Bacillus

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 determined by the reverse passive latex agglutination test . Sixteen different ribotypes were found among B . cereus strains from board machines (n = 16) and from packaging board (n = 16), indicating many different sources of B . cereus contamination in board mills . Strains originating from packaging board had predominantly different ribotypes from those of dairy and dairy product originating strains . Nine (53%) out of 17 strains from a single dairy process shared the same ribotype whereas strains from milk and milk products from different dairies had different ribotypes indicating that B . cereus group populations were dairy specific . Twenty-two percent of strains isolated from the paperboard industry on non-selective medium were lecithinase negative, including enterotoxin producing strains . This stresses the importance of other detection methods not based on a positive lecithinase reaction.

Cancer Detect Prev, 1999, 23(2), 163 - 71
Biomarkers in monitoring for efficacy of immunotherapy and chemoprevention of bladder cancer with dimethylsulfoxide; Hemstreet GP 3rd et al.; This study correlated biomarkers expressed in tumor and epithelial field with clinical response and recurrence . Of 25 bladder cancer patients, 11 received 6 weeks of intravesical Bacille Calmette-Guerin (BCG), and 14 were treated weekly with intravesical dimethylsulfoxide (DMSO) for 4 weeks to further modulate biomarker expression . G-actin, DNA aneuploidy, and p300 tumor antigen were evaluated by quantitative fluorescence image analysis on uroepithelial cells from bladder wash samples prior to and immediately following treatment . Excluding patients who did not respond to BCG (and who had persistently abnormal p300 and DNA markers), recurrence correlated with persistent abnormal G-actin findings . Of patients who were G-actin negative following therapy, only 25% recurred during follow-up in contrast to 67% in patients who were positive (p < 0.03 by Fisher's exact test) . The odds ratio for recurrence was 6.00 (95% confidence interval: 1.3-28.6) . Cytosolic G-actin levels can be an important intermediate end point marker for chemoprevention.

Appl Environ Microbiol, 1999 Apr, 65(4), 1413 - 9
Integrative model for binding of Bacillus thuringiensis toxins in susceptible and resistant larvae of the diamondback moth (Plutella xylostella); Ballester V et al.; Insecticidal crystal proteins from Bacillus thuringiensis in sprays and transgenic crops are extremely useful for environmentally sound pest management, but their long-term efficacy is threatened by evolution of resistance by target pests . The diamondback moth (Plutella xylostella) is the first insect to evolve resistance to B . thuringiensis in open-field populations . The only known mechanism of resistance to B . thuringiensis in the diamondback moth is reduced binding of toxin to midgut binding sites . In the present work we analyzed competitive binding of B . thuringiensis toxins Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F to brush border membrane vesicles from larval midguts in a susceptible strain and in resistant strains from the Philippines, Hawaii, and Pennsylvania . Based on the results, we propose a model for binding of B . thuringiensis crystal proteins in susceptible larvae with two binding sites for Cry1Aa, one of which is shared with Cry1Ab, Cry1Ac, and Cry1F . Our results show that the common binding site is altered in each of the three resistant strains . In the strain from the Philippines, the alteration reduced binding of Cry1Ab but did not affect binding of the other crystal proteins . In the resistant strains from Hawaii and Pennsylvania, the alteration affected binding of Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F . Previously reported evidence that a single mutation can confer resistance to Cry1Ab, Cry1Ac, and Cry1F corresponds to expectations based on the binding model . However, the following two other observations do not: the mutation in the Philippines strain affected binding of only Cry1Ab, and one mutation was sufficient for resistance to Cry1Aa . The imperfect correspondence between the model and observations suggests that reduced binding is not the only mechanism of resistance in the diamondback moth and that some, but not all, patterns of resistance and cross-resistance can be predicted correctly from the results of competitive binding analyses of susceptible strains.

Biotechnol Bioeng, 1999 May 5, 63(3), 344 - 55
Effect of temperature on the saccharide composition obtained after alpha-amylolysis of starch
Marchal LM, van de Laar AM, Goetheer E, Schimmelpennink EB, Bergsma J, Beeftink HH, Tramper J.
The hydrolysis of starch to low-molecular-weight products (normally characterised by their dextrose equivalent (DE), which is directly related to the number-average molecular mass) was studied at different temperatures . Amylopectin potato starch, lacking amylose, was selected because of its low tendency towards retrogradation at lower temperatures . Bacillus licheniformis alpha-amylase was added to 10% {w/w} gelatinised starch solutions . The hydrolysis experiments were done at 50, 70, and 90 degrees C . Samples were taken at defined DE values and these were analysed with respect to their saccharide composition . At the same DE the oligosaccharide composition depended on the hydrolysis temperature . This implies that at the same net number of bonds hydrolysed by the enzyme, the saccharide composition was different . The hydrolysis temperature also influenced the initial overall molecular-weight distribution . Higher temperatures led to a more homogenous molecular weight distribution . Similar effects were observed for alpha-amylases from other microbial sources such as Bacillus amyloliquefaciens and Bacillus stearothermophilus . Varying the pH (5.1, 6.2, and 7.6) at 70 degrees C did not significantly influence the saccharide composition obtained during B . licheniformis alpha-amylase hydrolysis . The underlying mechanisms for B . licheniformis alpha-amylase were studied using pure linear oligosaccharides, ranging from maltotriose to maltoheptaose as substrates . Activation energies for the hydrolysis of individual oligosaccharides were calculated from Arrhenius plots at 60, 70, 80, and 90 degrees C . Oligosaccharides with a degree of polymerisation exceeding that of the substrate could be detected . The contribution of these oligosaccharides increased as the degree of polymerisation of the substrate decreased and the temperature of hydrolysis increased . The product specificity decreased with increasing temperature of hydrolysis, which led to a more equal distribution between the possible products formed . Calculations with the subsite map as determined for the closely related alpha-amylase from B . amyloliquefaciens reconfirmed this finding of a decreased substrate specificity with increased temperature of hydrolysis .

Biotechnol Bioeng, 1999 Apr 5, 63(1), 110 - 5
Biosynthesis and ultrasonic degradation of bacterial poly(gamma-glutamic acid); Perez-Camero G et al.; A study of the production of poly(gamma-glutamic acid) (PGGA) by Bacillus licheniformis NCIMB 11709 grown on medium E in shake flasks at 30 degrees C is reported . The enantiomeric composition of PGGA was found to be highly sensitive to the concentration of Mn++, especially when the ion is present in small amounts (</= 20 microM) . Polymers with D-unit contents ranging from 10 to 90% and Mw between 0.4 and 2.0 million g mol-1 were obtained for {Mn++} ranging from 0 to 1230 microM . Ultrasonic degradation was proven to be an effective method to reduce both the molecular weight and the polydispersity of naturally produced PGGA without disturbing the chemical constitution of the polymer .

Biotechnol Bioeng, 1999 Feb 5, 62(3), 348 - 57
The effect of process conditions on the alpha-amylolytic hydrolysis of amylopectin potato starch: An experimental design approach; Marchal LM et al.; The hydrolysis of amylopectin potato starch with Bacillus licheniformis alpha-amylase (Maxamyl) was studied under industrially relevant conditions (i.e . high dry-weight concentrations) . The following ranges of process conditions were chosen and investigated by means of an experimental design: pH {5.6-7.6}; calcium addition {0-120 microg/g}; temperature {63-97 degrees C}; dry-weight concentration {3-37% {w/w}}; enzyme dosage {27.6-372.4 microL/kg} and stirring {0-200 rpm} . The rate of hydrolysis was followed as a function of the theoretical dextrose equivalent . The highest rate (at a dextrose equivalent of 10) was observed at high temperature (90 degrees C) and low pH (6) . At a higher pH (7.2), the maximum temperature of hydrolysis shifted to a lower value . Also, high levels of calcium resulted in a decrease of the maximum temperature of hydrolysis . The pH, temperature, and the amount of enzyme added showed interactive effects on the observed rate of hydrolysis . No product or substrate inhibition was observed . Stirring did not effect the rate of hydrolysis . The oligosaccharide composition after hydrolysis (at a certain dextrose equivalent) did depend on the reaction temperature . The level of maltopentaose {15-24% {w/w}}, a major product of starch hydrolysis by B . licheniformis alpha-amylase, was influenced mostly by temperature .

Biotechnol Bioeng, 1998 Dec 20, 60(6), 729 - 38
Optimization of a heterogeneous reaction system for the production of optically active D-amino acids using thermostable D-hydantoinase; Lee DC et al.; A thermostable D-hydantoinase from Bacillus stearothermophilus SD-1 was previously mass-produced by batch cultivation of the recombinant E . coli harboring the gene encoding the enzyme (Lee et al., 1997) . In this work, we attempted to optimize the process for the production of N-carbamoyl-D-p-hydroxyphenylglycine, which is readily hydrolyzed to D-p-hydroxyphenylglycine under acidic conditions, from 5-(4-hydroxyphenyl)hydantoin using the mass-produced D-hydantoinase . In an effort to overcome the low solubility of the substrate, enzyme reaction was carried out in a heterogeneous system consisting of a high substrate concentration up to 300 g/L . In this reaction system, most of substrate is present in suspended particles . Optimal temperature and pH were determined to be 45 degrees C and 8.5, respectively, by taking into account the reaction rate and conversion yield . When the free enzyme was employed as a biocatalyst, enzyme loading higher than 300 unit/g-substrate was required to achieve maximum conversion . Use of whole cell enzyme resulted in maximum conversion even at lower enzyme loadings than the free enzyme, showing 96% conversion yield at 300 g/L substrate . The heterogeneous reaction system used in this work might be applied to the enzymatic production of other valuable compounds from a rarely water-soluble substrate .

Biotechnol Bioeng, 1998 Dec 5, 60(5), 534 - 40
Kinetic modeling of omega-transamination for enzymatic kinetic resolution of alpha-methylbenzylamine; Shin JS et al.; A kinetic model for omega-transaminase from Bacillus thuringiensis JS64 was developed by using the King-Altman method to simulate the kinetic resolution of alpha-methylbenzylamine (alpha-MBA) . Starting from a ping-pong bi-bi mechanism, a complete kinetic model including substrate inhibition only in the reverse reaction (i.e., transamination between acetophenone and L-alanine) was developed . The asymmetric synthesis of (S)-alpha-MBA proved to be difficult due to a much lower maximum reverse reaction rate than the maximum forward reaction rate, thermodynamically exergonic forward reaction (i.e., transamination between (S)-alpha-MBA and pyruvate), and the severe product and substrate inhibition of the reverse reaction . Experimental values for kinetic parameters show that the product inhibition constant of (S)-alpha-MBA is the most important parameter on determining the resolution reaction rate, suggesting that the resolution reaction rate will be very low unless (S)-alpha-MBA strongly inhibits the reverse reaction . Using the kinetic model, the kinetic resolution of alpha-MBA in aqueous buffer was simulated, and the simulation results showed a high degree of consistency with experimental data over a range of reaction conditions . Various simulation results suggest that the crucial bottleneck in the kinetic resolution of alpha-MBA lies mainly in the accumulation of acetophenone in reaction media as the reaction proceeds, whereas L-alanine exerts a little inhibitory effect on the reaction . The model predicts that removing acetophenone produced during the reaction can enhance the reaction rate dramatically . Indeed, the biphasic reaction system is capable of extracting acetophenone from the aqueous phase, showing a much higher reaction rate compared to a monophasic reaction system . The kinetic model was also useful in predicting the properties of other, better enzymes as well as the optimal concentrations of amino acceptor and enzyme in the resolution reaction .

Biotechnol Bioeng, 1998 Sep 20, 59(6), 786 - 91
From detergent additive to semisynthetic peroxidase-simplified and up-scaled synthesis of seleno-subtilisin
Haring D, Schreier P.
A simplified and up-scaled synthesis of the semisynthetic peroxidase seleno-subtilisin was developed . Highly purified to technical grade subtilisin preparations from Bacillus licheniformis and Bacillus amyloliquefaciens were applied as starting materials . Activation of Ser 221 with phenylmethanesulfonyl fluoride, nucleophilic substitution by sodium hydrogen selenide, and oxidation to the seleninic acid with hydrogen peroxide completed the chemical active-site modification . The reactions were accomplished with a minimum of simple work-up procedures in 10 g scale . Kinetics and enantioselectivity of the preparations were tested using 1-phenylethyl hydroperoxide . For the first time, an up-scaled synthesis of a semisynthetic enzyme is available .

Biotechnol Bioeng, 1998 Sep 5, 59(5), 576 - 81
Morphological change and enhanced pigment production of monascus when cocultured with saccharomyces cerevisiae or aspergillus oryzae
Shin CS, Kim HJ, Kim MJ, Ju JY.
When a Monascus isolate, a producer of Monascus pigments, was cocultured with either Saccharomyces cerevisiae or Aspergillus oryzae in a solid sucrose medium, there were significant morphological changes in Monascus culture . Cocultures exhibited cell mass increases of 2 times and pigment yield increases of 30 to 40 times compared to monocultures of Monascus . However, enhanced cell growth, an increase in pigment production, and morphological change did not occur in coculture with Bacillus cereus . Saccharomyces cerevisiae was more effective at enhancing pigment production than Asp . oryzae . Enhanced cell growth and increased pigment production occurred only in conjunction with morphological changes . Culture filtrates of S . cerevisiae were also effective in inducing morphology change in Monascus, similar to culture broths of S . cerevisiae . The hydrolytic enzymes produced by S . cerevisiae, such as amylase, and chitinase, are thought to be the effectors . The commercial enzymes alpha-amylase and protease from Asp . oryzae both caused a morphological change in Monascus and were effective in enhancing pigment production . However, lysozyme, alpha-amylase and protease from Bacillus species, protease from Staphylococcus, and chitinase from Streptomyces were not effective . The hydrolytic enzymes which cause a morphological change of Monascus culture and enhancement of pigment production are thought to be capable of degrading Monascus cell walls . An approximate 10-fold increase in pigment production was observed in liquid cocultures with S . cerevisiae .

Biotechnol Bioeng, 1998 Aug 5, 59(3), 386 - 91
An Escherichia coli host strain useful for efficient overproduction of secreted recombinant protein; Weikert C et al.; Periplasmic secretion of overexpressed Bacillus stearothermophilus alpha-amylase was analyzed in batch and fed-batch cultivations of Escherichia coli MG1655:pCSS4-p and the mutant strain CWML2:pCSS4-p . Under all conditions investigated, growth and product formation of MG1655:pCSS4-p were severely impaired by heterologous protein expression and/or processing, while E . coli CWML2:pCSS4-p was found to be more robust and to accumulate 2- to 3-fold higher maximum alpha-amylase levels . While this strain is itself potentially interesting for applications, its properties also illustrate the potential of the selection procedure that was employed to obtain it from its progenitor MG1655 (Weikert, C., Sauer, U., Bailey, J . E., 1997 . Microbiol . 143: 1567-1574 . Application of this procedure to existing industrial strains may lead to significantly improved process organisms .

Biotechnol Bioeng, 1998 Feb 20, 57(4), 430 - 7
Effects of glucose and glycerol on gamma-poly(glutamic acid) formation by Bacillus licheniformis ATCC 9945a; Ko YH et al.; Bacillus licheniformis ATCC 9945a is one of the bacterial strains that produce gamma-poly(glutamic acid) (gamma-PGA) . The use of carbohydrate medium components for gamma-PGA production was explored . Cells were grown in shake flasks or in controlled pH fermentors using medium formulations that contain different carbon sources . During the cultivations, aliquots were removed to monitor cell growth, carbon utilization, polymer production, and polymer molecular weight . Glucose was a better carbon source than glycerol for cell growth . Furthermore, glucose was utilized at a faster rate than glycerol, citrate, or glutamate . However, by using mixtures of glucose and glycerol in medium formulations, the efficiency of gamma-PGA production increased . For example, by increasing the glycerol in medium formulations from 0 to 40 g/L, the gamma-PGA broth concentration after 96 h increased from 5.7 to 20.5 g/L . Considering that glycerol utilization was low for the glucose/glycerol mixtures studied, it was unclear as to the mechanism by which glycerol leads to enhanced product formation . Cell growth and concomitant gamma-PGA production (12 g/L) at pH 6.5 was possible using glucose as a carbon source if trace amounts (0.5 g/L each) of citrate and glutamate were present in the medium . We suggested that citrate and glutamate were useful in preventing salt precipitation from the medium . In addition, glutamate may be preferred relative to ammonium chloride as a nitrogen source . The conversion of glucose to gamma-PGA by the strain ATCC 9945a was believed to occur by glycolysis of glucose to acetyl-CoA and tricarboxylic acid (TCA) cycle intermediates that were then metabolized via the TCA cycle to form alpha-ketoglutarate, which is a direct glutamate precursor .

Int J Food Microbiol, 1999 Feb 18, 46(3), 243 - 9
Influence of pH on heat resistance of spores of Bacillus coagulans in buffer and homogenized foods; Palop A et al.; The influence of pH of heating menstruum (McIlvaine buffer) on the heat resistance of Bacillus coagulans spores has been investigated and compared with the heat resistance in homogenized tomato and asparagus at pH 7 and 4 at a wide range of temperatures . Spores were less heat resistant in all menstrua at acid pH . The magnitude of this effect was greatest at the lowest heating temperatures tested . z values in buffer increased from 8.9 degrees C at pH 7 to 10.5 degrees C at pH 4 . pH of menstrua was the main influencing factor, but media composition also influenced heat resistance: at pH 7 heat resistance was similar in all menstrua (D111 degrees C = 1.6 min) but at pH 4 the heat resistance in homogenized foods (D111 degrees C = 0.26 min in tomato and D111 degrees C = 0.28 min in asparagus) was lower than in buffer (D111 degrees C = 0.49 min) . The reduced influence of the acidification of media on the heat resistance of B . coagulans at higher temperatures should be taken into account when a rise in the temperature of treatment for canned vegetables is considered to shorten duration of heat processes.

Microbiol Immunol, 1999, 43(1), 15 - 8
Growth conditions of and emetic toxin production by Bacillus cereus in a defined medium with amino acids; Agata N et al.; The growth and emetic toxin (cereulide) production of Bacillus cereus strains in defined culture media were studied . We found that a fully synthetic medium (CADM) allowed the production of emetic toxin and the addition of glucose enhanced it . By subtracting each amino acid from CADM, we found that only three amino acids, valine, leucine and threonine, were essential for growth and toxin production by B . cereus . The addition of high levels (50 mM) of leucine, isoleucine and glutamic acid decreased the toxin production . Other amino acids had no effect at this concentration.

FEBS Lett, 1999 Mar 5, 446(1), 81 - 5
Secondary structure of the C-terminal domain of the tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus: a novel type of anticodon binding domain?
Pintar A, Guez V, Castagne C, Bedouelle H, Delepierre M.
The tyrosyl-tRNA synthetase catalyzes the activation of tyrosine and its coupling to the cognate tRNA . The enzyme is made of two domains: an N-terminal catalytic domain and a C-terminal domain that is necessary for tRNA binding and for which it was not possible to determine the structure by X-ray crystallography . We determined the secondary structure of the C-terminal domain of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus by nuclear magnetic resonance methods and found that it is of the alpha+beta type . Its arrangement differs from those of the other anticodon binding domains whose structure is known . We also found that the isolated C-terminal domain behaves as a folded globular protein, and we suggest the presence of a flexible linker between the two domains.

Br J Cancer, 1999 Mar, 79(7-8), 1162 - 7
The association between CD2+ peripheral blood lymphocyte subsets and the relapse of bladder cancer in prophylactically BCG-treated patients; Reyes E et al.; We investigated the potential existence of differences in the distribution of T-lymphocyte subsets and in the proliferative response of these CD2+ cells to polyclonal mitogens in patients with transitional cell bladder carcinoma (SBTCC) treated with prophylactic intracavitary instillations of bacillus Calmette-Guerin (BCG) according to their clinical response to this treatment . Before BCG treatment, different subset distribution (CD8+ and CD3+ CD56+), activation antigen expression (CD3+ HLA- DR+) and proliferative response to mitogenic signals were found in CD2+ cells from SBTCC patients prophylactically treated with BCG who remained free of disease or those who had recurrence of tumour . Otherwise, the prophylactic intracavitary BCG instillations in SBTCC patients are associated with a transitory variation of T-lymphocyte subset distribution (CD4 and CD8) and activation antigens expression (CD25).

Acta Cytol, 1999 Mar-Apr, 43(2), 191 - 4
Fine needle aspiration cytology in the diagnosis of tuberculous mastitis; Gupta D et al.; OBJECTIVE: To study the relationship between granulomas in the breast and tuberculous mastitis . STUDY DESIGN: Retrospective analysis of 22 breast aspirates that showed epithelioid cell granulomas . The aspirates were reviewed and the cytomorphologic findings summarized . RESULTS: Aspiration cytology revealed epithelioid cell granulomas along with giant cells, necrosis and inflammatory cell infiltrate . Overall acid-fast bacillus (AFB) positivity was 22.7% . AFB positivity was greater in the presence of necrosis when epithelioid cells were absent . CONCLUSION: In a country like India, the diagnosis of granulomatous mastitis must be made with caution, even in the absence of AFB . Only after a sufficient trial of antituberculosis treatment has been given and the patient fails to respond should an alternative diagnosis be suggested.

J Nat Prod, 1999 Mar, 62(3), 502 - 3
A novel extracellular diterpenoid with antibacterial activity from the cyanobacterium Nostoc commune; Jaki B et al.; A novel extracellular metabolite with an unprecedented diterpenoid skeleton, 8-{(5-carboxy-2-hydroxy)benzyl}-2-hydroxy-1,1,4a,7, 8-pentamethyl-1,2,3,4,4a,6,7,8,8a,9,10,10a-dodecahydrophenanthrene , has been isolated from the culture medium of the terrestrial cyanobacterium Nostoc commune Vaucher (EAWAG 122b) by means of bioguided isolation . The molecule was designated as noscomin . The structure was determined by spectroscopic methods, mainly NMR and mass spectrometry . Noscomin exhibited antibacterial activity against Bacillus cereus, Staphylococcus epidermidis, and Escherichia coli.

Biochim Biophys Acta, 1999 Mar 19, 1444(3), 429 - 33
Cloning, expression, and purification of Bacillus stearothermophilus DNA primase and crystallization of the zinc-binding domain; Pan H et al.; The dnaG gene encoding DNA primase has been isolated from chromosomal DNA of Bacillus stearothermophilus and its entire nucleotide sequence determined . The deduced amino acid sequence comprised 597 amino acid residues and the molecular mass was calculated to be 67068 Da . B . stearothermophilus primase was overexpressed in Escherichia coli and purified to homogeneity . The N-terminal 12 kDa zinc-binding domain has been crystallized . The crystals are of the monoclinic space group P21 with cell dimensions a=36 A, b=59 A, c=46 A, beta=91.8 degrees and diffract to 1.7 A resolution.

Biochim Biophys Acta, 1999 Mar 19, 1444(3), 424 - 8
The Bacillus stearothermophilus replicative helicase: cloning, overexpression and activity; Bird LE et al.; As part of biochemical and structural studies of the primosome of a gram positive bacterial species, we describe the cloning of the Bacillus stearothermophilus replicative helicase, DnaB . The protein is 45% and 82% identical to the Escherichia coli and B . subtilis replicative helicases, respectively . Recombinant DnaB was purified and shown to be an active helicase.

J Immunother, 1999 Mar, 22(2), 166 - 74
Recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) and autologous melanoma vaccine mediate tumor regression in patients with metastatic melanoma; Leong SP et al.; In mice, significant immunoprotection was achieved using B16 melanoma cells transfected with granulocyte-macrophage colony-stimulating factor (GM-CSF) as vaccines (Dranoff G, Jaffee E, Lazenby A, et al . Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity . Proc Natl Acad Sci USA 1993;90:3539-43) . The aim of this study is to test the hypothesis that recombinant human GM-CSF (rhGM-CSF) injected with autologous melanoma vaccine may result in tumor rejection in melanoma patients . Twenty stage IV melanoma patients were treated as outpatients with multiple cycles of autologous melanoma vaccine and bacillus Calmette-Guerin (BCG) plus rhGM-CSF injection in the vaccine sites . Two patients (10%) showed a complete response, with one patient showing resolution of subcutaneous, hepatic, and splenic metastases . In the second patient, buccal, subcutaneous, pulmonary, paraaortic, hepatic, splenic, and retroperitoneal metastases regressed completely . Two patients (10%) showed partial response, with regression of a paraaortic metastasis in one patient . In the second patient, there was shrinkage (> 75%) of a large hepatic lesion . One patient has been rendered free of disease after resection of a single pulmonary metastatic nodule . Three patients (15%) had stable disease during treatment but subsequently developed progression of disease . In 12 patients (60%), the disease progressed . Side effects were minimal . In a separate pilot study, 15 stage IV melanoma patients were also treated with autologous melanoma vaccine with BCG but not with rhGM-CSF; none responded . The fact that four patients showed objective responses to active specific immunotherapy with rhGM-CSF demonstrates that melanoma patients bearing a significant tumor burden may respond specifically to their autologous melanoma.

Int J Tuberc Lung Dis, 1999 Mar, 3(3), 255 - 60
The effects of BCG immunization and human immunodeficiency virus infection on dual skin test reactions to purified protein derivative and Mycobacterium avium sensitin among adults in Zambia; Waddell RD et al.; SETTING: University hospital in Lusaka, Zambia . OBJECTIVE: To determine the effects of childhood bacille Calmette-Guerin (BCG) immunization and human immunodeficiency virus (HIV) infection on dual skin test reactions to purified protein derivative (PPD) and Mycobacterium avium sensitin (MAS) in a developing country . DESIGN: Descriptive cross-sectional study . RESULTS: Dual skin testing was performed on 112 adults, 58 HIV-positive and 54 HIV-negative . Forty-seven (42%) of 112 had PPD reactions > or =10 mm and 52 (46%) had MAS reactions > or =10 mm . PPD reactions > or =10 mm were present in 30 (63%) of 48 BCG-positive subjects compared to 17 (27%) of 64 BCG-negative subjects (P<0.001) . Nineteen (33%) of 58 PPD or MAS skin test positive subjects were PPD dominant, 15 (26%) were MAS dominant, and 24 (41%) were non-dominant . MAS dominant and non-dominant reactions were significantly reduced in HIV-positive subjects, and non-dominant reactions were increased in BCG-positive subjects . CONCLUSIONS: Childhood BCG immunization is associated with PPD reactions > or =10 mm among adults . Reduced PPD reaction rates in HIV-positive adults appear to be due to a loss of BCG-induced PPD reactivity . Prior infection with M . avium complex is detectable in a significant proportion of adults in a developing country.

Int J Tuberc Lung Dis, 1999 Jan, 3(1), 62 - 7
The validity of acid-fast smears of gastric aspirates as an indicator of pulmonary tuberculosis; Bahammam A et al.; SETTING: A tuberculosis referral hospital in Canada . OBJECTIVE: To determine the validity of acid-fast (AFB) smears of gastric aspirates (GA) in the diagnosis of pulmonary tuberculosis, and to assess the prevalence of nontuberculous mycobacteria (NTM) in GA isolates from such patients . DESIGN: A retrospective case review of our experience with AFB smears (Kinyoun) and cultures of GA and sputum over a 3-year period . RESULTS: From 1994 to 1996 inclusive, 1155 GA were performed in 889 patients . Mycobacteria were cultured from 109 (9%) GA . Thirteen of these were positive on smear (sensitivity 19%) . All GA that were positive on smear were culture positive for Mycobacterium tuberculosis . There were no false positive smears (specificity 100%) . The sensitivity and specificity of the sputum smear were 45% and 99%, respectively . Of the 96 culture positive, smear negative GA, 54 grew M . tuberculosis and 42 grew an NTM . Of 13 patients who had sputum and GA studied coincidentally, and in whom the sputum was both smear and culture positive, the GA culture was positive in 13 and the smear was positive in eight (66%) . CONCLUSION: AFB smear of GA is a relatively insensitive but highly specific indicator of pulmonary tuberculosis warranting institution of antituberculosis treatment . Gastric AFB smear positivity appears to reflect a high bacillary burden within the respiratory tract.

