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