Scand J Immunol, 1999 May, 49(5), 515 - 22 Identification of a novel protein antigen encoded by a Mycobacterium tuberculosis-specific RD1 region gene; Ahmad S et al.; A genomic DNA region, designated RD1, that is present in virulent and clinical strains of Mycobacterium tuberculosis and M . bovis, has been shown to be deleted in bacillus Calmette Guerin (BCG) . The DNA segments corresponding to three open reading frames (ORFs: ORF-10, ORF-14 and ORF-15) of the RD1 region, that are deleted in BCG strains, were amplified from M . tuberculosis genomic DNA by polymerase chain reaction (PCR), subcloned into pGEX-4T vector system and expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST) . The recombinant proteins appeared as major cellular proteins in SDS-PAGE gels at the expected molecular mass . The identity of each fusion protein was confirmed by reactivity with anti-GST antibodies in Western immunoblots . When pooled human sera from 11 tuberculosis (TB) patients were used as the source of antibodies, only GST-ORF-14 fusion protein reacted in Western immunoblots . The protein corresponding to ORF-14 was then purified to near homogeneity and isolated free of its fusion partner (GST) by treating the purified GST-ORF-14 fusion protein with thrombin protease . In Western immunoblots, the purified ORF-14 protein reacted with antibodies in 26 of 57 human sera (46%) from TB patients while no reactivity was seen with 11 sera from M . bovis BCG-vaccinated healthy subjects . Interestingly, sera from nine of 15 (60%) long-term contacts of TB patients also had antibodies reactive to the ORF-14 protein . These results suggest that the ORF-14 protein in combination with other immunodominant proteins could be useful in the serodiagnosis of individuals infected with M . tuberculosis. Mol Microbiol, 1999 May, 32(3), 657 - 68 MIC231, a naturally occurring mobile insertion cassette from Bacillus cereus; Chen Y et al.; Recent dissection of numerous plasmids and transposable elements has given more credence to the modular organization of these genetic and genomic entities . Although many variations on each theme exist, the number of basic functional cassettes is thought to be relatively limited . In this paper, a novel type of mobile cassette is described . A naturally occurring assemblage consisting of two left IS231 ends flanking a D-stereospecific endopeptidase (adp) gene was found in several natural isolates of Bacillus cereus . This 1.9 kb genetic entity was shown to transpose in the presence of IS231A transposase, not only in Escherichia coli but also in Bacillus . The acronym MIC231 is proposed for this mobile insertion cassette trans-activated (teletransposed) by IS231 . Using (D-Phe)4 tetrapeptide as substrate, the endopeptidase activity of the MIC231 adp gene could be demonstrated in E . coli and B . subtilis . Interestingly, this D-stereospecific endopeptidase activity was not limited to the original B . cereus isolates but was also detected in all but one of the 69 B . cereus sensu lato strains tested, indicating its important, yet dispensable, biological function . However, inactivation of the MIC231 adp gene in two B . cereus strains did not result in any detectable variation of their activity on (D-Phe)4, suggesting the presence of other distantly related adp gene(s) . Thus, although the exact role of MIC231 adp remains elusive, its presence inside a mobile cassette represents the archetype of a novel insertion sequence modular organization. Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 821 - 31 Gracilibacillus gen . nov., with description of Gracilibacillus halotolerans gen . nov., sp . nov.; transfer of Bacillus dipsosauri to Gracilibacillus dipsosauri comb . nov., and Bacillus salexigens to the genus Salibacillus gen . nov., as Salibacillus salexigens comb . nov; Waino M et al.; A Gram-positive, extremely halotolerant bacterium was isolated from the Great Salt Lake, Utah, USA . The strain, designated NNT (= DSM 11805T), was strictly aerobic, rod-shaped, motile by peritrichous flagella and spore-forming . Strain NNT grew at salinities of 0-20% (w/v) NaCl . A distinctive feature of strain NNT was its optimal growth in salt-free medium . The polar lipid pattern of strain NNT consisted of phosphatidyl glycerol, diphosphatidyl glycerol and two phospholipids of unknown structure . The G + C content of its DNA was 38 mol% . The morphological, physiological and, particularly, the 16S rDNA sequence data, showed that strain NNT was associated with 'Bacillus group 1' . However, the organisms showing the greatest degree of sequence similarity to strain NNT were members of the genus Halobacillus and the species Marinococcus albus, Virgibacillus pantothenticus, Bacillus salexigens and Bacillus dipsosauri . On the basis of chemotaxonomic data, strain NNT was shown to be chemically most similar to B . salexigens and B . dipsosauri, with the greatest degree of similarity being shown to the latter organism . This was consistent with the 16S rDNA sequence data . Members of the genus Halobacillus comprise a chemically distinct group and can easily be distinguished from all other organisms of 'Bacillus group 1' . On the basis of the 16S rDNA data, chemotaxonomy and the physiology of strain NNT, it is proposed that this organism is a member of a new species, within a new genus, for which the name Gracilibacillus halotolerans is proposed . It is also proposed that B . dipsosauri be transferred to this genus as Gracilibacillus dipsosauri comb . nov . and that B . salexigens be transferred to the genus Salibacillus gen . nov., as Salibacillus salexigens comb . nov . Finally, additional data is provided to support the transfer of Bacillus pantothenticus to the genus Virgibacillus, as Virgibacillus pantothenticus Heyndrickx et al . (1998). Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 795 - 802 Bacillus silvestris sp . nov., a new member of the genus Bacillus that contains lysine in its cell wall; Rheims H et al.; A Gram-positive, aerobic, rod-shaped, peritrichously flagellated, round-endospore-forming bacterium was isolated from a forest soil near Braunschweig, Lower Saxony, Germany, and designated strain HR3-23T (T = type strain) . Morphologically, strain HR3-23T shows the characteristics of a member of the genus Bacillus . The spore position is terminal in a swollen sporangium . Comparative analysis of the 16S rDNA sequence shows strain HR3-23T to be most closely related to Caryophanon tenue (95.8% 16S rRNA similarity) and to Bacillus sphaericus (95.4% 16S rRNA similarity) . Phylogenetically, the isolate clusters among species of Bacillus RNA group 2 . The DNA G + C content of isolate HR3-23T is 39.3 mol%, the peptidoglycan type is A4 alpha (L-Lys-D-Glu), the major respiratory lipoquinone is menaquinone MK-7 and the predominant fatty acid is of the iso-C15:0 type . Based on the morphological, chemotaxonomic, physiological and phylogenetic properties, a new species, Bacillus silvestris, is proposed; strain HR3-23T is the type strain (= DSM 12223T). Proc Natl Acad Sci U S A, 1999 May 11, 96(10), 5412 - 7 The crystal structure of a multifunctional protein: phosphoglucose isomerase/autocrine motility factor/neuroleukin; Sun YJ et al.; Phosphoglucose isomerase (PGI) plays a central role in both the glycolysis and the gluconeogenesis pathways . We present here the complete crystal structure of PGI from Bacillus stearothermophilus at 2.3-A resolution . We show that PGI has cell-motility-stimulating activity on mouse colon cancer cells similar to that of endogenous autocrine motility factor (AMF) . PGI can also enhance neurite outgrowth on neuronal progenitor cells similar to that observed for neuroleukin . The results confirm that PGI is neuroleukin and AMF . PGI has an open twisted alpha/beta structural motif consisting of two globular domains and two protruding parts . Based on this substrate-free structure, together with the previously published biological, biochemical, and modeling results, we postulate a possible substrate-binding site that is located within the domains' interface for PGI and AMF . In addition, the structure provides evidence suggesting that the top part of the large domain together with one of the protruding loops might participate in inducing the neurotrophic activity. Trends Plant Sci, 1999 Jan, 4(1), 9 - 13 toxin-mediated insect resistance in plants; de Maagd RA et al.; We are currently in an interesting phase of plant biotechnology releases, both for the scientists responsible for these innovations who are beginning to see their ideas realized, and for the biotechnology companies that are starting to see a return on their investment . One of the most notable examples, is the introduction of transgenic crops that are engineered to express a Bacillus thuringiensis toxin that confers resistance to insect predation . However, the picture is not altogether positive - there is concern that the introduction of this technology was premature or should not have happened at all, and that the valuable insecticidal properties of Bacillus thuringiensis will be lost. Immunology, 1999 Apr, 96(4), 517 - 23 An in vivo comparison of bacillus Calmette-Guérin (BCG) and cytokine-secreting BCG vaccines; Slobbe L et al.; A recombinant bacillus Calmette-Guerin (BCG) vaccine has been developed, which constitutively secretes interleukin (IL)-2 . Groups of deer were immunized with either normal BCG (Pasteur 1173 P2 strain) or recombinant BCG (rBCG/IL-2) and their immune responses were monitored over 3 months . Animals gained weight over this period and showed no signs of adverse reactions to either vaccine . Lymphocyte transformation responses did not differ significantly between the two groups . No antibody that was specific for BCG was detected in any animal . Intradermal skin-test responses to BCG antigens showed that the rBCG/IL-2 induced a smaller delayed-type hypersensitivity response than the normal BCG . Cytokine transcription was determined by reverse transcription-polymerase chain reaction (RT-PCR) . While IL-2 and interferon-gamma (IFN-gamma) levels did not differ significantly between the two groups, the level of IL-4 was found to be lower in the group given rBCG/IL-2 . This resulted in a strong interferon-gamma:IL-4 ratio, suggesting a skewing of the immune response towards a Type 1 response . The rate at which the vaccine was eliminated from the host was the same regardless of whether BCG or rBCG was used . At autopsy (3 months after vaccination) 99.99% of the organisms had been eliminated . The small number of organisms isolated from the draining lymph node of animals given rBCG/IL-2 were grown in antibiotic-containing media . They were shown to still contain the shuttle plasmid and to secrete biologically active IL-2, indicating that the plasmid was stably maintained despite the host's immune response and in the absence of antibiotic selection. Immunology, 1999 Apr, 96(4), 511 - 6 Co-immunization with DNA vaccines expressing granulocyte-macrophage colony-stimulating factor and mycobacterial secreted proteins enhances T-cell immunity, but not protective efficacy against Mycobacterium tuberculosis; Kamath AT et al.; The development of more effective antituberculosis vaccines would assist in the control of the global problem of infection with Mycobacterium tuberculosis . One recent vaccination strategy is immunization with DNA plasmids encoding individual microbial genes . Using the genes for the M . tuberculosis-secreted proteins, MPT64 (23 000 MW) and Ag85B (30 000 MW) as candidate antigens, we previously prepared DNA vaccines and demonstrated their ability to stimulate T-cell responses and confer protection in a mouse model of aerosol tuberculosis (TB) . The protective efficacy of the DNA vaccines was less than that promoted by the current vaccine Mycobacterium bovis bacille Calmette-Guerin (BCG) . To improve the immunogenicity and protective efficacy of these mycobacterial vectors, co-immunization of a plasmid expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) was investigated . Intramuscular immunization with DNA expressing MPT64 or Ag85B and GM-CSF enhanced the antigen-specific cellular immune response, with increased proliferative response and production of interferon-gamma (IFN-gamma) . The titre of antimycobacterial protein immunoglobulin G (IgG) antibodies was unchanged . Mice immunized with DNA vaccines showed reduced pulmonary bacterial load following an aerosol challenge of M . tuberculosis, but codelivery of the plasmid expressing GM-CSF did not increase the protective effect . Therefore, despite modifying the cellular immune response to DNA vaccines, GM-CSF does not improve their protective efficacy at the peak of infection after an aerosol challenge with 100 c.f.u . of M . tuberculosis. J Rheumatol, 1999 Apr, 26(4), 933 - 5 Bacillus Calmette-Guérin associated arthropathy mimicking undifferentiated spondyloarthropathy; Schwartzenberg JM et al.; The development of an inflammatory arthritis mimicking an undifferentiated spondyloarthropathy (SpA) was seen in a patient being treated for a superficial bladder cancer with intravesical bacillus Calmette-Guerin (BCG) . Physical findings included classic dactylitis of both feet . This is the fourth report identifying a patient with BCG induced articular findings suggestive of a SpA with dactylitis . Studies of BCG stimulated cytokine secretion from peripheral blood mononuclear cells showed the patient to have enhanced interleukin 6 (IL-6) levels and reduced interferon-gamma (IFN-gamma) levels . Spontaneous IL-6 secretion was markedly elevated for the patient, compared to the control subject, but IFN-gamma secretion was quite similar . No differences were apparent with IL-4. Am J Respir Crit Care Med, 1999 May, 159(5 Pt 1), 1629 - 37 Effect of stimulation of human macrophages on intracellular survival of Mycobacterium bovis Bacillus Calmette-Guerin . Evaluation with a mycobacterial reporter strain; Bonay M et al.; The mechanisms through which immune and inflammatory responses stimulate the expression of antimycobacterial activity by human macrophages remain poorly defined . To study this question, we developed a method permitting the rapid quantification of viable mycobacteria, based on the detection of luciferase activity expressed by a Mycobacterium bovis Bacillus Calmette-Guerin (BCG) reporter strain, and used this approach to evaluate mycobacterial survival in human monocyte-derived macrophages following stimulation with cytokines and through crosslinking of costimulatory molecules expressed on the cell surface . Modest proliferation, followed by persistence of mycobacteria, was observed in unpretreated macrophages as assessed both by measurement of luciferase activity and by the evaluation of colony forming units . Of the 19 cytokines tested, only granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) were found to improve the mycobactericidal activity of monocyte-derived macrophages . In both cases, this effect was observed only when macrophages were pretreated with the cytokines prior to infection . In contrast, pretreatment of human macrophages with interferon-gamma, either alone or in combination with other mediators (including tumor necrosis factor-alpha and 1,25{OH}2-vitamin D3), did not improve mycobacterial killing . The stimulation of macrophages through several different costimulatory molecules known to participate in macrophage-lymphocyte interactions (CD4, CD40, CD45, CD86, CD95 {Fas/Apo-1}) also failed to improve mycobactericidal activity . This study shows that GM-CSF and IL-3, cytokines whose receptors are known to share a common subunit and to use common second messengers, may contribute to the stimulation of mycobactericidal activity in humans . The ability to rapidly screen the effects of different macrophage stimuli on mycobacterial survival through the detection of luciferase activity should help define additional signals required for optimal antimycobacterial responses. Microb Ecol, 1999 Apr, 37(3), 218 - 224 Lead Resistance in Two Bacterial Isolates from Heavy Metal-Contaminated Soils; Roane TM; > Abstract Microorganisms have developed mechanisms of coping with a variety of toxic metals; however, few studies have explored microbial resistance to lead . In this study, the overall mechanisms of a lead-resistant Pseudomonas marginalis and a lead-resistant Bacillus megaterium isolated from two different metal-contaminated soils were investigated . The P.marginalis had a higher lead resistance level at 2.5mM total lead as compared to 0.6 mM for B . megaterium . Resistance to soluble lead was much lower, 0.3 and 0.1 mM, respectively . The degree of lead resistance and the mechanism of lead resistance for these two isolates corresponded with their environmental lead exposure . When viewed with transmission electron microscopy, P.marginalis, isolated from a soil contaminated with high total but undetectable soluble lead, showed extracellular lead exclusion . B.megaterium, from a soil with both high total and soluble lead levels, was less resistant with an intracellular cytoplasmic accumulation of lead as observed with TEM . Polarization microscopy indicated that while P.marginalis produced a high amount of an extracellular polymer implicated in the organism's mechanism of lead resistance, B.megaterium produced no discernable extracellular polymeric substances . The study of these two organisms demonstrated differences in how soil microorganisms respond to environmental lead exposure, including the novel mechanism of intracellular sequestration of lead. Biosci Biotechnol Biochem, 1999 Mar, 63(3), 563 - 6 Cloning of oxetanocin A biosynthetic and resistance genes that reside on a plasmid of Bacillus megaterium strain NK84-0128; Morita M et al.; Bacillus megaterium strain NK84-0218 produces a potent antiviral antibiotic, oxetanocin A, which has an oxetanosyl-N-glycoside linkage to an adenine moiety . However, the oxetanocin A productivity of the original strain was unstable and low . In this study, oxetanocin A productivity and resistance was shown to be lost simultaneously when a 51.5-kb plasmid, pOXT1, was cured during cultivation . The deficiency of oxetanocin A productivity and resistance was restored by re-introduction of the pOXT1 plasmid into the cured strain . By a cloning experiment it was shown that a 6.8-kb BglI-D fragment of the pOXT1 plasmid was responsible for oxetanocin A productivity and resistance. Appl Environ Microbiol, 1999 May, 65(5), 2049 - 53 Antagonism between Cry1Ac1 and Cyt1A1 toxins of bacillus thuringiensis del Rincon-Castro MC, Barajas-Huerta J, Ibarra JE. Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals . Some of the combinations clearly interact synergistically, like the toxins present in B . thuringiensis subsp . israelensis . In this paper we describe a novel joint activity of toxins from different strains of B . thuringiensis . In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B . thuringiensis subsp . kurstaki, Cyt1A1 from B . thuringiensis subsp . israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics . The 50% lethal concentrations (LC50s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium . When mixtures of these toxins in different proportions were assayed, eight different LC50s were obtained . All of these LC50s were significantly higher than the expected LC50s of the mixtures . In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins . The estimated mean LC50 of Cry1Ac1 was 2.46 ng/cm2 of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations . The estimated mean LC50s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm2 of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm2 of diet, respectively . These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo . Other joint-action analyses corroborated these results . Although this is the second report of antagonism between B . thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B . thuringiensis (B . thuringiensis subsp . kurstaki and B . thuringiensis subsp . israelensis) detected both in vivo and in vitro . Some possible explanations for this relationship are discussed. Appl Environ Microbiol, 1999 May, 65(5), 1900 - 3 Role of bacillus thuringiensis toxin domains in toxicity and receptor binding in the diamondback moth Ballester V V, Granero F, de Maagd RA, Bosch D, Mensua JL, Ferre J. The toxic fragment of Bacillus thuringiensis crystal proteins consists of three distinct structural domains . There is evidence that domain I is involved in pore formation and that domain II is involved in receptor binding and specificity . It has been found that, in some cases, domain III is also important in determining specificity . Furthermore, involvement of domain III in binding has also been reported recently . To investigate the role of toxin domains in the diamondback moth (Plutella xylostella), we used hybrid toxins with domain III substitutions among Cry1C, Cry1E, and Cry1Ab . Neither Cry1E nor G27 (a hybrid with domains I and II from Cry1E and domain III from Cry1C) was toxic, whereas Cry1C and F26 (the reciprocal hybrid) were equally toxic . H04 (a hybrid with domains I and II from Cry1Ab and domain III from Cry1C) showed toxicity that was of a similar level as that of Cry1Ab and significantly higher than that of Cry1C . Binding assays with 125I-Cry1C showed that Cry1C and F26 competed for the same binding sites on midgut membrane vesicles, whereas Cry1E, G27, and H04 did not bind to these sites . Our results show that, in contrast to findings in other insects for the toxins and hybrids used here, toxin specificity as well as specificity of binding to membrane vesicles in the diamondback moth is mediated by domain II (and/or I) and not by domain III. Proteins, 1999 May 1, 35(2), 195 - 205 Different sequence patterns in signal peptides from mycoplasmas, other gram-positive bacteria, and Escherichia coli: a multivariate data analysis; Edman M et al.; Signal peptides are essential N-terminal extensions in export proteins, and have a positively charged N-terminus, a hydrophobic central core, and a C-terminal cleavage region . They interact in a consecutive manner with different accessory proteins during the secretion process . Potential patterns or periodicity in the amino acid (aa) sequence were searched, using multivariate techniques, for a large number of signal peptides from mollicutes (mycoplasmas), other Gram-positive bacteria, and Escherichia coli . Mollicutes signal peptides were significantly different from the E . coli and Gram-positive ones by their N-terminal charge, peptide length, and especially, unique periodicities of side chain hydrophobicity and volumes . Their lipoprotein signal peptides were longer than for any other bacteria . Significant differences were also recorded between the other bacterial peptide groups . Specific aa patterns were more related within the signal peptides from several groups of secreted bacillus enzymes, than for all signal peptides from one bacillus species . In E . coli, signal peptides from proteins routed for the various destinations revealed significant and compartment-specific sequence patterns not evident by other methods . This was substantiated from a large number of signal peptide secretion mutants for the E . coli periplasmic space . It is proposed that the differences in aa patterns and side-chain properties are related to the secondary structure sidedness and topology of the signal peptides, and important for specific interactions during the secretion process. J Biol Chem, 1999 May 7, 274(19), 13242 - 9 Mono- and binuclear Zn2+-beta-lactamase . Role of the conserved cysteine in the catalytic mechanism; Paul-Soto R et al.; When expressed by pathogenic bacteria, Zn2+-beta-lactamases induce resistance to most beta-lactam antibiotics . A possible strategy to fight these bacteria would be a combined therapy with non-toxic inhibitors of Zn2+-beta-lactamases together with standard antibiotics . For this purpose, it is important to verify that the inhibitor is effective under all clinical conditions . We have investigated the correlation between the number of zinc ions bound to the Zn2+-beta-lactamase from Bacillus cereus and hydrolysis of benzylpenicillin and nitrocefin for the wild type and a mutant where cysteine 168 is replaced by alanine . It is shown that both the mono-Zn2+ (mononuclear) and di-Zn2+ (binuclear) Zn2+-beta-lactamases are catalytically active but with different kinetic properties . The mono-Zn2+-beta-lactamase requires the conserved cysteine residue for hydrolysis of the beta-lactam ring in contrast to the binuclear enzyme where the cysteine residue is not essential . Substrate affinity is not significantly affected by the mutation for the mononuclear enzyme but is decreased for the binuclear enzyme . These results were derived from kinetic studies on two wild types and the mutant enzyme with benzylpenicillin and nitrocefin as substrates . Thus, targeting drug design to modify this residue might represent an efficient strategy, the more so if it also interferes with the formation of the binuclear enzyme. Appl Environ Microbiol, 1999 May, 65(5), 1849 - 53 Subspecies-dependent regulation of Bacillus thuringiensis protoxin genes; Cheng P et al.; Bacillus thuringiensis accumulates, primarily during sporulation, large quantities of insecticidal protoxins which are deposited as crystalline, intracellular inclusions . Most subspecies contain several plasmid-encoded cry genes, each of which has a unique specificity . The overall toxicity profile of a subspecies depends not only on the array of cry genes present but also on the relative expression of the genes . In general, transcription depends on sporulation-specific sigma factors, but little is known about regulation of expression of the individual genes . In order to determine whether expression of a particular cry gene varies in different subspecies, lacZ fusions to the cry promoters of two protoxin genes (cry1 class) were constructed . Protoxin accumulation and mRNA contents were also measured by performing immunoblotting and Northern analyses, respectively . The expression of a cry1Ab-lacZ fusion, but not the expression of a cry1C-lacZ fusion, was three to four times lower in B . thuringiensis subsp . aizawai strains than in B . thuringiensis subsp . kurstaki or B . thuringiensis subsp . tolworthi . Also, the Cry1Ab antigen and steady-state mRNA contents of B . thuringiensis subsp . aizawai were lower . The regulation of the genes must involve regions upstream of the promoters which are unique to each cry gene since (i) mutations in the upstream region of the cry1Ab gene resulted in enhanced expression in B . thuringiensis subsp . aizawai and (ii) no differences were found when the lacZ fusions contained the cry1Ab promoters but no upstream sequences . The capacity to regulate each of the protoxin genes must be a factor in the overall protoxin composition of a subspecies and thus its toxicity profile. J Invertebr Pathol, 1999 May, 73(3), 255 - 9 Susceptibility of the taro beetle, Papuana uninodis (Coleoptera, Scarabaeidae) to two new Bacillus popilliae isolates from Papuana spp; Theunis W et al.; Two morphological types of Bacillus popilliae, causal agent of the milky disease, have been isolated from taro beetles (Papuana spp, Coleoptera: Scarabaeidae) . B . popilliae from P . woodlarkiana woodlarkiana (Papua New Guinea) was a type A1 with a small sporangium (4.1 x 1.6 microm) and a large spore (2.1 x 1.4 microm) and parasporal body (1.8 x 1.2 microm) that sometimes overlap . B . popilliae from P . uninodis and P . woodlarkiana laevipennis (Solomon Islands) was a type B2 with a small sporangium (2.8 x 1.3 microm), a small eccentric spore (1.1 x 0.7 microm), and no parasporal body . The infectivity