Int J Tuberc Lung Dis, 1999 Jan, 3(1), 23 - 30
Tuberculin reactivity in a pediatric population with high BCG vaccination coverage; Lockman S et al.; SETTING: The tuberculin skin test (TST) is often included in diagnostic algorithms for tuberculosis (TB) in children . TST interpretation, however, may be complicated by prior Bacillus Calmette-Guerin (BCG) vaccination . We assessed the prevalence of and risk factors for positive TST reactions in children 3 to 60 months of age in Botswana, a country with high TB rates and BCG coverage of over 90% . METHODS: A multi-stage cluster survey was conducted in one rural and three urban districts . Data collected included demographic characteristics, nutritional indices, vaccination status, and prior TB exposure . Mantoux TSTs were administered and induration measured at 48-72 hours . RESULTS: Of 821 children identified, 783 had TSTs placed and read . Of the 759 children with vaccination cards, 755 (99.5%) had received BCG vaccine . Seventy-nine per cent of children had 0 mm induration, 7% had > or =10 mm induration ('positive' TST), and 2% had > or =15 mm . A positive TST was associated with reported contact with any person with active TB (odds ratio {OR} 1.9; 95% confidence interval {CI} 1.02-3.6), or a mother (OR 5.1; 95% CI 2.1-12.4) or aunt (OR 5.3; 95% CI 2.0-14.0) with active TB . TSTs > or =5 mm (but not > or =10 mm) were associated with presence of a BCG scar . Positive reactions were not associated with age, time since BCG vaccination, clinical signs or symptoms of TB, nutritional status, crowding, or recent measles or polio immunization . CONCLUSION: The TST remains useful in identifying children with tuberculous infection in this setting of high TB prevalence and extensive BCG coverage.

Presse Med, 1999 Feb 27, 28(8), 429 - 34, 438
{Bartonella infection in humans}; Raoult D; BARTONELLA BACILLIFORMIS: Among the 3 species of Bartonella known to be human pathogens, B . bacilliformis causes Carriun's disease, which manifests an acute phase (Oroya fever) and a chronic phase marked by benign skin eruption with wart like macules of vascular origin . Until 1993, B . bacilliformis was considered to be the only species in Bartonella genus . In 1993, species formally in the Rochalimaea genus were designated as Bartonella species . BARTONELLA QUINTANA: This species causes trench fever . It is also the causal agent in cases of bacillary angiomatosis, septicemia, endocarditis with negative blood cultures, and chronic nodal infections, particularly in immunosuppressed patients . Trench fever is transmitted by body lice and is becoming more prevalent, particularly in the homeless . BARTONELLA HENSELAE: This agent causes bacillary angiomatosis, visceral peliosis, septicemia, endocarditis and cat-scratch disease . Transmitted by cats, and perhaps by lice, cat-scratch disease is one of the most frequent zoonoses . OTHER SPECIES: The spectrum of Bartonella infections has continued to widen these last 5 years . The role of B . elizabethae and C . clarridgeiae as human pathogens remains to be defined {abstract corrected}

Appl Microbiol Biotechnol, 1999 Feb, 51(2), 170 - 5
The amylopullulanase of Bacillus sp . DSM 405; Brunswick JM et al.; The amylopullulanse produced by Bacillus sp . DSM 405 was purified to homogeneity . It exhibited dual activity, cleaving the alpha 1-4 bonds in starch, releasing a range of malto-oligosaccharides, and also cleaving the alpha 1-6 bonds in pullulan, releasing maltotriose as the sole end-product . The enzyme was a glycoprotein and had a relative molecular mass of 126,000 and an isoelectric point of 4.3 . While the enzyme was optimally active on starch at pH 6.5 and at pH 6.0 on pullulan, activity on both substrates was maximal at 70 degrees C . Kinetic analyses of the enzyme in a system that contained both starch and pullulan as two competing substrates demonstrated the dual specificity of the enzyme . Chemical modification of the carboxyl groups within the active centre of the protein showed that one active site was responsible for hydrolysis of the alpha 1-4 and alpha 1-6 bonds in starch and pullulan respectively . This is the first comprehensive investigation of an amylopullulanse produced by an aerobic bacterium, showing a single active site responsible for both activities.

Microb Pathog, 1999 Feb, 26(2), 85 - 91
Catecholamine enhancement of Aeromonas hydrophila growth; Kinney KS et al.; Several species of bacteria have been shown to respond to the administration of norepinephrine and other catecholamines with increased growth (in culture) and virulence . In this study, we examined the effects of catecholamines on the growth of cultures of Aeromonas hydrophila, a Gram-negative bacillus found in brackish water . Bacterial cultures were maintained in tryptic soy both, then washed free of medium and transferred to a bovine serum-supplemented minimal salts medium . Treatment of A . hydrophila cultures with 10(-3)to 10(-5)M norepinephrine resulted in dramatic increases in growth at 24 h and longer, as assessed by spot plate analysis on tryptic soy agar plates . Norepinephrine-treated cultures had 4.5 log greater bacterial numbers than control cultures . Epinephrine, dopamine and isoproterenol were shown to be similarly effective in enhancing growth of A . hydrophila, over narrower concentration ranges . Acetylcholine supplementation of cultures did not alter the growth of A . hydrophila . Serotonin slightly enhanced Aeromonas growth when administered at very high concentrations (10(-3)M) . The increased growth observed after catecholamine administration may alter the capacity to infect an animal under stressful conditions, and is another potential mechanism by which a stress response can affect susceptibility to disease .

Biochemistry, 1999 Mar 23, 38(12), 3519 - 29
Alternative routes for entry of HgX2 into the active site of mercuric ion reductase depend on the nature of the X ligands; Engst S et al.; Wild-type mercuric ion reductase (CCCC enzyme) possesses four cysteines in each of its Hg(II) binding sites, a redox-active pair and a C-terminal pair . Mutation of the C-terminal cysteines to alanines (CCAA enzyme) leads to a loss of steady-state mercuric ion reductase activity using Hg(SR)2 substrates . However, CCCC and CCAA enzymes exhibit an equally high rate of binding and turnover using HgBr2 as substrate under pre-steady-state conditions {Engst and Miller (1998) Biochemistry 37, 11496-11507.} . Since the ligands in these HgX2 substrates differ both in size and in affinity for Hg(II), one or both of these properties may contribute to their different reactivities with CCAA enzyme . To further explore the importance of these two properties, we have examined the pre-steady-state reactions of CCCC and CCAA with Hg(CN)2, which has small, high-affinity ligands, and with Hg(Cys)2, which has bulky, high-affinity ligands . The results indicate that HgX2 substrates with small ligands can rapidly access the redox-active cysteines in the absence of the C-terminal cysteines, but those with large ligands require the C-terminal cysteines for rapid access . In addition, it is concluded that the C-terminal cysteines play a critical role in removing the high-affinity ligands before Hg(II) reaches the redox-active cysteines in the inner active site, since direct access of HgX2 substrates with high-affinity ligands leads to formation of an inhibited complex . Consistent with the results, both a narrow channel leading directly to the redox-active cysteines and a wider channel leading to the redox-active cysteines via initial contact with the C-terminal cysteines can be identified in the structure of the enzyme from Bacillus sp . RC607.

Lab Anim Sci, 1998 Jun, 48(3), 234 - 9
Antigenic analyses of cilia-associated respiratory (CAR) bacillus isolates by use of monoclonal antibodies; Hook RR Jr et al.; Mouse monoclonal antibodies (MAbs) developed to a rat isolate (R-3) of cilia-associated respiratory (CAR) bacillus were used to assess antigenic relationships among three rat and five rabbit CAR bacillus isolates . Evaluation of MAbs by enzyme-linked immunosorbent assays (ELISAs) indicated that 87 of 241 hybridomas secreted CAR bacillus-reactive antibodies that could be grouped into four major groups . Group-I MAbs reacted with epitopes expressed by all CAR bacillus isolates and at least two or more nonrelated species of bacteria . Group-II, -III, and -IV MAbs reacted with only one or more of the rat CAR bacillus isolates; no MAbs reacted only with rat and rabbit CAR bacillus isolates . Western blot analyses indicated that 41-, 50-, and 105-kDa peptides of rat CAR bacillus isolates expressed rat CAR bacillus group- and isolate-specific epitopes . Hyperimmune anti-CAR bacillus antiserum and serum specimens from a CAR bacillus histologically positive mouse and rat also reacted with the 41-, 50-, and 105-kDa peptides . Sera from CAR bacillus histologically negative rats did not react with these peptides . These results suggest that the 41-, 50-, and 105-kDa peptides may represent suitable antigens for development of a specific ELISA for detection of rodent CAR bacillus infections . Furthermore, these data indicate that use of crude CAR bacillus preparations for either rat or rabbit CAR bacillus ELISAs is inappropriate.

Acta Crystallogr D Biol Crystallogr, 1999 Mar, 55 ( Pt 3), 712 - 6
Preliminary characterization by X-ray diffraction and Raman spectroscopy of a crystalline complex of Bacillus stearothermophilus initiation factor 2 C-domain and fMet-tRNAfMet; Forster C et al.; Bacillus stearothermophilus translation initiation factor 2 (IF2) specifically binds initiator fMet-tRNAfMet and positions it into the ribosomal peptidyl site in the course of the initiation of protein biosynthesis . The isolated C-terminal domain of IF2 is capable of binding fMet-tRNAfMet, as shown by RNase A and hydrolysis protection experiments . In the presence of fMet-tRNAfMet, the IF2 C-domain yielded orthorhombic crystals of space group I222 (I212121) diffracting to 3.4 A resolution . The existence of equimolar amounts of tRNA and protein in the crystals was proven by Raman spectroscopy . The observed unit cell suggests the presence of two IF2 C- domain-fMet-tRNAfMet complexes per asymmetric unit of the crystal.

Acta Crystallogr D Biol Crystallogr, 1999 Mar, 55 ( Pt 3), 671 - 6
Crystallization of the regulatory and effector domains of the key sporulation response regulator Spo0A; Muchova K et al.; The key response-regulator gene of sporulation, spo0A, has been cloned from Bacillus stearothermophilus and the encoded protein purified . The DNA-binding and phospho-acceptor domains of Spo0A have been prepared by tryptic digestion of the intact protein and subsequently crystallized in forms suitable for X-ray crystallographic studies . The DNA-binding domain has been crystallized in two forms, one of which diffracts X-rays to beyond 2 . 5 A spacing . The crystals of the phospho-acceptor domain diffract X-rays beyond 2.0 A spacing using synchrotron radiation.

Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 287 - 90 Epub 1999 Jan 01.
Cloning, expression and crystallization of pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus; Zhou M et al.; Pyrimidine nucleoside phosphorylase (PYNP) from B . stearothermophilus has been cloned and purified for crystallization . Crystals of a potential protein-inhibitor complex have been prepared by co-crystallization techniques using the substrate analog pseudouridine . These crystals provide good-quality diffraction images to 2.7 A and belong to space group P21 . The asymmetric unit contains the dimer structure of PYNP with unit-cell parameters a = 53.9, b = 71.9, c = 123.3 A and beta = 96.9 degrees.

Acta Crystallogr D Biol Crystallogr, 1999 Feb, 55 ( Pt 2), 386 - 98
Refinement and structural analysis of barnase at 1.5 A resolution; Martin C et al.; The structure of Bacillus amyloliquefaciens ribonuclease (barnase), an extracellular 110-residue enzyme initially solved at 2.0 A resolution, has been refined at 1.5 A using synchrotron radiation and an imaging-plate scanner . Refinement with anisotropic atomic displacement parameters resulted in increased accuracy of the structure . The final model has a crystallographic R factor of 11.5% and an Rfree of 17.4% . The three independent molecules in the asymmetric unit, referred to as A, B and C, allowed detailed analysis of this final model and meaningful comparison with structures of barnase complexed either with nucleotide inhibitors or with its natural intracellular inhibitor, barstar . The analysis of the overall solvent structure revealed a similar number of water molecules associated with each barnase molecule; among these were 16 equivalent buried solvent molecules, the locations of which are discussed in detail and classified on the basis of their structural role . The importance of the water molecules' contribution to the barnase-barstar interaction is also highlighted . The high accuracy of the present analysis revealed the presence of a Zn2+ ion mediating the contacts between pairs of symmetry-related A, B or C molecules; such an ion had previously only been identified for pairs of C molecules.

Acta Crystallogr D Biol Crystallogr, 1999 Apr, 55 ( Pt 4), 898 - 900
Crystallization and preliminary x-ray analysis of beta-amylase from Bacillus polymyxa; Yamane T et al.; A truncated beta-amylase (E.C . 3.2.1.2) from Bacillus polymyxa has been crystallized using the hanging-drop vapour-diffusion method at 277 K . The crystals belong to the orthorhombic space group P212121 with cell dimensions a = 64.6, b = 141.9, c = 155.1 A and diffract to 2.5 A resolution . The asymmetric unit containing three protein molecules was derived from an electron-density map calculated at 4 A resolution using MIR phases . This gives a Vm value of 2.36 A3 Da-1.

Acta Crystallogr D Biol Crystallogr, 1999 Apr, 55 ( Pt 4), 869 - 72
Crystallization and preliminary X-ray analysis of alpha-D-glucuronidase from Bacillus stearothermophilus T-6; Teplitsky A et al.; alpha-D-Glucuronidases cleave the alpha-1,2-glycosidic bond of the 4-O-methyl-alpha-D-glucuronic acid side chain in xylan . Of the xylan-debranching hydrolases, these enzymes are the least studied and characterized . The alpha-glucuronidase gene (aguA) from Bacillus stearothermophilus T-6 has been cloned, sequenced and overproduced in Escherichia coli . The gene encodes for a protein of 679 amino acids with a calculated molecular weight of 78480 and a pI of 5.42 . alpha-Glucuronidase T-6 shows high homology to the alpha-glucuronidases of Thermotoga maritima (60% identity) and of Tri-choderma reesei (44% identity) . Based on the amino-acid sequence similarity, it is likely that these enzymes represent a new class of glycosyl hydrolases . Crystallographic studies of alpha-glucuronidase T-6 were initiated to study the mechanism of catalysis, as well as to provide a structural basis for rational introduction of enhanced thermostability by site-specific mutagenesis . In this report, the crystallization and preliminary crystallographic characterization of the native alpha-glucuronidase T-6 enzyme is described . Two crystal forms were found suitable for detailed crystal structure analysis . The T1 form was obtained by the vapour-diffusion method using PEG 4000 as a precipitant and 2-propanol as an organic additive . The crystals belong to a primitive tetragonal crystal system (space group P41212 or P43212) with unit-cell dimensions a = b = 76.1 and c = 331.2 A . These crystals are mechanically strong, are stable in the X--ray beam and diffract X-rays to better than 2.4 A resolution . A full 3.0 A resolution diffraction data set (97.3% completeness, Rmerge 9.8%) has recently been collected on one crystal at room temperature using a rotating-anode X-ray source and an R-AXIS IIc imaging-plate detector . The M1 form was obtained and characterized by similar techniques . The best crystallization occurred at a slightly lower pH and a lower concentration of 2-propanol . The crystals belong to a primitive monoclinic crystal system (space group P21) with unit-cell dimensions a = 65.8, b = 127.4, c = 96.6 A and beta = 97.9 degrees . These crystals are also quite strong and stable, and diffract to better than 2.8 A resolution . A full 2.8 A resolution diffraction data set (96.2% completeness, Rmerge 7.6%) has recently been collected on one crystal at room temperature using the same R-AXIS IIc setup . Both forms are currently being used to obtain crystallographic phasing via isomorphous heavy-atom derivatives and selenomethionine MAD experiments.

Clin Rheumatol, 1999, 18(1), 74 - 6
Polyarthritis as a complication of intravesical bacillus Calmette-Guerin immunotherapy for bladder cancer; Onur O et al.; Bacillus Calmette-Guerin (BCG) is the most effective agent currently available for the treatment of superficial bladder cancer . However, this form of treatment is associated with some complications, including arthritis . In this report, we present a 69-year-old woman who developed inflammatory polyarthritis following BCG treatment for superficial bladder cancer . The arthritis resolved following treatment with a non-steroidal anti-inflammatory drug and chloroquinine.

J Pediatr Orthop, 1999 Mar-Apr, 19(2), 151 - 5
Tuberculous osteomyelitis in young children; Wang MN et al.; Twenty-three cases of tuberculous osteomyelitis in children were reviewed . Age at diagnosis ranged from 10 months to 11 years; 17 patients were younger than 3 years . At clinical presentation, patients were generally afebrile with local swelling and painful limb disability . Delay in diagnosis was common, with an average of 4.3 months . Laboratory data showed mild increase in white cell counts and erythrocyte sedimentation rates . However, C-reactive protein levels were all within normal limits except one . Roentgenograms demonstrated osteolytic lesions over metaphyseal areas with surrounding soft-tissue swelling . All patients had received BCG vaccinations at infancy . None of the patients had pulmonary tuberculosis . No familial or environmental history could be attributed to these victims, nor was any immunodeficient disease noted . Bacille Calmette-Guerin (BCG) vaccination was suspected to be the cause of tuberculosis in these young children . All patients received surgical debridement and oral antituberculosis chemotherapy for 1 year . After an average follow-up period of 71.4 months, all children had complete bony healing and excellent clinical results.

Arch Biochem Biophys, 1999 Apr 1, 364(1), 61 - 6
Purification and mode of action of an alkali-resistant endo-1, 4-beta-glucanase from Bacillus pumilus; Christakopoulos P et al.; Alkaline endo-1,4-beta-d-glucanase was secreted by Bacillus pumilus grown in submerged culture on a combination of oat spelt xylan and corn starch as carbon sources . The enzyme was purified to homogeneity by Sephacryl S-200 and Q-Sepharose column chromatography . The protein corresponded to molecular mass and pI values of 67 kDa and 3.7, respectively . The enzyme was optimally active at pH 7.0-8.0 and 60 degrees C and retained 50% of its optimum activity at pH 12 . The most notable characteristic of the endoglucanase was its high stability up to pH 12 for 20 h at 30 degrees C . The enzyme hydrolyzed carboxymethylcellulose (CMC) and cello-oligosaccharides but was inactive on cellobiose, cellotriose, Avicel, xylan, 4-nitrophenyl-beta-d-glucoside, 4-nitrophenyl-beta-d-cellobioside, and 4-nitrophenyl-beta-d-xyloside . Analysis of reaction mixtures by HPLC revealed that the enzyme produced almost exclusively cellotriose when acted on CMC and appeared to hydrolyze cello-oligosaccharides by successively releasing cellotriose . The use of 4-methylumbelliferyl cello-oligosaccharides and the determination of bond cleavage frequency revealed that the enzyme preferentially hydrolyzed the third glycosidic bond adjacent to the glycon . The enzyme mediated a decrease in the viscosity of CMC associated with a release of only small amounts of reducing sugar . The enzyme activity was not inhibited by metal ions, surfactants, and chelating agents used as components of laundry detergents .

Extremophiles, 1999 Jan, 3(1), 21 - 8
An improved physical and genetic map of the genome of alkaliphilic Bacillus sp . C-125; Takami H et al.; Among alkaliphilic bacteria reported so far, Bacillus sp . C-125 is the strain most thoroughly characterized physiologically, biochemically, and genetically . A physical map of the chromosome of this strain was constructed to facilitate further genome analysis, and the genome size was revised from 3.7 to 4.25Mb . Complete digestion of the chromosomal DNA with two rare cut restriction endonucleases, AscI and Sse8387I, each yielded 20 fragments ranging in size from 20 to 600 kb . Seventeen linking clones were isolated in each instance to join the adjacent AscI or Sse8387I fragments in the chromosomal map . All AscI linking clones isolated were sequenced and analyzed by comparison with the BSORF database to map the genes in the chromosome of strain C-125 . Several ORFs showing significant similarities to those of B . subtilis in the AscI linking clones were positioned on the physical map . The oriC region of the C-125 chromosome was identified by southern blot analysis with a DNA probe containing the gyrB region.

J Commun Dis, 1997 Dec, 29(4), 345 - 9
Effectiveness of Bacillus Calamette Guerin (BCG) vaccination against genital tuberculosis: a case-control study; Zodpey SP et al.; A hospital-based pair-matched case-control study was performed at Government Medical College Hospital, Nagpur, to estimate the effectiveness of Bacillus Calmette Guerin (BCG) vaccination against genital tuberculosis . The study included 48 cases of genital tuberculosis in the age group of 21-34 years and an equal number of controls, matched for age and socioeconomic status . The estimates of vaccine effectiveness and prevented fraction were higher for the subjects in the age group of 21-30 years and subjects from middle strata of socioeconomic class . The overall vaccine effectiveness and prevented fraction was estimated to be 75 (38.85-89.79) and 49.99 (17.46-74.55) per cent respectively . Results of this study thus indicate that BCG vaccination is effective against genital tuberculosis.

Infect Immun, 1999 Apr, 67(4), 2035 - 9
Characterization of exochelins of the Mycobacterium bovis type strain and BCG substrains; Gobin J et al.; Pathogenic mycobacteria must acquire iron in the host in order to multiply and cause disease . To do so, they release abundant quantities of siderophores called exochelins, which have the capacity to scavenge iron from host iron-binding proteins and deliver it to the mycobacteria . In this study, we have characterized the exochelins of Mycobacterium bovis, the causative agent of bovine and occasionally of human tuberculosis, and the highly attenuated descendant of M . bovis, bacillus Calmette-Guerin (BCG), widely used as a vaccine against human tuberculosis . The M . bovis type strain, five substrains of M . bovis BCG (Copenhagen, Glaxo, Japanese, Pasteur, and Tice), and two strains of virulent Mycobacterium tuberculosis all produce the same set of exochelins, although the relative amounts of individual exochelins may differ . Among these mycobacteria, the total amount of exochelins produced is greatest in M . tuberculosis, intermediate in M . bovis, and smallest in M . bovis BCG.

J Am Mosq Control Assoc, 1998 Dec, 14(4), 472 - 6
Bacterial control of mosquito larvae: investigation of stability of Bacillus thuringiensis var . israelensis and Bacillus sphaericus standard powders; Thiery I et al.; Bacillus thuringiensis var . israelensis and Bacillus sphaericus products were assayed against their respective reference powders IPS82 and SPH88 . Since their production in 1982 and 1988, the potency and larvicidal activity of these standard powders have been regularly checked on their test insects Aedes aegypti (for IPS82) or Culex pipiens (for SPH88) . Over the 16-year evaluation period of IPS82 and 10-year evaluation period of SPH88, their potencies were considered stable . The global mean of each year's mean showed a coefficient of variation of less than 20% . Larval rearing was the most important factor in the reproducibility of the bioassay, although some variation also originated from the person performing the bioassay . This study demonstrated that the SPH88 standard could be kept in a stock suspension at 4 degrees C for 3 years without loss of potency . Moreover, after 9 years of storage in suspension, only a 2-fold decrease in the potency of SPH88 was detected.

J Am Mosq Control Assoc, 1998 Dec, 14(4), 463 - 6
Acute toxicity of selected pesticides to the Pacific blue-eye, Pseudomugil signifer (Pisces); Brown MD et al.; Because the larvivorous fish Pseudomugil signifer is native to southeastern Queensland and is abundant in shallow estuarine habitats, intertidal marshes, wetland habitats, and freshwater streams, it was chosen as an indicator species for toxicologic studies with pesticides . Acute toxicity studies with 2 organophosphorus pesticides (pirimiphos-methyl and temephos) and 3 alternate compounds under evaluation for registration in Australia (Bacillus thuringiensis var . israelensis, s-methoprene, and pyriproxyfen), were tested in 96-h laboratory trials . Pirimiphos-methyl was the most toxic compound, with a median lethal concentration (LC50) of 0.091 ppm (0.3 times the estimated field concentration {EFC} for a 15-cm-deep pool) . Temephos had an LC50 value of 0.594 ppm (9.9 times the EFC) . Bacillus thuringiensis var . israelensis and pyriproxyfen produced LC50 values of 6.1 x 10(11) International Toxic Units (477 times the EFC) and 0.854 ppm (106 times the EFC), respectively . s-Methoprene was the least toxic compound, with no mortality recorded at 500 times the EFC.

J Am Mosq Control Assoc, 1998 Dec, 14(4), 457 - 62
Field trials of biolarvicide Bacillus thuringiensis var . israelensis strain 164 and the larvivorous fish Aplocheilus blocki against Anopheles stephensi for malaria control in Goa, India; Kumar A et al.; Severe outbreaks of malaria occurred in the coastal villages of the Candolim Primary Health Centre (PHC) of Goa, India, in 1993 and 1994 . These outbreaks were associated with accelerated construction activity with an influx of migrant laborers . The weekly application of Bacillus thuringiensis var . israelensis (B.t.i.) strain 164 at 1 g/m2 and introduction of the indigenous larvivorous fish Aplocheilus blocki in major breeding habitats of Anopheles stephensi replaced ongoing DDT spraying and pyrethrum fogging in June 1994 . The objective of this study was to evaluate the effects of B.t.i . and larvivorous fish on An . stephensi and subsequent transmission of malaria in the Candolim PHC, Goa, India . In 1995 the populations of an . stephensi in larger habitats (habitats with immatures: t = 5.19, P = 0.0017; immature density: t = 3.57, P = 0.007) and smaller habitats (habitats with immature: t = 3.86, P = 0.005; immature density: t = 4.93, P = 0.002) and malaria incidence declined substantially (malaria cases: chi 2 = 712, P < 0.001; slide positivity rate: chi 2 = 10.36, P < 0.001; annual parasite index; chi 2 = 15.1, P < 0.001), whereas the incidence of malaria continued to increase in other nearby towns.

Bull World Health Organ, 1999, 77(2), 138 - 43
Can vector control play a useful supplementary role against bancroftian filariasis?
Maxwell CA, Mohammed K, Kisumku U, Curtis CF.
A single campaign of mass treatment for bancroftian filariasis with diethylcarbamazine (DEC) in Makunduchi, a town in Zanzibar, United Republic of Tanzania, combined with elimination of mosquito breeding in pit latrines with polystyrene beads was followed by a progressive decline over a 5-year period in the microfilarial rate from 49% to 3% . Evidence that vector control had contributed to this long-term decline was obtained by comparison with another town, Moga, where a DEC campaign was used without vector control and where resurgence of microfilariae could be observed 3-6 years after the campaign . In Zanzibar town, treatment of 3844 wet pit latrines and cesspits with polystyrene beads reduced the adult mosquito population in houses by about 65% . Supplementary treatment of open drains and marshes with Bacillus sphaericus produced little or no additional reduction compared to a sector of the town where only pit treatment with polystyrene was carried out . The cost and effort of achieving the 65% reduction in mosquito population could hardly be justified for its impact on filariasis alone, but its noticeable impact on biting nuisance might help to gain community support for an integrated programmePIP: With chemotherapy schedules for bancroftian filariasis having become far more convenient to use, the World Health Organization currently emphasizes chemotherapy against the disease, and vector control in only a supplementary role . Data are presented from studies conducted in urban areas of Zanzibar, Tanzania, where the vector of bancroftian filariasis is Culex quinquefasciatus, to determine whether sustainable vector control contributes usefully to the suppression of the disease and is feasible in different types of breeding sites without the use of organophosphorus insecticides . Data were obtained from a follow-up study conducted in the late 1980s in the highly filaria-endemic town of Makunduchi . A single campaign in early 1988 provided mass treatment of the population of 12,000 with diethylcarbamazine (DEC) at the dose of 72 mg/kg body weight . DEC treatment, together with the elimination of mosquito breeding in pit latrines using polystyrene beads, was followed by a progressive decline over a 5-year period in the microfilarial rate from 49% to 3% . Evidence that vector control contributed to this long-term decline was obtained by comparison with another town, Moga, where a DEC campaign was used without vector control and where resurgence of microfilariae could be observed 3-6 years after the campaign . In Zanzibar town, treatment of 3844 wet pit latrines and cesspits with polystyrene beads reduced the adult mosquito population in houses by about 65% . The supplementary treatment of open drains and marshes with Bacillus sphaericus produced little or no additional reduction compared to a sector of the town where only pit treatment with polystyrene was carried out .

