellulase genes from alkalophilic Bacillus sp . strain N-4 and their strong homology; Fukumori F et al.; Two genes for cellulases of alkalophilic Bacillus sp . strain N-4 (ATCC 21833) have been sequenced . From the DNA sequences the cellulases encoded in the plasmids pNK1 and pNK2 consist of 488 and 409 amino acids, respectively . The DNA and protein sequences of the pNK1-encoded cellulase are related to those of the pNK2-encoded cellulase . The pNK2-encoded cellulase lacks the direct repeat sequence of a stretch of 60 amino acids near the C-terminal end of the pNK1-encoded cellulase . The duplication of the cellulase genes and the formation of the direct repeat in the pNK1-encoded cellulase occurred at almost the same time. Infect Immun, 1986 Nov, 54(2), 347 - 53 Entry of Bartonella bacilliformis into erythrocytes; Benson LA et al.; Bartonella bacilliformis, which causes the human diseases Oroya fever and verruga peruana, binds to human erythrocytes in vitro and produces substantial and long-lasting deformations in erythrocyte membranes, including cone-shaped depressions, trenches, and deep invaginations . The deforming force is probably provided by the polar flagella of these highly motile bacteria . Deep invaginations containing bacteria are commonly seen, and membrane fusion at the necks of the invaginations leads to the formation of intracellular vacuoles containing bacteria . Fluorescent compounds present externally render the vacuoles fluorescent and, occasionally, lightly fluorescent cells are seen, suggesting that the vacuoles sometimes rupture to admit the bacteria to the cytoplasm . Vacuoles present in fluorescent erythrocytes prepared by preloading the erythrocytes with fluorescent compounds are seen as dark areas from which the fluorescent marker is excluded . Entry of the bacteria appears to be the result of a process of forced endocytosis. J Allergy Clin Immunol, 1986 Nov, 78(5 Pt 1), 877 - 86 Detection of antibodies to proteases used in laundry detergents by the radioallergosorbent test; Dor PJ et al.; Two proteases, Esperase and Alcalase, derived from Bacillus licheniformis and B . subtilis, respectively, are used in laundry products . In testing for the prevalence of IgE antibodies to these enzymes in sera among 300 laundry product workers, we experienced two problems in the establishment of a reliable RAST for these antigens . The first problem was the propensity of the allergen, Esperase, to undergo autolysis, suggesting that solid-phase Esperase might also lose reactivity through degradation . Treatment of Esperase with phenylmethylsulfonyl fluoride stabilized the enzyme and permitted the synthesis of a stable solid-phase antigen . The second problem was the finding that sera reactive with Esperase in the RAST were also reactive with Savinase, an enzyme from B . licheniformis to which the workers were not exposed . Immunochemical analyses of the three enzymes with specific rabbit antisera by gel diffusion and by two-site immunoradiometric assay demonstrated that they were not cross contaminated to any appreciable extent . RAST inhibition demonstrated that solid-phase Esperase possessed unique allergenic determinants in that the reactivity of IgE antibodies was inhibited by low concentrations of Esperase and only by very high concentrations of Alcalase and Savinase . In contrast, the reactivity of solid-phase Alcalase was occasionally inhibited equally well by Esperase and Alcalase . Most strikingly, the reaction of IgE antibodies with solid-phase Savinase was always inhibited by comparable quantities of Esperase, Alcalase, and Savinase . Thus, the establishment of the RAST for these proteases appears to require the use of phenylmethylsulfonyl fluoride to retard autolysis, and the results must be interpreted with caution because IgE antibodies in certain sera demonstrate cross-reactivity with Alcalase and Savinase. Cancer Res, 1986 Nov, 46(11), 5963 - 8 Intratumoral Bacillus Calmette-Guérin immunotherapy prior to surgery for carcinoma of the lung: results of a prospective randomized trial; Matthay RA et al.; A prospective randomized trial of preoperative intratumoral therapy with Bacillus Calmette-Guerin (BCG) was conducted in non-small cell lung cancer patients . Eighty-eight patients (48 BCG-treated and 40 control subjects) were entered into the study; three control subjects were removed from data analysis because histology revealed pathology other than non-small cell lung cancer . There were no differences between BCG-treated and control patients in sex, age, cigarettes smoked per day, pack-years of cigarette smoking, white blood cell count, or number of peripheral blood lymphocytes . Toxicity of BCG was limited to transient malaise and fever (average peak temperature, 38.7 degrees C) . There was no significant difference in outcome (recurrence or survival) between BCG-treated and control groups with Stage I or Stage III tumors; there were too few Stage II tumors for separate statistical analysis . Outcome was not affected within or between the two treatment groups by tuberculin skin test status . Combining both treatment groups, Stage III patients had a worse outcome than did Stage I-II patients, non-squamous cell tumor patients (large cell and adenocarcinoma) had worse outcomes than did squamous cell tumor patients, and men had a worse outcome than women . We conclude that, although preoperative intratumoral BCG therapy is safe, it does not lengthen disease-free interval or prolong survival in patients with non-small cell lung cancer. Arch Fr Pediatr, 1986 Nov, 43(9), 719 - 21 {Post-angina septicemia caused by Fusobacterium necrophorum in a 7-year-old child}; Dufillot D et al.; A fusobacterium necrophorum septicemia due to a neglected peritonsillar abscess is reported in a 7 year-old boy with no significant past medical history . Osteo-articulary, hepatic and pleuro-pulmonary septic localizations, with an otherwise favourable outcome left severe orthopedic sequelae in the right hip . This resembles the post-peritonsillar abscess septicemia described by Lemierre in 1936 which was due to an anaerobic bacillus (fusobacterium) . The reappearance of this pathology should lead to systematic anaerobic blood and abscess studies . Penicillin G and Metronidazole are still efficient in controlling this organism. J Gen Microbiol, 1986 Nov, 132 ( Pt 11), 3047 - 53 Subdivision of daughter strains of bacille Calmette-Guérin (BCG) according to secreted protein patterns; Abou-Zeid C et al.; In order to identify proteins secreted by live organisms, daughter strains of the Bacillus Calmette-Guerin (BCG) were grown for 4-7 d in a defined medium containing {35S}methionine . Secreted components were then separated by polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions, and analysed by autoradiography and in an Ambis beta-scanner . The results indicate that BCG daughter strains can be subdivided into two groups according to their secretion of a 46 kDa protein dimer consisting of two similar 23 kDa subunits . High-producer strains (Japanese, Brazilian and Russian) secrete very large quantities of this material, which constitutes approximately 23% of all secreted protein . These findings correlate with earlier studies in which degradation products of the protein dimer may have been identified, and with the data from patterns of cell wall lipids. Mikrobiologiia, 1986 Nov-Dec, 55(6), 1045 - 7 {Cleavage of DNA from 2 phages