of these B . popilliae to Papuana uninodis larvae was compared with two B . popilliae samples from Popillia japonica in injection tests . The hemolymph of P . uninodis supported the germination and growth of isolates from Papuana and P . japonica . Results were similar in third instars and adults . Highest infection (spores present) and mortality was caused by the isolates from Papuana: mortality reached almost 100% 4 weeks after injection of the B2 type B . popilliae with 40% of larvae and 52% of adults infected . Injection of type A1 caused lower mortality but a similar percentage infected . Of two A1 B . popilliae from P . japonica, one caused a mortality comparable to type A1 from Papuana but lower infection; an older isolate resulted in low mortality and only one infected larva . B . popilliae type A1 from P . woodlarkiana was produced in the Solomon Islands by injection of spores in P . uninodis . Thirty four percent of the injected larvae and 31% of the adults produced spores with an average yield of 3.2 and 0.8 x 10(9) spores/insect, respectively . Oral application of a single dose of 10(7) spores of the B . popilliae isolates from P . uninodis or P . japonica did not cause infection and similarly inoculation of the food with spores of B . popilliae type B2 did not result in infections . However, when different rates were applied to the food of second- and third-instar P . uninodis, the B . popilliae type A1 from P . woodlarkiana caused up to 15% infection and concentration-related mortality . Anal Biochem, 1999 May 1, 269(2), 359 - 66 A continuous spectrophotometric assay for P450 BM-3, a fatty acid hydroxylating enzyme, and its mutant F87A; Schwaneberg U et al.; Cytochrome P450 BM-3 from Bacillus megaterium catalyzes the subterminal hydroxylation of medium- and long-chain fatty acids at the positions omega-1, omega-2, and omega-3 . A rapid and continuous spectrophotometric activity assay for cytochrome P450 BM-3 based on the conversion of p-nitrophenoxycarboxylic acids (pNCA) to omega-oxycarboxylic acids and the chromophore p-nitrophenolate was developed . In contrast to the commonly used activity assays for this enzyme, relying on the consumption of oxygen or NADPH or the use of 14C-labeled carboxylic acids, the pNCA assay can even be used with crude extracts of the recombinant enzyme from lysed Escherichia coli cells . The kinetics of p-nitrophenolate formation are directly measured at a wavelength of 410 nm using a spectrophotometer or microtiter plate reader . Sensitivity of the assay is greatly enhanced if p-nitrophenoxydodecanoic or p-nitrophenoxypentadecanoic acid are used with the F87A mutant instead of the wild-type P450 BM-3 enzyme . Drug Saf, 1999 Mar, 20(3), 207 - 12 Immunisation and type 1 diabetes mellitus: is there a link? Hiltunen M, Lonnrot M, Hyoty H. Recent evidence from animal studies has raised the possibility that immunisation by vaccines can influence the pathogenesis of type I (insulin-dependent) diabetes mellitus . In non-obese diabetic mice and biobreeding rats, complete Freund's adjuvant and bacillus Calmette-Guerin (BCG) vaccine have successfully been used to interrupt the development of diabetes mellitus . This effect is probably mediated by nonspecific suppression of the autoimmune process . A number of attempts have also been made to assess the impact of parenteral immunisation on type 1 diabetes mellitus in humans . Epidemiological evidence has not indicated any clear link between BCG vaccination and the development of diabetes mellitus in humans . Some reports have suggested that natural mumps or mumps vaccinations can induce islet cell autoimmunity, but there is no evidence that mumps-measles-rubella mass vaccination programmes have changed the incidence of diabetes mellitus in any population . An independent protective role of measles virus has been suggested in one study . Recent studies have indicated that enterovirus infections may induce beta cell autoimmunity and clinical diabetes . The only currently available enterovirus vaccine is the poliovirus vaccine which, in theory, could modulate the protection against other enteroviruses by inducing cross-reactive T cell immune responses; however, this hypothesis has not been tested so far . In conclusion, there is no clear evidence that any currently used vaccine can prevent or induce diabetes in humans . However, only a few studies are available on the subject and therefore the possibility of a link between vaccination and diabetes mellitus cannot be excluded. Ned Tijdschr Geneeskd, 1999 Feb 20, 143(8), 388 - 92 {Whipple's disease}; Zaaijer HL et al.; Whipple's disease is characterized by malabsorption, weight loss, diarrhoea and abdominal pain, often preceded by a long period of migrating arthralgias . Instead of the intestine the heart, brain, eyes, lungs or blood vessels may be affected . Whipple's disease is caused by Tropheryma whippelii, a bacillus found inside phagocytes . A specific defect in the immune system of the host appears to play a part . The diagnosis is based on microscopic examination of periodic-acid-Schiff(PAS)-stained slides and on polymerase chain reaction (PCR) analysis of affected tissue . Recently a method for culturing T . whippelii was described . Prolonged treatment with cotrimoxazole, preceded or not by two weeks of penicillin and streptomycin, often cures the disease, but relapses do occur. FEMS Microbiol Lett, 1999 Apr 1, 173(1), 217 - 22 Expression of the genes for guanyl-specific ribonucleases from Bacillus intermedius and Bacillus pumilus is regulated by the two component signal transduction system PhoP-PhoR in B . subtilis; Znamenskaya LV et al.; Promoters of the genes for guanyl-specific ribonucleases, secreted by B . intermedius (binase) and B . pumilus (Rnase Bp) in phosphate deficient conditions, contain regions similar to appropriate consensus sequences in promoters of the PHO regulated genes of B . subtilis . A number of genes expressed in response to phosphate starvation in B . subtilis are regulated by the two component signal transduction system PhoP-PhoR . Expression of recombinant genes for binase and RNase Bp in B . subtilis strains with mutations in the regulatory protein genes of the PHO regulon was studied . Their expression is strongly regulated by the regulatory proteins of the B . subtilis PHO regulon. Proc Natl Acad Sci U S A, 1999 Apr 27, 96(9), 4786 - 90 Genetic basis in plants for interactions with disease-suppressive bacteria; Smith KP et al.; Plant health depends, in part, on associations with disease-suppressive microflora, but little is known about the role of plant genes in establishing such associations . Identifying such genes will contribute to understanding the basis for plant health in natural communities and to new strategies to reduce dependence on pesticides in agriculture . To assess the role of the plant host in disease suppression, we used a genetic mapping population of tomato to evaluate the efficacy of the biocontrol agent Bacillus cereus against the seed pathogen Pythium torulosum . We detected significant phenotypic variation among recombinant inbred lines that comprise the mapping population for resistance to P . torulosum, disease suppression by B . cereus, and growth of B . cereus on the seed . Genetic analysis revealed that three quantitative trait loci (QTL) associated with disease suppression by B . cereus explained 38% of the phenotypic variation among the recombinant inbred lines . In two cases, QTL for disease suppression by B . cereus map to the same locations as QTL for other traits, suggesting that the host effect on biocontrol is mediated by different mechanisms . The discovery of a genetic basis in the host for interactions with a biocontrol agent suggests new opportunities to exploit natural genetic variation in host species to enhance our understanding of beneficial plant-microbe interactions and develop ecologically sound strategies for disease control in agriculture. Biochemistry, 1999 Apr 27, 38(17), 5643 - 50 Folding of the multidomain ribosomal protein L9: the two domains fold independently with remarkably different rates; Sato S et al.; The folding and unfolding behavior of the multidomain ribosomal protein L9 from Bacillus stearothermophilus was studied by a novel combination of stopped-flow fluorescence and nuclear magnetic resonance (NMR) spectroscopy . One-dimensional 1H spectra acquired at various temperatures show that the C-terminal domain unfolds at a lower temperature than the N-terminal domain (Tm = 67 degrees C for the C-terminal domain, 80 degrees C for the N-terminal domain) . NMR line-shape analysis was used to determine the folding and unfolding rates for the N-terminal domain . At 72 degrees C, the folding rate constant equals 2980 s-1 and the unfolding rate constant equals 640 s-1 . For the C-terminal domain, saturation transfer experiments performed at 69 degrees C were used to determine the folding rate constant, 3.3 s-1, and the unfolding rate constant, 9.0 s-1 . Stopped-flow fluorescence experiments detected two resolved phases: a fast phase for the N-terminal domain and a slow phase for the C-terminal domain . The folding and unfolding rate constants determined by stopped-flow fluorescence are 760 s-1 and 0.36 s-1, respectively, for the N-terminal domain at 25 degrees C and 3.0 s-1 and 0.0025 s-1 for the C-terminal domain . The Chevron plots for both domains show a V-shaped curve that is indicative of two-state folding . The measured folding rate constants for the N-terminal domain in the intact protein are very similar to the values determined for the isolated N-terminal domain, demonstrating that the folding kinetics of this domain is not affected by the rest of the protein . The remarkably different rate constants between the N- and C-terminal domains suggest that the two domains can fold and unfold independently . The folding behavior of L9 argues that extremely rapid folding is not necessarily functionally important. Biochemistry, 1999 Apr 27, 38(17), 5588 - 95 Structure of the flavocoenzyme of two homologous amine oxidases: monomeric sarcosine oxidase and N-methyltryptophan oxidase; Wagner MA et al.; Monomeric sarcosine oxidase (MSOX) and N-methyltryptophan oxidase (MTOX) are homologous enzymes that catalyze the oxidative demethylation of sarcosine (N-methylglycine) and N-methyl-L-tryptophan, respectively . MSOX is induced in various bacteria upon growth on sarcosine . MTOX is an E . coli enzyme of unknown metabolic function . Both enzymes contain covalently bound flavin . The covalent flavin is at the FAD level as judged by electrospray mass spectrometry . The data provide the first evidence that MTOX is a flavoprotein . The following observations indicate that 8alpha-(S-cysteinyl)FAD is the covalent flavin in MSOX from Bacillus sp . B-0618 and MTOX . FMN-containing peptides, prepared by digestion of MSOX or MTOX with trypsin, chymotrypsin, and phosphodiesterase, exhibited absorption and fluorescence properties characteristic of an 8alpha-(S-cysteinyl)flavin and could be bound to apo-flavodoxin . The thioether link in the FMN-containing peptides was converted to the sulfone by performic acid oxidation, as judged by characteristic absorbance changes and an increase in flavin fluorescence . The sulfone underwent a predicted reductive cleavage reaction upon treatment with dithionite, releasing unmodified FMN . Cys315 was identified as the covalent FAD attachment site in MSOX from B . sp . B-0618, as judged by the sequence obtained for a flavin-containing tryptic peptide (GAVCMYT) . Cys315 aligns with a conserved cysteine in MSOX from other bacteria, MTOX (Cys308) and pipecolate oxidase, a homologous mammalian enzyme known to contain covalently bound flavin . There is only one conserved cysteine found among these enzymes, suggesting that Cys308 is the covalent flavin attachment site in MTOX. Biochemistry, 1999 Apr 27, 38(17), 5346 - 54 Sugar ring distortion in the glycosyl-enzyme intermediate of a family G/11 xylanase; Sidhu G et al.; The 1.8 A resolution structure of the glycosyl-enzyme intermediate formed on the retaining beta-1,4-xylanase from Bacillus circulans has been determined using X-ray crystallographic techniques . The 2-fluoro-xylose residue bound in the -1 subsite adopts a 2,5B (boat) conformation, allowing atoms C5, O5, C1, and C2 of the sugar to achieve coplanarity as required at the oxocarbenium ion-like transition states of the double-displacement catalytic mechanism . Comparison of this structure to that of a mutant of this same enzyme noncovalently complexed with xylotetraose {Wakarchuk et al . (1994) Protein Sci . 