Biochim Biophys Acta, 1999 Mar 14, 1427(1), 121 - 32
Active site characterization of the exo-N-acetyl-beta-D- glucosaminidase from thermotolerant Bacillus sp . NCIM 5120: involvement of tryptophan, histidine and carboxylate residues in catalytic activity; Amutha B et al.; The exo-N-acetyl-beta-d-glucosaminidase (EC 3.2.1.30) from thermotolerant Bacillus sp . NCIM 5120 is a homotetramer with a molecular mass of 240000 kDa . Chemical modification studies on the purified exo-N-acetyl-beta-d-glucosaminidase revealed the involvement of a single tryptophan, histidine and carboxylate, per monomer, in the catalytic activity of the enzyme . Spectral analysis and maintenance of total enzyme activities indicated that N-acetylglucosamine (competitive inhibitor) and p-nitrophenyl-N-acetyl-beta-d-glucosaminide (substrate) prevented the modification of a single essential tryptophan, histidine and carboxylate residue . Kinetic parameters of partially inactivated enzyme (by NBS/HNBB) showed the involvement of tryptophan in substrate binding while that of histidine (by photooxidation/DEPC) and carboxylate (by EDAC/WRK) in catalysis . The Bacillus sp . NCIM 5120 exo-N-acetyl-beta-d-glucosaminidase deviates from the reported N-acetyl-beta-d-glucosaminidases and beta-hexosaminidases that utilize anchimeric assistance in their hydrolytic mechanism.

Protein Sci, 1998 Aug, 7(8), 1671 - 80
Improving the binding affinity of an antibody using molecular modeling and site-directed mutagenesis; Casipit CL et al.; Activated Factor X releases F1.2, a 271-amino acid peptide, from the amino terminus of prothrombin during blood coagulation . A nine-amino acid peptide, C9 (DSDRAIEGR), corresponding to the carboxyl terminus of F1.2 was synthesized and used to produce a monoclonal antibody, TA1 (K(D)) 1.22 x 10(-6) M) . To model the TA1 antibody, we entered the sequence information of the cloned TA1 Fv into the antibody modeling program, ABM, which combines homology methods, conformational search procedures, and energy screening and has proved to be a reliable and reproducible antibody modeling method . Using a novel protein fusion procedure, we expressed the C9 peptide fused to the carboxyl terminus of the PENI repressor protein from Bacillus licheniformis in Escherichia coli . We constructed fusion proteins containing alanine substitutions for each amino acid in the C9 epitope . Binding studies, using the C9 alanine mutants and TA1, and spatial constraints predicted by the modeled TA1 binding cleft enabled us to establish a plausible conformation for C9 complexed with TA1 . Furthermore, based on binding results of conservative amino acid substitutions in C9 and mutations in the antibody, we were able to refine the complex model and identify antibody mutations that would improve binding affinity.

J Urol, 1999 Apr, 161(4), 1124 - 7
5-year followup of a randomized prospective study comparing mitomycin C and bacillus Calmette-Guerin in patients with superficial bladder carcinoma . Swedish-Norwegian Bladder Cancer Study Group; Malmstrom PU et al.; PURPOSE: We report the 5-year followup of a randomized comparison of mitomycin C and bacillus Calmette-Guerin (BCG) in patients with superficial bladder carcinoma . Recurrence, progression and survival rates, crossover results, prognostic factors and long-term side effects were analyzed . MATERIALS AND METHODS: A total of 261 patients were enrolled in the study, and the inclusion criteria were primary Tis, dysplasia G2, T1 G3 and multiple recurrent Ta/T1 G1-2 disease . Intravesical instillations of 40 mg . mitomycin C and 120 mg . Pasteur BCG, Danish strain 1331, were given for 2 years . RESULTS: After a median followup of 64 months 101 of the 250 evaluable patients (42%) were disease-free . A significant difference was noted in disease-free survival with BCG (p = 0.04), which was most pronounced for stage Tis disease . No difference in tumor progression, or crude or corrected survival was found between the 2 arms . Crossover treatment was successful in 39% of patients with second line BCG and 19% with second line mitomycin C . Independent risk factors for progression were initial p53 status and stage . Only the completion of treatment was predictive of outcome for patients treated with BCG . Bladder shrinkage occurred in 2.4% of patients . CONCLUSIONS: Therapy with BCG was superior to mitomycin C for recurrence prophylaxis but no difference was found for progression and survival.

Biochem Biophys Res Commun, 1999 Mar 24, 256(3), 474 - 9
The IalA invasion gene of Bartonella bacilliformis encodes a (de)nucleoside polyphosphate hydrolase of the MutT motif family and has homologs in other invasive bacteria; Cartwright JL et al.; The product of the ialA invasion gene of Bartonella bacilliformis has been expressed as a thioredoxin fusion protein . It is a (di)nucleoside polyphosphate hydrolase of the MutT motif protein family with strong sequence similarity to plant diadenosine tetraphosphate hydrolases . It hydrolyses nucleoside and dinucleoside polyphosphates with four or more phosphate groups, always producing an NTP as one product . Diadenosine tetraphosphate (Ap4A) is the preferred substrate with a Km of 10 microM and a kcat of 3.0 s-1 . It is inhibited by Ca2+ and F- (Ki = 30 microM) . Hydrolysis of Ap4A in H218O yielded {18O}AMP as the only labelled product . In terms of sequence, reaction mechanism and properties, IalA is very similar to eukaryotic Ap4A hydrolases and unlike previously described bacterial Ap4A hydrolases . Homologs are present in the genomes of other invasive pathogens . They may function to reduce stress-induced dinucleotide levels during invasion and so enhance pathogen survival .

Biochem Biophys Res Commun, 1999 Mar 16, 256(2), 333 - 40
Characterization of psychrophilic alanine racemase from Bacillus psychrosaccharolyticus; Okubo Y et al.; A psychrophilic alanine racemase gene from Bacillus psychrosaccharolyticus was cloned and expressed in Escherichia coli SOLR with a plasmid pYOK3 . The gene starting with the unusual initiation codon GTG showed higher preference for codons ending in A or T . The enzyme purified to homogeneity showed the high catalytic activity even at 0 degrees C and was extremely labile over 35 degrees C . The enzyme was found to have a markedly large Km value (5.0 microM) for the pyridoxal 5'-phosphate (PLP) cofactor in comparison with other reported alanine racemases, and was stabilized up to 50 degrees C in the presence of excess amounts of PLP . The low affinity of the enzyme for PLP may be related to the thermolability, and may be related to the high catalytic activity, initiated by the transaldimination reaction, at low temperature . The enzyme has a distinguishing hydrophilic region around the residue no . 150 in the deduced amino acid sequence (383 residues), whereas the corresponding regions of other Bacillus alanine racemases are hydrophobic . The position of the region in the three dimensional structure of C atoms of the enzyme was predicted to be in a surface loop surrounding the active site . The region may interact with solvent and reduce the compactness of the active site.

Clin Immunol, 1999 Feb, 90(2), 230 - 7
Evaluation of lymphocytic responses after treatment with Bacillus Calmette-Guerin and interferon-alpha 2b for superficial bladder cancer; Gan YH et al.; Bladder wash-derived lymphocytes from superficial bladder cancer patients involved in high dose BCG, low dose BCG, and low dose BCG with IFN-alpha treatments were examined . We found an increasing trend in the percentage of CD3 T cells with each weekly intravesical instillation and the proportion of CD3 T cells expressing the gammadelta T cell receptor was significantly higher in patients receiving standard dose BCG than those receiving low dose BCG or low dose BCG plus IFN-alpha . Most patients had a predominance of CD4 T cells, while some had more CD8 T cells . The CD4/CD8 ratio did not vary much during the instillations . Surprisingly, both patients and normal control individuals had high percentages of CD69- and CD45RO-positive lymphocytes in the bladder wash and this was not reflected in lymphocytes from peripheral blood collected in parallel . We found no differences in lymphocyte phenotypes, cytokine production, and clinical outcome in the patients from three arms . This may reflect that the qualitative and quantitative immune responses elicited from the three treatments are similar . However, the lymphokine-activated killing ability of peripheral blood lymphocytes against allogeneic cell-lines from the cancer patients was depressed compared to normal individuals and the cytotoxicity appeared to be enhanced after intravesical treatment .

Neth J Med, 1999 Feb, 54(2), 76 - 9
Melioidosis; Beeker A et al.; Melioidosis is an infection with the gram-negative bacillus Burkholdria pseudomallei, previous known as Pseudomonas pseudomallei . We present a case report of a man with Non-Hodgkin's Lymphoma, who after visiting Indonesia presented himself with a urosepsis, with positive cultures to B . pseudomallei . Melioidosis is endemic in areas of southeast Asia and the northern part of Australia . Infection with the B . pseudomallei has a high mortality rate . Diagnosis can be made on the basis of cultures, the bacteria grow on standard media . Treatment should be based up on sensitivity studies . The optimum duration of therapy and choice of antibiotics remain to be determined . Due to the increase in travel, the infection will be seen more frequently in other parts of the world.

Biochemistry, 1999 Mar 16, 38(11), 3293 - 301
Structure of a Michaelis complex analogue: propionate binds in the substrate carboxylate site of alanine racemase; Morollo AA et al.; The structure of alanine racemase from Bacillus stearothermophilus with the inhibitor propionate bound in the active site was determined by X-ray crystallography to a resolution of 1.9 A . The enzyme is a homodimer in solution and crystallizes with a dimer in the asymmetric unit . Both active sites contain a pyridoxal 5'-phosphate (PLP) molecule in aldimine linkage to Lys39 as a protonated Schiff base, and the pH-independence of UV-visible absorption spectra suggests that the protonated PLP-Lys39 Schiff base is the reactive form of the enzyme . The carboxylate group of propionate bound in the active site makes numerous interactions with active-site residues, defining the substrate binding site of the enzyme . The propionate-bound structure therefore approximates features of the Michaelis complex formed between alanine racemase and its amino acid substrate . The structure also provides evidence for the existence of a carbamate formed on the side-chain amino group of Lys129, stabilized by interactions with one of the residues interacting with the carboxylate group of propionate, Arg136 . We propose that this novel interaction influences both substrate binding and catalysis by precisely positioning Arg136 and modulating its charge.

Mikrobiol Z, 1998 Sep-Oct, 60(5), 3 - 18
{The ecological, taxonomic and biotechnology aspects of research on bacteria and higher plants that produce biologically active substances}; Smirnov VV; The paper deals with the basic results of scientific activity of the Department of Antibiotics at the Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine for the last two decades . Results of fundamental ecological and taxonomical investigations of bacteria of the Pseudomonas and Bacillus genera, study of regularities in formation of antibiotics and other biologically active substances by microorganism of these taxa and by higher plants have been presented; new antibiotics, probiotics and other biopreparations developed at the Department during this period have been characterized.

FEMS Microbiol Lett, 1999 Feb 15, 171(2), 191 - 201
Involvement of host cell tyrosine phosphorylation in the invasion of HEp-2 cells by Bartonella bacilliformis; Williams-Bouyer NM et al.; We have provided evidence that exposure of human cells to protein kinase inhibitors results in decreased invasion of these cells by Bartonella bacilliformis in a dose-dependent manner . Preincubation of human laryngeal epithelial cells in the presence of genistein, a tyrosine protein kinase inhibitor, decreased the invasion of these cells by B . bacilliformis significantly . Further, exposure of normal human umbilical vein endothelial cells to staurosporine, a potent inhibitor of protein kinase C and some tyrosine protein kinases, resulted in a considerable reduction in the number of organisms internalized by these cells . Moreover, Bartonella infection of HEp-2 cells induced tyrosine phosphorylation of several Triton X-100 soluble proteins with approximate molecular masses of 243, 215 179, 172 (doublet), 160, 145 and 110 kDa that were absent or reduced in the presence of genistein in cells after 1 h of infection . Exposure of HEp-2 cell monolayers to anti-alpha 5 and anti-beta 1 chain integrin monoclonal antibodies resulted in a moderate decrease in the invasion of these cells, suggesting a possible role of alpha 5 beta 1 integrins in the uptake of Bartonella into nucleated cells.

Appl Microbiol Biotechnol, 1999 Jan, 51(1), 65 - 70
Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface; Murai T et al.; The construction of a whole-cell biocatalyst with its sequential reaction has been performed by the genetic immobilization of two amylolytic enzymes on the yeast cell surface . A recombinant strain of Saccharomyces cerevisiae that displays glucoamylase and alpha-amylase on its cell surface was constructed and its starch-utilizing ability was evaluated . The gene encoding Rhizopus oryzae glucoamylase, with its own secretion signal peptide, and a truncated fragment of the alpha-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast alpha factor, respectively, were fused with the gene encoding the C-terminal half of the yeast alpha-agglutinin . The constructed fusion genes were introduced into the different loci of chromosomes of S . cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter . The glucoamylase and alpha-amylase activities were not detected in the culture medium, but in the cell pellet fraction . The transformant strain co-displaying glucoamylase and alpha-amylase could grow faster on starch as the sole carbon source than the transformant strain displaying only glucoamylase.

Biol Pharm Bull, 1999 Feb, 22(2), 197 - 9
Lactoferrin inhibits Bacillus cereus growth and heme analogs recover its growth; Sato N et al.; The growth of Bacillus cereus was markedly inhibited by the addition of lactoferrin and was recovered by the addition of FeCl3 . The growth inhibition was also reversed by the addition of erythrocytes and hemoglobin . B . cereus can use heme or heme-protein complex (hemoglobin-haptoglobin and hematin-albumin complexes) as iron sources in iron deficient conditions . Therefore, B . cereus uses these heme or heme-protein complexes to prevent the growth inhibition of lactoferrin in vivo.

FEMS Immunol Med Microbiol, 1999 Feb, 23(2), 149 - 58
Immunity to tuberculosis: a delicate balance between protection and pathology; Ehlers S; The currently used tuberculosis (TB) vaccine (Bacillus Calmette-Guerin) does not consistently prevent pulmonary infection . Novel vaccine strategies require an in-depth characterization of the cellular and molecular mechanisms of antituberculous protection . This review summarizes relevant data obtained in animal models of TB infection . In mycobacterial infections, immunologically mediated protection is intrinsically associated with tissue damage in the form of granuloma formation . The implications of this finding for future vaccine design and evaluation are discussed.

J Microbiol Methods, 1999 Feb, 35(1), 13 - 21
Method for purification of bacterial endospores from soils: UV resistance of natural Sonoran desert soil populations of Bacillus spp . with reference to B . subtilis strain 168; Nicholson WL et al.; Endospores of Bacillus spp . were purified from three Sonoran desert soil samples by Chelex extraction and NaBr density gradient centrifugation and their UV resistances compared with that of B . subtilis strain 168 . Natural spore populations exhibited tight adherence to soil particles which was not readily overcome by the extraction and purification procedure . It was observed that spores purified from soil exhibited 2-3 fold higher resistance to UV (as measured by the 90% lethal dose, LD90) than did B . subtilis strain 168 grown on NSM, a standard laboratory sporulation medium, and purified by the same extraction procedure . Cultivation of spore-forming bacteria isolated from soil on NSM resulted in production of spores with essentially identical UV resistance as strain 168, suggesting that spore UV resistance is influenced by the environment in which spores are produced.

EMBO J, 1999 Mar 15, 18(6), 1459 - 67
The three-dimensional structure of the RNA-binding domain of ribosomal protein L2; a protein at the peptidyl transferase center of the ribosome; Nakagawa A et al.; Ribosomal protein L2 is the largest protein component in the ribosome . It is located at or near the peptidyl transferase center and has been a prime candidate for the peptidyl transferase activity . It binds directly to 23S rRNA and plays a crucial role in its assembly . The three-dimensional structure of the RNA-binding domain of L2 from Bacillus stearothermophilus has been determined at 2.3 A resolution by X-ray crystallography using the selenomethionyl MAD method . The RNA-binding domain of L2 consists of two recurring motifs of approximately 70 residues each . The N-terminal domain (positions 60-130) is homologous to the OB-fold, and the C-terminal domain (positions 131-201) is homologous to the SH3-like barrel . Residues Arg86 and Arg155, which have been identified by mutation experiments to be involved in the 23S rRNA binding, are located at the gate of the interface region between the two domains . The molecular architecture suggests how this important protein has evolved from the ancient nucleic acid-binding proteins to create a 23S rRNA-binding domain in the very remote past.

J Mol Biol, 1999 Mar 19, 287(1), 33 - 45
The antibiotic micrococcin acts on protein L11 at the ribosomal GTPase centre; Porse BT et al.; Micrococcin-resistant mutants of Bacillus megaterium that carry mutations affecting ribosomal protein L11 have been characterised . The mutants fall into two groups . "L11-minus" strains containing an L11 gene with deletions, insertions or nonsense mutations which grow 2.5-fold slower than the wild-type strain, whereas other mutants carrying single-site substitutions within an 11 amino acid residue segment of the N-terminal domain of L11 grow normally . Protein L11 binds to 23 S rRNA within the ribosomal GTPase centre which regulates GTP hydrolysis on ribosomal factors . Micrococcin binding within the rRNA component of this centre was probed on wild-type and mutant ribosomes, in vivo, using dimethyl sulphate where it generated an rRNA footprint indistinguishable from that produced in vitro, even after the cell growth had been arrested by treatment with either kirromycin or fusidic acid . No drug-rRNA binding was detected in vivo for the L11-minus mutants, while reduced binding (approximately 30-fold) was observed for two single-site mutants P23L and P26L . For the latter, the reduced drug affinity alone did not account for the resistance-phenotype because rapid cell growth occurred even at drug concentrations that would saturate the ribosomes . Micrococcin was also bound to complexes containing an rRNA fragment and wild-type or mutant L11, expressed as fusion proteins, and they were probed with proteinases . The drug produced strong protection effects on the wild-type protein and weak effects on the P23L and P26L mutant proteins . We infer that inhibition of cell growth by micrococcin, as for thiostrepton, results from the imposition of a conformational constraint on protein L11 which, in turn, perturbs the function(s) of the ribosomal factor-guanosine nucleotide complexes .

J Med Entomol, 1999 Jan, 36(1), 62 - 7
Experimental formulations of Bacillus sphaericus and B . thuringiensis israelensis against Culex quinquefasciatus and Anopheles gambiae (Diptera: Culicidae) in Burkina Faso; Skovmand O et al.; Efficacies of locally produced, sustained release granular formulations of Bacillus sphaericus (BS) strain 2362 and B . thuringiensis israelensis (BTI) were compared with commercial liquid concentrates of the same bacteria in cesspits and rain-filled puddles in Ouagadougou, Burkina Faso . Duration of control was dependent on the product, the transient nature of some sites, and the target mosquito larvae . BS granules applied at the rate of 3.0 g/m2 (30 kg/ha) reduced Culex quinquefasciatus Say 99% for at least 28 d in cesspits, whereas the same dosage of 2 BTI granules and commercial liquid formulations of BS and BTI gave 95% control for 8-14 d . The levels of control obtained with the 2 liquid products were not different . Accordingly, the reported inferiority of BTI to BS in polluted water was attributed to low dosages of BTI . Because products were compared at equal application rates, recycling seemed to play a minor role compared with product formulation . BTI was more broad spectrumed than BS also killing Cx decens Theobald, Cx cinereus Granpre & Charmoy, and psychodid larvae . Depending on the method of estimation, granular and liquid BS gave 60-97% control of Anopheles gambiae s.l . Giles for 10 d in rain puddles . The transient nature of the rain puddles was probably more important for the duration of control than formulation type . Cesspits and puddles, respectively, were the most important larval habitats for these 2 species in Ouagadougou during the rainy season, and these trials showed that An . gambiae was not controlled easily with larvicides alone . However, biological larvicides may play a central role in the control of Cx . quinquefasciatus provided that most breeding sites are treated.

J Med Entomol, 1999 Jan, 36(1), 41 - 9
Interruption of chemical mosquito control and evolution of insecticide resistance genes in Culex pipiens (Diptera: Culicidae); Eritja R et al.; Within the Llobregat Delta (Barcelona, Spain), Culex pipiens L . has been the target of organophosphate insecticide (OP) control for 10 yr (1982-1992) . Between 1991 and 1992, OPs were replaced by Bacillus-based toxins in all the mosquito control programs within > 150 km of this area . The distribution of several OP-resistance genes was surveyed within the Llobregat Delta and neighboring populations (< 25 km) during the 2 yr following this regional pesticide change to investigate how the change in selection pressure affected the dynamics of OP-resistance genes . The immigration failure of the A2-B2 resistant esterases and the observed difference in OP-resistance dynamics between isolated and nonisolated populations may indicate fitness disadvantages associated with OP-resistance genes, hence a tendency for a decrease in OP-resistance . In contrast, one OP-resistance gene further increased in frequency, whereas the frequencies of some others were maintained . These unexpected results question the importance of pesticides from sources other than mosquito control, and the variability of pleiotropic fitness costs among pesticide resistance genes.

Biopolymers, 1999 Jan, 49(1), 29 - 40
Conformational analysis of peptide fragments derived from the peripheral subunit-binding domain from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: evidence for nonrandom structure in the unfolded state; Spector S et al.; There is currently a great deal of interest in the early events in protein folding . Two issues that have generated particular interest are the nature of the unfolded state under native conditions and the role of local interactions in folding . Here, we report the results of a study of a set of peptides derived from a small two-helix protein, the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex . Five peptides of overlapping sequence were prepared, including sequences corresponding to each of the helices and to the region connecting them . The peptides were characterized by CD and, where possible, nmr . A peptide corresponding to the second helix is between 12 and 17% helical at neutral pH . CD also indicates a lower percentage of helical structure in the peptide corresponding to the first alpha-helix, although the values of the alpha-proton chemical shifts suggest some preference for nonrandom structure . Peptides corresponding to the interhelical loop, which in the full domain contains two overlapping beta-turns and a 5-residue 3(10)-helix, are less structured . There is no significant change in the helicity of any of these peptides with pH . To test for fragment complementation, CD spectra of the two peptides derived from each helix and the long connecting peptide were compared to the spectra of each possible pair, as well as to a mixture containing all three . No increase in structure was observed . We complement our peptide studies by characterizing a point mutant, D34V, which disrupts a critical hydrogen bonding network . This mutant is unable to fold and provides a useful model of the denatured state . The mutant is between 9 and 16% helical as judged by CD . The modest amount of helical structure formed in some of the peptide fragments and in the point mutant suggests that the denatured state of the peripheral subunit binding domain is not completely unstructured . This may contribute to the very rapid folding observed for the intact protein.

Insect Biochem Mol Biol, 1999 Jan, 29(1), 19 - 24
Minimum structure of peptidoglycan required for induction of antibacterial protein synthesis in the silkworm, Bombyx mori; Iketani M et al.; Various peptidoglycan fragments, different in mode of cross-linking and molecular size, were isolated, and the elicitor activity was tested for induction of antibacterial protein synthesis in larvae of Bombyx mori . Linear uncross-linked peptidoglycans from Bacillus licheniformis and Micrococcus luteus were effective elicitors, similar to the directly cross-linked peptidoglycan from B . licheniformis cell wall . The fragments of uncross-linked peptidoglycan with a sugar chain length of four or more were active elicitors, but the disaccharide unit had no elicitor activity . The minimum structure of peptidoglycan required for induction of antibacterial protein synthesis was determined to be two repeating N-acetylglucosamine-N-acetylmuramic acid units with peptide side chains.

Curr Microbiol, 1999 Apr, 38(4), 224 - 7
Characterization of the mbl determinant and cloning of the spoIIID gene from Bacillus cereus ATCC 10987; Lindback T et al.; To examine the transcriptional start site of the putative Bacillus cereus mbl gene, we have cloned and sequenced the upstream region of the previously reported B . cereus mbl determinant, which showed sequence similarity to the mreB morphogene from Escherichia coli . Primer extension analysis revealed the transcriptional start site of mbl to 259 bp upstream of the putative translational start site and showed that the mbl gene was expressed at 3, 6, and 10 h of growth after inoculation . Antibodies against B . cereus Mbl showed that even though mbl mRNA was present in amounts detectable with Northern blot analysis exclusively in the early logarithmic growth phase, the amount of protein was constant during the cell cycle . Immunogold labeling cryo-transmission electron microscopy indicates that B . cereus Mbl is a cytoplasmic protein . The upstream sequence of mbl revealed two open reading frames, spoIIID and orf1 . The amino acid sequence of B . cereus SpoIIID is identical to the SpoIIID sequence from Bacillus thuringiensis . Primer extension showed that mbl is not cotranscribed with spoIIID.

Curr Microbiol, 1999 Apr, 38(4), 199 - 204
Behavior of pythium Torulosum zoospores during their interaction with tobacco roots and Bacillus cereus; Shang H et al.; Bacillus cereus UW85 suppresses seedling damping-off diseases caused by Oomycetes and produces antibiotics that inhibit development of Oomycetes in culture . The goal of this study was to determine how UW85 and its antibiotics affected the behavior of an Oomycete, Pythium torulosum, in its interaction with plant roots . We studied tobacco seedlings inoculated with zoospores of P . torulosum and UW85 culture, culture filtrate, washed cells, antibiotics (zwittermicin A or kanosamine), purified from cultures of UW85, and UW030, a mutant of UW85 that does not suppress disease and does not produce the antibiotics . Microscopic observation revealed that all of the treatments inhibited zoospore activity around roots and encystment on roots . Treatment with UW85 culture, culture filtrate, zwittermicin A, or kanosamine delayed cyst germination and the elongation rate of germ tubes, whereas treatment with UW030 or washed UW85 cells did not . In an in vitro seedling bioassay of disease suppression, the antibiotics, zwittermicin A and kanosamine, suppressed disease singly or together, although UW85 culture suppressed disease more effectively than did the antibiotics . The results show that B . cereus cultures affect zoospore behavior in the presence of roots, and B . cereus-produced antibiotics, zwittermicin A and kanosamine, contribute to disease suppression and inhibition of germ tube elongation in the presence of the plant root.