of Bacillus thuringiensis by EcoRI and HindIII endonucleases}; Kochkina ZM; The DNA of two Bacillus thuringiensis phages was restricted by endonucleases EcoRI and HindIII and the electrophoretic distribution of the fragments in agarose gel was studied . EcoRI was shown to restrict the DNA of phage 1-97A into 8 fragments and the DNA of phage 1-97B into 12 fragments . Restriction with HindIII results in the formation of 22 and 9 fragments for phage 1-97A and phage 1-97B, respectively . The molecular mass of the DNAs determined by summing up EcoRI restricts is 80.87 MDa for phage 1-97A and 32.45 MDa for phage 1-97B. Am J Vet Res, 1986 Nov, 47(11), 2329 - 36 Detection of latent pseudorabies virus in porcine tissue, using a DNA hybridization dot-blot assay; McFarlane RG et al.; A DNA-hybridization dot-blot technique was developed to detect the presence of pseudorabies virus (PRV) DNA in porcine tissue . Seven 32P-nick translated probes of high specific activity were prepared from transformed Escherichia coli plasmids into which Bacillus amyloliquefaciens H (Bam HI) restriction fragments of PRV-DNA had been inserted . Samples of DNA that had been extracted from porcine tissue or from PRV grown in tissue culture were transferred to nitrocellulose paper, using a microsample filtration manifold and were hybridized to the probes under high-stringency conditions . Under optimal hybridization conditions, the minimum detection amount of PRV-DNA was 10(-11) g, which is equivalent to 1 copy of the PRV genome/80 host cells . Four probes did not show cross hybridization with DNA extracted from tissues of known PRV-negative swine, and these were subsequently used to detect PRV-DNA in infected porcine tissues . Generally, correlation between virus isolation and hybridization data was good for tissues from swine that had died of acute PRV infection . Furthermore, PRV-DNA was present in specific tissues of all 4 seropositive swine that had recovered from pseudorabies and in which no infective virus or viral products were detected at necropsy . Pseudorabies virus DNA was present in the rostralis cerebral cortex (n = 2) or in the medulla oblongata (n = 1) and trigeminal ganglion (n = 1) . This probably indicated the portal of entry of the virus into the CNS . In another seropositive pig, there was evidence of a productive infection in the tonsils, although virus was not isolated in a tissue culture system.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1986 Nov, 168(2), 839 - 42 Expression of alpha-amylase in Bacillus licheniformis; Rothstein DM et al.; In Bacillus licheniformis, alpha-amylase production varied more than 100-fold depending on the presence or absence of a catabolite-repressing carbon source in the growth medium . alpha-Amylase was produced during the growth phase and not at the onset of the stationary phase . Induction of alpha-amylase correlated with synthesis of mRNA initiating at the promoter of the alpha-amylase gene. Appl Environ Microbiol, 1986 Nov, 52(5), 1046 - 54 DNA-damaging activity of patulin in Escherichia coli; Lee KS et al.; At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli . At 50 micrograms/ml, double-strand breaks were observed also . Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source . The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium . When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication . The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis . In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml . Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed . Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity. J Bacteriol, 1986 Nov, 168(2), 1023 - 5 Cloning and nucleotide sequence of the Bacillus megaterium gene coding for small, acid-soluble spore protein B; Hackett RH et al.; The Bacillus megaterium gene coding for small, acid-soluble spore protein (SASP) B was cloned and its nucleotide sequence was determined . The amino acid sequence predicted from the DNA sequence was identical to that determined previously for SASP B, with the exception of the amino-terminal methionine predicted from the gene sequence which is presumably removed posttranslationally and an asparagine residue predicted at position 21 which was originally identified as an aspartate residue . The mRNA encoded by the SASP B gene is synthesized for only a discrete period midway in sporulation, in parallel with mRNAs coding for other SASPs . The small size of the SASP B mRNA (365 nucleotides) indicated that the mRNA is monocistronic . The SASP B gene itself hybridized strongly to only one band in Southern blots of restriction enzyme digests of B . megaterium DNA, suggesting that the SASP B gene is not a member of a highly conserved multigene family, as is the case for other SASP genes. Biochemistry, 1986 Oct 21, 25(21), 6603 - 8 Free energy of hydrolysis of tyrosyl adenylate and its binding to wild-type and engineered mutant tyrosyl-tRNA synthetases; Wells TN et al.; The equilibrium constant for the formation of tyrosyl adenylate and pyrophosphate from ATP and tyrosine in solution has been measured by applying the Haldane relationship to wild-type and three mutant tyrosyl-tRNA synthetases from Bacillus stearothermophilus . The formation constant (={Tyr-AMP} {PPi}/{ATP} {Tyr}) at pH 7.78, 25 degrees C, and 10 mM MgCl2 is (3.5 +/- 0.5) X 10(-7) . This corresponds to a free energy of hydrolysis of tyrosyl adenylate at pH 7.0 and 25 degrees C of -16.7 kcal mol-1 . All necessary rate constants had been determined previously for the calculations apart from the dissociation constant of tyrosyl adenylate from its enzyme-bound complex . This was measured by taking advantage of the 100-fold difference in hydrolysis rates of the tyrosyl adenylate when sequestered by the enzyme and when free in solution . These are technically difficult measurements because the dissociation constants are so low and the complexes unstable . The task was simplified by using mutants prepared by site-directed mutagenesis . These were designed to have different rate and equilibrium constants for dissociation of tyrosyl adenylate from the enzyme-bound complexes . The dissociation constants were in the range (3.5-38) X 10(-12) M, with that for wild type at 13 X 10(-12) M . The four enzymes all gave consistent data for the formation constant of tyrosyl adenylate in solution . This not only improves the reliability of the measurement but also provides confirmation of the reliability of the measured kinetic constants for the series of enzymes. J Mol Biol, 1986 Oct 20, 191(4), 713 - 20 Crystallographic structure of allosterically inhibited phosphofructokinase at 7 A resolution; Evans PR et al.; The structure of the allosterically inhibited form of phosphofructokinase from Bacillus stearothermophilus has been determined by X-ray crystallography to 7 A resolution by molecular replacement using the known structure of the active state as a starting model . Comparing the inhibited state with the active state, the tetramer is twisted about its long axis such that one pair of subunits in the tetramer rotates relative to the other pair by about 8 degrees around one of the molecular dyad axes . This rotation partly closes the binding site for the co-operative substrate fructose-6-phosphate, explaining its weaker binding to this conformational state . Within the subunit, one domain rotates relative to the other by 4.5 degrees, which further closes the