3, 467-475} reveals a number of differences beyond the distortion of the sugar moiety . Most notably, a bifurcated hydrogen bond interaction is formed in the glycosyl-enzyme intermediate involving Heta of Tyr69, the endocyclic oxygen (O5) of the xylose residue in the -1 subsite, and Oepsilon2 of the catalytic nucleophile, Glu78 . To gain additional understanding of the role of Tyr69 at the active site of this enzyme, we also determined the 1.5 A resolution structure of the catalytically inactive Tyr69Phe mutant . Interestingly, no significant structural perturbation due to the loss of the phenolic group is observed . These results suggest that the interactions involving the phenolic group of Tyr69, O5 of the proximal saccharide, and Glu78 Oepsilon2 are important for the catalytic mechanism of this enzyme, and it is proposed that, through charge redistribution, these interactions serve to stabilize the oxocarbenium-like ion of the transition state . Studies of the covalent glycosyl-enzyme intermediate of this xylanase also provide insight into specificity, as contacts with C5 of the xylose moiety exclude sugars with hydroxymethyl substituents, and the mechanism of catalysis, including aspects of stereoelectronic theory as applied to glycoside hydrolysis. Toxicon, 1999 May, 37(5), 801 - 13 The membranotropic activity of cyclic acyldepsipeptides from bacterium Bacillus pumilus, associated with the marine sponge Ircinia sp; Prokof'eva NG et al.; The isolate of Bacillus pumilus associated with the marine sponge Ircinia sp . produced the surfactin-like lipopeptides, cyclic acyldepsipeptides . The hemolytic activity of individual cyclic acyldepsipeptides, bacircines (BI) 2, 3, 4, 5 and 5A having different acyl side chain structures (anteiso-C13, iso-C14, normal-C14, anteiso-C15, and iso-C15, respectively) was studied . The hemolytic power of bacircines depended on both the structure of the side chain (n->iso->anteiso-) and pH values (5.6 and 6.5 > 7.4) . Hemolytic potency as a function of BI 5 concentration was given for pH 6.5; 7.4; 8.0; 9.0 . pH dependent hemolysis induced by BI 5 was shown to be reversible . The membrane damaging potential of bacircine 5 (5 microM) at pH 6.5 was characterized by a higher rate of hemolysis and by a shorter time between the introduction of BI 5 solution into the RBC samples and the onset of hemolysis . Under this condition, BI 5 decreased abnormally the microviscosity of erythrocyte ghosts bilayer . The damaging potency of BI 5 decreased with an increase pH from 6.5 to 7.4 or its decrease from 6.5 to 4.9 . It was shown that fatty acid bacircine fragment penetrated into the lipid bilayer to a depth of minimum 7 carbon atoms . Constants of dissociation of the Asp (pK 4.75) and Glu (pK 6.65) residues of bacircine in the lipid bilayer were obtained . These results showed that at pH 6.5 BI 5 possessed membranotropic activity in the monoionic form. Acta Crystallogr D Biol Crystallogr, 1999 May, 55(5), 1083 - 5 Crystallization and preliminary X-ray diffraction studies of the 51 kDa protein of the mosquito-larvicidal binary toxin from Bacillus sphaericus; Chiou C et al.; Certain strains of Bacillus sphaericus produce a highly toxic mosquito-larvicidal protein during sporulation which is active against vectors of dengue, encephalitis and malaria . This toxin is initially expressed as 51 and 42 kDa proteins and is converted to 43 and 39 kDa proteins, respectively, which form the active heterodimer complex . For a better understanding of the toxicity mechanism at the molecular level, the 51 kDa protein of the binary toxin of B . sphaericus strain 2297 was expressed as a glutathione-S-transferase fusion protein and purified by affinity chromatography . Protein crystals were grown from an amorphous precipitate in five months using the hanging-drop vapor-diffusion method . The protein crystals were dissolved and were found to be composed of a proteolytically modified 45.2 kDa derivative similar to the active form of this protein . The crystals form in space group P43212 (or P41212) and diffract to 2.6 A, with unit-cell dimensions a = b = 133.48, c = 69 . 76 A. Microbiology, 1999 Mar, 145 ( Pt 3), 549 - 59 The mycelium-associated Streptomyces reticuli catalase-peroxidase, its gene and regulation by FurS; Zou P et al.; During early stages of growth, Streptomyces reticuli synthesizes a hyphae-associated, haem-containing enzyme which exhibits catalase and peroxidase activities with broad substrate specificity (CpeB) . The purified dimeric enzyme (160 kDa) consists of two identical subunits . Using anti-CpeB antibodies and an expression- as well as a mini-library, the corresponding cpeB gene was identified and sequenced . It encodes a protein of 740 aa with a molecular mass of 81.3 kDa . The deduced protein shares the highest level of amino acid identity with KatG from Caulobacter crescentus and Mycobacterium tuberculosis, and PerA from Bacillus stearothermophilus . Streptomyces lividans transformants carrying cpeB and the upstream-located furS gene with its regulatory region on the bifunctional vector pWHM3 produced low or enhanced levels of CpeB in the presence or absence of Fe ions, respectively . An in-frame deletion of the major part of furS induces increased CpeB synthesis . The data imply that FurS regulates the transcription of cpeB . The deduced FurS protein is rich in histidine residues, contains a putative N-terminally situated helix-turn-helix motif and has a molecular mass of 15.1 kDa . It shares only 29% amino acid identity with the Escherichia coli ferric uptake regulator (Fur) protein, but about 64% with FurA deduced from the genomic sequences of several mycobacteria . The predicted secondary structures of FurS and FurA are highly similar and considerably divergent from those of the E . coli Fur . In contrast to some Gram-negative bacteria, within several mycobacteria an intact furA gene or a furA pseudogene is upstream of a catalase-peroxidase (katG) gene predicted to encode a functional or a non-functional (Mycobacterium leprae) enzyme . Thus the data obtained for Streptomyces reticuli are expected to serve as an additional model to elucidate the regulation of mycobacterial catalase-peroxidase genes. C R Acad Sci III, 1999 Apr, 322(4), 311 - 22 Detection of nitrosylated epitopes in Trypanosoma brucei gambiense by polyclonal and monoclonal anti-conjugated-NO-cysteine antibodies; Mnaimneh S et al.; Activated macrophages with the Calmette/Guerin bacillus (BCG) have a cytotoxic/cytostatic effect on the extracellular parasite, Trypanosoma brucei gambiense . This effect was inhibited when the NO-synthase inhibitor NG-monomethyl-L-arginine (NMMA; 0.5 mM) was added to the culture media . Using an immunocytochemical method with rabbit polyclonal or mouse monoclonal antibodies directed against conjugated nitroso-epitopes (anti-conjugated-NO-cysteine), nitrosylated antigens were visualized in fixed trypanosomes . These results suggest that NO was synthesized by the activated macrophages and that it reacted with some parasitic proteins containing cysteine . The release of NO bound to parasitic proteins may cause the killing of trypanosomes . The immunoreactivity was positive when the trypanosomes were obtained from the supernatant of the BCG-activated macrophages that contains BSA (4 mg/mL) . In contrast, the parasites cocultured with non-activated macrophages remained completely viable, and, the immunoreactivity was completely negative. Biochim Biophys Acta, 1999 Apr 19, 1427(2), 145 - 54 Enzymatic properties and deduced amino acid sequence of a high-alkaline pectate lyase from an alkaliphilic Bacillus isolate; Kobayashi T et al.; A high-alkaline pectate lyase (pectate trans-eliminase, EC 4.2.2.2.) from alkaliphilic Bacillus sp . strain KSM-P7, designated Pel-7, was purified to homogeneity . The purified Pel-7 had a molecular mass of approximately 33 kDa as determined by SDS-polyacrylamide gel electrophoresis . The isoelectric point was close to or higher than pH 10.5 . In the presence of Ca2+ ions, Pel-7 trans-eliminated polygalacturonate in random manner to generate oligogalacturonides; it exhibited optimal activity at pH 10.5 and around at 60 to 65 degrees C in glycine-NaOH buffer . Mn2+ and Sr2+ ions can serve as cofactors at almost the same level of Ca2+ ions . It also exhibited a protopectinase-like activity, liberating soluble pectin and/or oligogalacturonides from cotton fibers . The pel gene was cloned and sequenced, and the deduced amino acid sequence of mature Pel-7 (302 amino acids, 33, 355 Da) showed some conserved regions in Pel superfamily, although homology to amino acid sequences of known Pels with 27 to 32% identity . Furthermore, Pel-7 appears to have similar core structure of parallel beta-helix and active site topology with other Pels as revealed by secondary structure prediction in the Pel proteins . These results suggest that Pel-7 is basically grouped into Pel superfamily although the enzymatic and molecular properties are different. Appl Environ Microbiol, 1998 Feb, 64(2), 789 - 92 Purification and characterization of an acetyl xylan esterase from Bacillus pumilus; Degrassi G et al.; Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan . The enzyme catalyzing this reaction has been purified to homogeneity and characterized . The enzyme was secreted, and its production was induced by corncob powder and xylan . Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa . The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55 degrees C and pH 8.0 . The activity was inhibited by most of the metal ions, while no enhancement was observed . The Michaelis contant (Km) and Vmax for alpha-naphthyl acetate were 1.54 mM and 360 micromol min-1 mg of protein-1, respectively. J Appl Microbiol, 1999 Apr, 86(4), 660 - 72 Updating the H-antigen classification of Bacillus thuringiensis; Lecadet MM et al.; The classification of Bacillus thuringiensis strains has been revised and updated based on flagellar antigens which have been in use for many years . Sixty-nine serotypes and 13 sub-antigenic groups have now been identified, giving 82 serovars among the 3500 B . thuringiensis isolates of the IEBC Collection . The number of serovars has gradually increased with the total number of strains . The biochemical characters used have also been investigated and their value assessed for identification of B . thuringiensis at the subspecies level . A crystal analysis was carried out in terms of morphology, delta-endotoxin profiles and larvicidal activity for the newly identified serovars . It was found that atypical crystals, some with novel components, are becoming more common . No insect susceptible to these serovars has been discovered among known target species . The number of cross-reacting H-antigens among B . cereus strains is increasing and may be of biological significance. Lab Invest, 1999 Apr, 79(4), 379 - 86 Role of tumor necrosis factor-alpha in Mycobacterium-induced granuloma formation in tumor necrosis factor-alpha-deficient mice; Kaneko H et al.; To study the role of TNF-alpha in mycobacterial infection, we generated TNF-alpha-knockout (KO) mice, in which the third and fourth exons of the TNF-alpha gene were disrupted . The C57BL/6 KO mice were injected with virulent Mycobacterium tuberculosis strain Kurono or avirulent bacillus Calmette-Guerin (BCG) Pasteur (10(6) colony-forming units), through the tail veins . The major organs were removed at weekly intervals, and morphologic observation, assays of IL-1, IL-12, IFN-gamma, and inducible nitric oxide synthase mRNA expression, and colony counts in the lungs and spleen were performed . Peritoneal macrophages from BCG- and H37Rv strain-treated mice produced significant levels of nitric oxide after stimulation in vitro . Formation of abscesses was seen only in the Kurono-treated groups, and these abscesses contained large numbers of mycobacteria . The administration of recombinant TNF-alpha significantly ameliorated the mycobacterial lesions . IFN-gamma mRNA was expressed significantly in virulent H37Rv-treated groups with time, and the number of mycobacterial colonies per unit weight increased remarkably with time . Nitric oxide production was not observed in H37Rv-treated groups but was seen in BCG-treated groups . We concluded that TNF-alpha played an important role in protective immunity against virulent mycobacteria . Because avirulent mycobacteria did not induce granulomas in TNF-alpha-KO mice, TNF-alpha played an indirect role in granuloma formation. Arthritis Rheum, 1999 Apr, 42(4), 812 - 7 Whipple's arthritis: direct detection of Tropheryma whippelii in synovial fluid and tissue; O'Duffy JD et al.; We describe 2 patients presenting with polyarthritis in whom the synovial fluid (1 patient) or synovial tissue (1 patient) was positive for Tropheryma whippelii, the Whipple's disease-associated bacillus, when examined by polymerase chain reaction (PCR) and DNA sequencing . Histopathologic findings were consistent with articular Whipple's disease in the synovial fluid of 1 patient and the synovial tissue of the other . In both patients, bowel mucosal specimens were negative for Whipple's disease features by histologic and PCR methods . One patient was positive for T whippelii in the peripheral blood . Control synovial fluid specimens from 40 patients with other arthritides, including Lyme arthritis, were negative . Sequencing of a 284-basepair region of the 16S ribosomal RNA gene confirmed that the sequence is closely related to the known T whippelii sequence . Both patients responded to treatment with antibiotics. Hybridoma, 1999 Feb, 18(1), 99 - 102 Combined surgical and immunotherapeutic treatment of patients with fourth stage colon cancer; Tarasov VA et al.; Sixty-five patients with the fourth stage colon cancer were subjected to the combined surgical and immunotherapy . The following conclusions are made: (1) surgical elimination of the bulk of tumor mass is a necessary prerequisite for effective immunotherapy; (2) vaccination with autological tumor cells accompanied with bacille bilie de Calmette-Guerin (BCG) as the adjuvant and with interleukin-2 as the immunostimulator effectively prevents metastasizing after successful surgery; (3) the vaccine must necessary contain living tumor cells adequately presenting tumor antigens; and (4) in some cases, immunotherapy causes undesirable autoimmune complications . They can be registered by corresponding inflammation control methods. BJU Int, 1999 Mar, 83(4), 429 - 31 Intravesical bacille Calmette-Guérin in the treatment of carcinoma in situ or high-grade superficial bladder carcinoma after radiotherapy for bladder carcinoma; Palou J et al.; OBJECTIVE: To evaluate the effectiveness and tolerance of endovesical bacille Calmette-Guerin (BCG) after pelvic radiation therapy for bladder cancer in patients with recurrence as carcinoma in situ (CIS) and/or high-grade superficial bladder cancer . PATIENTS AND METHODS: In a prospective study, 13 patients were treated with weekly instillations of 81 mg BCG Connaught for 6 weeks . for CIS and/or high-grade superficial bladder carcinoma . All had been treated previously with radical