Prep Biochem Biotechnol, 1999 Feb, 29(1), 35 - 47
A single step purification process for cyclodextrin glucanotransferase from a Bacillus sp . isolated from soil; Thatai A et al.; Cyclodextrin glucanotransferase (CGTase) is a commercially important enzyme which catalyses the formation of cyclodextrins (CDs) from starch . A CGTase producing bacterium was isolated from soil which gave a fairly high enzyme activity of 7.5 U mL(-1) after 24 h of growth which was further increased to 22 U mL(-1) by proper media design . The enzyme was purified to homogeneity by a novel single affinity precipitation step which resulted in a very high recovery of more than 90% . The molecular weight, as determined by SDS-PAGE, was found to be 68 kDa . The pH optimum was found to be 6.6 while a temperature optimum of 65 degrees C was observed . The enzyme exhibited a fairly high degree of functional stability with little loss of activity, even after 8 h of incubation at 65 degrees C . Ca++ had little effect on the activity of the enzyme while urea at 10 mM concentration increased the activity of the enzyme by more than 200%, suggesting that it is a unique enzyme.

Int Immunol, 1999 Feb, 11(2), 209 - 16
Characterization of the culture filtrate-specific cytotoxic T lymphocyte response induced by Bacillus Calmette-Guérin vaccination in H-2b mice; Denis O et al.; Although CD8+ T cells are supposed to play an important role in protective immunity to mycobacteria, cytotoxic T lymphocyte (CTL) responses in this infection remain poorly characterized . We previously demonstrated that bacillus Calmette-Guerin (BCG) immunization of H-2b mice induced CTL able to recognize and kill macrophages incubated with proteins from mycobacterial culture supernatant {culture filtrate (CF) antigens} . In the present study, we have further characterized the lytic activity of these CTL and the processing pathway used for the presentation of CF proteins . We show that they use the degranulation pathway (secretion of perforins and granzymes) as the main lytic mechanism of cytotoxicity and also secrete IFN-gamma upon incubation with CF-pulsed macrophages . The in vitro presentation of CF proteins to CTL required a processing step inhibited in the cold but insensitive to Brefeldin A . Transporter-associated protein (TAP)-2-deficient RMA-S cells were efficiently recognized and killed by CF-specific CTL, demonstrating the lack of TAP requirement for this presentation . However, recognition of target cells by CTL was abolished when carried out in the presence of chloroquine . These results indicate that a non-classical MHC class I-processing pathway allows the recognition of a CF protein by CTL in BCG-vaccinated H-2b mice.

J Dairy Sci, 1999 Feb, 82(2), 305 - 14
Bacillus cereus spores in raw milk: factors affecting the contamination of milk during the grazing period; Christiansson A et al.; Psychrotrophic Bacillus cereus is a limiting factor for the shelf-life of pasteurized milk, particularly during the grazing season . Potential sources of contamination and factors that might affect the spore content of milk were studied in detail for a group of eight cows during three 2-wk study periods from June to September over 2 yr . The spore content of milk was strongly associated with the degree of contamination of the teats with soil . High water content of soil, low evaporation of water and dirty access alloys were the most important factors correlating with high spore concentrations . The spore content of soil varied from < 50 to 380,000/g, depending on time and sampling site . The milking equipment did not contribute significantly to the contamination . The spore contents in air during milking (< 100 cfu/m3) and in feed (silage, hay, fresh grass, and concentrates) were too low to be of importance for contamination . The spore content in dung was also low . Further support that soil was the major contamination source was found by comparison of genetic fingerprints by random amplified polymorphic DNA polymerase chain reaction of isolates of B . cereus from soil and milk and by teat cleansing experiments, which resulted in reduced contamination levels in milk.

Arch Biochem Biophys, 1999 Mar 15, 363(2), 259 - 66
The active site of phosphorylating glyceraldehyde-3-phosphate dehydrogenase is not designed to increase the nucleophilicity of a serine residue; Boschi-Muller S et al.; Changing a catalytic cysteine into a serine, and vice versa, generally leads to a dramatic decrease in enzymatic efficiency . Except a study done on thiol subtilisin, no extensive study was carried out for determining whether the decrease in activity is due to a low nucleophilicity of the introduced amino acid . In the present study, Cys149 of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus was converted into a Ser residue . This leads to a drastic reduction of the kcat value . The rate-limiting step occurs before the hydride transfer step . Selective, but slow, inactivation is observed with specific, structurally different, inhibitors of serine protease . The esterolytic activity of serine mutant towards activated esters is also strongly decreased . The rate-limiting step of the esterase reaction also shifts from deacylation in the wild type to acylation in the mutant . Altogether, these results strongly suggest that the low catalytic efficiency of the Ser mutant is due to a poor nucleophilicity of the hydroxyl serine group within the active site of the enzyme . The fact that (1) the apo --> holo transition does not change esterolytic and inactivating efficiencies, and (2) Ser149 Asn176 double mutant exhibits the same chemical reactivity and esterolytic catalytic efficiency compared to the Ser149 single mutant indicates that the serine residue is not subject to His176 general base catalysis . A linear relationship between the catalytic dehydrogenase rate, the kcat/KM for esterolysis, and the concentration of OH- is observed, thus supporting the alcoholate entity as the attacking reactive species . Collectively this study shows that the active site environment of GAPDH is not adapted to increase the nucleophilicity of a serine residue . This is discussed in relation to what is known about Ser and Cys protease active sites .

Vaccine, 1999 Feb 26, 17(7-8), 915 - 22
A historical and molecular phylogeny of BCG strains; Behr MA et al.; Bacillus Calmette-Guerin (BCG) is the name given to a family of vaccines derived in 1921 by the in-vitro attenuation of Mycobacterium bovis . Subsequently, BCG seed lots were distributed globally, and both phenotypic and genotypic differences between strains have been described . As a step to understanding BCG diversity, we have reviewed the English and French historical record on 13 strains and DNA was extracted from these strains for restriction-fragment-length-polymorphism study . The written record suggests "early strains" obtained from the Pasteur Institute between 1924 and 1926 and "later strains" obtained after 1931 . Molecular typing resulted in 3 clades, based on variability in IS6110-typing and the presence of mpt64 gene . With two exceptions, these clades correspond with strains obtained in 1924-26 (IS6110-2/mpt64+), 1926-31 (IS6110-1/mpt64+), and 1931 or later (IS6110-1/mpt64-) This analysis demonstrates that BCG has undergone genetic changes since 1921, consistent with ongoing in-vitro evolution.

Vaccine, 1999 Feb 26, 17(7-8), 904 - 14
Safety and immunogenicity of recombinant Bacille Calmette-Guérin (rBCG) expressing Borrelia burgdorferi outer surface protein A (OspA) lipoprotein in adult volunteers: a candidate Lyme disease vaccine; Edelman R et al.; This phase I clinical trial was designed to determine the feasibility of using rBCG as a live bacterial vaccine vector for the outer surface protein A (OspA) of Borrelia burgdorferi and as model for other vaccines based on a rBCG vector . To construct the vaccine, a signal peptide derived from a mycobacterial lipoprotein was used to direct the export, and membrane-associated surface expression, of OspA in a standard strain of BCG (Connaught) . The rBCG OspA vaccine was safe and immunogenic in several animal species, and protective in a mouse model of Lyme borreliosis . An intradermal injection (0.1 ml) of rBCG OspA was administered to 24 healthy adult volunteers sequentially at one of four dose levels, ranging from 2.0 x 10(4) CFU to 2 x 10(7) CFU, using a dose-escalation design . All volunteers were initially PPD-skin test and OspA antibody negative, and they were monitored for 2 years after immunization . Three volunteers had mild flu-like reactions 1-2 days after vaccination . Local ulceration and drainage at the site of injection, which occurred in 50% and 83% of volunteers in the two highest dose groups, persisted for 1-70 days before the ulcers healed . Most of the drainage samples yielded rBCG colonies that contained the OspA plasmid . Thirteen of 24 vaccinees, principally in the two highest dose groups, converted their PPD skin tests from negative to positive . None of the 24 volunteers developed OspA antibody . In conclusion, the current rBCG vaccine construct, the first such construct tested in humans, had a safety profile comparable to that of licensed BCG, but it did not elicit primary humoral responses to the vectored antigen.

Kekkaku, 1999 Jan, 74(1), 27 - 32
{Tuberculous meningitis developed during treatment for systemic lupus erythematosus (SLE)}; Tsushima K et al.; A 51-year-old woman was admitted to our hospital complaining of fever and general fatigue . Physical examination revealed butterfly-like erythema in face, facial edema and diffuse purpura all over her body . Laboratory data showed renal dysfunction, nephrotic syndrome and active phase of SLE . She was administered first methylprednisolone (1g/day/3 days by intravenous drip) then prednisolone (60 mg/day/month, orally) and had immune adsorption therapy for eight times . However, 14 days after the last session of immune adsorption, she developed fever of 39 degrees C and mild headache, and then 3 days later, she gradually became unconscious . Brain CT showed hydrocephalus . We diagnosed her as having tuberculous meningitis based on the detection of acid-fast bacillus in cerebrospinal fluid, and began treatment with antituberculous agents . We suspected that tuberculous meningitis had caused hydrocephalus . We tried percutaneous drainage of the left ventricle for hydrocephalus . Brain MRI showed a tuberculoma depicted as a mass of low intensity in the right cerebellum on the T1-weighted image, and of high intensity on the T2-weighted image, and the meninx in the basal cistern was enhanced . After treatment with antituberculous agents, we performed serial brain MRI and examined cerebrospinal adenosine deaminase activity (ADA) . Despite treatment with antituberculous agents, new intracerebral tuberculomas had developed in some areas, whereas they had disappeared in other areas . After treatment for 4 months, the level of cerebrospinal ADA became normal, and the patient recovered consciousness despite the presence of multiple tuberculomas . Both the cell counts and the level of ADA in cerebrospinal fluid are the good indicators of the activity of tuberculous meningitis and reflected its clinical course . Furthermore, the level of ADA in cerebrospinal fluid changed with brain MRI image . Serial brain MRI and examination of ADA in cerebrospinal fluid were useful to know the activity of tuberculous meningitis and to evaluate the response to treatment.

Nihon Kokyuki Gakkai Zasshi, 1998 Dec, 36(12), 1053 - 7
{A case of cervical-mediastinal lymph node tuberculosis progressed to pulmonary lesion through a bronchial fistula}; Kawamoto H et al.; The patient was a 25-year-old man who had been admitted to a local hospital due to fever and trachelophyma . Tubercle bacillus was detected in pus culture obtained by biopsy of the trachelophyma, but not in sputum culture . Because combined therapy with 3 antituberculous drugs (RFP, INH and SM) failed to reduce the fever or drainage from the biopsy region, the patient was transferred to our hospital . Chest X-ray films taken on admission revealed dilatation of the superior mediastinal shadow; chest CT images revealed cervical and mediastinal lymphadenopathy and an anterior mediastinal abscess, but no pulmonary lesion . About 2 months after admission, cough developed and Gaffky type 2 was detected in the patients sputum . Bronchoscopy and bronchography revealed a bronchomediastinal fistula . Forty days after the onset of cough, reticulogranular shadows were observed in the right upper lobe on chest X-ray films, and a diffuse centrilobular lesion was observed in the right upper lobe on chest CT images . From these clinical observations, the patient was given a diagnosis of cervical-mediastinal lymph node tuberculosis, which had progressed to pulmonary lesion through a bronchial fistula due to lymphadenitis.

Eur J Immunol, 1999 Feb, 29(2), 650 - 9
Induction of IFN-gamma-producing CD4+ natural killer T cells by Mycobacterium bovis bacillus Calmette Guérin; Emoto M et al.; The CD4+ natural killer (NK)T cells in the liver are potent IL-4 producers and hence may promote Th2 cell development . Following Mycobacterium bovis bacillus Calmette Guerin (BCG) infection, IL-4-producing CD4+ NKT cells become undetectable in liver mononuclear cells of normal density (interface between 40 and 70% Percoll) by flow cytometry . The present study shows that M . bovis BCG infection changes the density of liver CD4+ NKT cells and shifts cytokine production from IL-4 to IFN-gamma . The number of CD4+ NK1+ TCR alpha/beta(intermediate) cells increased in the low-density fraction (<40% Percoll density gradient) in parallel to the reduction of this cell population in the fraction of normal density . The number of IL-4-producing cells, however, was small and high frequencies of IFN-gamma-secreting cells were identified in the low-density fraction after TCR/CD3 ligation . Accordingly, selected low-density CD4+ NKT cells encompassed high numbers of IFN-gamma producers and minute numbers of IL-4-secreting cells . Induction of low-density CD4+ NKT cells by M . bovis BCG was abrogated by endogenous IL-12 neutralization which also caused increased bacterial growth in the liver . We assume that M . bovis BCG infection changes cytokine secretion by the CD4+ NKT cell population from IL-4 to IFN-gamma through IL-12 induction . Thus, CD4+ NKT cells may contribute to host resistance against intracellular bacteria prior to conventional IFN-gamma-producing Th1 cells.

J Appl Microbiol, 1999 Feb, 86(2), 311 - 24
Inhibition of Bacillus licheniformis spore growth in milk by nisin, monolaurin, and pH combinations; Mansour M et al.; The effects of nisin and monolaurin, alone and in combination, were investigated on Bacillus licheniformis spores in milk at 37 degrees C . In the absence of inhibitors, germinated spores developed into growing vegetative cells and started sporulation at the end of the exponential phase . In the presence of nisin (25 IU ml-1), spore outgrowth was inhibited (4 log10 reduction at 10 h) . Regrowth appeared between 10 and 24 h and reached a high population level (1.25 x 10(8) cfu ml-1) after 7 d . Monolaurin (250 micrograms ml-1) had a bacteriostatic effect during the first 10 h but thereafter, regrowth occurred slowly with a population level after 7 d (4 x 10(5) cfu ml-1) lower than that of nisin . Different combined effects of nisin (between 0 and 42 IU ml-1), monolaurin (ranging from 0 to 300 micrograms ml-1), pH values (between 5.0 and 7.0) and spore loads (10(3), 10(4), 10(5) spores ml-1) were investigated using a Doehlert matrix in order to study the main effects of these factors and the different interactions . Results were analysed using the Response Surface Methodology (RSM) and indicated that nisin and monolaurin had no action on spores before germination; only pH values had a significant effect (P < or = 0.001), i.e . spore count decreased as the pH value increased in relation to germination . Sublethal concentrations of nisin (30 IU ml-1) and monolaurin (100 micrograms ml-1) in combination acted synergistically on outgrown spores and vegetative cells, showing total inhibition at pH 6.0, without regrowth, within 7 d at 37 degrees C.

J Appl Microbiol, 1999 Feb, 86(2), 266 - 74
The certification of a reference material for the evaluation of methods for the enumeration of Bacillus cereus; in 't Veld PH et al.; A reference material containing Bacillus cereus was certified by the Community Bureau of Reference (BCR) for its number of colony-forming particles (cfp) in 0.1 ml reconstituted capsule solution . To this end, a batch of approximately 15,000 capsules was produced and tested for its homogeneity and stability . The variation in the number of cfp between capsules (homogeneity) was not found to be significantly different from a Poisson distribution . Stability was tested for extended periods at storage temperature (-20 degrees C) and at various higher temperatures up to 37 degrees C for 4 weeks to simulate transport conditions . Only at 37 degrees C did a small but significant decrease in the number of cfp occur . At -20 degrees C, no decrease in the number of cfp was observed over a period of about 4 years . For certification, 12 laboratories determined the number of cfp on two agars: Mannitol Egg-Yolk Polymyxin agar (MEYP, incubated at 30 degrees C) and Polymyxin pyruvate Egg-yolk Bromothymol blue Agar (PEMBA, incubated at 37 degrees C) . The certified geometric mean value on MEYP after 24 h of incubation was 53.4 cfp 0.1 ml-1 of the reconstituted capsule solution (95% confidence interval 51.7-55.2) and on PEMBA, 55.0 (95% confidence interval 52.8-57.4) . Based on these certified values, user tables were constructed specifying the 95% confidence limits when testing a smaller number of capsules, as would be done in individual laboratories . Based on the information on homogeneity, stability and the certification study, the BCR decided to certify the material as CRM 528.

Biosci Biotechnol Biochem, 1999 Jan, 63(1), 199 - 201
Separation and properties of two acetylacetoin reductases from Bacillus cereus YUF-4; Hosaka T et al.; The separation and purification of two kinds of acetylacetoin reductases (AACRs) from Bacillus cereus YUF-4 were examined . NADPH-linked AACR (AACR I) and NADH-linked AACR (AACR II) were separated from each other by ammonium sulfate fractionation, DEAE-cellulose chromatography, and hydroxyapatite chromatography . The former was purified 3.4-fold with a yield of 10.0%, and the latter was purified 29-fold with a yield of 15.6% . The two enzymes differ from each other in some enzymic properties such as substrate specificity.

Biosci Biotechnol Biochem, 1999 Jan, 63(1), 96 - 103
Cloning of Bacillus stearothermophilus ctaA and heme A synthesis with the CtaA protein produced in Escherichia coli; Sakamoto J et al.; The Bacillus stearothermophilus ctaA gene, which is required for heme A synthesis, was found upstream of the ctaBCDEF/caaEABCD gene cluster as in B . subtilis and B . firmus . The deduced protein sequence indicate that CtaA is a 35-kDa intrinsic membrane protein with seven hydrophobic segments . Alignment of CtaA sequences showed conserved residues including histidines that may be involved in heme B binding and substrate binding . Expression of ctaA in E . coli resulted in increased formation of a membrane-bound b-type cytochrome, heme A production, and severe growth inhibition . Furthermore, B . stearothermophilus CtaA produced in E . coli was found to catalyze the conversion of heme O to heme A in vitro.

Biosci Biotechnol Biochem, 1999 Jan, 63(1), 65 - 72
Purification and properties of a low-molecular-weight, high-alkaline pectate lyase from an alkaliphilic strain of Bacillus; Kobayashi T et al.; A low-molecular-weight, high-alkaline pectate lyase (pectate transeliminase, EC 4.2.2.2) was found in an alkaline culture of Bacillus sp . strain KSM-P15, purified to homogeneity, and crystallized . The enzyme had a relative molecular weight of approximately 20,300 as measured by sedimentation equilibrium, with a sedimentation coefficient (s20,w0) of 1.73 S . It was a basic protein with an isoelectric point of pH 10.3, and the alpha-helical content was only 6.6% . In the presence of Ca2+ ions, the enzyme degraded polygalacturonic acid in a random manner to yield 4,5-unsaturated oligo-galacturonides and had its optimal activity around pH 10.5 and 50-55 degrees C . It also had a protopectinase-like activity on cotton fibers . The N-terminal amino acid sequences of the intact protein (28 amino acids) and its two lysyl endopeptidase-cleaved peptide fragments (8 and 12 amino acids) had very low sequence similarity with pectate lyases reported to date . These results strongly suggest that the pectate lyase of Bacillus sp . strain KSM-P15 may be a novel enzyme and belongs in a new family.

Proc Natl Acad Sci U S A, 1999 Mar 2, 96(5), 1840 - 5
Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects; Kota M et al.; Evolving levels of resistance in insects to the bioinsecticide Bacillus thuringiensis (Bt) can be dramatically reduced through the genetic engineering of chloroplasts in plants . When transgenic tobacco leaves expressing Cry2Aa2 protoxin in chloroplasts were fed to susceptible, Cry1A-resistant (20,000- to 40,000-fold) and Cry2Aa2-resistant (330- to 393-fold) tobacco budworm Heliothis virescens, cotton bollworm Helicoverpa zea, and the beet armyworm Spodoptera exigua, 100% mortality was observed against all insect species and strains . Cry2Aa2 was chosen for this study because of its toxicity to many economically important insect pests, relatively low levels of cross-resistance against Cry1A-resistant insects, and its expression as a protoxin instead of a toxin because of its relatively small size (65 kDa) . Southern blot analysis confirmed stable integration of cry2Aa2 into all of the chloroplast genomes (5, 000-10,000 copies per cell) of transgenic plants . Transformed tobacco leaves expressed Cry2Aa2 protoxin at levels between 2% and 3% of total soluble protein, 20- to 30-fold higher levels than current commercial nuclear transgenic plants . These results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt-resistance development in the field.

Appl Environ Microbiol, 1999 Mar, 65(3), 898 - 902
Growth characteristics of Heterosigma akashiwo virus and its possible use as a microbiological agent for red tide control; Nagasaki K et al.; The growth characteristics of Heterosigma akashiwo virus clone 01 (HaV01) were examined by performing a one-step growth experiment . The virus had a latent period of 30 to 33 h and a burst size of 7.7 x 10(2) lysis-causing units in an infected cell . Transmission electron microscopy showed that the virus particles formed on the peripheries of viroplasms, as observed in a natural H . akashiwo cell . Inoculation of HaV01 into a mixed algal culture containing four phytoplankton species, H . akashiwo H93616, Chattonella antiqua (a member of the family Raphidophyceae), Heterocapsa triquetra (a member of the family Dinophyceae), and Ditylum brightwellii (a member of the family Bacillariophyceae), resulted in selective growth inhibition of H . akashiwo . Inoculation of HaV01 and H . akashiwo H93616 into a natural seawater sample produced similar results . However, a natural H . akashiwo red tide sample did not exhibit any conspicuous sensitivity to HaV01, presumably because of the great diversity of the host species with respect to virus infection . The growth characteristics of the lytic virus infecting the noxious harmful algal bloom-causing alga were considered, and the possibility of using this virus as a microbiological agent against H . akashiwo red tides is discussed.

Nucleic Acids Res, 1999 Mar 15, 27(6), 1421 - 8
Plasmid replication initiator protein RepD increases the processivity of PcrA DNA helicase; Soultanas P et al.; The replication initiator protein RepD encoded by the Staphylococcus chloramphenicol resistance plasmid pC221 stimulates the helicase activity of the Bacillus stearothermophilus PcrA DNA helicase in vitro . This stimulatory effect seems to be specific for PcrA and differs from the stimulatory effect of the Escherichia coli ribosomal protein L3 . Whereas L3 stimulates the PcrA helicase activity by promoting co-operative PcrA binding onto its DNA substrate, RepD stimulates the PcrA helicase activity by increasing the processivity of the enzyme and enables PcrA to displace DNA from a nicked substrate . The implication of these results is that PcrA is the helicase recruited into the replisome by RepD during rolling circle replication of plasmids of the pT181 family.

J Econ Entomol, 1999 Feb, 92(1), 246 - 52
Evaluation of transgenic sweet corn hybrids expressing CryIA (b) toxin for resistance to corn earworm and fall armyworm (Lepidoptera: Noctuidae); Lynch RE et al.; Many of the lepidopterous insects which attack sweet corn, Zea mays L., are susceptible to insecticidal proteins produced by Bacillus thuringiensis ssp . kurstaki (Berliner) (Btk) . Transgenic sweet corn expressing a synthetic cry gene for production of a Btk-insecticidal protein may provide a more environmentally acceptable means of sweet corn production . Eight transgenic sweet corn hybrids containing a synthetic gene for CryIA(b) protein production (BT11 event) were evaluated for resistance to the corn earworm, Helicoverpa zea (Boddie), and fall armyworm, Spodoptera frugiperda (J . E . Smith) . Laboratory tests revealed that all Btk sweet corn hybrids were highly resistant to leaf and silk feeding by neonate 3 and 6 d old corn earworm larvae . Ear damage in the field to the Btk sweet corn hybrids caused by corn earworm was negligible . All Btk sweet corn hybrids, except Btk 95-0901, were moderately resistant to leaf and silk feeding by the fall armyworm . Survival and weight gain were reduced when neonates were fed excised whorl leaves of the Btk plants . Weight gain, but not survival, was reduced when 3- and 6-d-old fall armyworm larvae were fed excised whorl leaves of the Btk plants . Btk sweet corn hybrids appear to be ideal candidates for use in integrated pest management (IPM) programs for both the fresh and processing sweet corn markets, and their use should drastically reduce the quantity of insecticides currently used to control these pests in sweet corn . With appropriate cultural practices, it is highly unlikely that Btk sweet corn will contribute to the development of resistance to Btk proteins in these insects because of the high toxicity of the Cry proteins expressed in these sweet corn hybrids and the harvest of sweet corn ears from fields before larvae can complete development.

J Steroid Biochem Mol Biol, 1998 Dec, 67(5-6), 451 - 65
Transformation of steroids by Bacillus strains isolated from the foregut of water beetles (Coleoptera:Dytiscidae): I . Metabolism of androst-4-en-3,17-dione (AD); Schaaf O et al.; Two Bacillus strains were isolated from the foregut of the water beetle Agabus affinis (Payk.) and tested for their steroid transforming ability . After incubation with androst-4-en-3,17-dione (AD), 13 different transformation products were detected . AD was hydroxylated at C6, C7, C11 and C14, resulting in formation of 6beta-, 7alpha-, 11alpha- and 14alpha-hydroxy-AD . One strain also produced small amounts of 6beta,14alpha-dihydroxy-AD . Partly, the 6beta-hydroxy group was further oxidized to the corresponding 6-oxo steroids . In addition, a specific reduction of the delta4-double bond was observed, leading to the formation of 5alpha-androstane derivatives . In minor yields the carbonyl functions at C3 and C17 were reduced leading to the formation of 3zeta-OH or 17beta-OH steroids . EI mass spectra of the trimethylsilyl and O-methyloxime trimethylsilyl ether derivatives of some transformation products are presented for the first time.

Arq Neuropsiquiatr, 1998 Dec, 56(4), 772 - 7
{Prognostic factors of tuberculous meningoencephalitis lethality}; Nunes C et al.; In order to describe the lethality predictors of patients with tuberculous meningoencephalitis, records of 231 patients were analysed . Ages ranged from less than 1 year to 68 years . Ninety-seven patients (42%) were four years old or less . Apart from 73.2% of patients whose diagnosis was performed by clinical and epidemiologic criteria associated with response to specific therapy, 26.8% had diagnostic confirmation through cerebrospinal fluid (culture, bacilloscopy, PCR) or necropsy . The lethality predictors were: less than 4 years of age, seizures, and severe alterations of consciousness.

Mol Microbiol, 1999 Jan, 31(2), 463 - 71
Domain III of the Bacillus thuringiensis delta-endotoxin Cry1Ac is involved in binding to Manduca sexta brush border membranes and to its purified aminopeptidase N; de Maagd RA et al.; Three types of binding assays were used to study the binding of Bacillus thuringiensis delta-endotoxin Cry1Ac to brush border membrane vesicle (BBMV) membranes and a purified putative receptor of the target insect Manduca sexta . Using hybrid proteins consisting of Cry1Ac and the related Cry1C protein, it was shown that domain III of Cry1Ac is involved in specificity of binding as observed by all three techniques . In ligand blotting experiments using SDS-PAGE-separated BBMV proteins as well as the purified putative receptor aminopeptidase N (APN), the presence of domain III of Cry1Ac in a hybrid with Cry1C was necessary and sufficient for specific binding to APN . Using the surface plasmon resonance (SPR) technique with immobilized APN, it was shown that the presence of domain III of Cry1Ac in a hybrid is sufficient for binding to one of the two previously identified Cry1Ac binding sites, whereas the second site requires the full Cry1Ac toxin for binding . In addition, the role of domain III in the very specific inhibition of Cry1Ac binding by the amino sugar N-acetylgalactosamine (GalNac) was determined . Both in ligand blotting and in surface plasmon resonance experiments, as well as in binding assays using intact BBMVs, it was shown that the presence of domain III of Cry1Ac in a toxin molecule is sufficient for the inhibition of binding by GalNAc . These and other results strongly suggest that domain III of delta-endotoxins play a role in insect specificity through their involvement in specific binding to insect gut epithelial receptors.