fructose-6-phosphate site, without closing the cleft between the domains of the same subunit: this motion causes little change to the catalytic site . This T-state model is consistent with the simple allosteric kinetic scheme in which the active and the inhibited conformations differ in their affinities for fructose-6-phosphate, but not in their catalytic rates . It does not explain the heterotropic allosteric effects. Biochem J, 1986 Oct 15, 239(2), 473 - 6 Metabolism of 17-hydroxyprogesterone by a Bacillus species; Mahato SB et al.; Fermentation of 17-hydroxyprogesterone with a Bacillus species (IICB-301) in a modified nutrient medium under aerobic conditions yielded androst-4-ene-3,17-dione and 15 alpha,17-dihydroxypregn-4-ene-3,20-dione in addition to a new pregnane analogue, 6 beta,17,20 alpha-trihydroxypregn-4-ene-3-one . Each microbial metabolite was characterized by the application of various spectroscopic techniques . The availability of the new metabolite, 6 beta,17,20 alpha-trihydroxypregn-4-ene-3-one, enabled complete elucidation of its 13C-n.m.r . spectrum. Biochem Biophys Res Commun, 1986 Oct 15, 140(1), 114 - 9 Antibodies prepared to Bacillus cereus phospholipase C crossreact with a phosphatidylcholine preferring phospholipase C in mammalian cells; Clark MA et al.; Antibodies against Bacillus cereus phospholipase C were prepared in rabbits and used to affinity purify a phosphatidylcholine-preferring phospholipase C from a human monocytic cell line . Affinity chromatography resulted in an approximately 3000-fold, one-step enrichment of phospholipase C . The human enzyme had an apparent molecular mass of 40,000 daltons as determined by SDS gel electrophoresis . Western blotting analysis demonstrated that this protein interacted specifically with the rabbit antibody raised against bacterial phospholipase C . The purified enzyme preferred phosphatidylcholine as a substrate, was neutral pH active and was inhibited by EGTA . These studies demonstrate that antibodies raised against bacterial phospholipase C may be useful in purifying phospholipase C from a human source. J Biol Chem, 1986 Oct 5, 261(28), 13216 - 23 Analogs of diaminopimelic acid as inhibitors of meso-diaminopimelate decarboxylase from Bacillus sphaericus and wheat germ; Kelland JG et al.; Analogs (1----6) of diaminopimelic acid have been synthesized and tested for inhibition of meso-diaminopimelate decarboxylases from Bacillus sphaericus IFO 3525 and from wheat germ (Triticum vulgaris) . Difluoromethyl diaminopimelate 1 does not irreversibly inactivate or strongly competitively inhibit either enzyme . Lanthionine sulfoxides (2ab, 2c, and 2d) are good competitive inhibitors (about 50% inhibition at 1 mM) of both decarboxylases . The meso and LL-isomers of lanthionine sulfone (3ab and 3c) and lanthionine (6ab and 6c) are weaker competitive inhibitors (about 50% inhibition at 10-20 mM) . The corresponding DD-isomers (3d and 6d) are less effective . The N-modified analogs are the most potent competitive inhibitors . The inhibition constant (Ki) values for B . sphaericus and wheat germ decarboxylases with N-hydroxydiaminopimelate 4 (mixture of isomers) are 0.91 and 0.71 mM, respectively; for the N-aminodiaminopimelate 5 (mixture of isomers) the Ki values are 0.10 and 0.084 mM, respectively . These N-modified analogs do not effectively inhibit L-lysine decarboxylase . None of the compounds showed any time-dependent inactivation of the decarboxylases, in contrast to behavior of other pyridoxal phosphate-dependent enzymes with analogous substrate derivatives . Possible mechanisms of inhibition are discussed . In preliminary tests for antibiotic activity 4 and 5 both gave 75% growth inhibition of Bacillus megaterium at 20 micrograms/ml in defined media . Other analogs (1----3) showed essentially no antibacterial activity. J Biol Chem, 1986 Oct 5, 261(28), 12911 - 4 Interplay between lipids and viral glycoproteins during hemolysis and fusion by influenza virus; Huang RT et al.; Since mixtures of lipids alone are known to elicit membrane fusion without participation of fusion proteins, the role of viral lipids in the so-called virus-induced hemolysis and cell fusion has been investigated, using as a model the fowl plague virus (influenza A/FPV/Rostock/H7N1) . The experiments were planned in a way that allowed quantitative modification of viral lipids without changing envelope glycoproteins . Under the conditions employed, cholesterol oxidase of Nocardia erythropolis and phospholipase C of Bacillus cereus were shown to completely modify their substrates in the virus without altering virus-associated hemagglutinating and neuraminidase activities . It was found with such enzyme treatment that virus-induced hemolysis and cell fusion are greatly influenced by cholesterol and phospholipids of the envelope . It became clear, that hemolysis and fusion are differently dependent on the nature of lipid components even though mediated by the same viral glycoproteins. J Appl Bacteriol, 1986 Oct, 61(4), 275 - 85 Factors contributing to the seasonal variation of Bacillus spp . in pasteurized dairy products; Phillips JD et al.; The seasonal variation in the spoilage of pasteurized products, especially double cream, by spore-forming bacteria was due to a number of factors . By far the most important was the seasonal variation in the types of organisms isolated from raw milks . Psychrotrophic spore-formers predominated in the summer-autumn months and these strains were able to germinate rapidly and grow in refrigerated dairy products . There was evidence that the concentration of one or more factors which promoted germination of psychrotrophic strains of Bacillus spp . in milk was higher during the summer than in the winter . This again may contribute to seasonal differences in spoilage by spore-forming bacteria . Post-heat treatment contamination by spores of Bacillus spp . may also be more prevalent in the summer-autumn period and evidence was obtained that spores associated with post-pasteurization contamination could germinate and grow more rapidly than those introduced into the product from the raw material . Thus, the increased spoilage of pasteurized products by Bacillus spp . observed in the June to October period may be due to a combination of factors . The relative contribution that each makes is not easily resolved. Appl Environ Microbiol, 1986 Oct, 52(4), 758 - 64 Sporulation-associated activation of Bacillus sphaericus larvicide; Broadwell AH et al.; Preparations of the larvicidal crystal from 46-h cultures of Bacillus sphaericus 2362 contain 125-, 110-, 63-, and 43-kilodalton (kDa) proteins (P . Baumann, B . M . Unterman, L . Baumann, A.H . Broadwell, S.J . Abbene, and R.D . Bowditch, J . Bacteriol . 163:738-747, 1985) . The 63- and 43-kDa proteins, which have been purified, are not immunologically cross-reactive, and only the 43-kDa protein is toxic to mosquito larvae . Since antigenic determinants of the two smaller proteins have been detected in the higher-molecular-weight proteins (125 and 110 kDa), it has been suggested that the latter are precursors of the 43- and 63-kDa peptides . In the present study, purified 110-kDa protein was found to be toxic to the larvae of Culex pipiens (50% lethal concentration = 115 ng/ml) . A luciferase-luciferin assay for intracellular ATP as well as an assay based on the exclusion of Trypan Blue by live cells indicated that the 110-kDa protein had no effect on tissue-culture-grown cells of C . quinquefasciatus, while cells exposed to the 43-kDa protein rapidly lost viability (50% lethal concentration = 54 microgram(s)/ml by the intracellular ATP assay) . These findings suggested that the 110-kDa protein and, by extension, the 125-kDa protein are protoxins which are activated during sporulation by cleavage to a 43-kDa toxin . To further investigate the origins and relationships of the crystal proteins of B . sphaericus, we analyzed samples during the growth and sporulation of the culture . Synthesis of crystal proteins was initiated at the end of exponential growth and was completed after about 7 h.