radiation therapy for bladder carcinoma . RESULTS: Five patients had no recurrences and six patients retained their bladders, within a median follow-up of 74.5 months . Five patients progressed; two underwent radical surgery and are alive after 75 months of follow-up, and three died from the disease (two were high-risk surgical patients and one had metastatic disease) . Another two patients died from intercurrent disease without bladder cancer . Eight patients were alive at a mean (SD range) follow-up of 85 (12, 65-97) months . CONCLUSION: Intravesical BCG is useful for controlling CIS and/or high-grade superficial bladder carcinoma in irradiated bladders and has an acceptable local tolerance: more than a third of patients were free of disease and preserved their bladders . This proportion is acceptable in patients currently scheduled for cystectomy. J Urol, 1999 May, 161(5), 1702 - 6 Effects of acetylic salicylic acid and pentoxifylline on the efficacy of intravesical BCG therapy in orthotopic murine bladder cancer (MB49); Gunther JH et al.; PURPOSE: Intravesical immunotherapy with bacillus Calmette-Guerin (BCG), which has become the gold standard in the adjuvant treatment of superficial bladder cancer, is hampered by local side effects . Anti-inflammatory drugs may be helpful, but as an undesired side effect, therapeutic efficacy of BCG might be impaired . Therefore, we investigated the effects of anti-inflammatory drugs on the efficacy of intravesical BCG in an animal model . MATERIALS AND METHODS: Syngenic tumor cells were implanted into the bladders of 75 mice according to our modification of the method . Mice were randomized to 5 groups with 15 animals each and treated with phosphate buffered saline (PBS), group 1; BCG, group 2; BCG + acetylic salicylic acid (ASA), group 3; BCG + pentoxifylline (POF), group 4; autoclaved BCG (aBCG), group 5 . Intravesical instillation of 1.35 mg . BCG was initiated one day after tumor inoculation and repeated in weekly intervals for 4 instillations altogether . ASA and POF in doses of 200 mg./kg . and 150 mg./kg., respectively, were given continuously with the drinking water starting at the first instillation . Autoclaved BCG served as control for the importance of viability and was given at the same dose as viable BCG . Mice were monitored for survival, gross hematuria and body weight and after 28 days evaluated for bladder weight and tumor occurrence . RESULTS: Autoclaved BCG and PBS had no effect on tumor growth, whereas animals treated with viable BCG alone and in combination with POF and ASA, respectively, showed a significant reduction in bladder weight: PBS, 248 mg.; BCG, 140 mg . (p = 0.0009); BCG + ASA, 123 mg . (p = 0.0001); BCG + POF, 145 mg . (p = 0.0004); autoclaved BCG, 283 mg . (p = 0.21) . Mice treated with BCG, BCG + ASA and BCG + POF showed a significantly higher proportion of survival until day 28 as compared to PBS alone . Autoclaved BCG had no therapeutic efficacy (Kaplan-Meier method/log rank test: BCG, p = 0.0053; BCG + ASA, p = 0.0044; BCG + POF, p = 0.0027; aBCG, p = 0.33) . No significant differences among the 3 groups treated with viable BCG, with or without anti-inflammatory drugs, regarding bladder weight and survival were detectable . CONCLUSIONS: The efficacy of BCG therapy in murine orthotopic bladder cancer is dependent on BCG viability and is not compromised by ASA or POF . Clinical trials to evaluate the efficacy of routine ASA or POF to reduce BCG side effects in patients, using self-assessment criteria, should be initiated. Am J Med Sci, 1999 Apr, 317(4), 263 - 5 Bacillus popilliae endocarditis with prolonged complete heart block; Wu YJ et al.; Bacillus popilliae, a fastidious, aerobic, gram-positive, spore-forming bacillus, has never been reported as a pathogen in human infectious diseases . We report the first case of a human infected by the pathogen B . popilliae, which presented as endocarditis involving the bicuspid aortic valve and complicated with prolonged (> 30 days; to our knowledge, the longest in the literature) complete heart block . Although surgery may be warranted by previous reports, the patient was successfully managed by medical treatment instead, because of the absence of evidence from various approaches that support the existence of perivalvular extension of infection. Carbohydr Res, 1998 Dec 15, 313(3-4), 235 - 46 Transglycosylation reactions of Bacillus stearothermophilus maltogenic amylase with acarbose and various acceptors; Park KH et al.; It was observed that Bacillus stearothermophilus maltogenic amylase cleaved the first glycosidic bond of acarbose to produce glucose and a pseudotrisaccharide (PTS) that was transferred to C-6 of the glucose to give an alpha-(1-->6) glycosidic linkage and the formation of isoacarbose . The addition of a number of different carbohydrates to the digest gave transfer products in which PTS was primarily attached alpha-(1-->6) to D-glucose, D-mannose, D-galactose, and methyl alpha-D-glucopyranoside . With D-fructopyranose and D-xylopyranose, PTS was linked alpha-(1-->5) and alpha-(1-->4), respectively . PTS was primarily transferred to C-6 of the nonreducing residue of maltose, cellobiose, lactose, and gentiobiose . Lesser amounts of alpha-(1-->3) and/or alpha-(1-->4) transfer products were also observed for these carbohydrate acceptors . The major transfer product to sucrose gave PTS linked alpha-(1-->4) to the glucose residue . alpha,alpha-Trehalose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) . Maltitol gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the glucopyranose residue . Raffinose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the D-galactopyranose residue . Maltotriose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the nonreducing end glucopyranose residue . Xylitol gave PTS linked alpha-(1-->5) as the major product and D-glucitol gave PTS linked alpha-(1-->6) as the only product . The structures of the transfer products were determined using thin-layer chromatography, high-performance ion chromatography, enzyme hydrolysis, methylation analysis and 13C NMR spectroscopy . The best acceptor was gentiobiose, followed closely by maltose and cellobiose, and the weakest acceptor was D-glucitol. Res Microbiol, 1999 Mar, 150(2), 153 - 60 Phenotypic and genetic diversity among Bacillus sphaericus strains isolated in Brazil, potentially useful as biological control agents against mosquito larvae; da Silva KR et al.; Thirty mosquitocidal strains of Bacillus sphaericus isolated from different sources and localities in Brazil were characterized phenotypically and genetically to determine their relationship . Among the strains tested, 93.3% were shown to be resistant to lincomycin, 96.6% to novobiocin, 60% to chloramphenicol and all strains were resistant to streptomycin . Resistance to HgCl2, NiSO4.6H2O and CuSO4 was observed in 83.3, 86.6 and 100% of the strains, respectively . All strains were inhibited by the presence of CoSO4 . Tolerance to ethanol and variable responses to different amounts of creolin, phenol and xylol was also observed . Amplification of DNA of each of 30 isolates using repetitive primers allowed the identification of 5 groups of similar strains in BOX-PCR and 8 groups in REP-PCR . Using cloned toxin genes from B . sphaericus as probes in hybridization studies, 83% of the strains studied hybridized to the bin probe and 90% to the mtx probe . A comparison of the 30 strains by similarity matrix analysis using the data obtained in all approaches used in this study resulted in 22 groups (16 groups among the 24 high-toxicity strains) at 100% similarity, indicating a high degree of diversity among the strains tested . Some of the strains studied here, which are resistant to different stress conditions, should be considered for further ecological studies. Mol Microbiol, 1999 Mar, 31(6), 1653 - 64 Characterization and modelling of VanT: a novel, membrane-bound, serine racemase from vancomycin-resistant Enterococcus gallinarum BM4174; Arias CA et al.; Sequence determination of a region downstream from the vanXYc gene in Enterococcus gallinarum BM4174 revealed an open reading frame, designated vanT, that encodes a 698-amino-acid polypeptide with an amino-terminal domain containing 10 predicted transmembrane segments . The protein contained a highly conserved pyridoxal phosphate attachment site in the C-terminal domain, typical of alanine racemases . The protein was overexpressed in Escherichia coli, and serine racemase activity was detected in the membrane but not in the cytoplasmic fraction after centrifugation of sonicated cells, whereas alanine racemase activity was located almost exclusively in the cytoplasm . When the protein was overexpressed as a polypeptide lacking the predicted transmembrane domain, serine racemase activity was detected in the cytoplasm . The serine racemase activity was partially (64%) inhibited by D-cycloserine, whereas host alanine racemase activity was almost totally inhibited (97%) . Serine racemase activity was also detected in membrane preparations of constitutively vancomycin-resistant E . gallinarum BM4174 but not in BM4175, in which insertional inactivation of the vanC-1 D-Ala:D-Ser ligase gene probably had a polar effect on expression of the vanXYc and vanT genes . Comparative modelling of the deduced C-terminal domain was based on the alignment of VanT with the Air alanine racemase from Bacillus stearothermophilus . The model revealed that almost all critical amino acids in the active site of Air were conserved in VanT, indicating that the C-terminal domain of VanT is likely to adopt a three-dimensional structure similar to that of Air and that the protein could exist as a dimer . These results indicate that the source of D-serine for peptidoglycan synthesis in vancomycin-resistant enterococci expressing the VanC phenotype involves racemization of L- to D-serine by a membrane-bound serine racemase. Biochim Biophys Acta, 1999 Apr 14, 1418(1), 106 - 16 Stabilizing effect of an S-layer on liposomes towards thermal or mechanical stress; Mader C et al.; Isolated subunits of the crystalline cell surface layer (S-layer) protein of Bacillus stearothermophilus PV72/p2 were recrystallized on positively charged unilamellar liposomes . Liposomes were composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol and hexadecylamine (HDA) in a molar ratio of 10:5:4 and they were prepared by the dehydration-rehydration method followed by an extrusion procedure . The S-layer protein to DPPC ratio was 5.7 nmol/micromol which approximately corresponds to the theoretical value estimated by using the areas occupied by the S-layer lattice and the lipid membrane . Coating of the positively charged liposomes with S-layer protein resulted in inversion of the zeta-potential from +29.1 mV to -27.1 mV . Covalent crosslinking of the recrystallized S-layer protein was achieved with glutaraldehyde . Chemical analysis revealed that almost all amino groups (>95%) from HDA in the liposomal membrane were involved in the reaction . To study the influence of an S-layer lattice on the stability of the liposomes, the hydrophilic marker carboxyfluoresceine (CF) was encapsulated and its release was determined for plain and S-layer-coated liposomes in the course of mechanical and thermal challenges . In comparison to plain liposomes, S-layer-coated liposomes released only half the amount of enclosed CF upon exposure to shear forces or ultrasonication as mechanical stress factors . Furthermore, temperature shifts from 25 degrees C to 55 degrees C and vice versa induced considerably less CF release from S-layer-coated than from plain liposomes . A similar stabilizing effect of the S-layer lattice was observed after glutaraldehyde treatment of plain and S-layer-coated liposomes. Gen Comp Endocrinol, 1999 May, 114(2), 191 - 205 Endocrine pancreatic cells from Xenopus laevis: light and electron microscopic studies; Lozano MT et al.; Insulin, glucagon, pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), somatostatin (SST)-28 (1-12), salmon (s) SST-25, and SST-14 immunoreactivities were demonstrated in the pancreatic endocrine cells of Xenopus laevis using light and electron microscopic immunocytochemistry . Insulin-, SST-28 (1-12)/SST-14-, and PYY-immunoreactive (ir) cells were found throughout the pancreas either isolated in small clusters of a single cell type or, except in the case of PYY-ir cells, forming islets consisting of various cell types . Anti-sSST-25 serum detected the invariant SST-14 form . Cells that were only immunoreactive to glucagon were isolated or clustered in the duodenal lobe, while in the splenic lobe cells immunoreactive to both glucagon and PP were observed in isolation, clustered, or in the periphery of the islets . There were no cells that were immunoreactive only to PP or to NPY . Ultrastructurally, the endocrine cells were characterized by their secretory granules, which were immunogold labeled with the corresponding antisera . Insulin cells had large round secretory granules with a round, irregular, or crystalline-like dense core . Glucagon-ir cells had round secretory granules with a dense core and a clear halo . Glucagon/PP- and PYY-ir cells showed round, ovoid, or pear-shaped secretory granules, which were larger and less electron dense in the latter cell type . The secretory granules of SST-ir cells were ovoid or bacillary with a medium electron-dense content . A sixth cell type with very small secretory granules could only be characterized by conventional electron microscopy, since it did not immunoreact with any of the antisera applied in this study . Atherosclerosis, 1999 Mar, 143(1), 105 - 13 Immunization with bacillus Calmette-Guerin vaccine increases aortic atherosclerosis in the cholesterol-fed rabbit; Lamb DJ et al.; New Zealand White rabbits were injected subcutaneously with either a human dose of