Biochemistry, 1999 Feb 9, 38(6), 1772 - 9
Reconstitution of functional 50S ribosomes from in vitro transcripts of Bacillus stearothermophilus 23S rRNA; Green R et al.; In vitro transcripts of Bacillus stearothermophilus 23S rRNA can be reconstituted into catalytically active 50S ribosomal subunits with an efficiency only 3-4-fold lower than that of natural 23S rRNA . Thus, post-transcriptional modifications in 23S rRNA are not essential for the assembly or function of the 50S subunit of the ribosome . This reconstitution sytem has been used to characterize the peptidyl transferase activity of site-directed mutations in 23S rRNA at positions G2252, U2506, U2584, and A2602 (Escherichia coli numbering), demonstrating its potential for the analysis of the role played by 23S rRNA in the function of the 50S subunit of the ribosome.

J Urol, 1999 Mar, 161(3), 772 - 5; discussion 775-6
13-year experience with percutaneous management of upper tract transitional cell carcinoma; Clark PE et al.; PURPOSE: We determined the immediate and long-term results of percutaneous management of upper trace transitional cell carcinoma in regard to rates of tumor recurrence and preservation of renal function . MATERIALS AND METHODS: Since July 1985, 12 men and 5 women 50 to 86 years old (mean age 72.2) years old underwent percutaneous management of upper tract transitional cell carcinoma . Of the patients 12 (71%) had a solitary kidney and 1 was treated bilaterally . In 16 of the 18 treated renal units (89%) definitive percutaneous resection of the tumor was followed by 6 weekly percutaneous installations of bacillus Calmette-Guerin . RESULTS: Complete resection was accomplished in 17 of the 18 renal units . Of the 18 renal units 15 (83.3%) had documented stage pTa lesions and 14 (77.8%) had grade 1/3 or 2/3 disease . Followup for all patients ranged from 1.7 to 75.5 months (mean 20.5) . At the latest followup 11 patients (64.7%) are alive with no evidence of disease, and 6 (35.3%) died, 3 of whom (17.6%) had metastatic transitional cell carcinoma . Of the 13 patients undergoing treatment to solitary kidneys or bilaterally followup ranged from 1.7 to 75.5 months (mean 23.6) . Serum creatinine ranged from 1.1 to 3.5 mg./dl . (mean 1.6) before percutaneous tumor resection and from 1.1 to 2.2 mg./dl . (mean 1.6) at the latest followup . Only 1 of these 13 patients (7.7%) with a solitary kidney has required dialysis . Ipsilateral local recurrence developed in 6 of the 18 renal units (33%), and in 4 of these 6 patients (67%) the tumor was grade 2/3 or 3/3 at initial resection . These recurrences were treated endoscopically in 4 patients, 3 of whom are currently without evidence of disease, and with nephroureterectomy in 2 . Of the 17 patients only 1 (5.9%) with high grade (3/3), invasive (pT2) primary tumor at initial resection died of locally persistent or recurrent disease . CONCLUSIONS: Percutaneous management of upper tract transitional cell carcinoma is technically feasible and applicable in a significant number of patients in whom nephron sparing management is otherwise warranted . In carefully selected patients the results are at least comparable to other forms of "conservative" management in terms of tumor control and preservation of renal function.

J Urol, 1999 Mar, 161(3), 977 - 83
Effects of bacillus Calmette-Guèrin and interferon alpha-2B on cytokine production in human bladder cancer cell lines; Zhang Y et al.; PURPOSE: To determine the effects of live BCG, autoclaved BCG and interferon alpha-2b on cytokine production in human bladder cancer cell lines . MATERIALS AND METHODS: The release of nine cytokines from the human bladder cancer cell lines, RT4, RT112, SD, MGH and J82, was measured by ELISA assay . The mRNA level of IL-6 and GM-CSF was determined by RT-PCR . RESULTS: BCG and/or interferon alpha-2b differentially increased IL-1beta, IL-6, IL-8, GM-CSF and TNF-alpha production in the bladder cancer cells . High grade cell lines were more responsive to BCG whereas low grade lines were more sensitive to interferon alpha-2b . This correlated with cytotoxicity and growth inhibition induced by these agents . BCG could also induce low levels of IFN-alpha production in all the cell lines . Compared with live BCG, autoclaved BCG had no antiproliferative effect on MGH cells and was less effective in stimulating the production of IL-6, IL-8 and GM-CSF . However, autoclaved BCG was as effective as live BCG in inhibiting growth and stimulating IL-6 and TNF-alpha production of J82 cells . The combination of BCG and interferon alpha-2b also completely suppressed TGF-beta1 production in the MGH and RT112 cell lines . CONCLUSIONS: The combination of BCG and interferon alpha-2b has additive effects in cytokine production from bladder cancer cells . This correlates with cytotoxicity and growth inhibition induced by these agents.

J Urol, 1999 Mar, 161(3), 792 - 8
Correlation and prognostic significance of p53, p21WAF1/CIP1 and Ki-67 expression in patients with superficial bladder tumors treated with bacillus Calmette-Guerin intravesical therapy; Zlotta AR et al.; PURPOSE: We determine if, before intravesical bacillus-Calmette Guerin (BCG) therapy, p53, p21WAF-1-CIP1 (a critical downstream effector of p53 pathway of cell growth control, inhibiting cyclin dependent kinases) and the cell proliferation marker Ki-67 (MIB-1) could be used as prognostic markers of response to BCG in patients with superficial bladder tumors . MATERIALS AND METHODS: The study included 47 patients with superficial bladder tumors at high risk for recurrence or progression treated with 6 weekly intravesical BCG instillations . We analyzed p53, p21 and Ki-67 on paraffin embedded samples by immunohistochemistry and the percentage of positive cells was determined in a blinded fashion . Quantitative immunostaining was analyzed in relation to time to recurrence and progression using univariate or multivariate analysis and the Kaplan-Meier method . RESULTS: During a mean followup of 24.6 months 23 of the 47 patients (48.9%) presented with tumor recurrence and 10 (21.2%) had later progression to invasive disease . A p21 over expression (greater than 10%) was observed in 23 tumors (48.9%) and positively correlated with p53 (p = 0.0097) but not with Ki-67 (p = 0.327) . Of the tumors 18 (38.2%) were p53 and p21 negative . Among p21 positive tumors 15 (65.2%) were p53 and p21 positive, suggesting that p21 may also be regulated by p53 independent pathways . However, p53 did not act as a predictor of recurrence or progression . In contrast, using Kaplan-Meier curves p21 over expression (greater than 10%) and Ki-67 at a 25% cutoff were associated with shorter recurrence-free survival (both p = 0.02 log rank test) but they did not predict additional information about risk of progression . However, multivariate analysis failed to demonstrate any independent prognostic value for p21 or Ki-67 in contrast to tumor stage . CONCLUSIONS: Our results indicate that p21WAF-1-CIP1 seems to be regulated by p53 independent pathways in superficial bladder cancer . The present study did not indicate an independent prognostic significance in patients treated with BCG for p53, p21WAF-1-CIP1 or Ki-67 markers . Larger prospective studies are needed to evaluate further the independent value of these biological markers in superficial bladder cancer management.

Philos Trans R Soc Lond B Biol Sci, 1998 Oct 29, 353(1376), 1787 - 95
Myths, models and mitigation of resistance to pesticides; Hoy MA; Resistance to pesticides in arthropod pests is a significant economic, ecological and public health problem . Although extensive research has been conducted on diverse aspects of pesticide resistance and we have learned a great deal during the past 50 years, to some degree the discussion about 'resistance management' has been based on 'myths' . One myth involves the belief that we can manage resistance . I will maintain that we can only attempt to mitigate resistance because resistance is a natural evolutionary response to environmental stresses . As such, resistance will remain an ongoing dilemma in pest management and we can only delay the onset of resistance to pesticides . 'Resistance management' models and tactics have been much discussed but have been tested and deployed in practical pest management programmes with only limited success . Yet the myth persists that better models will provide a 'solution' to the problem . The reality is that success in using mitigation models is limited because these models are applied to inappropriate situations in which the critical genetic, ecological, biological or logistic assumptions cannot be met . It is difficult to predict in advance which model is appropriate to a particular situation; if the model assumptions cannot be met, applying the model sometimes can increase the rate of resistance development rather than slow it down . Are there any solutions? I believe we already have one . Unfortunately, it is not a simple or easy one to deploy . It involves employing effective agronomic practices to develop and maintain a healthy crop, monitoring pest densities, evaluating economic injury levels so that pesticides are applied only when necessary, deploying and conserving biological control agents, using host-plant resistance, cultural controls of the pest, biorational pest controls, and genetic control methods . As a part of a truly multi-tactic strategy, it is crucial to evaluate the effect of pesticides on natural enemies in order to preserve them in the cropping system . Sometimes, pesticide-resistant natural enemies are effective components of this resistance mitigation programme . Another name for this resistance mitigation model is integrated pest management (IPM) . This complex model was outlined in some detail nearly 40 years ago by V . M . Stern and colleagues . To deploy the IPM resistance mitigation model, we must admit that pest management and resistance mitigation programmes are not sustainable if based on a single-tactic strategy . Delaying resistance, whether to traditional pesticides or to transgenic plants containing toxin genes from Bacillus thuringiensis, will require that we develop multi-tactic pest management programmes that incorporate all appropriate pest management approaches . Because pesticides are limited resources, and their loss can result in significant social and economic costs, they should be reserved for situations where they are truly needed--as tools to subdue an unexpected pest population outbreak . Effective multi-tactic IPM programmes delay resistance (= mitigation) because the number and rates of pesticide applications will be reduced.

J R Coll Surg Edinb, 1998 Dec, 43(6), 410 - 1
The painless fracture: could it be TB?
Reading AD, Stother IG.
Tuberculosis is one of the commonest infections world-wide, with one third of the world's population carrying the bacillus . Since the 1980s the decline in notification rates in the UK has stopped and recently reversed . The reasons for this are multifactorial and are discussed briefly here . We present the case of an unusual presentation of tuberculosis in the metatarsal of an elderly Caucasian gentleman . This serves as a reminder to include tuberculosis in the differential diagnoses of unusual musculoskeletal presentations.

Biochim Biophys Acta, 1999 Jan 11, 1429(2), 342 - 50
The oligomeric state of Bacillus thuringiensis Cry toxins in solution; Guereca L et al.; The molecular mass of different Cry toxins produced by Bacillus thuringiensis bacteria was estimated by size-exclusion chromatography and non-denaturing polyacrylamide gel electrophoresis at neutral and alkaline pH in order to assess the existence of oligomers in solution . We found that Cry1Aa, Cry1Ac, Cry1C, Cry1D and Cry3A toxins exist in solution as a mixture of monomer and high molecular mass aggregates with an apparent molecular mass greater than 600 kDa, that depend on the time elapsed between toxin activation and analysis . Aggregation of toxins by disulfide bonds is unlikely because aggregates are also observed in samples incubated with DTT . These data show that the Cry toxins studied do not form oligomers of less than ten subunits in solution and suggest that oligomer formation may occur after the toxin binds to the receptor and inserts into the membrane.

J Biol Chem, 1999 Feb 19, 274(8), 4754 - 63
The Bacillus stearothermophilus mannitol regulator, MtlR, of the phosphotransferase system . A DNA-binding protein, regulated by HPr and iicbmtl-dependent phosphorylation; Henstra SA et al.; D-Mannitol is taken up by Bacillus stearothermophilus and phosphorylated via a phosphoenolpyruvate-dependent phosphotransferase system (PTS) . The genes involved in the mannitol uptake were recently cloned and sequenced . One of the genes codes for a putative transcriptional regulator, MtlR . The presence of a DNA binding helix-turn-helix motif and two antiterminator-like PTS regulation domains, suggest that MtlR is a DNA-binding protein, the activity of which can be regulated by phosphorylation by components of the PTS . To demonstrate DNA binding of MtlR to a region upstream of the mannitol promoter, by DNA footprinting, MtlR was overproduced and purified . EI, HPr, IIAmtl, and IICBmtl of B . stearothermophilus were purified and used to demonstrate that MtlR can be phosphorylated and regulated by HPr and IICBmtl, in vitro . Phosphorylation of MtlR by HPr increases the affinity of MtlR for its binding site, whereas phosphorylation by IICBmtl results in a reduction of this affinity . The differential effect of phosphorylation, by two different proteins, on the DNA binding properties of a bacterial transcriptional regulator has not, to our knowledge, been described before . Regulation of MtlR by two components of the PTS is an example of an elegant control system sensing both the presence of mannitol and the need to utilize this substrate.

J Infect Dis, 1999 Feb, 179 Suppl 1, S263 - 7
Interventions to control virus transmission during an outbreak of Ebola hemorrhagic fever: experience from Kikwit, Democratic Republic of the Congo, 1995; Kerstiens B et al.; On 6 May 1995, the Medecins sans Frontieres (MSF) coordinator in Kinshasa, Democratic Republic of the Congo (DRC), received a request for assistance for what was believed to be a concurrent outbreak of bacillary dysentery and viral hemorrhagic fever (suspected Ebola hemorrhagic fever {EHF}) in the town of Kikwit, DRC . On 11 May, the MSF intervention team assessed Kikwit General Hospital . This initial assessment revealed a nonfunctional isolation ward for suspected EHF cases; a lack of water and electricity; no waste disposal system; and no protective gear for medical staff . The priorities set by MSF were to establish a functional isolation ward to deal with EHF and to distribute protective supplies to individuals who were involved with patient care . Before the intervention, 67 health workers contracted EHF; after the initiation of control measures, just 3 cases were reported among health staff and none among Red Cross volunteers involved in body burial.

Vaccine, 1999 Jan 21, 17(3), 245 - 51
Host immune responses to ribosome, ribosomal proteins, and RNA from Mycobacterium bovis bacille de Calmette-Gúerin; Miyazaki C et al.; The ribosomes from BCG strongly induced delayed type hypersensitivity (DTH) skin reactions in guinea pigs immunized with live BCG or heat killed Mycobacterium tuberculosis H37Rv, and also induced lymphocyte proliferative response in mice immunized with ribosomes . In contrast, neither ribosomal proteins nor RNA alone induced both DTH skin reactions and lymphocyte proliferative responses . Particle form consisted of ribosomal proteins and RNAs might be absolutely required for the activation of immune responses.

Transplantation, 1999 Jan 27, 67(2), 296 - 8
Bacillary angiomatosis in a renal transplant recipient; Cline MS et al.; A case of bacillary angiomatosis infection presenting as a skin nodule in a renal transplant recipient was found . The patient was taking cyclosporine, prednisone, and mycophenolate mofetil at the time of presentation . The bacillary angiomatosis responded to 6 months of therapy with oral erythromycin.

Microb Pathog, 1999 Jan, 26(1), 1 - 11
Hormonal modulation of phagocytosis and intracellular growth of Mycobacterium avium ss . paratuberculosis In bovine peripheral blood monocytes; Feola RP et al.; In this study, we evaluated the effects of several hormones (i.e . growth hormone, prolactin, vitamin D3, luteinizing hormone, oxytocin) on the phagocytosis and intracellular survival of Mycobacterium avium ss . paratuberculosis within bovine peripheral blood monocytes . Phagocytosis of M . avium ss . paratuberculosis declined in a dose-dependent manner when monocytes were exposed to increasing amounts of recombinant bovine growth hormone, with little phagocytosis occurring at a growth hormone concentration of 50 ng/ml . The other hormones tested had little effect on phagocytosis . Continuous exposure of bovine monocytes to bovine growth hormone (10 ng per ml) resulted in enhanced intracellular bacillary growth . This was detected within 3 days of monocyte infection, and resulted in a 1 Log10 greater number of M . avium ss . paratuberculosis in growth hormone treated, than control, monocytes at 12 days of infection . When monocytes were incubated with growth hormone for only the first 5 days of a 12 day incubation period, a further increase in bacillary multiplication was observed . A similar increase in bacillary multiplication was observed when M . avium ss . paratuberculosis monocytes were incubated with prolactin for the first 5 days of a 12 day incubation period . These data indicate that varying levels of growth hormone and prolactin can affect the intracellular multiplication of M . avium ss . paratuberculosis in bovine monocytes .

J Mol Biol, 1999 Feb 19, 286(2), 375 - 87
Sites of interaction of streptogramin A and B antibiotics in the peptidyl transferase loop of 23 S rRNA and the synergism of their inhibitory mechanisms; Porse BT et al.; Streptogramin antibiotics contain two active A and B components that inhibit peptide elongation synergistically . Mutants resistant to the A component (virginiamycin M1 and pristinamycin IIA) were selected for the archaeon Halobacterium halobium . The mutations mapped to the universally conserved nucleotides A2059 and A2503 within the peptidyl transferase loop of 23 S rRNA (Escherichia coli numbering) . When bound to wild-type and mutant haloarchaeal ribosomes, the A and B components (pristinamycins IIA and IA, respectively) produced partially overlapping rRNA footprints, involving six to eight nucleotides in the peptidyl transferase loop of 23 S rRNA, including the two mutated nucleotides . An rRNA footprinting study, performed both in vivo and in vitro, on the A and B components complexed to Bacillus megaterium ribosomes, indicated that similar drug-induced effects occur on free ribosomes and within the bacterial cells . It is inferred that position 2058 and the sites of mutation, A2059 and A2503, are involved in the synergistic inhibition by the two antibiotics . A structural model is presented which links A2059 and A2503 and provides a structural rationale for the rRNA footprints .

J Immunol, 1999 Feb 15, 162(4), 2204 - 16
Mycobacterium bovis bacille Calmette-Guérin enhances pathogenicity of simian immunodeficiency virus infection and accelerates progression to AIDS in macaques: a role of persistent T cell activation in AIDS pathogenesis; Zhou D et al.; It has recently been proposed that Mycobacterium tuberculosis may enhance the pathogenicity of HIV infections and accelerate the course of HIV disease . This hypothesis has been tested in the present study using a simian immunodeficiency virus of macaques (SIVmac)/Mycobacterium bovis bacille Calmette-Guerin (BCG)-coinfected macaque model . Naive and chronically SIVmac-infected monkeys were evaluated . Following BCG inoculation, the SIVmac-infected monkeys exhibited the dominant responses of TCR-beta complementarity-determining region 3-restricted T cell subpopulations . This BCG-driven T cell activation correlated with a marked increase in viral loads in SIVmac-infected monkeys . Moreover, the prolonged T cell activation coincided with the enhanced decline of CD4+ PBL counts and the accelerated progression to clinical AIDS in the coinfected monkeys, suggesting that Mycobacterium-driven T cell activation may be the mechanism underlying the enhanced pathogenicity of AIDS virus infection in the coinfected individuals . Within 2 to 7 mo after BCG coinfection, all chronically SIVmac-infected monkeys died from SIV-induced AIDS including tuberculosis-like disease . Surprisingly, the naive monkeys manifested a T cell activation-related toxic shock syndrome and a profound depletion of CD4+ lymphocytes 2 wk after simultaneous SIVmac/BCG inoculation . These naive animals died 2 mo after SIVmac/BCG inoculation, with the evidence of the persistent SIV p27 antigenemia and SIVmac-induced disease . In contrast, the normal monkeys not infected with SIVmac survived BCG infection; the control SIVmac-infected animals showed a natural course of chronic SIV infection . Thus, results from this SIV/BCG coinfection model strongly support the hypothesis that active coinfection with HIV and Mycobacterium can impact remarkably on the AIDS virus-induced disease.

Biosci Biotechnol Biochem, 1998 Dec, 62(12), 2463 - 6
Synthesis of a novel phosphate ester of a vitamin E derivative and its antioxidative activity; Miyamoto S et al.; A novel phosphate ester containing a chromanol structure was synthesized from 1,2-diacyl-sn-glycero-3-phospho-2'-hydroxyethyl-2',5',7',8'-tetramethyl- 6' -hydroxychroman (PCh) by hydrolysis catalyzed by phospholipase C from Bacillus cereus . The structure of the product was found by spectral analyses to be 2-(2',5',7',8'-tetramethyl-6'-hydroxychromanyl)ethylphosphate++ + (Ch-P) . Ch-P was highly soluble in the aqueous phase at neutral pH values and exerted higher antioxidative activity than alpha-tocopherol and PCh in the Fe(III)/ascorbic acid-catalyzed peroxidation of a fish oil emulsion and the autoxidation of a rat brain homogenate.

Biosci Biotechnol Biochem, 1998 Nov, 62(11), 2107 - 14
Chitosanase activity of the enzyme previously reported as beta-1,3-1,4-glucanase from Bacillus circulans WL-12; Mitsutomi M et al.; Chitosanases 33 kDa and 40 kDa in size were detected in the culture supernatant of Bacillus circulans WL-12 . One of the two chitosanases, chitosanse 40 (40-kDa chitosanase) has been shown to be identical to the enzyme which has been reported previously as a beta-1,3-1,4-glucanase by Bueno et al . The enzyme has been classified into family 8 glycosyl hydrolases together with the enzymes formally known as cellulase family D . This enzyme named chitosanase 40/beta-1,3-1,4-glucanase hydrolyzed both chitosan and beta-1,3-1,4-glucan with similar efficiency . However, the production of the enzyme was induced with chitosan but not by beta-1,3-1,4-glucan . Therefore, it seems possible that the major substrate of this enzyme is chitosan rather than beta-1,3-1,4-glucan . Analysis of degradation products generated from partially N-acetylated chitosan showed that chitosanase 40/beta-1,3-1,4-glucanse hydrolyzes GlcN-GlcN and GlcN-GlcNAc linkages but not GlcNAc-GlcNAc nor GlcNAc-GlcN . The specificity for hydrolyzing linkages of this enzyme is similar to that of the chitosanase from S . griseus HUT6037.

J Infect Dis, 1999 Mar, 179(3), 637 - 45
Human T cell responses to the ESAT-6 antigen from Mycobacterium tuberculosis; Ravn P et al.; Human T cell responses to ESAT-6 and eight synthetic overlapping peptides were investigated in tuberculosis (TB) patients and control subjects from regions of high and low endemicity for TB . ESAT-6 was recognized by 65% of all tuberculin purified protein derivative-responsive TB patients, whereas only 2 of 29 bacille Calmette-Guerin-vaccinated Danish healthy donors recognized this molecule . In Ethiopia, a high frequency (58%) of healthy contacts of TB patients recognized ESAT-6 . All of the peptides were recognized by some donors, indicating that the molecule holds multiple epitopes . Danish and Ethiopian patients differed in the fine specificity of their peptide responses . Recognition of the C-terminal region (aa 72-95) was predominant in Danish patients, whereas recognition of aa 42-75 was predominant in Ethiopia . The relationship of these differences to the distribution of HLA types in the two populations is discussed . This study demonstrates that ESAT-6 is frequently recognized during early infection and holds potential as a component of a future TB-specific diagnostic reagent.

J Vasc Surg, 1999 Feb, 29(2), 377 - 81
Mycotic vascular infections of large arteries with Mycobacterium bovis after intravesical bacillus Calmette-Guérin therapy: case report; Seelig MH et al.; Disseminated infection after intravesical bacille Calmette-Guerin instillation for bladder cancer is a rare but potential complication . Vascular infection is an additional serious complication but is seldom reported . We present the first report of a small series of patients with vascular infections after intravesical bacille Calmette-Guerin instillation, and we review the related literature.

J Hosp Infect, 1999 Jan, 41(1), 19 - 22
An outbreak of Bacillus cereus respiratory tract infections on a neonatal unit due to contaminated ventilator circuits; Gray J et al.; An outbreak of Bacillus cereus respiratory tract infections affecting six ventilated preterm neonates over a two-week period is described . Reusable ventilator circuits were identified as the cause of the outbreak . Ordinarily these were reprocessed on the Neonatal Unit (NNU), first through a washing machine and then through a low-temperature steam (LTS) disinfector . The onset of the outbreak coincided with a breakdown of the LTS facility, which necessitated sending the washed circuits off site for LTS disinfection . The washing machine was shown to be contaminated with the same serovars of B . cereus as those isolated from patients . Two critical steps in the off site LTS disinfection process allowed exsporulation and multiplication of B . cereus: the circuits were inadequately dried after processing, whilst return of the moist circuits to the NNU was often delayed . The outbreak was terminated by withdrawal of the heat-disinfected ventilator circuits . This outbreak emphasizes the need for high standards where medical equipment is reprocessed, especially for use in vulnerable patients.

Novartis Found Symp, 1998, 217, 73 - 87; discussion 87-98
Immunological and endocrinological characteristics of tuberculosis that provide opportunities for immunotherapeutic intervention; Rook GA et al.; Immunity to tuberculosis requires a T helper 1 (Th1) response which can be compromised by excessive release of inflammatory cytokines or Th2 activity . Environmental saprophytes can protect against tuberculosis by inducing Th1 recognition of the common antigens, or make mice more susceptible to tuberculosis than unimmunized controls by evoking a Th2 response . A mixed Th1 + Th2 response increases the local toxicity of tumour necrosis factor alpha (TNF-alpha) . Some saprophytes are potent immunogens . A killed preparation of Mycobacterium vaccae can cause systemic activation of spontaneously Th1 cytokine-secreting cells in humans, and can non-specifically suppress pre-existing IgE formation and interleukin 5 (IL-5) production in murine models of allergy . These effects, and the Th2-inducing effects of other species, may explain the variable efficacy of Bacillus Calmette-Guerin, and suggest the need for new approaches to the screening of vaccines before trial in humans . The balance of Th1 to Th2 and the function of inflammatory cytokines are also regulated by cortisol . Glucocorticoid metabolism is abnormal in tuberculosis, suggesting overactivity of 11-beta-hydroxysteroid reductase enzymes . The reductase activity of these enzymes is enhanced by TNF-alpha and IL-1 beta . The roles of Th2, inflammatory cytokines, common antigens and changes in cortisol metabolism suggest several strategies for immunotherapy, and several sites where genetic polymorphisms may affect susceptibility.

Zh Mikrobiol Epidemiol Immunobiol, 1998 Nov-Dec, (6), 68 - 70
{Safety of bacteria of the genus Bacillus, forming the base of some probiotics}; Osipova IG et al.; The study of the safety of bacillary strains forming the base of new probiotics (biosporin and subalin) was made . For control, the safety of Bacillus sp . IP5832 (the base of the preparation of bactisubtil) was studied . The results obtained in this investigation demonstrated that the strains contained in biosporin and subalin were safe when injected intravenously and intraperitoneally into animals in dose of 5 x 10(9) cells per 0.5 ml of physiological saline . The introduction of Bacillus sp . IP5832 in the same dose produced 100% lethal effect.

J Biol Chem, 1999 Feb 12, 274(7), 4189 - 94
Role of lysine 39 of alanine racemase from Bacillus stearothermophilus that binds pyridoxal 5'-phosphate . Chemical rescue studies of Lys39 --> Ala mutant; Watanabe A et al.; The lysine residue binding with the cofactor pyridoxal 5'-phosphate (PLP) plays an important role in catalysis, such as in the transaldimination and abstraction of alpha-hydrogen from a substrate amino acid in PLP-dependent enzymes . We studied the role of Lys39 of alanine racemase (EC 5.1.1.1) from Bacillus stearothermophilus, the PLP-binding residue of the enzyme, by replacing it site-specifically with alanine and characterizing the resultant K39A mutant enzyme . The mutant enzyme turned out to be inherently inactive, but gained an activity as high as about 0.1% of that of the wild-type enzyme upon addition of 0.2 M methylamine . The amine-assisted activity of the mutant enzyme depended on the pKa values and molecular volumes of the alkylamines used . A strong kinetic isotope effect was observed when alpha-deuterated D-alanine was used as a substrate in the methylamine-assisted reaction, but little effect was observed using its antipode . In marked contrast, only L-enantiomer of alanine showed a solvent isotope effect in deuterium oxide in the methylamine-assisted reaction . These results suggest that methylamine serves as a base not only to abstract the alpha-hydrogen from D-alanine but also to transfer a proton from water to the alpha-position of the deprotonated (achiral) intermediate to form D-alanine . Therefore, the exogenous amine can be regarded as a functional group fully representing Lys39 of the wild-type enzyme . Lys39 of the wild-type enzyme probably acts as the base catalyst specific to the D-enantiomer of alanine . Another residue specific to the L-enantiomer in the wild-type enzyme is kept intact in the K39A mutant.