(ABSTRACT TRUNCATED AT 250 WORDS) Appl Environ Microbiol, 1986 Oct, 52(4), 650 - 3 Analysis of mosquito larvicidal potential exhibited by vegetative cells of Bacillus thuringiensis subsp . israelensis; Walther CJ et al.; Vegetative Bacillus thuringiensis subsp . israelensis cells (6 X 10(5)/ml) achieved 100% mortality of Aedes aegypti larvae within 24 h . This larvicidal potential was localized within the cells; the cell-free supernatants did not kill mosquito larvae . However, they did contain a heat-labile hemolysin which was immunologically distinct from the general cytolytic (hemolytic) factor released during solubilization of B . thuringiensis subsp . israelensis crystals . The larvicidal potential of the vegetative cells was not due to poly-beta-hydroxybutyrate . Instead, it correlated with the ability of vegetative cells to sporulate during the bioassays . No toxicity was observed when bioassays were conducted in the presence of chloramphenicol or streptomycin . It is unlikely that the vegetative cells sporulate in the alkaline (pH 9.5 to 10.5) larval guts after ingestion . B . thuringiensis subsp . israelensis is not an alkalophile; we have been unable to grow it in culture at pH values of greater than or equal to 9.5 . Moreover, we have been unable to demonstrate formation of a protective capsule . However, bacteria may replicate in the gut fluids of dead or dying mosquito larvae because their alkaline gut pH values drop markedly after exposure to the B . thuringiensis subsp . israelensis crystal toxins. Am J Pathol, 1986 Oct, 125(1), 16 - 27 Long-term evolution of BCG- and CFA-induced granulomas in rat lungs . Correlation of histologic features with cells in bronchoalveolar lavage samples; Chang JC et al.; Granulomatous inflammation was induced in the lungs of rats for assessment of the suitability of this animal species for long-term study of granuloma development and resolution and for comparison of the histologic changes with the cellular profile in bronchoalveolar lavage (BAL) samples . It was found that after a single intravenous injection of bacillus Calmette-Guerin (BCG) organisms suspended in saline-0.01% Triton, complete Freund's adjuvant (CFA), or BCG suspended in CFA (BCG + CFA), distinct pathologic patterns developed in the lungs during the acute stages . BCG alone caused numerous small epithelioid granulomas associated with interstitial infiltration; CFA alone caused large discrete granulomas with minimal interstitial changes, and BCG + CFA caused large granulomas associated with diffuse interstitial infiltration . Following CFA or BCG + CFA the maximum number and size of granulomas were reached approximately 4 weeks after injection . By 8 weeks collagen was seen in the larger granulomas in sections stained with hematoxylin and eosin from animals given CFA or BCG + CFA . Maximum collagen deposition was seen at 16 weeks . From that point onward the degree of collagen deposition decreased, so that by approximately 42 weeks collagen was no longer seen . At 52 weeks residual granulomatous lesions consisted of a few foci of foamy macrophages surrounded by several layers of lymphocytes or epithelioid cells surrounded by dense layers of lymphocytes . Bronchoalveolar lavage samples revealed that at all time periods the number of leukocytes was increased and that the increase was due primarily to an influx of macrophages and lymphocytes . The increase in number of lymphocytes was so striking that at the peak of granulomatous changes following injection of BCG + CFA up to 50% of the total cells were found to be lymphocytes . The total number of leukocytes in BAL samples and the absolute number of lymphocytes closely paralleled the intensity of histologic changes seen in microscopic sections . It is concluded that the intravenous injection of BCG, CFA, or BCG + CFA in rats causes distinct and profound granulomatous patterns that are associated with increased cellularity in BAL samples . The findings suggest that the rat may be an excellent model for study of the mechanisms of granuloma development and resolution. Lab Anim Sci, 1986 Oct, 36(5), 504 - 6 Naturally occurring Bacillus piliformis infection (Tyzzer's disease) in guinea pigs; Waggie KS et al.; Two juvenile male Hartley guinea pigs (Cavia porcellus) were found dead 36 hours after receipt from a commercial source . Both guinea pigs had dependent, subcutaneous edema and excess serous fluid in their thoracic and abdominal cavities . Their livers were mottled and the cecal walls were reddened focally . Histopathologic exam revealed nests of Bacillus piliformis within the absorptive epithelial cells of the ileum, cecum and colon . Vegetative organisms and spore-like structures were observed in the cytoplasm of intestinal epithelial cells by electron microscopy . A diagnosis of Tyzzer's disease was made. Lab Anim Sci, 1986 Oct, 36(5), 492 - 5 Experimentally induced Tyzzer's disease in the African white-tailed rat (Mystromys albicaudatus); Waggie KS et al.; Tyzzer's disease was induced experimentally in nonimmunosuppressed, weanling Mystromys albicaudatus by oral inoculation with Bacillus piliformis spores . Focal areas of necrosis bordered by cells containing B . piliformis were observed first in the tunica muscularis of the intestine and in the periportal region of the liver 3 days post-inoculation, in the ventricular myocardium 7 days post-inoculation and in the brainstem 9 days post-inoculation . Healing in the tunica muscularis, liver and myocardium was accompanied by granuloma formation . The findings indicate that Mystromys are susceptible to B . piliformis infection . This is, to our knowledge, the first time brain lesions have been reported in any species following oral inoculation with B . piliformis . Tyzzer's disease should be considered as a possible diagnosis in Mystromys with hepatoenteritis, myocarditis, or indications of central nervous system disorders. J Bacteriol, 1986 Oct, 168(1), 303 - 8 Three-dimensional structure of the T-layer of Bacillus sphaericus P-1; Lepault J et al.; The three-dimensional structure of the regular surface layer of Bacillus sphaericus P-1 (T-layer) was determined to a resolution of ca . 2.5 nm by electron microscopy and image analysis . The T-layer has P4 symmetry, a lattice constant of 13 +/- 0.2 nm, and a thickness of ca . 