bacillus Calmette Guerin (BCG) vaccine (n = 7) or saline (n = 7) . A further half dose of BCG or saline was injected after a further 4 weeks . The animals were subsequently fed a 0.25-1% cholesterol diet for 10 weeks, 8 weeks after the first injection . The rabbits were killed and perfusion fixed with 4% paraformaldehyde . The integrated plasma cholesterol levels did not differ significantly between the groups (P > 0.05) . Plasma levels of anti-mycobacterial antibodies rose following BCG immunization, reaching a peak after 8 weeks (P < 0.05) compared to basal titers and the control group . BCG immunization was also associated with increased peripheral lymphocyte and monocyte activation, as evidenced by increased surface expression of IL-2 receptor (CD25) (P < 0.02) and MAC-I (CD11b) (P < 0.05), respectively . Significantly more mononuclear cells bound to the aortic endothelium of BCG immunized, cholesterol-fed rabbits (1.93+/-0.77 mononuclear cells/1000 endothelial cells) than to that of saline immunized rabbits (0.08+/-0.08 mononuclear cells/1000 endothelial cells; P < 0.01) . The aortic intimal:medial ratio was greater in the BCG immunized rabbits (0.19+/-0.08) than those treated with saline (0.04+/-0.03; P < 0.05) . This suggests that BCG immunization enhances peripheral leucocyte activation, aortic monocyte recruitment and atherogenesis in the cholesterol-fed rabbit. Microbiology, 1999 Jan, 145 ( Pt 1), 159 - 67 A low-molecular-mass protein from Methylococcus capsulatus (Bath) is responsible for the regulation of formaldehyde dehydrogenase activity in vitro; Tate S et al.; An 8.6 kDa protein, which the authors call a modifin, has been purified from Methylococcus capsulatus (Bath) and has been shown to alter the substrate specificity and kinetics of NAD+-linked formaldehyde dehydrogenase (FDH) isolated from the same organism . Purification methods for both the modifin and FDH are presented which reliably produced pure protein for further analysis . Analysis of the molecular mass and N-terminal sequence of both FDH and the modifin indicate that they are unique proteins and show no similarity to alcohol or aldehyde dehydrogenase enzymes isolated from methylotrophic bacteria . Substrate specificity studies demonstrated that FDH oxidized formaldehyde exclusively in the presence of the modifin; a diverse range of aldehydes and alcohols were oxidized by FDH in the absence of the modifin . No formaldehyde oxidation was detected in the absence of the modifin . Attempts to replace the modifin with glutathione or high concentrations of methanol to stimulate formaldehyde oxidation failed . With acetaldehyde as substrate, FDH showed standard Michaelis-Menten kinetics; interaction of FDH with the modifin using formaldehyde as substrate altered the kinetics of the reaction to sigmoidal . Kinetic analysis during turnover experiments indicated that the FDH may be associated with bound formaldehyde following enzyme isolation and that NAD may also be associated with the enzyme but in a form that is less tightly bound than found with the methanol dehydrogenase from Bacillus methanolicus . Data are presented which indicate that the modifin may play an important role in regulating formaldehyde concentration in vivo. Acta Trop, 1999 Mar 15, 72(2), 185 - 201 Immunochemotherapy with interferon-gamma and multidrug therapy for multibacillary leprosy; Barral-Netto M et al.; Treatment for multibacillary leprosy is presently performed with a multidrug therapy (MDT) scheme maintained for 2 years . Leprosy treatment however can benefit from the reduction of length . The lack of interferon-gamma (IFN-gamma) production by lepromatous leprosy (LL) patients' lymphocytes lead us to use this cytokine in the treatment of multibacillary leprosy associated with MDT in the treatment of multibacillary leprosy, and monitor several clinical and immunological parameters during the course of treatment . A total of 20 multibacillary leprosy patients were evaluated, 10 treated with MDT alone, and 10 treated with MDT + 10 daily doses of 2 x 10(6) international units (IU) of recombinant human IFN-gamma/m2 followed by 10 daily doses of 10(7) IU IFN-gamma/m2, intramuscularly, during the first 20 days of MDT . IFN-gamma was well tolerated and did not cause any increase in the rate of leprosy reactions development during treatment . Decrease of bacillary load, fall of anti-Mycobacterium leprae IgG serum antibodies, changes of histological pattern, as well as changes in lymphocyte proliferation assay in response to mitogens (PHA or PWM), M . leprae antigen or PPD was similar in both groups of patients . Among several soluble immunological markers measured before and 30 days after beginning of treatment, levels of soluble IL-2R receptor increased in patients treated with MDT plus IFN-gamma whereas decreased in patients treated with MDT alone . Soluble ICAM-1 levels decreased in the MDT group but did not change in the MDT + IFN-gamma treated patients . Soluble CD4 and soluble CD8 markers did not change significantly in either group of patients . Neopterin, a marker of macrophage activation, increased in all but one patient treated with MDT + IFN-gamma but in none treated with MDT alone, indicating that IFN-gamma was active in vivo . Our findings indicate that despite being able to promote macrophage activation in multibacillary leprosy patients a short course of systemically administered IFN-gamma is not able to change the clinical course of a long standing disease such as leprosy. Biochem Mol Biol Int, 1999 Feb, 47(2), 171 - 5 Cloning of a tellurite resistance determinant from Bacillus stearothermophilus V in Escherichia coli; Vasquez C et al.; A potassium tellurite-resistance determinant was isolated from Bacillus stearothermophilus V and cloned in Escherichia coli . Transformed cells formed black colonies when grown on solid media containing permissive tellurite concentrations . The resistance determinant was contained in a B . stearothermophilus V chromosomal DNA fragment of 7 kb. Probl Tuberk, 1999, (1), 9 - 12 {Results of DOTS use in Russia}; Grishina TA; At present, the DOT programme has been introduced in Russia, which is supported by the following foreign organizations: MERLIN (British non-governmental agency; WHO, Finnish Association of Pulmonary Diseases; Norwegian Association of Patients with Tuberculosis; New York Institute of Health, "Physicians Without Borders" (Belgium) . The main point of the programme is to identify bacillary patients with tuberculosis in the clinical diagnostic laboratories among those who see doctors for complaints of suspected tuberculosis . While implementing the programme, the work of a general health care laboratory could be activated in identifying patients with tuberculosis. Probl Tuberk, 1999, (1), 4 - 8 {DOTS strategy and its application in Russia}; Khomenko AG; Leading principles of DOTS strategy for Russia are outlined . It has been introduced in Russia since 1994 in Ivanovo, Tomsk Regions, Mary El Republic . New territories (Leningrad and Arkhangelsk Regions) have recently joined the project . Methods of detecting bacillary patients, scheme of chemotherapy for different tuberculosis patients and of bacteriological and x-ray follow-up control are presented . Positive aspects of the program are analyzed as well as causes of its insufficient benefit under conditions of Russian Federation. Infect Dis Clin North Am, 1999 Mar, 13(1), 169 - 85, vii-viii New vaccines against tuberculosis . The status of current research; Orme IM; The increasing realization that the current vaccine for tuberculosis, bacille Calmette-Guerin (BCG), is of varying effectiveness, and is less protective in adults than in children, has prompted new research for a replacement . New research has resulted in innovative approaches, including the use of sub-unit vaccines, auxotropic vaccines, DNA vaccines, and recombinant vaccines, among others . This article reviews these approaches and test results in animal models, and discusses their potential for use in humans. Structure Fold Des, 1999 Apr 15, 7(4), 435 - 48 Crystal structures of Bacillus caldovelox arginase in complex with substrate and inhibitors reveal new insights into activation, inhibition and catalysis in the arginase superfamily; Bewley MC et al.; BACKGROUND: Arginase is a manganese-dependent enzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea . In ureotelic animals arginase is the final enzyme of the urea cycle, but in many species it has a wider role controlling the use of arginine for other metabolic purposes, including the production of creatine, polyamines, proline and nitric oxide . Arginase activity is regulated by various small molecules, including the product L-ornithine . The aim of these structural studies was to test aspects of the catalytic mechanism and to investigate the structural basis of arginase inhibition . RESULTS: We report here the crystal structures of arginase from Bacillus caldovelox at pH 5.6 and pH 8.5, and of binary complexes of the enzyme with L-arginine, L-ornithine and L-lysine at pH 8.5 . The arginase monomer comprises a single compact alpha/beta domain that further associates into a hexameric quaternary structure . The binary complexes reveal a common mode of ligand binding, which places the substrate adjacent to the dimanganese centre . We also observe a conformational change that impacts on the active site and is coupled with the occupancy of an external site by guanidine or arginine . CONCLUSIONS: The structures reported here clarify aspects of the active site and indicate key features of the catalytic mechanism, including substrate coordination to one of the manganese ions and an orientational role for a neighboring histidine residue . Stereospecificity for L-amino acids is found to depend on their precise recognition at the active-site rim . Identification of a second arginine-binding site, remote from the active site, and associated conformational changes lead us to propose a regulatory role for this site in substrate hydrolysis. Structure Fold Des, 1999 Apr 15, 7(4), 399 - 411 The crystal structure of pyroglutamyl peptidase I from Bacillus amyloliquefaciens reveals a new structure for a cysteine protease; Odagaki Y et al.; BACKGROUND: The N-terminal pyroglutamyl (pGlu) residue of peptide hormones, such as thyrotropin-releasing hormone (TRH) and luteinizing hormone releasing hormone (LH-RH), confers resistance to proteolysis by conventional aminopeptidases . Specialized pyroglutamyl peptidases (PGPs) are able to cleave an N-terminal pyroglutamyl residue and thus control hormonal signals . Until now, no direct or homology-based three-dimensional structure was available for any PGP . RESULTS: The crystal structure of pyroglutamyl peptidase I (PGP-I) from Bacillus amyloliquefaciens has been determined to 1.6 A resolution . The crystallographic asymmetric unit of PGP-I is a tetramer of four identical monomers related by noncrystallographic 222 symmetry . The protein folds into an alpha/beta globular domain with a hydrophobic core consisting of a twisted beta sheet surrounded by five alpha helices . The structure allows the function of most of the conserved residues in the PGP-I family to be identified . The catalytic triad comprises Cys144, His168 and Glu81 . CONCLUSIONS: The catalytic site does not have a conventional oxyanion hole, although Cys144, the sidechain of Arg91 and the dipole of an alpha helix could all stabilize a negative charge . The catalytic site has an S1 pocket lined with conserved hydrophobic residues to accommodate the pyroglutamyl residue . Aside from the S1 pocket, there is no clearly defined mainchain substrate-binding region, consistent with the lack of substrate specificity . Although the overall structure of PGP-I resembles some other alpha/beta twisted open-sheet structures, such as purine nucleoside phosphorylase and cutinase, there are important differences in the location and organization of the active-site residues . Thus, PGP-I belongs to a new family of cysteine proteases. J Bacteriol, 1999 Apr, 181(8), 2624 - 30 Correlation of 16S ribosomal DNA signature sequences with temperature-dependent growth rates of mesophilic and psychrotolerant strains of the Bacillus cereus group; Pruss BM et al.; Sequences of the 16S ribosomal DNA (rDNA) from psychrotolerant and mesophilic strains of the Bacillus cereus group revealed signatures which were specific for these two thermal groups of bacteria . Further analysis of the genomic DNA from a wide range of food and soil isolates showed that B . cereus group strains have between 6 and 10 copies of 16S rDNA . Moreover, a number of these environmental strains have both rDNA operons with psychrotolerant signatures and rDNA operons with mesophilic signatures . The ability of these isolates to grow at low temperatures correlates with the prevalence of rDNA operons with psychrotolerant signatures, indicating specific nucleotides within the 16S rRNA to play a role in psychrotolerance. J Laryngol Otol, 1998 Nov, 112(11), 1038 - 41 Study of ethmoid sinus involvement in multibacillary leprosy; Srinivasan S et al.; The nasal mucosal involvement in lepromatous leprosy is well recognized . Currently interest has centred around the involvement of paranasal sinuses in leprosy . They act as a reservoir and constant source of reinfection to the nasal mucosa . In the present prospective study 25 untreated patients with multi-bacillary leprosy were included . Clinical examination, computed tomography (CT) scan of paranasal sinuses, ethmoid sinus endoscopy and biopsy were carried out in all patients, to investigate the involvement of the paranasal sinuses in leprosy . Ethmoid sinus involvement was noted in 20 patients on CT scan . Bilateral involvement was more common (65 per cent) . Anterior ethmoids were more commonly affected (65 per cent) . On ethmoid sinus endoscopy abnormal mucosa was noted