Int J Lepr Other Mycobact Dis, 1998 Sep, 66(3), 309 - 15
Effectiveness of bacillus Calmette-Guerin (BCG) vaccination in the prevention of leprosy; a case-finding control study in Nagpur, India; Zodpey SP et al.; A hospital-based, pair-matched, casecontrol study was carried out at Government Medical College Hospital in Nagpur in central India to estimate the effectiveness of BCG vaccination in the prevention of leprosy . The study included 314 incidence cases of leprosy {diagnosed by World Health Organization (WHO) criteria} below the age of 32 years . Each case was pair matched with one control for age, sex and socioeconomic status . Controls were selected from subjects attending this hospital for conditions other than tuberculosis and leprosy . A significant protective association between BCG and leprosy was observed (OR 0.29, 95% CI 0.21-0.41) . The vaccine effectiveness (VE) was estimated to be 71% (95% CI 59-79) . The BCG effectiveness against multibacillary and paucibacillary leprosy was 79% (95% CI 60-89) and 67% (95% CI 45-78), respectively . It was more effective during the first decade of life (VE 74%; 95% CI 38-90), among females (VE 82%; 95% CI 64-90), and in the lower socioeconomic strata (VE 75%; 95% CI 32-92) . The prevented fraction was calculated to be 51% (95% CI 38-62) . In conclusion, this study has identified a beneficial role of BCG vaccination in the prevention of leprosy in central India.

Aten Primaria, 1998 Dec, 22(10), 649 - 54
{Epidemic outbreak in a home for the aged caused probably by Bacillus cereus}; Pena Gonzalez P et al.; OBJECTIVES: To identify the causative agent and the factors precipitating the outbreak . DESIGN: Observational, crossover study . SETTING: Las Delicias Health district, Jerez de la Frontera . PATIENTS AND OTHER PARTICIPANTS: The population exposed, belonging to an elderly persons' home . MEASUREMENTS AND MAIN RESULTS: The total number of people exposed was the 425 persons living in an elderly persons' home in Jerez in November 1995 . The clinical histories were reviewed, and a specific questionnaire used to interview 77 ill persons and 77 healthy ones . The criteria for ill cases were presence of vomiting and/or diarrhoea . 32.6% of the ill people had fundamentally vomiting; 24.67% diarrhoea, 37.66% vomiting plus diarrhoea, and 100% ran no temperature . Positive and significant OR were detected in various foods (from 2.36 to 10.52 OR) . We isolated 3,000,000, and up to 5,600,000, colonies of Bacillus cereus per gram in several foods . We observed incorrect practices in the conservation and handling of foods . CONCLUSIONS: Epidemiological, microbiological and clinical indications placed us before an outbreak of food poisoning probably caused by Bacillus cereus . The intervention at critical points, inter-institution coordination and communication in time and with data between professionals (microbiologist, doctors, nurses, vets and epidemiologist) were decisive in solving the outbreak.

Gene, 1999 Jan 21, 226(2), 297 - 305
A vector for promoter trapping in Bacillus cereus; Dunn AK et al.; We constructed a promoter-trap plasmid, pAD123, for Bacillus cereus . This plasmid contains a promoterless gene that encodes a mutant version of the green fluorescent protein, GFPmut3a, that is optimized for fluorescence-activated cell sorting {Cormack, B.P., Valdivia, R.H., Falkow, S., 1996 . FACS-optimized mutants of the green fluorescent protein (GFP) . Gene 173, 33-38.} . The plasmid replicates and confers drug resistance in both Escherichia coli and B . cereus . We constructed a library in pAD123, which consists of 29000 clones containing chromosomal DNA from B . cereus strain UW85 . A portion of the library (988 clones) was screened for GFP expression in B . cereus UW85 using a 96-well microtiter dish assay . GFP expression was detected by visual inspection with a fluorimager . We identified 21 clones as fluorescing in the initial screen, and further characterized these clones by restriction analysis, sequencing, and quantification of fluorescence intensity . Flow cytometry and cell sorting efficiently separated B . cereus cells expressing GFP from a 10000-fold excess of non-expressing cells . Selected clones provided useful markers to follow B . cereus populations on plant surfaces . Our results indicate that GFP and pAD123 are useful tools for identifying regulatory sequences in Bacillus cereus, and that flow cytometry and cell sorting is a useful method for screening large libraries constructed in this vector.

Gene, 1998 Dec 28, 225(1-2), 143 - 51
Mycobacteriophage D29 integrase-mediated recombination: specificity of mycobacteriophage integration; Pena CE et al.; Mycobacteriophage D29 is a lytic phage that infects both fast- and slow-growing species of the mycobacteria . D29 forms clear plaques on lawns of Mycobacterium smegmatis and Mycobacterium bovis bacille Calmette-Guerin (BCG) in which a very high proportion of infected cells are killed . However, genomic analysis of D29 demonstrates that it is a close relative of the temperate mycobacteriophage L5, and is presumably a non-temperate derivative of a temperate parent . The D29 genome encodes a putative integrase protein with a primary amino acid sequence similar to that of the L5 integrase; the corresponding int genes fall in colinear positions within the D29 and L5 genomes, immediately flanking and transcribed away from their associated attP sites . We show here that the D29 integrase is functional and catalyzes integrative recombination between the D29 attP site and the M . smegmatis attB site in vitro in an mIHF-dependent manner . D29 integrase also mediates recombination between the L5 attP site and attB DNA and, reciprocally, L5 integrase catalyzes recombination with D29 attP DNA . However, in both in-vitro and in-vivo assays, the D29-encoded integrase recombines the D29 attP more efficiently than the L5 attP, and vice versa, suggesting that each integration system has evolved a degree of specificity of attP recognition . We also present the sequences of the putative attP site and integrase protein of the cryptic prophage-like element phiRv2, and compare them to those of mycobacteriophages L5 and D29.

Biochim Biophys Acta, 1999 Jan 18, 1444(1), 131 - 7
cDNA cloning and expression of Bacillus thuringiensis Cry1Aa toxin binding 120 kDa aminopeptidase N from Bombyx mori; Yaoi K et al.; Bacillus thuringiensis Cry1Aa toxin binds to a 120 kDa putative receptor protein in the Bombyx mori midgut . Recently, this protein was purified and identified as glycosyl-phosphatidylinositol (GPI) anchored aminopeptidase N (APN) . In this study, a full-length cDNA thought to encode this 120 kDa APN was isolated and sequenced . It has a 2958 bp ORF encoding 986 amino acids . In the deduced amino acid sequence, we identified GPI-anchor and zinc-metallopeptidase signals, which are the same as those of APNs of other insects that are reported to be putative Cry1 toxin receptors . The B . mori APN amino acid sequence also has a high similarity with those of the other APNs . Subsequently, the recombinant APN was expressed by Escherichia coli and its Cry1Aa toxin binding ability was analyzed . Ligand blotting showed that Cry1Aa toxin bound to the recombinant APN.

Biochem J, 1999 Feb 15, 338 ( Pt 1), 185 - 93
Biochemical characterization of Bacillus thuringiensis cytolytic toxins in association with a phospholipid bilayer; Du J et al.; The interaction of two Bacillus thuringiensis cytolytic toxins, CytA and CytB, with a phospholipid bilayer and their structure in the membrane-bound state were investigated by proteolysis using phospholipid vesicles as a model system . A toxin conformational change upon membrane binding was detected by comparing the proteolytic profile of membrane-bound toxin and saline-solubilized toxin . When membrane-bound toxin was exposed to protease K or trypsin, novel cleavage sites were found between the alpha-helical N-terminal half and beta-strand C-terminal half of the structure at K154 and N155 in CytA and at I150 and G141 in CytB . N-terminal sequencing of membrane-protected fragments showed that the C-terminal half of the toxin structure comprising mainly beta-strands was inserted into the membrane, whereas the N-terminal half comprising mainly alpha-helices was exposed on the outside of the liposomes and could be removed when liposomes with bound toxin were washed extensively after proteolysis . The C-termini of the membrane-inserted proteolytic fragments were also located by a combination of N-terminal sequencing and measurement of the molecular masses of the fragments by electrospray MS . Using a liposome glucose-release assay, the membrane-inserted structure was seen to retain its function as a membrane pore even after removal of exposed N-terminal segments by proteolysis . These data strongly suggest that the pores for glucose release are assembled from the three major beta-strands (beta-5, beta-6 and beta-7) in the C-terminal half of the toxin.

J Struct Biol, 1998 Dec 1, 124(1), 99 - 101
Crystallization and preliminary X-ray crystallographic study of a 23S rRNA binding domain of the ribosomal protein L2 from Bacillus stearothermophilus; Nakashima T et al.; Ribosomal protein L2 from Bacillus stearothermophilus, a single polypeptide chain with 275 amino acid residues, is a primary 23S rRNA-binding protein in the large ribosomal subunit . Crystals of a 23S rRNA binding domain (BstL2-RBD: positions 60-201) of the ribosomal protein L2 from B . stearothermophilus overexpressed in Escherichia coli have been grown in 0.1 M MES (pH 6.5) containing 15% polyethylene glycol 20 000 . The crystals diffract to 2.3-A resolution on a synchrotron X-ray source . The crystal belongs to the space group P1 and the unit cell axes are a = 28.05, b = 36.20, c = 69.74 A, alpha = 99.58 degrees, beta = 95.86 degrees, and gamma = 102.62 degrees . There are two molecules of the BstL2-RBD in the asymmetric unit .

J Mol Biol, 1999 Feb 12, 286(1), 247 - 55
Molecular directionality of beta-chitin biosynthesis; Sugiyama J et al.; The molecular packing in beta-chitin unit cells was experimentally determined by a combination of unidirectional degradation by Bacillus circulans chitinase A1 and microdiffraction electron crystallography using highly crystalline beta-chitin microfibrils from the protective tubes secreted by Lamellibrachia satsuma . The mode of chain packing was found to be identical with that of the previously published crystal model for beta-chitin, despite a controversial definition of the unit cell parameters . Here, a "parallel-down" packing was determined, where the reducing ends of chains point in an opposite direction to the crystallographic c-axis . Microdiffraction analyses of nascent beta-chitin microfibrils generated from diatom Thalassiosira sp . showed that the c-axis of the crystal was directed toward the diatoms, and therefore the reducing end of a growing chain pointed away from the locus of biosynthesis . This mechanism agreed well with what we found recently in the cellulose biosynthesis system, and provides strong evidence that the polymerization by the processive glycosyl transferase takes place at the non-reducing end of the growing polysaccharide chains .

Prikl Biokhim Mikrobiol, 1998 Nov-Dec, 34(6), 617 - 21
{Absorption of nitric oxide by a strain of Bacillus stearothermophilus and its use in a bioreactor for purifying air}; Lebedeva EV et al.; A new Bacillus stearothermophilus strain, INMI 50, was isolated and identified . Cells of this strain immobilized on a ceramic carrier demonstrated a high NO uptake in a bioreactor . The bioreactor volume was 4 l; air flow, 100 l/h; initial NO concentration, 5 ppm; and temperature, 60 degrees C . Glycerol or 1,2-propanediol was used as carbon and energy source . The uptake of NO was 60-90% of the initial concentration over six months of continuous operation of the bioreactor . The developed procedure can be used for removal of nitrogen oxide from products of combustion of diesel fuel or from air in production areas.

Ann N Y Acad Sci, 1998 Dec 13, 864, 118 - 30
Screening of inhibitory monoclonal antibodies . A critical step for producing anti-idiotypic catalytic antibodies; Avalle B et al.; In accord with the original approach that we proposed, catalytic antibodies may be produced by using the anti-idiotypic pathway according to antigen/antibody complementarity rules . The generation and screening of the idiotypic Ab1, the central point on which are anchored the interactions with both the antigen (enzyme) and the anti-idiotypic abzyme, represent a crucial step for the success of this approach . We herein propose to describe a strategy for which we have developed a number of assays, aiming at selecting the proper Ab1, with desired features, likely to elicit an anti-idiotypic catalytic antibody . beta-Lactamase from Bacillus cereus was chosen as the example illustrating our arguments.

Appl Environ Microbiol, 1999 Feb, 65(2), 694 - 7
Purification and properties of a xylan-binding endoxylanase from alkaliphilic bacillus sp . Strain K-1; Ratanakhanokchai K et al.; An alkaliphilic bacterium, Bacillus sp . strain K-1, produces extracellular xylanolytic enzymes such as xylanases, beta-xylosidase, arabinofuranosidase, and acetyl esterase when grown in xylan medium . One of the extracellular xylanases that is stable in an alkaline state was purified to homogeneity by affinity adsorption-desorption on insoluble xylan . The enzyme bound to insoluble xylan but not to crystalline cellulose . The molecular mass of the purified xylan-binding xylanase was estimated to be approximately 23 kDa . The enzyme was stable at alkaline pHs up to 12 . The optimum temperature and optimum pH of the enzyme activity were 60 degreesC and 5.5, respectively . Metal ions such as Fe2+, Ca2+, and Mg2+ greatly increased the xylanase activity, whereas Mn2+ strongly inhibited it . We also demonstrated that the enzyme could hydrolyze the raw lignocellulosic substances effectively . The enzymatic products of xylan hydrolysis were a series of short-chain xylooligosaccharides, indicating that the enzyme was an endoxylanase.

Appl Environ Microbiol, 1999 Feb, 65(2), 457 - 64
Toxicity, binding, and permeability analyses of four bacillus thuringiensis cry1 delta-endotoxins using brush border membrane vesicles of spodoptera exigua and spodoptera frugiperda; Luo K et al.; The binding and pore formation properties of four Bacillus thuringiensis Cry1 toxins were analyzed by using brush border membrane vesicles from Spodoptera exigua and Spodoptera frugiperda, and the results were compared to the results of toxicity bioassays . Cry1Fa was highly toxic and Cry1Ac was nontoxic to S . exigua and S . frugiperda larvae, while Cry1Ca was highly toxic to S . exigua and weakly toxic to S . frugiperda . In contrast, Cry1Bb was active against S . frugiperda but only marginally active against S . exigua . Bioassays performed with iodinated Cry1Bb, Cry1Fa, and Cry1Ca showed that the effects of iodination on toxin activity were different . The toxicities of I-labeled Cry1Bb and Cry1Fa against Spodoptera species were significantly less than the toxicities of the unlabeled toxins, while Cry1Ca retained its insecticidal activity when it was labeled with 125I . Binding assays showed that iodination prevented Cry1Fa from binding to Spodoptera brush border membrane vesicles . 125I-labeled Cry1Ac, Cry1Bb, and Cry1Ca bound with high-affinities to brush border membrane vesicles from S . exigua and S . frugiperda . Competition binding experiments performed with heterologous toxins revealed two major binding sites . Cry1Ac and Cry1Fa have a common binding site, and Cry1Bb, Cry1C, and Cry1Fa have a second common binding site . No obvious relationship between dissociation of bound toxins from brush border membrane vesicles and toxicity was detected . Cry1 toxins were also tested for the ability to alter the permeability of membrane vesicles, as measured by a light scattering assay . Cry1 proteins toxic to Spodoptera larvae permeabilized brush border membrane vesicles, but the extent of permeabilization did not necessarily correlate with in vivo toxicity.

Appl Environ Microbiol, 1999 Feb, 65(2), 873 - 6
A comparative study of methods to validate formaldehyde decontamination of biological safety cabinets; Munro K et al.; Methods of validation of formaldehyde decontamination of biological safety cabinets were compared . Decontamination of metal strips inoculated with Mycobacterium bovis, poliovirus, or Bacillus spp . spores was compared with the results obtained with three biological indicators . Conditions for successful decontamination, particularly relative humidity, were defined . The Attest 1291 biological indicator was the only biological indicator which was an aid in the detection of gross decontamination failure.

Syst Appl Microbiol, 1998 Dec, 21(4), 520 - 9
Contribution to phenotypic and genotypic characterization of Bacillus licheniformis and description of new genomovars; Manachini PL et al.; A study of phenotypic and genotypic characteristics was carried out on 182 strains isolated from soil of different geographical areas; the type strains were B . licheniformis, B . subtilis, B . pumilus, B . cereus and B . coagulans . The results showed, primarily on the basis of phenotypic features, that all the isolates belonged to the B . licheniformis species, however DNA relatedness studies revealed only 161 to be genetically related to B . licheniformis, the DNA relatedness levels ranging from 66 to 100% . The other 21 isolates appeared to be genetically distinct not only from B . licheniformis but also from B . subtilis and B . pumilus, where there were low levels of DNA relatedness (from 4 to 37%) . Nevertheless the ARDRA results indicate that the 21 atypical isolates were phylogenetically related to B . licheniformis . Our data and the phenotypic homogeneity found suggest the presence of three different genomovars.

Rev Panam Salud Publica, 1998 Oct, 4(4), 223 - 32
{Public laboratories for vaccine production: a new paradigm}; Homma A et al.; In Latin America and the Caribbean, public laboratories that produce vaccines have contributed in varying degrees to the control and eradication of vaccine-preventable diseases, and several of them are manufacturing vaccines that are routinely applied in national immunization programs, such as the vaccine against tuberculosis (made with the bacillus of Calmette-Guerin, BCG), the triple vaccine against diphtheriatetanus-pertussis (DTP), tetanus toxoid (TT), the vaccine against measles and the oral vaccine against polio . Thanks to recent scientific strides, one can foresee an important increase in the number of safe and effective vaccines that will be available in the near future for use in routine vaccination programs . However, there are high costs involved in developing such vaccines and in protecting the intellectual property rights involved, and few laboratories in Latin America have the technical capacity to research and develop these vaccines . Such factors will affect the speed with which they are assimilated into vaccination programs in countries of the Region . Currently, public laboratories that manufacture vaccines in the Region are not equipped to compete in this new scenario and run the risk of being completely outmarketed . Thus, they must radically change their style of management and their scientific and technical capabilities, backed by a commitment from governments to improve and strengthen those political and financial aspects that can assure that national laboratories participate in the sustainable supply of vaccines to immunization programs, as well as in researching, developing, and producing new vaccines.

J Virol Methods, 1998 Dec, 76(1-2), 121 - 6
Differentiation of rice tungro bacilliform virus strains by restriction analysis and DNA hybridization; Cabauatan PQ et al.; Rice tungro bacilliform virus (RTBV) is one of the two viruses that cause tungro disease . Four RTBV strains maintained in the greenhouse for 4 years, G1, G2, Ic, and L, were differentiated by restriction fragment length polymorphism (RFLP) analysis of the native viral DNA . Although strains G1 and Ic had identical restriction patterns when cleaved with Pst1, BamHI, EcoRI, and EcoRV, they can be differentiated from strains G2 and L by EcoRI and EcoRV digestion . These same endonucleases also differentiate strain G2 from strain L . When total DNA extracts from infected plants were used instead of viral DNA, and digested with EcoRV, identical restriction patterns for each strain (G2 and L) were obtained from roots, leaves, and leaf sheaths of infected plants . The restriction patterns were consistent from plant to plant, in different varieties, and at different times after inoculation . This technique can be used to differentiate RTBV strains and determine the variability of a large number of field samples.

Curr Microbiol, 1999 Mar, 38(3), 163 - 7
Introduction and expression of the cry1Ac gene of Bacillus thuringiensis in a cereal-associated bacterium, Bacillus polymyxa; Sudha SN et al.; The abilities of Bacillus polymyxa and Bacillus thuringiensis to survive on the rice phyllospere were compared; it was found that B . polymyxa colonizes the crop better . This study also showed that B . polymyxa inoculation to rice plants increased the shoot and the root growth of the crop . Efforts were made to introduce the cry1Ac gene of B . thuringiensis subsp . kurstaki into B . polymyxa so that the application of such transgenic B . polymyxa strains would prove to be dually beneficial to rice crops both as a biopesticide and as a biofertilizer . Immunoblot analysis of the recombinant organism containing the cry1Ac gene, strain BP113, indicated efficient expression of this gene in the heterologous host . Bioassays with the first instar larvae of the yellow stem borer of rice (Scirpophaga incertulas) revealed that the protein preparations from BP113 were toxic.

J Food Prot, 1999 Jan, 62(1), 57 - 64
Effect of shift in growth temperature on tolerance of psychrotrophic and mesophilic strains of Bacillus cereus to heat and sodium chloride; Mahakarnchanakul W et al.; A shift in growth temperature of a psychrotrophic (F3802A/84) strain and a mesophilic strain (B4ac-1) of Bacillus cereus grown at 30 degrees C for 10 h, then at 37 degrees C or 40 degrees C for 14 h, enhanced thermotolerance . Sodium chloride, at concentrations of 2.0 and 4.0% in brain heart infusion (BHI) broth, had no effect on thermotolerance of strain B4ac-1 heated at 50 degrees C, whereas the same concentrations of NaCl caused a decrease in thermotolerance of strain F3802A/84 heated at 48 degrees C . A downshift in growth temperature from 30 degrees C to 10 degrees C followed by incubation for 3 to 9 days increased thermotolerance of strain F3802A/84 but not strain B4ac-1 heated in BHI broth containing 2.0 or 4.0% NaCl compared to thermotolerance in BHI broth containing 0.5% NaCl . Protein analysis using one-dimensional gel electrophoresis revealed an increase in proteins with molecular weights of 54, 50, 44, and 42 kDa in cells of strain F3802A/84 and 83 and 69 kDa in cells of strain B4ac-1 subjected to an upshift in growth temperature from 30 degrees C to 37 degrees C or 40 degrees C, respectively . A downshift in growth temperature from 30 degrees C to 10 degrees C resulted in substantial amounts of proteins with molecular weights of 63, 40, and 29 kDa produced by strain F3802A/84 and 63 kDa to be produced by strain B4ac-1 . Proteins produced in response to upshift or downshift in growth temperature are suspected to play an important role in heat resistance of the psychrotrophic and mesophilic strains of B . cereus examined in this study . Changes in resistance to heat or refrigeration temperatures, as well as tolerance to NaCl, as affected by previous exposure of cells to temperature shifts may influence the ability of B . cereus to grow in minimally processed foods during distribution and storage.

Ann Biol Clin (Paris), 1999 Jan-Feb, 57(1), 29 - 36
{Bartonellosis . II . Other Bartonella responsible for human diseases}; Piemont Y et al.; In addition to Bartonella henselae, five other Bartonella species were involved in human pathology . As for B . henselae, ectoparasites seem to be responsible for the transmission of most or all these bacterial species . B . bacilliformis is responsible for Carrion's disease that occurs in some valleys of Colombia, Ecuador and Peru . This disease is transmitted by biting of infected sandflies . The bacterial reservoir is constituted by humans only . That disease occurs either as an acute form with severe infectious hemolytic anemia (or Oroya fever), or as benign cutaneous tumors, also called verruga peruana . Healthy blood carriers of the bacterium exist . Trench fever was described during the First World War . This non-lethal disease is constituted of recurrent febrile attacks associated particularly with osseous pains . The causative agent of the disease is B . quintana, transmitted by the body louse . Humans seem to be the reservoir of that bacterium . In some patients, B . quintana can be responsible for endocarditis, bacillary angiomatosis and chronic or recurrent bacteremia . Other human infections due to Bartonella sp . have been described: B . vinsonii, isolated from blood of small rodents, and B . elizabethae, the reservoir of which is currently unknown, can be responsible for endocardites . B . clarridgeiae (isolated from blood of 5% of pet cats and 17% of stray cats) may be responsible for human cat scratch disease . All these bartonelloses are diagnosed by non-standard blood culture or by in vitro DNA amplification or by serological testing . Their treatment requires tetracyclines or chloramphenicol or macrolides.

Nat Biotechnol, 1999 Jan, 17(1), 58 - 61
Evolutionary molecular engineering by random elongation mutagenesis; Matsuura T et al.; We describe a new method of random mutagenesis that employs the addition of peptide tails with random sequences to the C-terminal of enzyme molecules . A mutant population of catalase I from Bacillus stearothermophilus prepared by this method has a diversity in thermostability and enzyme activity equal to that obtained after random point mutagenesis . When a triple mutant of catalase I (I108T/D130N/1222T)-the thermostability of which is much lower than that of the wild type-was subjected to random elongation mutagenesis, we generated a mutant population containing only mutants with higher thermostability than the triple mutant . Some had an even higher stability than the wild-type enzyme, whose thermostability is considered to be optimized . These results indicate that peptide addition expands the protein sequence space resulting in a new fitness landscape . The enzyme can then move along the routes of the new landscape until it reaches a new optimum . The combination of random elongation mutagenesis with random point mutagenesis should be a useful approach to the in vitro evolution of proteins with new properties.

Atherosclerosis, 1999 Jan, 142(1), 57 - 66
Macrophage released proteoglycans are involved in cell-mediated aggregation of LDL; Maor I et al.; Aggregated low density lipoprotein (LDL) is taken up by macrophages at enhanced rate, leading to macrophage cholesterol accumulation and foam cell formation . Since macrophages were shown to mediate self aggregation of modified forms of LDL, we sought to study the effect of macrophages on the susceptibility of native LDL to aggregation . Incubation of LDL (100 microg of protein/ml) with J-774A.1 macrophage-like cell line for 18 h at 37 degrees C, led to a 114 and 56% enhanced susceptibility of LDL to aggregation by vortexing and by Bacillus cereus SMase respectively . Macrophage conditioned media (MCMs) that were obtained from J-774A.1 cells also enhanced the susceptibility of LDL to aggregation by vortexing and SMase by 134 and 75% respectively, suggesting the involvement of macrophage secretory products in the enhanced aggregation of LDL . As proteoglycans were shown to be involved in lipoprotein aggregation, we analyzed the possible involvement of macrophage-released proteoglycans in LDL aggregation . Incubation of LDL (100 microg protein/ml) with 25 microg of proteoglycans that were isolated from MCM led to a dose-dependent enhanced susceptibility of LDL to aggregation by vortexing or by SMase by up to 62 and 77% respectively . The stimulatory effect of the MCMs on LDL aggregation was markedly reduced upon MCMs treatment with the glycosaminoglycan hydrolyzing enzyme chondroitinase ABC, chondroitinase AC, but not heparinase . On the contrary, incubation of LDL (100 microg of protein/ml) with increasing concentrations (up to 50 microg/ml) of chondroitin sulfate, or heparan sulfate enhanced the susceptibility of LDL to aggregation by up to 98 or by only 18% respectively, in comparison with non-treated LDL . Since macrophages under atherogenic conditions (cholesterol-loading, cellular lipid peroxidation and activation) demonstrate enhanced secretion of proteoglycans, we finally studied the effect of J-774A.1 macrophages on the susceptibility of native LDL to aggregation under the above atherogenic conditions . Incubation of LDL with cholesterol-loaded macrophages led to a 62% enhanced susceptibility of LDL to undergo aggregation by vortexing, in comparison with LDL that was incubated with non-loaded cells . Macrophage activation with phorbol myristate acetate (5 microM of PMA) also significantly increased cell-mediated aggregation of LDL by 50%, in comparison with non-activated cells . Lipid peroxidized macrophages obtained by cell treatment with either FeSO4 (50 microM), or angiotensin II (10(-7) M) enhanced the susceptibility of LDL to aggregation by 22 or by 39% respectively . These results suggest that under atherogenic conditions, macrophages release proteoglycans, and mainly chondroitin sulfate, which can contribute to cell-mediated formation of aggregated LDL, a potent inducer of macrophage foam cells which are the hallmark of early atherogenesis.