8 nm . The reconstruction revealed three distinct domains: a major, a minor, and an arm domain . In the z-direction, the domains are arranged in two planes creating two different surface reliefs. Infect Immun, 1986 Oct, 54(1), 228 - 32 Characterization of monoclonal antibodies to a crystal protein of Bacillus thuringiensis subsp . kurstaki; Huber-Lukac M et al.; Ten monoclonal antibodies were produced against a k-1-type crystal protein of Bacillus thuringiensis subsp . kurstaki . Eight of the antibodies belong to the immunoglobulin G1 (IgG1) subclass, with pI values ranging from 5.5 to 8.6, one could be assigned to the IgG2b subclass, and one could be assigned to the IgM class . Competitive antibody-binding assays and analysis of antibody specificity indicated that the 10 antibodies recognized at least nine distinct antigenic determinants . Eight antibodies bound to both protoxin and toxin, whereas the other two interacted with protoxin only . One antibody completely inhibited the biological activity of the delta-endotoxin, five antibodies reduced it by 15 to 82%, and four antibodies did not affect it at all . Based on cross-reaction studies, homologies and differences in the crystal protein structures of different B . thuringiensis subspecies were revealed . All of the monoclonal antibodies strongly cross-reacted with crystal proteins from strains of B . thuringiensis subsp . tolworthi, B . thuringiensis subsp . galleriae, B . thuringiensis subsp . dendrolimus, B . thuringiensis subsp . sotto, and B . thuringiensis subsp . subtoxicus . Some antibodies interacted only weakly with crystal proteins from strains of B . thuringiensis subsp . morrisoni and B . thuringiensis subsp . entomocidus, and some of these did not interact with B . thuringiensis subsp . kenyae and B . thuringiensis subsp . darmstadiensis . No cross-reaction was found with the parasporal inclusion protein of B . thuringiensis subsp . israelensis . Studies with the monoclonal antibodies also disclosed that crystal proteins from strains of the same subspecies can exhibit substantial differences in antigenic structure . In particular, striking strain-specific differences in the protoxins of B . thuringiensis subsp . kurstaki and B . thuringiensis subsp . thuringiensis were observed. Exp Parasitol, 1986 Oct, 62(2), 247 - 53 Trichostrongylus colubriformis: isolation and characterization of ovicidal activity from Bacillus thuringiensis israelensis; Bone LW et al.; Bioassay of media fractions from cultivation of Bacillus thuringiensis israelensis revealed that ovicidal activity for eggs of the ruminant nematode Trichostrongylus colubriformis was found in microbial crystals, but was not released into culture medium . The purified delta-endotoxin of B . t . israelensis, composed of two 25 kDa proteins, had no effect on nematode eggs . A fraction that had high ovicidal activity for eggs of T . colubriformis was isolated by high performance liquid chromatography from crystals of B . t . israelensis . Retention of the compound(s) on size exclusion columns indicated a mol wt of 1510 when compared to standards . The LD50 of this fraction for nematode eggs was not altered significantly by 5 mM calcium chloride with and without ethylenediaminetetraacetic acid or the enzyme inhibitor phenylmethylsulfonyl fluoride (10(-6) M) . The fraction was susceptible to proteolytic hydrolysis by bacterial protease VI whereas the fungal protease XIII slightly decreased the ovicidal effects of the fraction . The ovicidal activity was stable for 2 days at ambient temperature or 2 months at 0 C, but declined after 7 days at ambient temperature . Little activity was lost after heating to 100 C for 60 min . The ovicidal effects were also pH dependent with increased toxicity at alkaline pH values . The fraction, however, had no effect on larvae of the mosquito Aedes aegypti or mice after intraperitoneal injection. Clin Immunol Immunopathol, 1986 Oct, 41(1), 75 - 90 Leukocyte migration inhibition in bacillus Calmette-Guérin vaccinated healthy adults and in tuberculosis assayed by a rapid photoelectric procedure; Gauthier-Rahman S et al.; A sensitive photoelectric method was used for reading migrations of human peripheral blood leukocytes from agarose microdroplets . Its rapidity enabled the use of a wide range (12 logs) of in vitro concentrations of antigen . Inhibition of migration of leukocytes (LMI) of healthy subjects having had BCG vaccination in childhood was found to occur in two zones of PPD concentrations, one high, from 10(-1) to 1000 micrograms/ml with a peak at 100 micrograms/ml, and one low, from 10(-8) to 10(-2) micrograms/ml . While three-fifths of subjects showed high zone LMI, in one-fifth it was bizonal and in another fifth observed only in the low zone . In patients with active pulmonary tuberculosis LMI, prior to all treatment, was reduced, absent, or replaced by enhanced migration, particularly in the low zone or in both zones . One and a half to four months after treatment LMI was found to be bizonal, enhanced migration having disappeared . These observations suggest the participation of two cell populations with widely different sensitivity to PPD in LMI in tuberculous and BCG vaccinated subjects and the presence of migration stimulatory lymphokine(s) during active tuberculosis. J Antimicrob Chemother, 1986 Oct, 18 Suppl C, 51 - 6 Genetic and biochemical basis of tetracycline resistance; Chopra I; Properties of several, well characterized, tetracycline resistance determinants were compared . The determinants in Tn1721 and Tn10 (both from Gram-negative bacteria) each contain two genes; one encodes a repressor that regulates both its own transcription and that of a membrane protein that confers resistance by promoting efflux of the drug . Determinants from Gram-positive bacteria also encode efflux proteins, but expression of resistance is probably regulated by translational attenuation . The likely tetracycline binding site (a common dipeptide) in each efflux protein was predicted . The presence of the common binding site is consistent with the ability of an efflux protein originating in Bacillus species to be expressed in Escherichia coli. Antibiot Med Biotekhnol, 1986 Oct, 31(10), 743 - 8 {Comparative analysis of 2 Bacillus polymyxa strains differing in the spectrum of exogenous polysaccharides produced}; Glukhova EV et al.; The characteristic features of the development of 2 Bacillus polymyxa strains differing in the spectrum of the produced extracellular heteropolysaccharides were compared . The strains were grown on solid media and under submerged conditions . Electron microscopy revealed marked differences in the ultrastructure of the developing cultures . It was noted that the mutant variant B formed a vigorous fibrillar and capsular layer of the polysaccharide nature strictly oriented in the space perpendicularly to the cell surface . Changes in the mutant growth processes were characterized by a lower growth rate of the cell population . In the process of growth the initial culture did not form structured capsules and was lysogenic . Pronounced differences in the proteolytic activity were indicative of a definite interrelation between sporulation of the strains and activity of their exoproteases . The regularities of the development of the mutant strain of B . polymyxa allowed one to define the fundamental approaches to development of optimal cultivation conditions for the organism producing extracellular polysaccharides. Appl Environ Microbiol, 1986 Oct, 52(4), 644 - 9 Immunological relationships among proteins making up the Bacillus thuringiensis subsp . israelensis crystalline toxin; Pfannenstiel MA et al.; The immunological relationships among the proteins of the mosquito larvicidal toxin produced by Bacillus thuringiensis subsp . israelensis have been investigated by using polyclonal antisera specific