in 17 patients (68 per cent) . Ethmoid sinus biopsy was confirmative in 16 patients (64 per cent) . Statistically significant correlation was found between CT findings, sinus endoscopy and sinus biopsy findings. Lett Appl Microbiol, 1999 Mar, 28(3), 221 - 5 Detection of toxigenic strains of Bacillus cereus and other Bacillus spp . with an improved cytotoxicity assay; Beattie SH et al.; An improved qualitative cell cytotoxicity assay for the detection of Bacillus cereus emetic and enterotoxin is described . The presence of toxin in culture supernatant fluids was detected by measurement with the tetrazolium salt MTT, as it adversely affects the metabolic status of cultured CHO cells . Psychrotrophic B . cereus isolates (65) were assessed for toxin production using the cytotoxicity assay, and 91% of culture supernatant fluids were cytotoxic . Toxin assessment using BCET-RPLA and ELISA immunoassays indicated that 51% and 85% of the cultures, respectively, were toxigenic . There were pronounced strain differences in the amount of toxin produced by the B . cereus isolates . Some isolates of B . circulans, B . laterosporus/cereus, B . lentus, B . licheniformis, B . mycoides, B . subtilis and B . thuringiensis were also toxigenic. J Appl Microbiol, 1999 Mar, 86(3), 477 - 86 Unique activity associated with non-insecticidal Bacillus thuringiensis parasporal inclusions: in vitro cell-killing action on human cancer cells; Mizuki E et al.; Parasporal inclusion proteins from a total of 1744 Bacillus thuringiensis strains, consisting of 1700 Japanese isolates and 44 reference type strains of existing H serovars, were screened for cytocidal activity against human leukaemia T cells and haemolytic activity against sheep erythrocytes . Of 1684 B . thuringiensis strains having no haemolytic activity, 42 exhibited in vitro cytotoxicity against leukaemia T cells . These non-haemolytic but leukaemia cell-toxic strains belonged to several H-serovars including dakota, neoleonensis, shandongiensis, coreanensis and other unidentified serogroups . Purified parasporal inclusions of the three selected strains, designated 84-HS-1-11, 89-T-26-17 and 90-F-45-14, exhibited no haemolytic activity and no insecticidal activity against dipteran and lepidopteran insects, but were highly cytocidal against leukaemia T cells and other human cancer cells, showing different toxicity spectra and varied activity levels . Furthermore, the proteins from 84-HS-1-11 and 89-T-26-17 were able to discriminate between leukaemia and normal T cells, specifically killing the former cells . These findings may lead to the use of B . thuringiensis inclusion proteins for medical purposes. Chin J Biotechnol, 1998, 14(2), 59 - 65 Expression of delta-endotoxin cryIA(c) gene of Bacillus thuringiensis in Escherichia coli and Streptomyces lividans; Yang R et al.; The cryIA(c) gene of Bacillus thuringiensis was isolated from plasmid pOS1000 . In order to obtain a proper cloning site and open reading frame, some DNA sequences preceding the initiating codon of the gene were replaced by synthetic oligonucleotide sequences . The isolated cryIA(c) was cloned into E . coli expression vector pKK223-3, and production of CryIA(c) protein was detected after induction by IPTG . A recombinant plasmid, pHZ1256, was constructed by insertion of the cryIA(c) gene into Streptomyces vector pHZ1272 . pHZ1256 was introduced into Streptomyces lividans, and the production of CryIA(c) protein was confirmed by Western blotting after thiostrepton induction . A bioassy experiment showed that the CryIA(c) protein produced by E . coli and S . lividans caused 93% and 57% mortality to Plutella xylostella, respectively. J Struct Biol, 1999 Mar, 125(1), 19 - 24 Structural studies of a novel germination protease from spores of Bacillus megaterium; Ponnuraj K et al.; The amino acid sequence-specific protease (termed GPR) in the bacterium Bacillus megaterium initiates the rapid degradation of small, acid-soluble spore proteins during the germination of spores of this organism . GPR is synthesized during spore formation as an inactive zymogen termed P46, which later autoprocesses to a smaller active form termed P41, which acts during spore germination . However, GPR exhibits no obvious mechanistic or amino acid sequence similarity to any of the known classes of proteases . To initiate the determination of the mechanisms of P46 to P41 conversion, P46 inactivity, and P41 catalysis, B . megaterium GPR has been overexpressed in Escherichia coli and purified to homogeneity by anion-exchange and size exclusion chromatography, and crystals of both P46 and P41 have been obtained by the vapor diffusion method . P46 crystals diffracted x rays to 3.5 A but the crystals of P41 diffracted x rays to only 6.5 A . A native x-ray diffraction data set of P46 has been collected; the unit cell parameters are a = b = 76.8, c = 313.1 A, alpha = beta = gamma = 90 degrees; the space group is tetragonal P41212 or P43212 . The asymmetric unit contains two monomeric molecules with a crystal volume per unit protein mass of 2 . 85 A3/Da and a solvent content of about 57% . An isomorphous heavy atom derivative data set has also been obtained for P46 crystals with potassium dicyanoaurate (I) . Int Urol Nephrol, 1998, 30(6), 713 - 22 Possible factors affecting response to intravesical bacillus Calmette-Guérin (Tokyo 172 strain) therapy for carcinoma in situ of the bladder: a multivariate analysis; Takashi M et al.; To evaluate clinicopathological factors affecting response to intravesical instillation therapy with the bacillus Calmette-Guerin (BCG) Tokyo 172 strain for carcinoma in situ (CIS) of the bladder, we reviewed data for 84 patients treated between 1985 and 1996 . Median follow-up was 56 months . The patients comprised three groups: primary (only the in situ lesion, 31 patients), subsequent (found after treatment of a gross neoplasm, 20), and concomitant (found together with a gross neoplasm, 33) . A complete response was found in 62 (74%) of the 84 patients . Intravesical BCG therapy eradicated tumour cells in 74% of the primary group, 70% of the subsequent group, and 76% of the concomitant group . Multivariate logistic regression analysis revealed that the presence of gross haematuria and patient age were significantly associated with a complete response to the intravesical BCG therapy (p<0.05) . On the other hand, gender, irritative bladder symptoms, type of extent of CIS, histological grade of CIS, BCG dose, and number of times BCG was given did not exert any significant influence . The 5-year recurrence rate was 33% for the 62 patients for whom a complete response was once achieved . Patients aged 60 or older had a higher probability of recurrence than those less than 60 years of age (p<0.05) . Disease progression was found in 13% of the 84 patients and total cystectomy was performed in 19% . The present finding that patient age is related to the response to intravesical BCG therapy may point to a role for the reduced host immunocompetence in elderly individuals. Vaccine, 1999 Mar 5, 17(9-10), 1272 - 81 Construction and murine immunogenicity of recombinant Bacille Calmette Guérin vaccines expressing the B subunit of Escherichia coli heat labile enterotoxin; Hayward CM et al.; Three recombinant strains of Mycobacterium bovis Bacille Calmette Guerin (rBCG) were prepared in which the immunogenic B subunit of human Escherichia coli heat labile enterotoxin (LT-Bh) was expressed either as a cytoplasm protein, a cell wall associated lipoprotein or a secreted protein . Intraperitoneal immunisation of mice with these rBCG induced IgG and IgA antibodies to LT-Bh and shifted the serum IgG subclass response to subsequent challenge with purified LT-Bh from IgG1 to an IgG2a . Oral administration of recombinant BCG induced mucosal and serum IgA antibodies to LT-Bh which peaked four months after immunisation . Antibody responses were greater when LT-Bh was expressed as a secreted protein or lipoprotein rather than in the cytoplasm . Oral vaccination with recombinant BCG may be an effective approach, particularly to induce mucosal IgA and prime for a serum TH1 recall response. Biochemistry, 1999 Apr 6, 38(14), 4613 - 9 Thermodynamic stability of a cold-active alpha-amylase from the Antarctic bacterium Alteromonas haloplanctis; Feller G et al.; The thermal stability of the cold-active alpha-amylase (AHA) secreted by the Antarctic bacterium Alteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and differential scanning calorimetry . It was found that this heat-labile enzyme is the largest known multidomain protein exhibiting a reversible two-state unfolding, as demonstrated by the recovery of DeltaHcal values after consecutive calorimetric transitions, a DeltaHcal/DeltaHeff ratio close to unity, and the independence of unfolding thermodynamic parameters of scan rates . By contrast, the mesophilic alpha-amylases investigated here (from porcine pancreas, human salivary glands, yellow meal beetle, Bacillus amyloliquefaciens, and Bacillus licheniformis) unfold irreversibly according to a non-two-state mechanism . Unlike mesophilic alpha-amylases, the melting point of AHA is independent of calcium and chloride binding while the allosteric and structural functions of these ions are conserved . The thermostability of AHA at optimal conditions is characterized by a Tm of 43.7 degrees C, a DeltaHcal of 238 kcal mol-1, and a DeltaCp of 8.47 kcal mol-1 K-1 . These values were used to calculate the Gibbs free energy of unfolding over a wide range of temperatures . This stability curve shows that (a) the specific DeltaGmax of AHA {22 cal (mol of residue)-1} is 4 times lower than that of mesophilic alpha-amylases, (b) group hydration plays a crucial role in the enzyme flexibility at low temperatures, (c) the temperature of cold unfolding closely corresponds to the lower limit of bacterial growth, and (d) the recombinant heat-labile enzyme can be expressed in mesophilic hosts at moderate temperatures . It is also argued that the cold-active alpha-amylase has evolved toward the lowest possible conformational stability of its native state. Biochemistry, 1999 Apr 6, 38(14), 4403 - 8 Catalytic cycle of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus . Solvent viscosity, deuterium isotope effects, and proton inventory studies; Martin SF et al.; The phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLCBc) is a 28.5 kDa enzyme with three zinc ions in its active site . Although much is known about the roles that various PLCBc active site amino acids play in binding and catalysis, there is little information about the rate-determining step of the PLCBc-catalyzed hydrolysis of phospholipids and the catalytic cycle of the enzyme . To gain insight into these aspects of the hydrolysis, solvent viscosity variation experiments were conducted to determine whether an external step (substrate binding or product release) or an internal step (hydrolysis) is rate-limiting . The data indicate that the PLCBc-catalyzed reaction is unaffected by changes in solvent viscosity . This observation is inconsistent with the notion of substrate binding or product release being rate-determining and supports the hypothesis that a chemical step is rate-limiting . Furthermore, a deuterium isotope effect of 1.9 and a linear proton inventory plot indicate one proton is transferred in the rate-determining step . These data may be used to formulate a comprehensive catalytic cycle that is for the first time based on experimental evidence . In this mechanism, Asp55 of PLCBc activates an active site water molecule for attack on the phosphodiester bond, the hydrolysis of which is rate-limiting . The phosphorylcholine product is the first to leave the active site, followed by diacylglycerol. Biochemistry, 1999 Mar 30, 38(13), 4128 - 36 Nativelike structure and stability in a truncation mutant of a protein minidomain: the peripheral subunit-binding domain; Spector S et al.; Despite its small size, the peripheral subunit-binding domain from the dihydrolipoamide acetyltransferase component of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex adopts a unique, compact structure . To determine whether the full 43 residue sequence is required for the domain to adopt a stable, nativelike structure, 3 proteins of different lengths were prepared . Psbd41 corresponds to residues 3-43 of the domain, psbd36 spans residues 6-41, and psbd33 comprises residues 7-39 . Psbd41 folds in a cooperative, two-state fashion with a Tm of 53 degrees C and a stability at 25 degrees C of 2.2 kcal mol-1 . Psbd36 is nearly as stable with a Tm of 48 degrees C and a stability of 1.8 kcal mol-1 . Similar m-values and heat capacities suggest that psbd36 and psbd41 bury approximately the same surface area . Minimal differences in CalphaH and NH chemical shifts between psbd41 and psbd36 show that the two sequences adopt the same tertiary fold . On a per residue basis, DeltaH degrees and DeltaC degrees p fall within the range typical for single-domain globular proteins . Psbd33 is significantly less stable . It is not fully folded at 25 degrees C, and at all temperatures it shows broadened NMR lines . ANS titrations provide evidence that this is due to an equilibrium between nativelike and unfolded molecules rather than formation of a molten globule . The fraction of psbd33 molecules which are folded appear to adopt the same structure as the full-length domain . Thus, although more than the 33 residue core is required to form a fully stable native structure, the entire sequence is not required for folding. Biochemistry, 1999 Mar 30, 38(13), 4121 - 7 Transition state of the rate-limiting step of heat denaturation of Cry3A delta-endotoxin; Potekhin SA et al.; Heat denaturation of Cry3A delta-endotoxin from Bacillus thuringiensis var . tenebrionis and its 55 kDa fragment was studied by differential scanning microcalorimetry at low pH . Analysis of the calorimetric data has shown that denaturation of Cry3A delta-endotoxin is a nonequilibrium process at heating rates from 0 . 