Biochim Biophys Acta, 1998 Dec 8, 1429(1), 176 - 86
Identification by site-directed mutagenesis of amino acid residues in ribosomal protein L2 that are essential for binding to 23S ribosomal RNA; Harada N et al.; The ribosomal protein L2 (BstL2) from Bacillus stearothermophilus is a primary 23S rRNA binding protein . We made use of site-directed mutagenesis to identify essential basic and aromatic amino acid residues for 23S rRNA binding . Four mutants, R68Q, K70Q, R86Q, and R155Q, in which Arg-68, Lys-70, Arg-86, and Arg-155, respectively, are replaced by the Gln residue . showed reduced binding affinities as compared with that of the wild type BstL2 (a binding constant K=8.93 microM(-1)): K values of these mutants range between 0.24 and 1.86 microM(-1) . As for aromatic amino acids, replacements of Phe-66, Tyr-95 or Tyr-102 by alanine significantly abolished the binding affinities . CD analysis of the mutant proteins indicated that the mutations of four basic residues (Arg-68, Lys-70, Arg-86 and Arg-155) did not affect protein structure, whereas those of aromatic residues (Phe-66, Tyr-95, and Tyr-102) appeared to cause slight structural perturbations . These results, together with sequence comparison of L2 family proteins, suggest that Arg-86 and Arg-155 in BstL2 may act as positively charged recognition groups for negatively charged phosphate backbone of the 23S rRNA, and that Phe-66, Tyr-95, and Tyr-102 may be candidate residues which stabilize the BstL2-23S rRNA interaction through intramolecular interactions.

Epidemiol Mikrobiol Imunol, 1998 Dec, 47(4), 150 - 3
{Sporicidal effect of Presept and Chloramine B on Bacillus cereus spores}; Hlavacek O et al.; The sporicidal effect of Presept was compared with Chloramine B on the spores of Bacillus cereus . Either compound was calibrated to the same concentration of active chlorine . While a portion of spore population after 4 hrs of treatment by Chloramine germinated and started to divide in a rich nutrient medium, the optical density of the culture inoculated with spores treated by Presept did not increase even after 7 hrs when exposed to the nutrient medium . Significant morphological differences were found in either population of spores . Spores treated by Presept lost the impermeability within 3 hrs in the nutrient medium but almost no postgerminative development was observed . However, a portion of spores treated by Chloramine B developed after germination within 3 hrs into vegetative cells . It seems that Presept does not block germination and/or loss of impermeability of spores, but prevents their postgerminative development and division.

Indian J Med Res, 1998 Dec, 108, 260 - 4
Efficacy of a Bacillus sphaericus formulation as influenced by the quality of Culex quinquefasciatus breeding waters; Gunasekaran K et al.; To understand the physico-chemical factors that influence the efficacy of B . sphaericus formulation in the breeding sites of Culex quinquefasciatus, a study was carried out in Mayiladuturai area of Tamil Nadu (India) . The factors studied were hydrogen ion concentration (pH), acidity, alkalinity, chlorides, phosphates, total hardness, sulphates, total solids, dissolved solids, suspended solids, nitrate nitrogen, ammoniacal nitrogen, dissolved oxygen, biological oxygen demand (BOD) and chemical oxygen demand (COD) . Efficacy of the formulation was assessed in terms of reduction in larval population in the treated habitats . pH of water in the treated sites was around neutral range (mean +/- SD 7.65 +/- 0.23) . Phosphate content was low (2.27 +/- 1.34 ppm) whereas chlorides (326.1 +/- 55.8 ppm) and sulphates (38.9 +/- 23.8 ppm) were high . Total hardness ranged from 206 to 462.5 ppm with a mean of 312.1 +/- 80.5 ppm . The chlorides and sulphates, though present in considerable quantity, did not have any influence on the efficacy of B . sphaericus formulation . However, the proportion of insoluble chlorides and sulphates which contribute to total hardness seemed to influence the formulation adversely.

Virology, 1999 Jan 20, 253(2), 319 - 26
Rice tungro bacilliform virus open reading frame 3 encodes a single 37-kDa coat protein; Marmey P et al.; Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus and a member of the Caulimoviridae family and closely related to viruses in the Badnavirus genus . The coat protein of RTBV is part of the large polyprotein encoded by open reading frame 3 (ORF3) . ORF3 of an RTBV isolate from Malaysia was sequenced (accession no . AF076470) and compared with published sequences for the region that encodes the coat protein or proteins . Molecular mass of virion proteins was determined by mass spectrometry (matrix-assisted laser desorption/ionization-TOF) performed on purified virus particles from three RTBV isolates from Malaysia . The N- and C-terminal amino acid sequences of the coat protein were deduced from the mass spectral analysis, leading to the conclusion that purified virions contain a single coat protein of 37 kDa . The location of the coat protein domain in ORF3 was reinforced as a result of immunodetection reactions using antibodies raised against six different segments of ORF3 using Western immunoblots after SDS-PAGE and isoelectrofocusing of proteins purified from RTBV particles . These studies demonstrate that RTBV coat protein is released from the polyprotein as a single coat protein of 37 kDa .

Can J Vet Res, 1999 Jan, 63(1), 56 - 61
Intracellular fate of Mycobacterium avium subspecies paratuberculosis in monocytes from normal and infected, interferon-responsive cows as determined by a radiometric method; Zhao BY et al.; The ability of Mycobacterium avium subsp . paratuberculosis to survive in bovine monocytes was studied using radiometric (BACTEC) culture, standard plate counting and microscopic counting of acid-fast stained monocyte monolayers . Results of microscopic counts sharply contrasted with results of viable counts determined both by plate counting and radiometric counting . We observed an early phase (the first 6 d after in vitro infection) of intracellular bacillary growth, followed by a later phase of mycobacteriostasis or killing (up to 12 d after in vitro infection) in monocytes from non-infected cows . The data suggest that multiplication and death of M . avium subsp . paratuberculosis occur simultaneously in bovine monocytes infected in vitro . Using the BACTEC method, we compared the ability of bovine monocytes from normal cows and cows infected with M . avium subsp . paratuberculosis and showing evidence of a strong Thl-like cellular immune response to ingest and inhibit the intracellular growth of M . avium subsp . paratuberculosis . There was a trend toward greater phagocytosis and faster killing of Mycobacterium avium subsp . paratuberculosis by monocytes from the infected, immune responder cows . However, the observed numbers of viable M . avium subsp . paratuberculosis at each time after monocyte infection were not significantly different between normal and infected cows.

Kansenshogaku Zasshi, 1998 Dec, 72(12), 1325 - 9
{Bacillus cereus septicemia in a patient with severe aplastic anemia}; Ueda K et al.; A 78-year-old female was admitted with complaints of malaise and fatigue in the legs . The patient was diagnosed as severe aplastic anemia and treatment was started with metenolone and steroid pulse therapy . Administration of antibiotics and granulocyte-colony stimulating factor which led to a resolution of the high fever . About four months after admission, the patient developed vomiting and abdominal pain with a spiking fever . The next day after suddenly losing consciousness, she died . B . cereus was isolated from blood cultures . Autopsy specimens of the liver, cardiac muscle and lung showed changes due to B . cereus . This pathogen is widely distributed in nature . We should not overlook B . cereus as a contamination, but rather should consider it a potential pathogen in immunocompromised hosts, when it is isolated from blood cultures.

Infect Immun, 1999 Feb, 67(2), 760 - 71
Differential posttranslational processing confers intraspecies variation of a major surface lipoprotein and a macrophage-activating lipopeptide of Mycoplasma fermentans; Calcutt MJ et al.; The malp gene of Mycoplasma fermentans is shown to occur in single copy but to encode two discrete translated forms of lipid-modified surface protein that can be differentially expressed on isolates within this species: MALP-2, a 14-amino-acid (2-kDa) lipopeptide with potent macrophage-stimulatory activity (P . F . Muhlradt, M . Kiess, H . Meyer, R . Sussmuth, and G . Jung, J . Exp . Med . 185:1951-1958, 1997), and MALP-404, an abundant, full-length (404-amino-acid) surface lipoprotein of 41 kDa, previously designated P41 (K . S . Wise, M . F . Kim, P . M . Theiss, and S.-C . Lo, Infect . Immun . 61:3327-3333, 1993) . The sequences, transcripts, and translation products of malp were compared between clonal isolates of strains PG18 (known to express P41) and II-29/1 (known to express high levels of MALP-2) . Despite conserved malp DNA sequences containing full-length open reading frames and expression of full-length monocistronic transcripts in both isolates, Western blotting using a monoclonal antibody (MAb) to the N-terminal MALP-2 peptide revealed marked differences in the protein products expressed . Whereas PG18 expressed abundant MALP-404 with detectable MALP-2, II-29/1 revealed no MALP-404 even in samples containing a large comparative excess of MALP-2 . Colony immunoblots with the MAb showed uniform surface expression of MALP-2 in II-29/1 populations . A second MAb to an epitope of MALP-404 outside the MALP-2 sequence predictably failed to stain II-29/1 colonies but uniformly stained PG18 populations . Collectively, these results provide evidence for novel posttranscriptional (probably posttranslational) processing pathways leading to differential intraspecies expression of a major lipoprotein, and a potent macrophage-activating lipopeptide, on the surface of M . fermentans . In the course of this study, a striking conserved motif (consensus, TD-G--DDKSFNQSAWE--), designated SLA, was identified in MALP-404; this motif is also distributed among selected lipoproteins and species from diverse bacterial genera, including Bacillus, Borrelia, Listeria, Mycoplasma, and Treponema . In addition, malp was shown to flank a chromosomal polymorphism . In eight isolates of M . fermentans examined, malp occurred upstream of an operon encoding the phase-variable P78 ABC transporter; but, in three of these isolates, a newly discovered insertion sequence, IS1630 (of the IS30 class), was located between these genes.

N Engl J Med, 1999 Jan 21, 340(3), 184 - 9
Chronic Bartonella quintana bacteremia in homeless patients; Brouqui P et al.; BACKGROUND: Infection with Bartonella quintana can cause trench fever, endocarditis, bacillary angiomatosis, and peliosis . An outbreak of bacteremia due to B . quintana has been reported among homeless people in Seattle, and the seroprevalence is high among homeless people in both the United States and Europe . Body lice are known to be the vectors of B . quintana . METHODS: We studied all the homeless people who presented in 1997 to the emergency departments of the University Hospital, Marseilles, France . Blood was collected for microimmunofluorescence testing for antibodies against B . quintana and for culture of the bacterium . Body lice were collected and analyzed by the polymerase chain reaction and sequencing of a portion of the citrate synthase gene of B . quintana . RESULTS: In 10 of 71 homeless patients (14 percent), blood cultures were positive for B . quintana, and 21 of the patients (30 percent) had high titers of antibody against the organism . A total of 17 patients (24 percent) had evidence of recent infection (bacteremia or seroconversion) . Tests of lice from 3 of the 15 patients from whom they were collected were positive for B . quintana . The homeless people with B . quintana bacteremia were more likely to have been exposed to lice (P=0.002), were more likely to have headaches (P=0.03) and severe leg pain (P<0.001), and had lower platelet counts (P=0.006) than the homeless people who were seronegative for B . quintana and did not have bacteremia; 8 of the 10 patients with bacteremia were afebrile . Five patients had chronic bacteremia, as indicated by positive blood cultures over a period of several weeks . CONCLUSIONS: In an outbreak of urban trench fever among homeless people in Marseilles, B . quintana infections were associated with body lice in patients with nonspecific symptoms or no symptoms.

Biochem J, 1999 Feb 1, 337 ( Pt 3), 453 - 60
Lipoarabinomannans: characterization of the multiacylated forms of the phosphatidyl-myo-inositol anchor by NMR spectroscopy; Nigou J et al.; Lipoarabinomannans, which exhibit a large spectrum of immunological activities, emerge as the major antigens of mycobacterial envelopes . The lipoarabinomannan structure is based on a phosphatidyl-myo-inositol anchor whose integrity has been shown to be crucial for lipoarabinomannan biological activity and particularly for presentation to CD4/CD8 double-negative alphabetaT cells by CD1 molecules . In this report, an analytical approach was developed for high-resolution 31P-NMR analysis of native, i.e . multiacylated, lipoarabinomannans . The one-dimensional 31P spectrum of cellular lipoarabinomannans, from Mycobacterium bovis Bacillus Calmette-Guerin, exhibited four 31P resonances typifying four types of lipoarabinomannans . Two-dimensional 1H-31P heteronuclear multiple-quantum-correlation/homonuclear Hartmann-Hahn analysis of the native molecules showed that these four types of lipoarabinomannan differed in the number and localization of fatty acids (from 1 to 4) esterifying the anchor . Besides the three acylation sites previously described, i.e . positions 1 and 2 of glycerol and 6 of the mannosyl unit linked to the C-2 of myo-inositol, we demonstrate the existence of a fourth acylation position at the C-3 of myo-inositol . We report here the first structural study of native multiacylated lipoarabinomannans, establishing the structure of the intact phosphatidyl-myo-inositol anchor . Our findings would help gain more understanding of the molecular basis of lipoarabinomannan discrimination in the binding process to CD1 molecules.

Mikrobiologiia, 1998 Sep-Oct, 67(5), 619 - 25
{Biosynthesis of extracellular guanyl-specific ribonuclease from Bacillus circulans}; Znamenskaia LV et al.; Biosynthesis of extracellular alkaline guanyl-specific RNase by Bacillus circulans (RNase Bci) was studied . Synthesis of the enzyme by the culture started in the late exponential phase and was inhibited by inorganic phosphate and glucose, in contrast to the biosynthesis of its structural and functional homologue, RNase Ba (barnase) of B . amyloliquefaciens . It is suggested that differences in the regulation of the biosynthesis of RNase Bci and Ba are related to different structures of their gene promoters.

Biochemistry, 1999 Jan 5, 38(1), 2 - 10
Identification of a calcium binding site in Staphylococcus hyicus lipase: generation of calcium-independent variants; Simons JW et al.; In this study we have identified the presence of a high-affinity binding site for calcium in the lipase from Staphylococcus hyicus . By means of isothermal titration calorimetry we showed that the enzyme binds one calcium per molecule of enzyme with a dissociation constant of 55 microM . The residual activity of the apoenzyme compared to the activity in the presence of calcium ions varies from 65% at 10 degreesC to nearly zero at 40 degreesC . On the basis of primary sequence alignment with other staphylococcal lipases and the lipases from Bacillus thermocatenulatus and from Pseudomonas glumae in combination with site-directed mutagenesis, aspartates 354 and 357 could be identified as calcium ligands . Kinetic measurements with the D357E variant showed that replacement of Asp357 by a glutamate decreased the affinity for calcium ions 30-fold . Introduction of a lysine, an asparagine, or an alanine at position 357 and of a lysine or an asparagine at position 354 resulted in calcium-independent variants . Isothermal titration calorimetry confirmed the loss of calcium binding . Although the D357K, D357N, and D357A variants did not bind calcium, at room temperature they were nearly as active as wild-type lipase in the presence of calcium, but at elevated temperatures these calcium-independent lipases showed a reduced activity . Over the whole temperature range the activities of the D354K and D354N variants are significantly lower than wild-type enzyme in the presence of calcium and are comparable to the activity of the wild-type apoenzyme . Our results show that binding of calcium is important for the structural stabilization of staphylococcal lipases (and possibly other lipases) and that it is possible to engineer calcium-independent variants on the basis of limited structural homology with another lipase.

Insect Biochem Mol Biol, 1998 Dec, 28(12), 1013 - 23
Spruce budworm elastase precipitates Bacillus thuringiensis delta-endotoxin by specifically recognizing the C-terminal region; Milne R et al.; A gut juice protein from Choristoneura fumiferana (spruce budworm) larvae that precipitates certain delta-endotoxins shows a unique specificity for the C-terminal amino acid sequence . Using homolog scanning mutants, we have identified a contiguous region of the Cry1Aa toxin which interacts with the 75-kDa toxin precipitating protein (TPP-75)' resulting in precipitation . The contiguous region from Cry1Aa can be transferred to Cry1Ac and results in an identical precipitation reaction . The precipitation reaction occurs rapidly and is unique in that the ratio of precipitating protein to toxin is low (estimated at 0.01), unlike antibody-antigen reactions which exhibit mole ratios close to 1 . TPP-75 has been characterized as an elastase-like serine protease . We have taken advantage of this serine protease character and incorporated a radiolabel using an irreversible inhibitor . The radiolabel has allowed us to show the coincidence of the catalytically-inhibited TPP-75 with the toxin in a blotting assay and to follow the degradation of TPP-75 during storage . TPP-75 represents the first evidence that gut juice proteins may selectively attenuate the activity of delta-endotoxins, prior to binding to putative receptors on susceptible cells . TPP-75 should be evaluated as a possible resistance mechanism for those larvae that do not exhibit a receptor-based resistance.

Urology, 1999 Jan, 53(1), 82 - 7
Intravesical therapy for transitional cell carcinoma of the bladder: the community practice; Obek C et al.; OBJECTIVES: To assess how the community urologist employs intravesical therapy in patients with transitional cell carcinoma (TCC) of the bladder because most data on intravesical therapy reflect the experience of major referral centers . METHODS: The medical records of 234 consecutive patients with TCC were reviewed . Sixty-nine patients received intravesical treatment before referral . The initial pathologic findings, the indication for treatment (eg, grade and stage, initial versus recurrent tumor), the schedule of intravesical therapy, and the drug selected for each course of treatment were assessed . RESULTS: A total of 1 39 courses of intravesical treatment were given to 69 patients; thus, the avarage number of courses was 2.02 per patient . The drug used was bacillus Calmette-Guerin (BCG) in 81 (58%), mitomycin C in 34 (24%), thiotepa in 16 (12%), Adriamycin in 4 (3%), and unknown in 4 (3%) . Intravesical treatment was given after transurethral resection of the initial tumor in 33 patients; the initial pathologic finding was high grade (ie, grade 3 or carcinoma in situ) and/or Stage T1 in 22, TaG1-G2 in 9, and unknown in 2 . One course of treatment was administered to 34 patients (49%) and two or more courses to 35 patients (51%) . Eleven patients with TaG 1 -2 tumors were treated repetitively despite failure, with an average of 3.5 courses per patient; the drug used was BCG in 44% . Nineteen percent of patients received maintenance therapy . Intravesical therapy had to be discontinued in 10 patients because of side effects; 8 patients (12%) developed small contracted bladders and severe irritative symptoms, 3 required cystectomy despite the lack of bladder cancer . CONCLUSIONS: Intravesical therapy in community practice conforms with the generally accepted indications for high-grade and T1 disease . However, the use of BCG for low-grade TCC appears to be quite common . Repeated courses may result in significant side effects . We emphasize that excessive treatment should be avoided for low-grade, Ta lesions and BCG reserved for patients with TaG3, carcinoma in situ, or T1 TCC.

Southeast Asian J Trop Med Public Health, 1998 Jun, 29(2), 285 - 8
Effectiveness of Bacillus Calmette Guerin (BCG) vaccination in the prevention of childhood pulmonary tuberculosis: a case control study in Nagpur, India; Zodpey SP et al.; A hospital-based, pair matched, case control study was carried out to estimate the effectiveness of BCG vaccination in the prevention of childhood pulmonary tuberculosis . The study included 126 incident cases of pulmonary tuberculosis (diagnosed by WHO criteria) below/equal the age of 12 years . Each case was pair matched with one control for age, sex, socio-economic status . Controls were selected from subjects attending study hospital for conditions other than tuberculosis and leprosy . The significant protective association between BCG and childhood pulmonary tuberculosis was observed (OR = 0.39, 95% CI = 0.22, 0.68) . The overall vaccine effectiveness was 61% (95% CI = 32%, 78%) . BCG was nonsignificantly more effective in underfives, among males and in upper-middle socioeconomic strata . The overall prevented fraction was estimated to be 47.53% (95% CI = 21.41%, 67.25%) . Results of this study thus demonstrated a moderate effectiveness of BCG vaccination in prevention of childhood pulmonary tuberculosis in a Central India population.

Nippon Rinsho, 1998 Dec, 56(12), 3199 - 204
{Treatment and prognosis of nontuberculous mycobacterial pulmonary infection}; Kurashima A; In performing NTM chemotherapy, particularly to mycobacterium avium-intracellulare complex (MAC) infection, we find difficulty in planning a rational protocol, and thus depend on previous experiences . We have studied retrospectively the effects of previous combination chemo-therapy cases of pulmonary MAC infections at National Tokyo Hospital . We selected 135 cases which had received the same chemotherapy continuously over six months . Having set a mean CFU of 3 times sputum culture before treatment as 100%, we calculated a six-months sequential bacillary response to a regimen and plotted the bacillary response curves . The response curves of various regimens of multidrug chemotherapy indicate that combinations of more than 3 drugs including aminoglycoside and clarithromycin are effective . The survival curve of 104 cases which could be observed over 10 years showed that median survival time is about 10 years.

Br J Urol, 1998 Dec, 82(6), 870 - 6
Mycobacterium vaccae (SRL172): a potential immunological adjuvant evaluated in rat prostate cancer; Hrouda D et al.; OBJECTIVE: To evaluate the potential of heat-killed Mycobacterium vaccae (SRL172) as a nonspecific immunostimulant and as an adjuvant to whole tumour cell vaccination in the rat model of prostate cancer . MATERIALS AND METHODS: SRL172 was used as a vaccine in the prevention and treatment of subcutaneous tumours in rats . Prevention experiments were conducted using subcutaneous MAT-LyLu tumours in Copenhagen rats, comparing vaccination with SRL172 alone, SRL172 plus autologous cells, and bacille Calmette-Guerin (BCG) plus autologous cells before tumour implantation . Treatment experiments were conducted using subcutaneous MAT-LyLu tumours in the Copenhagen rat and subcutaneous PAIII tumours in the Lobund-Wistar rat . Tumours were induced by subcutaneous injection with tumour cells . Animals were then vaccinated with autologous cells, autologous cells plus SRL172, or SRL172 alone . RESULTS: SRL172 was effective as an adjuvant to autologous whole tumour cell vaccination in the prevention of MAT-LyLu tumours and the survival benefit was equivalent to that provided when the adjuvant was live-attenuated BCG . SRL172 alone did not reduce tumour take or tumour growth in this model and neither strategy was effective in delaying the growth of established MAT-LyLu tumours . In the Lobund-Wistar rat vaccination with autologous whole tumour cells and SRL172 significantly delayed the growth of established tumours . CONCLUSION: Mycobacterium vaccae deserves further evaluation as an adjuvant to whole tumour cell vaccination in a phase I clinical trial in patients with prostate cancer.

J Bacteriol, 1999 Jan, 181(2), 454 - 61
Protein-DNA complexes in mycobacteriophage L5 integrative recombination; Pena CE et al.; The temperate mycobacteriophage L5 integrates site specifically into the genomes of Mycobacterium smegmatis, Mycobacterium tuberculosis, and Mycobacterium bovis bacillus Calmette-Guerin . This integrative recombination event occurs between the phage L5 attP site and the mycobacterial attB site and requires the phage-encoded integrase and mycobacterial-encoded integration host factor mIHF . Here we show that attP, Int-L5, and mIHF assemble into a recombinationally active complex, the intasome, which is capable of attB capture and formation of products . The arm-type integrase binding sites within attP play specialized roles in the formation of specific protein-DNA architectures; the intasome is constructed by the formation of intramolecular integrase bridges between one pair of sites, P4-P5, and the attP core, while an additional pair of sites, P1-P2, is required for interaction with attB.

Biol Pharm Bull, 1998 Dec, 21(12), 1263 - 6
CytA protein, a delta-endotoxin of Bacillus thuringiensis subsp . israelensis is associated with DNA; Yokoyama Y et al.; CytA protein (27 kDa) is produced by Bacillus thuringiensis subsp . israelensis (BTI) and is contained in its inclusion bodies . We previously reported the isolation of 25 kDa portion of CytA protein (p25-CytA protein) and its strong cytotoxic activity to mammalian cells . When p25-CytA protein was applied to an anion-exchange column for further purification, three fractions (M1, M2 and M3) were separated . M1 and M2 fractions were both shown to be 25 kDa protein, while M3 was a high molecular weight complex composed of 25 kDa protein and DNA . Purification and amino acid sequence analysis showed that M1 and M2 fractions were proteins lacking 29 and 31 N-terminal amino acids from CytA protein, respectively, and M3 was M1 protein associated with DNA . DNA was detected in BTI cells co-localizing with inclusion bodies . Both M1 and M2 proteins could bind to double-stranded DNA of BTI genome in vitro; the DNA binding ability of M1 protein was higher than that of M2 protein . These results suggested that CytA protein has DNA binding ability and is associated with DNA in the mother cell.

Eur J Radiol, 1998 Oct, 28(3), 235 - 42
CT-guided percutaneous treatment of inoperable pulmonary aspergillomas: a study of 40 cases; Giron J et al.; OBJECTIVE: To treat symptomatic pulmonary aspergilloma in patients who were not considered to be operable . MATERIAL AND METHODS: Forty patients were treated by CT-guided percutaneous injection of amphotericin paste, the aim being to fill the cavity completely and create an anaerobic environment for the aspergillus . The aspergillomas had developed after bacillary infection and pulmonary fibrosis . Surgery was contra-indicated in these patients because of severe respiratory failure . The authors detail the method of preparation of the paste and the technique of percutaneous injection . RESULTS: Hemoptysis ceased in all 40 patients, with a follow-up ranging from 6 to 28 months; six patients were also treated with bronchial embolization . In 26 patients, the aspergilloma disappeared and serum tests for aspergillus became negative . Complete disappearance of both the aspergilloma and the cavity was obtained in three patients . CONCLUSION: This technique appears to be a valuable contribution to non-surgical treatment of inoperable patients with pulmonary aspergilloma, but study should be continued in a larger series to define the exact indications and the interaction with other treatments which have recently been introduced.

J Biochem (Tokyo), 1999 Jan, 125(1), 58 - 63
Hydrophobic interactions of Val75 are critical for oligomeric thermostability of inorganic pyrophosphatase from Bacillus stearothermophilus; Shinoda H et al.; To determine the role of Val75 in the oligomeric structure of trimeric inorganic pyrophosphatase (PPase) {EC 3.6.1.1} from Bacillus stearothermophilus (Bst.), we used site-directed mutagenesis to prepare variants in which Val75 was replaced by Ala, Phe, Leu, Ile, Lys, Gln, and Asp . As a result, the variants in which valine is replaced by hydrophobic residues such as Ala, Phe, Leu, and Ile (V75A, F, L, and I) show almost the same level of enzyme activity and thermostability as the wild type enzyme, whereas variants with hydrophilic residue replacements such as Lys, Gln, and Asp (V75K, Q, and D) showed gross reductions in enzyme activity and thermostability . The dissociation of V75K and V75D from trimer to monomers occurred rapidly as the temperature rose, while V75F, V75L, and V75I dissociated more slowly than the wild type . There was no particular effect of heat treatment on the dissociation of V75A or V75Q, but these variants were slightly dissociated even in the native state . Thus, we conclude that Val75 may locate at the interface between the monomers and its hydrophobic interactions with its surroundings may play a key role in the thermostability and oligomeric subunit interactions of the enzyme.