for the 28-, 70-, and 135-kilodalton proteins . Each of these proteins was immunologically distinct . There was no cross-reaction among the three proteins and the two non-homologous antisera . Treatment of toxin proteins with larval gut enzymes for 20 h identified protease-resistant domains at approximately 65, 38, and 22 kilodaltons . Similar domains were generated by treatment with trypsin and chymotrypsin . Our immunological and kinetic data indicate that the 28-kilodalton protein is degraded successively to protein bands at 26, 25, 23, and 22 kilodaltons, the 70-kilodalton protein is degraded to a protein at 38 kilodaltons, and the 135-kilodalton protein is degraded successively to protein bands at 94, 72, and, probably, 65 kilodaltons . Solubilized toxin possesses two biological activities, larvicidal and general cytolytic (hemolytic) . We used nondenaturing gel electrophoresis to show that the hemolytic activity resides in the 28-kilodalton protein . However, higher-molecular-weight proteins are required to achieve the level of toxicity observed in intact toxin. Clin Immunol Immunopathol, 1986 Oct, 41(1), 108 - 15 Inhibition of murine bladder tumor growth by bacille Calmette-Guerin: lack of a role of natural killer cells; Ratliff TL et al.; Intravesical instillation of bacille Calmette Guerin (BCG) currently is considered the most effective treatment for recurrent superficial bladder cancer, but little is known about the mechanism of action . We have adapted a model in which the mouse bladder tumor, MBT-2, is implanted directly into the bladder to examine the mechanism by which BCG inhibits tumor growth . The intravesical administration of BCG inhibited MBT-2 implantation in a dose-dependent manner . Concomitantly, natural killer (NK) activity was augmented in a dose-dependent manner . Conversely, BCG doses which did not augment NK activity lacked antitumor activity . Linear regression analysis showed a significant correlation between the antitumor activity of BCG and modulation of NK activity (correlation coefficient, 0.991) . Additional studies were performed in which NK activity was abrogated by administration of anti-asialo-GM1 serum . NK activity was depressed in spleens and lymph nodes of both normal and BCG-treated mice . Abrogation of NK activity did not alter the efficacy of BCG therapy suggesting that NK cells are not a major contributor to the antitumor activity of BCG. J Ultrastruct Mol Struct Res, 1986 Oct-Dec, 97(1-3), 73 - 88 Characterization of the ultrastructure and the self-assembly of the surface layer of Bacillus stearothermophilus strain NRS 2004/3a; Messner P et al.; The ultrastructure of the crystalline surface layer (S-layer) of Bacillus stearothermophilus strain NRS 2004/3a has been characterized by electron microscopy supplemented by optical and computer image analysis . The S-layer, composed of glycoprotein subunits, has oblique symmetry, and can be extracted by guanidine hydrochloride . Upon dialysis, this extract produced both flat and cylindrical mono- and double-layer self-assembly products . Optical diffraction analysis of negatively stained preparations showed five types of double-layered assembly products . Computer filtering separated the double-layer complexes and revealed them to be composed of a common monolayer with p2-symmetry (a = 9.4 nm, b = 11.6 nm, and gamma = ca . 78 degrees) . By analysis of freeze-dried and heavy metal-shadowed self-assemblies the surface topography and the characteristic "handedness" of the morphological units have been determined . Labeling with polycationic ferritin has shown that each surface of the S-layer possessed a different net charge . The results indicate that S-layers in vivo could prevent autoagglutination of cells. Can J Microbiol, 1986 Oct, 32(10), 822 - 5 Biosynthetic studies on N-acetylmannosaminuronic acid containing teichuronic acid in Bacillus megaterium; Arakawa H et al.; The particulate enzymes obtained from four strains of Bacillus megaterium AHU 1240, AHU 1373, AHU 1375, and T catalyzed the synthesis of a polysaccharide and glycolipids from UDP-N-acetylmannosaminuronic acid, UDP-N-acetylglucosamine, and UDP-glucose . Chemical studies involving Smith degradation, acid hydrolysis, and N-acetylation revealed that the polysaccharide product has a backbone made up of trisaccharide repeating units comprising glucose, N-acetylmannosaminuronic acid, and N-acetylglucosamine and that the main oligosaccharide moieties of the glycolipids were identical with N-acetylmannosaminuronosyl-N-acetylglucosamine and glucosyl-N-acetylmannosaminuronosyl-N-acetylglucosamine . Incubation of the disaccharide-linked lipid with each particulate enzyme in the presence of UDP-glucose produced the trisaccharide-linked lipid and a polysaccharide . It is therefore suggested that in this polysaccharide-synthesizing system the repeating unit is formed on a carrier lipid from appropriate nucleotide derivatives first and the polymerization of the units then occurs to synthesize the backbone while the growing chain remains in pyrophosphate linkage to the carrier lipid presumed to be undecaprenol. J Appl Bacteriol, 1986 Oct, 61(4), 347 - 56 Numerical taxonomy of Bacillus isolated from orally administered drugs; Gil MC et al.; Numerical taxonomy procedures were used to study 118 strains of Bacillus isolated from non-sterile drugs prepared for oral administration . Similarities between pairs of strains were calculated by the simple matching coefficient of Sokal and Michener (SSM) . Each strain was tested for 60 unit characters and three clusters were defined . The strains in each cluster presented a similarity level of at least 60% . Cluster A comprised the strains identified as Bacillus cereus (SSM = 93.13%), cluster B contained three subgroups corresponding to the species B . pumilus, B . subtilis and B . licheniformis (SSM = 84.35%) and cluster C also included three subgroups that belonged to the species B . firmus, B . lentus and B . badius (SSM = 80.14%) . The most discriminating tests were selected to differentiate the clusters from the subgroups . The feature with the highest discriminating power between clusters A and B was the lack of acid production from arabinose and mannitol . The Voges-Proskauer, methyl red tests and sensitivity to polymyxin B clearly distinguished cluster A from C . The Voges-Proskauer test and acid production from arabinose were the best to differentiate between B and C . Bacillus pumilus and B . subtilis differed in starch hydrolysis and B . licheniformis in growing anaerobically . To discriminate B . firmus from B . lentus the most important tests were the acid production from glucose and sucrose; intermediate strains were found . Bacillus badius was differentiated from B . firmus by 10 tests, and from B . lentus by the production of urease. J Appl Bacteriol, 1986 Oct, 61(4), 295 - 8 Immunodiffusion analysis shows that Mycobacterium paratuberculosis and other mycobactin-dependent mycobacteria are variants of Mycobacterium avium; McIntyre G et al.; Antigenic analysis by immunodiffusion has been applied to 74 strains of mycobactin-dependent mycobacteria . Thirty-eight strains were of Mycobacterium paratuberculosis from cases of Johne's disease of cattle or goats . The remaining cultures were obtained from a variety of animals and included the wood pigeon bacillus . Rabbit antisera were raised to some of the strains and these, together with antisera