125 to 2 K/min . This means that the stability of delta-endotoxin (the apparent temperature of denaturation Tm) under these conditions is under kinetic control rather than under thermodynamic control . It has been shown that heat denaturation of this protein is a one-step kinetic process . The enthalpy of the process and its activation energy were measured as functions of temperature . The data obtained allow confirmation of the fact that the conformation of delta-endotoxin at the transition state only slightly differs from its native conformation with respect to compactness and extent of hydration . The comparison of the activation energy for intact delta-endotoxin and the 55 kDa fragment showed that the transition of the molecule to a transition state does not cause any changes in the conformation of three N-terminal alpha-helices . Complete removal of the N-terminal domain of delta-endotoxin and 40 amino acids from the C-terminus beta-sheet domain III causes an irreversible loss of the tertiary structure . Thus, during protein folding the nucleation core determining protein stability does not involve its three initial alpha-helices but may include the remaining alpha-helices of the N-terminal domain . The functional significance of peculiarities of structure arrangement of the delta-endotoxin molecule is discussed. Biochemistry, 1999 Mar 30, 38(13), 4058 - 65 Evidence for a two-base mechanism involving tyrosine-265 from arginine-219 mutants of alanine racemase; Sun S et al.; A positively charged residue, R219, was found to interact with the pyridine nitrogen of pyridoxal phosphate in the structure of alanine racemase from Bacillus stearothermophilus {Shaw et al . (1997) Biochemistry 36, 1329-1342} . Three site-directed mutants, R219K, R219A, and R219E, have been characterized and compared to the wild type enzyme (WT) to investigate the role of R219 in catalysis . The R219K mutation is functionally conservative, retaining approximately 25% of the WT activity . The R219A and R219E mutations decrease enzyme activity by approximately 100- and 1000-fold, respectively . These results demonstrate that a positively charged residue at this position is required for efficient catalysis . R219 and Y265 are connected through H166 via hydrogen bonds . The R219 mutants exhibit similar kinetic isotope effect trends: increased primary isotope effects (1.5-2-fold) but unchanged solvent isotope effects in the L --> D direction and increased solvent isotope effects (1.5-2-fold) but unchanged primary isotope effects in the D --> L direction . These results support a two-base racemization mechanism involving Y265 and K39 . They additionally suggest that Y265 is selectively perturbed by R219 mutations through the H166 hydrogen-bond network . pH profiles show a large pKa shift from 7.1-7.4 (WT and R219K) to 9 . 5-10.4 (R219A and R219E) for kcat/KM, and from 7.3 to 9.9-10.4 for kcat . The group responsible for this ionization is likely to be the phenolic hydroxyl of Y265, whose pKa is electrostatically perturbed in the WT by the H166-mediated interaction with R219 . Accumulation of an absorbance band at 510 nm, indicative of a quinonoid intermediate, only in the D --> L direction with R219E provides additional evidence for a two-base mechanism involving Y265. An Med Interna, 1999 Feb, 16(2), 59 - 64 {The risk-benefit of the treatment of tuberculosis with a high bacilloscopy sensitivity in a provincial hospital}; Geijo Martinez MP et al.; BACKGROUND: To know the incidence and type of hepatic toxicity (HTX) of the tuberculous chemotherapy and to value the risk-benefit of treatment in our elderly population in a high sensibility context of the bacilloscopy . PATIENTS AND METHODS: Prospective study of 161 tuberculous patients with standards of 6 months, from January 1989 to December 1994 . 75 patients with (INH, FR, PZ and ETB) and 83 patients with (INH, RF and PZ) . It was accomplished clinical, analytical and microbiological control to all the patients during 24 months . RESULTS: 28% of the patients had more than 65 years and a 26% HIV infection . The tuberculosis (TBC) was disseminated in a 41% . A 74% of the patients ha positive bacilloscopy . The therapeutic fulfillment was correct in a 85% of the cases . A 48% of HTX was observed, with a 9% of serious HTX (associated with alcoholism and age greater tan 60 years) . In 14% of he patients was changed in a way definitive the therapeutic standard . There was a 17% of therapeutic failure (associated with disseminated TBC and HIV infection) and a 7% of relapses . The attributive mortality of TBC was of a 4% . CONCLUSIONS: The transient and moderate increase in transaminase activity is frequent and it does not require to modify the chemotherapy . In the greater patients of 65 years the benefit of trying outweigh the risk, if is accomplished a narrow follow-up with precocious suspension of the drugs in the event of serious toxicity. Biosci Biotechnol Biochem, 1999 Feb, 63(2), 452 - 5 Sequence analysis of a 32-kb region including the major ribosomal protein gene clusters from alkaliphilic Bacillus sp . strain C-125; Takami H et al.; Forty-one open reading frames (ORFs) were identified in a 32-kb DNA fragment of alkaliphilic Bacillus sp . C-125 . A similarity search using the BSORF database found 37 ORFs with significant sequence similarity to B . subtilis RNA polymerase subunits, elongation factor G, elongation factor Tu, and ribosomal proteins . Each ORF product showed more than 70% identity to those of B . subtilis . Gene organization in the region of str, S10, spc, and the alpha cluster was highly conserved among three strains, C-125, B . subtilis, and B . stearothermophilus. Nat Genet, 1999 Apr, 21(4), 370 - 8 A human IFNGR1 small deletion hotspot associated with dominant susceptibility to mycobacterial infection; Jouanguy E et al.; The immunogenetic basis of severe infections caused by bacille Calmette-Guerin vaccine and environmental mycobacteria in humans remains largely unknown . We describe 18 patients from several generations of 12 unrelated families who were heterozygous for 1 to 5 overlapping IFNGR1 frameshift small deletions and a wild-type IFNGR1 allele . There were 12 independent mutation events at a single mutation site, defining a small deletion hotspot . Neighbouring sequence analysis favours a small deletion model of slipped mispairing events during replication . The mutant alleles encode cell-surface IFNgamma receptors that lack the intra-cytoplasmic domain, which, through a combination of impaired recycling, abrogated signalling and normal binding to IFNgamma exert a dominant-negative effect . We thus report a hotspot for human IFNGR1 small deletions that confer dominant susceptibility to infections caused by poorly virulent mycobacteria. Eur J Clin Microbiol Infect Dis, 1999 Jan, 18(1), 23 - 9 Randomised study of the possible adjuvant effect of BCG vaccine on the immunogenicity of diphtheria-tetanus-acellular pertussis vaccine in Senegalese infants; Simondon F et al.; Following a study in Senegal (1990-1995) in which the relative efficacy of a diphtheria-tetanus-acellular pertussis vaccine (DTaP) was compared with that of a diphtheria-tetanus-whole-cell pertussis vaccine in children given a simultaneous injection of Bacille Calmette-Guerin (BCG) vaccine, this subsequent study was conducted to evaluate the possible adjuvant effect of the BCG vaccine on acellular pertussis vaccine components . A second objective was to compare the immunogenicity of these components when administered in accordance with a 2-4-6-month (spaced) schedule or an accelerated 2-3-4-month schedule . In all, 390 healthy Senegalese infants were randomly divided into three groups of 130 infants . Antibodies to acellular pertussis components were measured in serum samples obtained within 2 days of the first DTaP dose and 1 month after the third dose . BCG vaccine, given simultaneously with the DTaP vaccine, did not influence the immunogenicity of the acellular pertussis vaccine components when compared with separate administration of the two vaccines . Infants immunised according to a 2-4-6-month schedule had a significantly higher immune response than those immunised according to a 2-3-4-month schedule with respect to the response to pertussis toxoid assessed by seroneutralisation on Chinese hamster ovary cells (P<0.0001) . These results suggest that BCG and DTaP vaccines can be given simultaneously without interference or enhancement and that more optimal immunogenicity is achieved with an extended than with an accelerated schedule. Biochem J, 1999 Apr 15, 339 ( Pt 2), 371 - 9 Roles of key active-site residues in flavocytochrome P450 BM3; Noble MA et al.; The effects of mutation of key active-site residues (Arg-47, Tyr-51, Phe-42 and Phe-87) in Bacillus megaterium flavocytochrome P450 BM3 were investigated . Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate: R47A mutant, Km 859 microM, kcat 3960 min-1; Y51F mutant, Km 432 microM, kcat 6140 min-1; wild-type, Km 288 microM, kcat 5140 min-1) . A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (DeltaG) resulting from a smaller DeltaG of substrate binding . The side chain of Phe-42 acts as a phenyl 'cap' over the mouth of the substrate-binding channel . With mutant F42A, Km is massively increased and kcat is decreased for oxidation of both laurate (Km 2 . 08 mM, kcat 2450 min-1) and arachidonate (Km 34.9 microM, kcat 14620 min-1; compared with values of 4.7 microM and 17100 min-1 respectively for wild-type) . Amino acid Phe-87 is critical for efficient catalysis . Mutants F87G and F87Y not only exhibit increased Km and decreased kcat values for fatty acid oxidation, but also undergo an irreversible conversion process from a 'fast' to a 'slow' rate of substrate turnover {for F87G (F87Y)-catalysed laurate oxidation: kcat 'fast', 760 (1620) min-1; kcat 'slow', 48.0 (44.6) min-1; kconv (rate of conversion from fast to slow form), 4.9 (23.8) min-1} . All mutants showed less than 10% uncoupling of NADPH oxidation from fatty acid oxidation . The rate of FMN-to-haem electron transfer was shown to become rate-limiting in all mutants analysed . For wild-type P450 BM3, the rate of FMN-to-haem electron transfer (8340 min-1) is twice the steady-state rate of oxidation (4100 min-1), indicating that other steps contribute to rate limitation . Active-site structures of the mutants were probed with the inhibitors 12-(imidazolyl)dodecanoic acid and 1-phenylimidazole . Mutant F87G binds 1-phenylimidazole >10-fold more tightly than does the wild-type, whereas mutant Y51F binds the haem-co-ordinating fatty acid analogue 12-(imidazolyl)dodecanoic acid >30-fold more tightly than wild-type. Biochem J, 1999 Apr 15, 339 ( Pt 2), 309 - 17 Prediction and experimental testing of Bacillus acidocaldarius thioredoxin stability; Pedone E et al.; In order to investigate further the determinants of protein stability, four mutants of thioredoxin from Bacillus acidocaldarius were designed: K18G, R82E, K18G/R82E, and D102X, in which the last four amino acids were deleted . The mutants were constructed on the basis of molecular dynamic studies and the prediction of the structure of thioredoxin from B . acidocaldarius, performed by a comparative molecular modelling technique using Escherichia coli thioredoxin as the reference protein . The mutants obtained by PCR strategy were expressed in E . coli and then characterized . CD spectroscopy, spectrofluorimetry and thermodynamic comparative studies permitted comparison of the relative physicochemical behaviour of the four proteins with that of the wild-type protein . As predicted for the molecular dynamic analysis at 500 K in vacuo, the wild-type structure was more stable than that of the mutants; in fact the Tm of the four proteins showed a decrease of about 15 degrees C for the double and the truncated mutants, and a decrease of about 12 degrees C for the single mutants . A difference in the resistance of the proteins to denaturants such as guanidine HCl and urea was revealed; the wild-type protein always proved to be the most resistant . The results obtained show the importance of hydrogen bonds and ion pairs in determining protein stability and confirm that simulation methods are able to direct protein engineering in site-directed mutagenesis. Mol Gen Mikrobiol Virusol, 1999, (1), 37 - 8 {Plasmid pCL1 and antimicrobial activity of a strain of Bacillus sp . 62}; Pogosian GP et al.; The 60 kb plasmid pCL1 isolated from Bacillus sp . 62 confers antibacterial activity . Bacteria cured from pCL1 by ethidium bromide treatment loose the sign . Transformation of cured from by pCL1 restores the initial phenotype, while plasmid transfer into B . subtilis 168 does not confer antibacterial activity, which indicates the significance of chromosome locuses in the formation of this sign. Syst Appl Microbiol, 1999 Feb, 22(1), 133 - 44 Evaluation of methods for recognising strains of the Bacillus cereus group with food poisoning potential among industrial and environmental contaminants; Pirttijarvi TS et al.; Toxin production, biochemical properties and ribotypes of Bacillus cereus group (B . cereus, B . thuringiensis, B . mycoides) strains originating from industrial and environmental sources (n = 64), from food poisoning incidents (n = 22) and from reference sources (n = 7) were analysed . Forty ribotypes were found among the 93 strains . Eleven strains from food poisoning incidents produced emetic (mitochondrio) toxin, as determined by the boar spermatozoa toxicity test . These strains possessed closely similar ribotypes which were rare among strains of other origins . Sperm toxin producing (cereulide positive) strains did not hydrolyse starch and did not produce haemolysin BL, as determ