to M . avium and M . intracellulare, were used to examine sonicate preparations of all the cultures . All were found to be antigenically identical with M . avium and none were found to belong to M . intracellulare . A predominance of the cultures from Johne's disease belonged to the potential brunense subspecies of M . avium, and the remainder together with the majority of the other mycobactin-dependent strains belonged to the type subspecies . In view of these findings the separate species status of M . paratuberculosis is refuted and some difficulty remains in the nomenclature of strains giving rise to Johne's disease. J Bacteriol, 1986 Oct, 168(1), 334 - 40 Membrane lipid composition of obligately and facultatively alkalophilic strains of Bacillus spp; Clejan S et al.; The membrane lipids from two obligately and two facultatively alkalophilic strains of Bacillus spp . were characterized in a comparative study that included B . subtilis . Preparations of membrane lipids were made from pH 10.5-grown cells of all of the alkalophiles and from pH 7.5- or 7.0-grown cells of the two facultative strains and B . subtilis . The two obligate alkalophiles contained high ratios of membrane lipid to membrane protein, and the lipid fraction contained a high proportion of neutral lipid . These characteristics are probably not prerequisites for growth at very high pH since one or another of the facultative strains failed to show these properties at high pH . All of the alkalophiles contained appreciable amounts of squalene and C40 isoprenoids . Among the polar lipids, the alkalophiles all contained high concentrations of anionic phospholipids, including phosphatidylglycerol and especially large amounts of cardiolipin; phosphatidylethanolamine was the other major phospholipid . Small amounts of bis(monoacylglycero)phosphate were found in most, but not all, of the alkalophile preparations . Glycolipids and phosphoglycolipids were absent . The fatty acid composition of the total phospholipid and individual fractions revealed two features that distinguished between the obligate and facultative strains . Membranes from the obligately alkalophilic species contained a high concentration of branched-chain fatty acids, comparable to that in membranes from B . subtilis, as well as a relatively high content of unsaturated fatty acids . By contrast, the facultatively alkalophilic strains contained almost no unsaturated fatty acids and a lower concentration of branched-chain fatty acids than either the obligate alkalophiles or B . subtilis. J Gen Microbiol, 1986 Oct, 132 ( Pt 10), 2899 - 905 Methylation of spore DNA in Bacillus coagulans strain 26; Bueno A et al.; The modification status of DNA throughout the life cycle of Bacillus coagulans strain 26 was analysed by restriction analysis with methylation-sensitive enzymes . A significant fraction of the GATC sequences (dam target) in spore DNA contain N6-methyladenine, a modification that is lacking during the vegetative phase . From the modulation of the modification pattern of GATC sites, the existence of a de novo methylase may be inferred . Spore DNA was more sensitive than vegetative cell DNA to BamHI, HpaI, SalI and XhoI, indicating that the sites for these enzymes are modified during the vegetative growth phase. Arch Microbiol, 1986 Oct, 146(1), 7 - 11 Binding and activity of Bacillus thuringiensis delta-endotoxin to invertebrate cells; Hofmann C et al.; Fluorescein isothiocyanate was used as a label to detect delta-endotoxin of Bacillus thuringiensis subsp . thuringiensis and israelensis in binding studies with different in vitro cell systems . Protoxin of the subspecies thuringiensis could be labelled directly whereas the activated toxin had to be traced indirectly with labelled antibodies . Both protoxin and activated toxin bound to primary midgut cell cultures of Pieris brassicae larvae as well as to cells of an established culture of Drosophila melanogaster . No binding with either toxin form could be observed with hemocytes of P . brassicae . Biological activity as shown by the trypan blue viability assay was obtained only with the activated toxin against the midgut cells . Toxin of the subspecies israelensis reacted very unspecifically . Binding followed by rapid destruction was obtained with all the tested cultures. Immunobiology, 1986 Oct, 173(1), 12 - 22 Parallelism between superoxide production of peritoneal exudate cells and lung granulomatous response in mice vaccinated with BCG cell walls; Kimura T et al.; Since peritoneal macrophages are reported to be different from alveolar macrophages in their activated states, we examined whether O-2 production, one of the parameters of macrophage activation, in mouse peritoneal exudate cells (PEC) is enhanced under the condition in which lung granuloma, the accumulation of activated macrophages, is produced with Bacillus Calmette-Guerin (BCG) cell wall (CW) . As a result, we observed the enhanced O-2 production of PEC that occurs in parallel with lung granuloma formation; high responders, C56BL/6 mice, showed high O-2 production of PEC whereas low responders, C3H/He and DBA/1 mice showed low O-2 production of PEC, suggesting that enhanced O-2 production of PEC as well as lung granuloma formation is genetically controlled . Results from T cell-depleted mice and allogeneic bone marrow chimeric mice also showed the occurrence of this parallelism . From these findings, we presumed that circulating macrophage activating factor and other lymphokines produced by BCG CW-sensitized T cells may activate both peritoneal macrophages and lung macrophages. Mol Gen Genet, 1986 Oct, 205(1), 97 - 102 Molecular cloning and sequence of the Bacillus stearothermophilus translational initiation factor IF2 gene; Brombach M et al.; The structural gene for the Bacillus stearothermophilus initiation factor IF2 was localized to a 6 kb HindIII restriction fragment by cross-hybridization with the SstI-SmaI fragment of the Escherichia coli infB gene . This fragment corresponds to the central region of the molecule containing the GTP-binding domain which is homologous in E . coli IF2, EF-Tu, EF-G and the human ras1 oncogene protein . After cloning into pACYC177, the HindIII fragment was further analysed by restriction mapping and cross-hybridization . A smaller (2.2 kb) SphI-HindIII fragment, which showed cross-hybridization, was subcloned into M13 phage and sequenced by the dideoxy chain-terminating method . This fragment was found to contain the entire IF2 gene except for the region coding for the N-terminus . This remaining region, coding for 45 amino acids, was located by homologous hybridization on an overlapping ClaI-SstI fragment which was also subcloned and sequenced . Overall, the B . stearothermophilus IF2 gene codes for a protein of 742 amino acids (Mr = 82,043) whose primary sequence displays extensive homology with the C-terminal two-thirds (but little or no homology with the N-terminal one-third) of the corresponding E . coli IF2 molecule . When cloned into an expression vector under the control of the lambda PL promoter, the B . stearothermophilus IF2 gene, reconstituted by ligation of the two separately cloned pieces, could be expressed at high levels in E . coli cells. Surgery, 1986 Oct, 100(4), 621 - 8 Modified stage I (T1N0M0, T2N0M0), nonsmall cell lung cancer: treatment results, recurrence patterns, and adjuvant immunotherapy; Little AG et al.; We analyzed 96 patients who had surgery with T1N0M0 or T2N0M0 nonsmall cell lung cancer (NSCLC) to identify survival rates and recurrence patterns in well-staged patients and to evaluate adjuvant therapy . Preoperative staging included chest x-ray, gallium 67 scanning, and bronchoscopy in all patients . At thoracotomy, multiple mediastinal lymph node sites were routinely sampled . The results included an operative mortality rate of 5.2%, and the actuarial 5-year survival rate of all patients was 70.0% . Survival of T1N0 (n = 44) and T2N0 (n = 47) patients was 72.1% and 68.3%, respectively (p = NS) . Survival was not affected by type of surgery, cell type, sex, age, or race . Late death was due to recurrence in 12 patients, a new airway malignancy in three, and a noncancer problem in six . Disease recurred in 15 patients: four (9.1%) T1N0 patients versus 11 (23.4%) T2N0 patients, p less than 0.05 . Recurrence was local in four patients and distant in 11 . Second lung cancers developed in six patients at a mean interval of 65.7 months after resection . A prospective, randomized trial of systemic immunotherapy with bacillus Calmette-Guerin (BCG) skin scarification was carried out in 29 patients . Survival in those patients receiving BCG was 85.9% compared with 63.9% for control subjects (p = 0.075) and 69.6% for patients not in the study (p = 0.077) . The following conclusions can be made: Resection for well-staged, modified stage I NSCLC results in a 5-year survival rate of 70% . Nearly half the deaths are unrelated to recurrence of the original cancer . Recurrences are more frequent in T2N0 patients, but there is no survival difference compared with T1N0 patients . Systemic recurrences are more frequent than local recurrences, and there is an appreciable incidence of second lung cancers . Adjuvant chemotherapy or radiation therapy does not seem justified, but systemic immunotherapy holds sufficient promise to warrant further investigation. Neurochem Res, 1986 Oct, 11(10), 1447 - 62 Phospholipid requirement of Ca2+-stimulated, Mg2+-dependent ATP hydrolysis in rat brain synaptic membranes; Gandhi CR et al.; The phospholipid requirement for Ca2+-stimulated, Mg2+-dependent ATP hydrolysis (Ca2+/Mg2+-ATPase) and Mg2+-stimulated ATP hydrolysis (Mg2+-ATPase) in rat brain synaptosomal membranes was studied employing partial delipidation of the membranes with phospholipase A2 (Hog pancreas), phospholipase C (Bacillus cereus) and phospholipase D (cabbage) . Treatment with phospholipase A2 caused an increase in the activities of both Ca2+/Mg2+-ATPase and Mg2+-ATPase whereas with phospholipase C treatment both the enzyme activities were inhibited . Phospholipase D treatment had no effect on Ca2+/Mg2+-ATPase but Mg2+-ATPase activity was inhibited . Inhibition of Mg2+-ATPase activity after phospholipase C treatment was relieved with the addition of phosphatidylinositol-4,5-bisphosphate (PIP2) and to a lesser extent with phosphatidylinositol-4-phosphate (PIP) and phosphatidylcholine (PC) . Phosphatidylserine (PS), phosphatidic acid (PA), PIP and PIP2 brought about the reactivation of Ca2+/Mg2+-ATPase . Phosphatidylinositol (PI) and PA inhibited Mg2+-ATPase activity . Kms for Ca2+ (0.47 microM) and Mg2+ (60 microM) of the enzyme were found to be unaffected after treatment with the phospholipases. J Biol Chem, 1986 Sep 25, 261(27), 12828 - 33 DNA binding by the bacteriophage SPO1-encoded type II DNA-binding protein, transcription factor 1 . Site-specific binding requires 5-hydroxymethyluracil-containing DNA; Greene JR et al.; The bacteriophage SPO1-encoded Type II DNA-binding protein, transcription factor 1 (TF1), forms complexes with specific sites in SPO1 DNA . We have investigated the binding of TF1 to one of its preferred sites in which the normal 5-hydroxymethyluracil (hmUra) of SPO1 DNA has been replaced by thymine and have also investigated the binding of a bacterial Type II DNA-binding protein (from Bacillus stearothermophilus) to the hmUra- and thymine-containing forms of the same DNA segment . Our results show that TF1 binds selectively to this high affinity binding site only in hmUra-containing DNA and that the bacterial Type II DNA-binding protein interacts nonspecifically with both forms of DNA. J Biol Chem, 1986 Sep 25, 261(27), 12896 - 902 The membrane ATPase of alkalophilic Bacillus firmus RAB is an F1-type ATPase; Hicks DB et al.; At the optimal pH for growth (pH 10.5), alkalophilic Bacillus firmus RAB, an obligate aerobe, exhibits normal rates of oxidative phosphorylation despite the low transmembrane proton electrochemical gradient, about -60 mV (delta psi = -180 mV and delta pH = +120 mV) . This bioenergetic problem might be resolved by use of an Na+ coupled ATP synthase; otherwise an F1F0-ATPase must be able to utilize low driving forces in this organism . The ATPase activity was extracted from everted membrane vesicles by low ionic strength treatment and purified to homogeneity by hydrophobic interaction chromatography and sucrose density gradient centrifugation . The ATPase preparation had the characteristic F1-ATPase subunit structure, with Mr values of 51,500 (alpha), 48,900 (beta), 34,400 (gamma), 23,300 (delta), and 14,500 (epsilon); the identity of the alpha and beta subunits was confirmed by immunoblotting with anti-beta of Escherichia coli and anti-B . firmus RAB F1 . Methanol and octyl glucoside, agents that stimulated the low basal membrane ATPase activity 10- to 12-fold, dramatically elevated the MgATPase activity of the purified F1, more than 150-fold, to 50 mumol min-1 mg protein-1 . Anti-F1 inhibited membrane ATPase activity greater than or equal to 80% . The membranes exhibited no Na+-stimulated or vanadate-sensitive ATPase activity when prepared in the absence or presence of Na+ or ATP . These findings, which are consistent with previous studies, establish that in alkalophilic bacteria, ATP hydrolysis, and presumably ATP synthesis is catalyzed by an F1F0-ATPase rather than a Na+ ATPase. J Mol Biol, 1986 Sep 5, 191(1), 1 - 11 Bacillus thuringiensis var . israelensis delta-endotoxin . Nucleotide sequence and characterization of the transcripts in Bacillus thuringiensis and Escherichia coli; Ward ES et al.; The nucleotide sequence of a 1408 base-pair DNA fragment encoding the insecticidal 27,340 Mr delta-endotoxin of Bacillus thuringiensis var . israelensis has been determined by analysis of a recombinant plasmid from Escherichia coli . The hydropathy plot of the protein shows it to be highly hydrophobic, consistent with a postulated cytolytic mechanism of action for the toxin . In addition, the delta-endotoxin transcriptional start points that are used in B . thuringiensis and an E . coli recombinant have been determined . In B . thuringiensis var . israelensis, transcription initiates from a single start point, and gene-specific transcripts are not observed before stage II of sporulation . This is the stage at which delta-endotoxin antigen is first detected, indicating that control of expression is primarily at the transcriptional level for this protein . Analysis of gene-specific transcription in E . coli indicates that at least three start points are utilized in this organism . Interestingly, the highest level of delta-endotoxin mRNA is seen during mid-exponential growth of E . coli and the level appears to decrease as the cells enter the stationary phase of growth.
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