J Biol Chem, 1999 Jan 15, 274(3), 1203 - 6
The gene, ialA, associated with the invasion of human erythrocytes by Bartonella bacilliformis, designates a nudix hydrolase active on dinucleoside 5'-polyphosphates; Conyers GB et al.; ialA, one of two genes associated with the invasion of human red blood cells by Bartonella bacilliformis, the causative agent of several diseases, has been cloned and expressed in Escherichia coli . The protein, IalA, contains an amino acid array characteristic of a family of enzymes, the Nudix hydrolases, active on a variety of nucleoside diphosphate derivatives . IalA has been purified, identified, and characterized as an enzyme catalyzing the hydrolysis of members of a class of signaling nucleotides, the dinucleoside polyphosphates, with its highest activity on adenosine 5'-tetraphospho-5'-adenosine (Ap4A), but also hydrolyzing Ap5A, Ap6A, Gp4G, and Gp5G . In each case, a pyrophosphate linkage is cleaved yielding a nucleoside triphosphate and the remaining nucleotide moiety.

J Invertebr Pathol, 1999 Jan, 73(1), 107 - 12
Development and use of a PCR assay for detection of the reproductive virus in wild populations of helicoverpa zea (Lepidoptera: noctuidae)
Lupiani B, Raina AK, Huber C.
Helicoverpa zea reproductive virus (HzRV) is a nonoccluded bacilliform virus that affects both female and male moths of the corn earworm H . zea . In order to study the biology and host range of HzRV, a bioassay was previously developed to detect the presence of this virus in infected insects . A drawback of this bioassay is that it is time consuming and requires more than a month to complete . Here we describe the development of a polymerase chain reaction (PCR) assay for the rapid detection of HzRV in infected corn earworms . The genome of HzRV was digested with PstI and cloned into a plasmid vector . Sequences from two different clones, P4 and P13, were selected for designing two sets of primers . These primers were used for PCR and their sensitivity and specificity in detecting HzRV DNA were examined . Both sets of primers produced the expected amplification product in samples containing HzRV DNA but not in uninfected corn earworm samples, Spodoptera frugiperda (Sf-9) cells, or samples from the Autographa californica nuclear polyhedrosis virus . In addition, the primer pair of the clone P13 was sensitive enough to detect approximately 175 copies of viral DNA . We then used this assay to examine feral populations of H . zea from seven geographical locations in the United States . HzRV was detected primarily in the Mississippi populations and to a lesser extent in Iowa and Georgia, but none in Maryland, Missouri, and Texas populations . This PCR assay provides a highly specific, sensitive, and rapid way of detecting the presence of HzRV and will be useful in further studying the host range, tissue specificity, and incidence of this virus in wild populations of the corn earworm.

Mol Biochem Parasitol, 1998 Nov 30, 97(1-2), 123 - 31
Structure of the glycosylphosphatidylinositol-anchor of the trans-sialidase from Trypanosoma cruzi metacyclic trypomastigote forms; Agusti R et al.; Both, culture-derived and metacyclic trypomastigotes of Trypanosoma cruzi shed a glycoprotein, the shed acute phase antigen, that is responsible for the trans-sialidase activity . In the present work the structure of the glycosylphosphatidylinositol membrane anchor of the trans-sialidase isolated from metacyclic forms was determined . Parasites were metabolically labelled with {9, 10(n)3H}-palmitic acid and the glycoprotein was purified by immunoprecipitation with a monoclonal antibody directed against the repetitive aminoacid sequence . Treatment of the glycoprotein with phosphatidylinositol phospholipase C (PI-PLC) from Bacillus thuringiensis rendered a lipid that comigrated in TLC with a standard of ceramide . No alkylglycerol was detected in contrast with the results previously obtained for the trans-sialidase isolated from culture-derived trypomastigotes where both lipids were found . Chemical and chromatographic analysis showed that the lipid moiety is palmitoyldihydrosphingosine with a minor amount of stearoyldihydrosphingosine . The glycan constituent of the glycosylphosphatidylinositol-anchor was analysed by nitrous acid deamination of the aqueous phase of the PI-PLC treatment, followed by reduction with NaBH4 and hydrolysis of the phosphodiester with aqueous hydrofluoric acid . A major oligosaccharide was obtained and enzymatic treatment with exoglycosidases and further chromatography in a high pH anion exchange system showed that the trimannosyl core backbone is substituted by an alpha-galactose . A comparison between the lipid constituent of the glycosylphosphatidylinositol anchor of several proteins and their spontaneous shedding by the action of an endogenous PI-PLC was made.

J Vector Ecol, 1998 Dec, 23(2), 149 - 60
Effectiveness of control measures against mosquitoes at a constructed wetland in southern California; Walton WE et al.; The effectiveness of larvicide and adulticide treatments against mosquitoes at a constructed wetland in San Jacinto, California was assessed with larval surveys, trapping of emerging adults, and collections of host-seeking females by carbon dioxide-baited traps . Bacillus thuringiensis var . israelensis (Bti, Bactimos pellets) applied at a rate of 19 kg/ha did not demonstrably affect Culex larval and emergent adult populations . Larval populations in the seven marshes of the wetland decreased from approximately one third-fourth instar larva/dip to undetectable levels following two applications of Bacillus sphaericus (Vectolex CG) at a rate of either 19 or 23.6 kg/ha . The largest decline in the number of adult mosquitoes emerging per day from vegetated regions of the wetland occurred after B . sphaericus treatments . The Culex erythrothorax host-seeking population declined about 80-fold during September beginning three weeks after the first treatment with B . sphaericus; however, the Culex tarsalis host-seeking population did not decline abruptly until mid-October 1997 . This result suggests that immigration of females from other developmental sites might be an important factor influencing the Cx . tarsalis host-seeking population at the wetlands . Safety concerns required that insecticide applications were carried out during daylight hours, and two daytime applications of adulticide (Pyrenone) in early August were ineffective against mosquitoes resting in the thick vegetation.

Biochim Biophys Acta, 1999 Jan 4, 1426(1), 99 - 109
The production and characterisation of an immobilised chaperonin system; Preston NS et al.; Here we report a method of immobilising the chaperonins GroEL and GroES to a glass matrix . The immobilised chaperone system has been used to successfully refold target proteins denatured by guanidine hydrochloride and produce substantially higher levels of active protein than occur on dilution into aqueous solution alone . The chaperone system has been shown to refold proteins from each of the three categories of GroEL substrate . The refolding of the enzyme glycerol dehydrogenase from Bacillus stearothermophilus shows a two-fold increase in activity in the presence of immobilised GroEL compared to that in free solution . The lactate dehydrogenase from B . stearothermophilus also shows a two-fold higher yield of activity in the presence of the immobilised GroEL and ATP . The presence of immobilised GroEL in the absence of ATP arrests the refolding of LDH . The enzyme citrate synthetase from porcine heart demonstrates a three-fold increase in activity when refolded in the presence of immobilised GroEL, ATP and free GroES . Similar results are obtained in the presence of free GroEL, immobilised GroES and ATP . The matrix-bound chaperone can be removed from the refolding mixture by centrifugation, producing a reusable system that can be easily isolated and purified from the refolded substrate.

J Mol Biol, 1999 Jan 8, 285(1), 73 - 83
Functional domains of an NAD+-dependent DNA ligase; Timson DJ et al.; Limited proteolysis of the NAD+-dependent DNA ligase from Bacillus stearothermophilus with thermolysin results in two fragments which were resistant to further proteolysis . These fragments were characterised by N-terminal protein sequencing and electrospray mass spectrometry . The larger, N-terminal fragment consists of the first 318 residues and the smaller, C-terminal fragment begins at residue 397 and runs to the C terminus . Both fragments were over-expressed in Escherichia coli and purified to homogeneity from this source . The large fragment retains the full self-adenylation activity of the intact enzyme, has minimal DNA binding activity and vastly reduced ligation activity . The small fragment lacks adenylation activity but binds to nicked DNA with a similar affinity to that of the intact enzyme . It is unable to stimulate the ligation activity of the large fragment . Atomic absorption spectroscopy showed that the intact protein and the small fragment bind a zinc ion but the large fragment does not . No evidence of any interaction between the two fragments could be obtained . Thus, we conclude that NAD+-dependent DNA ligases consist of at least two discrete functional domains: an N-terminal domain which is responsible for cofactor binding and self adenylation, and a C-terminal DNA-binding domain which contains a zinc binding site .

J Invertebr Pathol, 1999 Jan, 73(1), 52 - 8
Toxicity and receptor binding properties of a Bacillus thuringiensis CryIC toxin active against both lepidoptera and diptera; Abdul-Rauf M et al.; This study describes an investigation into the relationship among toxicity, specificity, and binding of CryIC delta-endotoxin from Bacillus thuringiensis subspecies aizawai HD-229 . In vivo bioassays of larvae using inclusions (Aedes aegypti) or solubilized toxin (Spodoptera littoralis, Spodoptera exigua, Spodoptera frugiperda, and Manduca sexta) revealed that CryIC was toxic to both dipteran and lepidopteran larvae . In vitro saturation binding assays with CryIC toxin labeled in vivo with {35S}methionine revealed specific and saturable binding to brush border membrane vesicles from both resistant and susceptible insect species . In contrast, dissociation binding experiments showed that the relative toxicities and specificities of CryIC to the insect species tested were correlated with the extent of irreversible binding to the insects' midgut brush border membrane vesicles .

J Invertebr Pathol, 1999 Jan, 73(1), 45 - 51
Isolation and characterization of brush border membrane vesicles from whole Aedes aegypti larvae; Abdul-Rauf M et al.; Studies of the binding interactions of dipteran-specific Bacillus thuringiensis delta-endotoxins are hindered by the lengthy midgut dissection procedure needed for preparation of brush border membrane vesicles . In an attempt to resolve this problem, brush border membrane vesicles were isolated from homogenates of whole Aedes aegypti larvae by a modification of the method of MacIntosh et al . (1994) . These preparations were found to resolve well on SDS-PAGE and appeared as spherical vesicles of various sizes under electron microscopic examination . Specific activities of the brush border membrane marker enzymes alkaline phosphatase and leucine amino acid arylamidase were enriched 10.9- and 10.7-fold, respectively . Direct binding experiments using 35S-labeled B . thuringiensis CryIC toxin revealed a single class of high-affinity binding sites with a dissociation constant (Kd) of 27 +/- 0.6 nM and a maximum binding capacity (Bmax) of approximately 27 +/- 1.2 pmol/mg BBMV protein . These binding parameters are similar to those of vesicles prepared from isolated midguts, indicating that whole larval brush border membrane vesicles are suitable for in vitro membrane binding studies .

Cell Biol Int, 1998, 22(2), 137 - 44
Histopathological and histochemical changes in honeybee larvae (Apis mellifera L.) after infection with Bacillus larvae, the causative agent of American foulbrood disease; Gregorc A et al.; Morphological, histochemical and cytochemical changes were examined in honeybee larvae after infection with the bacterium Bacillus larvae . The results indicate cell necrosis in the midgut epithelium accompanied by increasing cell vacuolization and nuclear pyknosis following per os inoculation with B . larvae . Many autolysosomes were positive for acid phosphatase . Non-vacuolar acid phosphatase activity was also found in lysed cell compartments . No such activity was found in regenerative epithelial cells . Degradation of haemocytes, salivary glands and other tissues was also observed . Histochemical analyses after per cutaneous inoculation with B . larvae of three- and five-day-old honeybee larvae show intense non-vacuolar acid phosphatase activity followed by disintegration of infected salivary glands, epithelial cell cytoplasm and haemocytes .

J Pharm Pharmacol, 1998 Nov, 50(11), 1205 - 11
Comparative measurement of the molecular weight of an antineoplastic glucan from BCG vaccine; Farrugia IV et al.; Bacillus Calmette-Guerin (BCG) vaccine, developed originally for the prophylaxis of tuberculosis, is a potent immunostimulant used to treat superficial bladder carcinoma in man . The aim of this study was to compare the molecular weight and self-association properties of an antineoplastic glucan (PS1A1) extracted from BCG vaccine as determined by different techniques including diffusion, light-scattering and chromatographic methods . In the diffusion experiments, a semi-empirical relationship was derived between the effective diffusion coefficients, Dp, and the weight-average molecular weights, Mw, of several dextrans used as standards, according to the equation Dp = 2.233 x 10(-6) x Mw(-0.66) . On the basis of this relationship, the molecular weight of PS1A1 was found to be 57.4 kDa, although, unexpectedly, membrane association was high, most probably because of molecular branching . In the light-scattering experiment it was observed that, unlike dextran, PS1A1 undergoes concentration-dependent multimerization in water . However, the molecular weight of PS1A1 in 0.1 M sodium chloride ranged from 60 to 68 kDa, with a mean of 65 kDa, over the same concentration range . This value was in agreement with the molecular weight determined for PS1A1 by gel-filtration chromatography in previous studies, suggesting that 65 kDa represents the approximate monomeric size of the unassociated molecule . Thus, it was evident that the aggregation was suppressed by electrolyte . Elemental analysis by X-ray fluorescence showed that PS1A1 contained carbon, oxygen, hydrogen and phosphorus, indicating that hitherto unobserved ionized phosphate groups might promote electrostatic interactions.

Biophys J, 1999 Jan, 76(1 Pt 1), 458 - 68
Bacterial S-layer protein coupling to lipids: x-ray reflectivity and grazing incidence diffraction studies; Weygand M et al.; The coupling of bacterial surface (S)-layer proteins to lipid membranes is studied in molecular detail for proteins from Bacillus sphaericus CCM2177 and B . coagulans E38-66 recrystallized at dipalmitoylphosphatidylethanolamine (DPPE) monolayers on aqueous buffer . A comparison of the monolayer structure before and after protein recrystallization shows minimal reorganization of the lipid chains . By contrast, the lipid headgroups show major rearrangements . For the B . sphaericus CCM2177 protein underneath DPPE monolayers, x-ray reflectivity data suggest that amino acid side chains intercalate the lipid headgroups at least to the phosphate moieties, and probably further beyond . The number of electrons in the headgroup region increases by more than four per lipid . Analysis of the changes of the deduced electron density profiles in terms of a molecular interpretation shows that the phosphatidylethanolamine headgroups must reorient toward the surface normal to accommodate such changes . In terms of the protein structure (which is as yet unknown in three dimensions), the electron density profile reveals a thickness lz approximately 90 A of the recrystallized S-layer and shows water-filled cavities near its center . The protein volume fraction reaches maxima of >60% in two horizontal sections of the S-layer, close to the lipid monolayer and close to the free subphase . In between it drops to approximately 20% . Four S-layer protein monomers are located within the unit cell of a square lattice with a spacing of approximately 131 A.

J Food Prot, 1998 Dec, 61(12), 1629 - 35
Survival and growth of psychrotrophic Bacillus cereus in dry and reconstituted infant rice cereal; Jaquette CB et al.; The potential for growth of enterotoxigenic Bacillus cereus in reconstituted dry foods is a concern, especially when they are consumed by infants or the immunosuppressed . The ability of a four-strain mixture of spores or vegetative cells of psychrotrophic B . cereus to survive in a commercial, dry infant rice cereal as affected by water activity (a(w); 0.27 to 0.28, 0.52 to 0.55, and 0.75 to 0.78), pH (5.6 and 6.7), and temperature (5, 25, 35, and 45 degrees C) was investigated . The rate of death of vegetative cells in dry cereal stored for 36 weeks was not affected by a(w) or pH . Death of spores in cereal stored at 45 degrees C for up to 48 weeks was enhanced at a(w) 0.78 but was unaffected by pH; loss of viability at 5, 25, and 35 degrees C was largely unaffected by differences in a(w) . The effect of temperature (8, 15, 21, and 30 degrees C) on outgrowth of spores of B . cereus inoculated at three levels (0.14, 14, and 133 CFU/g, dry weight basis) into cereal reconstituted with apple juice and commercial pasteurized milk (2% fat) was also studied . Outgrowth of spores did not occur in cereal reconstituted with apple juice . Cereal reconstituted with milk and inoculated with 0.14, 14, and 133 spores per g contained >3 log CFU/g within 24, 9, and 6 h, respectively, at 21 degrees C . Populations in cereal reconstituted with milk and inoculated with 133 CFU of B . cereus spores per g reached 7.11, 7.72, and 7.40 log CFU/g within 12, 48, and 72 h when stored at 30, 21, and 15 degrees C, respectively . The organism grew in cereal reconstituted with milk and held at 8 degrees C for 72 h; however, enterotoxin was not detected . In reconstituted cereal inoculated with 133 spores per g, enterotoxin was detected (detection limit 16 ng/g) after 24, 48, and 72 h at 30, 21, and 15 degrees C, respectively, when the population of B . cereus reached >7 log CFU/g . It is recommended that reconstituted infant foods be either consumed immediately or held at < or = 8 degrees C and consumed within 48 h after preparation.

Eur J Biochem, 1998 Dec 1, 258(2), 502 - 14
Solution structure of an artificial Fe8S8 ferredoxin: the D13C variant of Bacillus schlegelii Fe7S8 ferredoxin; Aono S et al.; The solution structure of the D13C variant of the thermostable Fe7S8 ferredoxin from Bacillus schlegelii has been determined by 1H-NMR spectroscopy in its oxidized form . In a variable-temperature NMR study the D13C variant was as thermostable (up to 90 degrees C) as the wild-type protein (WT) . Seventy-five out of 77 amino acid residues and 81% of all theoretically expected proton resonances in the D13C Fe8S8 protein have been assigned . Its structure was determined through torsion angle dynamics calculations with the program DYANA, using 935 meaningful NOEs (from a total of 1251), hydrogen bond constraints, and NMR-derived dihedral angle constraints for the cluster-ligating cysteines . Afterwards, restrained energy minimization and restrained molecular dynamics were applied to each conformer of the family . The final family of 20 structures has RMSD values from the mean structure of 0.055 nm for the backbone atoms and of 0.099 nm for all heavy atoms . The overall folding of the WT is maintained in the mutant, except for the immediate vicinity of the new cysteine, which becomes much more similar to native Fe8S8 proteins . The two residues at positions 11 and 12, which constitute an insertion with respect to all known Fe8S8 proteins, assume a conformation that does not prevent the preceding and following residues from folding like in native Fe8S8 proteins . Clear evidence for the existence of two conformations involving almost half of the amino acid residues was found . The two conformations are structurally indistinguishable . Temperature-dependent NMR experiments show that one of them is thermodynamically more stable than the other.

Eur J Biochem, 1998 Dec 1, 258(2), 491 - 501
Expression of genes encoding the E2 and E3 components of the Bacillus stearothermophilus pyruvate dehydrogenase complex and the stoichiometry of subunit interaction in assembly in vitro; Lessard IA et al.; Genes encoding the dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase (PDH) multienzyme complex from Bacillus stearothermophilus were overexpressed in Escherichia coli . The E2 component was purified as a large soluble aggregate (molecular mass > 1 x 10(6) Da) with the characteristic 532 symmetry of an icosahedral (60-mer) structure, and the E3 as a homodimer with a molecular mass of 110 kDa . The recombinant E2 component in vitro was capable of binding either 60 E3(alpha2) dimers or 60 heterotetramers (alpha2beta2) of the pyruvate decarboxylase (E1) component (also the product of B . stearothermophilus genes overexpressed in E . coli) . Assembling the E2 polypeptide chain into the icosahedral E2 core did not impose any restriction on the binding of E1 or E3 to the peripheral subunit-binding domain in each E2 chain . This has important consequences for the stoichiometry of the assembled complex in vivo . The lipoyl domain of the recombinant E2 protein was found to be unlipoylated, but it could be correctly post-translationally modified in vitro using a recombinant lipoate protein ligase from E . coli . The lipoylated E2 component was able to bind recombinant E1 and E3 components in vitro to generate a PDH complex with a catalytic activity comparable with that of the wild-type enzyme . Reversible unfolding of the recombinant E2 and E3 components in 6 M guanidine hydrochloride was possible in the absence of chaperonins, with recoveries of enzymic activities of 95% and 85%, respectively . However, only 26% of the E1 enzyme activity was recovered under the same conditions as a result of irreversible denaturation of both E1alpha and E1beta . This represents the first complete post-translational modification and assembly of a fully active PDH complex from recombinant proteins in vitro.

Bioorg Med Chem Lett, 1998 Sep 22, 8(18), 2549 - 54
Substrate specificity of thermostable farnesyl diphosphate synthase with alkyl group homologs of isopentenyl diphosphate; Nagaki M et al.; 3-Alkyl group homologs of isopentenyl diphosphate were examined for the reactivity as substrates of the thermostable farnesyl diphosphate (FPP) synthase of Bacillus stearothermophilus . Even 3-n-propyl- and 3-n-butyl-but-3-enyl diphosphates, which are hardly acceptable by animal FPP synthases, are accepted by this bacterial enzyme as substrates to react with dimethylallyl- and geranyl diphosphates, yielding 7-methyl-3-n-propylocta-2,6-dienyl- and 7,11-dimethyl-3-n-propyldodeca-2,6,10-trienyl diphosphate, respectively.

Bioorg Med Chem Lett, 1998 Sep 8, 8(17), 2291 - 6
A combinatorial approach to chemical modification of subtilisin Bacillus lentus; Plettner E et al.; The reaction between methanethiosulfonate reagents and cysteine mutants of subtilisin is quantitative and can be used to prepare chemically modified mutant enzymes (CMMs) with novel properties . The virtually unrestricted structural variations possible for CMMs presents a preparative and screening challenge . To address this, a rapid combinatorial method for preparing and screening the activities of CMMs has been developed.

J Biol Chem, 1999 Jan 8, 274(2), 795 - 800
Purification and characterization of NAD:Penicillamine ADP transferase from Bacillus sphaericus . A novel NAD-dependent enzyme catalyzing phosphoramide bond formation; Yanagidani J et al.; A strain of Bacillus sphaericus isolated from a local soil sample has been found to use beta,beta-dimethyl-DL-cysteine (DL-penicillamine) as the sole nitrogen source . Crude cell extract of the bacterium showed potent penicillamine-consuming activity only in the presence of NAD, which, however, was not used as an electron acceptor . Characterization of reaction products revealed that penicillamine was derivatized to a phosphoramide adduct with the ADP moiety of NAD, whereas the nicotinamide-ribose group was released and hydrolyzed spontaneously to ribose and nicotinamide . The phosphoramide product, ADP-penicillamine, caused potent product inhibition on the purified enzyme, and adenylate deaminase was found to be effective in converting the inhibitory product into inosine-diphosphate-penicillamine and thereby maintained the catalysis for several hours . The novel enzyme, termed as NAD:penicillamine ADP transferase, showed a single band on SDS-polyacrylamide gel electrophoresis with a mass of approximately 42 kDa . The native enzyme was monomeric . The enzyme showed high substrate specificity to NAD (Km = 13.0 mM) and L-penicillamine (Km = 6.5 mM); other nucleotides such as NADP, NAD(P)H, AMP, ADP, and ADP-ribose did not substitute for NAD, and L-valine, L-cysteine, L-homocysteine, L-cystine, L-leucine, and L-isoleucine did not serve as the substrate . Kinetic studies suggested an Ordered Bi Bi mechanism, with NAD as the first substrate to bind and ADP-L-penicillamine as the last product released . The novel NAD-dependent enzyme may catalyze the first step in penicillamine degradation in the strain of B . sphaericus.

Bioorg Med Chem Lett, 1998 Mar 17, 8(6), 593 - 6
Enzymatic synthesis of a modified phospholipid and its evaluation as a substrate for B . cereus phospholipase C; Martin SF et al.; The novel phospholipid 2, which bears a tert-butyl moiety in place of the natural trimethyl ammonium group of phosphatidylcholine, has been enzymatically synthesized via a transphosphatidylation reaction mediated by phospholipase D . The change from the choline headgroup in 1 to the tert-butyl group in 2 reduced the efficiency of hydrolysis by the phosphatidylcholine-preferring phospholipase C from Bacillus cereus by a factor of greater than 10(3).

Curr Microbiol, 1999 Feb, 38(2), 86 - 91
In vivo and In vitro function of the intracellular proteolytic apparatus in nongrowing bacillus megaterium under heat stress
Kucerova H, Vachova L, Strnadova M, Votruba J, Chaloupka J.
In Bacillus megaterium sporulating at 35 degreesC, up to 90% of 10-min pulse-labeled proteins were degraded . Degradation proceeded in two waves . Short-lived proteins, i.e., intrinsically labile proteins and proteins made short-lived because of starvation, were mostly degraded during the reversible sporulation phase . Their amount corresponded to 20% or slightly more during 2 h . The second wave of protein degradation, which followed during the irreversible sporulation phase at 35 degreesC, increased the amount of total degradable pulse-labeled proteins to about 90% . This wave was absent in the isogenic asporogenic mutant 27-36 or in the wild strain, whose sporulation was inhibited by increased temperature . The proportion of degradable proteins was thus reduced to less than 40% in the asporogenic mutant incubated at 35 degreesC and to 46% in the wild strain whose sporulation was suppressed by the temperature of 47 degreesC . Unlike sporulating cells, these cells were thus capable of degrading short-lived and denatured proteins, but were not able to degrade most of other proteins . The in vitro protein degradation was substantially enhanced by increasing the Ca2+ concentration, suggesting a role of Ca2+-dependent proteinase(s) in the process.

Arch Microbiol, 1998 Dec, 171(1), 19 - 30
Bacillus arsenicoselenatis, sp . nov., and Bacillus selenitireducens, sp . nov.: two haloalkaliphiles from Mono Lake, California that respire oxyanions of selenium and arsenic; Switzer Blum J et al.; Two gram-positive anaerobic bacteria (strains E1H and MLS10) were isolated from the anoxic muds of Mono Lake, California, an alkaline, hypersaline, arsenic-rich water body . Both grew by dissimilatory reduction of As(V) to As(III) with the concomitant oxidation of lactate to acetate plus CO2 . Bacillus arsenicoselenatis (strain E1H) is a spore-forming rod that also grew by dissimilatory reduction of Se(VI) to Se(IV) . Bacillus selenitireducens (strain MLS10) is a short, non-spore-forming rod that grew by dissimilatory reduction of Se(IV) to Se(0) . When the two isolates were cocultured, a complete reduction of Se(VI) to Se(0) was achieved . Both isolates are alkaliphiles and had optimal specific growth rates in the pH range of 8.5-10 . Strain E1H had a salinity optimum at 60 g l-1 NaCl, while strain MLS10 had optimal growth at lower salinities (24-60 g l-1 NaCl) . Both strains have limited abilities to grow with electron donors and acceptors other than those given above . Strain MLS10 demonstrated weak growth as a microaerophile and was also capable of fermentative growth on glucose, while strain E1H is a strict anaerobe . Comparative 16S rRNA gene sequence analysis placed the two isolates with other Bacillus spp . in the low G+C gram-positive group of bacteria.

Mol Gen Genet, 1998 Nov, 260(4), 335 - 45
The genes encoding light-harvesting subunits of Cyclotella cryptica (Bacillariophyceae) constitute a complex and heterogeneous family; Eppard M et al.; Total RNA was isolated from the diatom Cyclotella cryptica and separated into poly(A)+ and poly(A)- fractions . These fractions were subjected to in vitro translation/immunoprecipitation experiments using an antiserum directed against the predominant light-harvesting complex of Cy . cryptica (ccry antiserum) and a heterologous antiserum raised against the light-harvesting complex of the cryptophyte Cryptomonas maculata (cmac antiserum) . From translation reactions programmed with poly(A)+ RNA the ccry-antiserum immunoprecipitated polypeptides with relative molecular weights (Mr) of 27000, 25000, 23000 and 21000, while the cmac-antiserum precipitated proteins with Mrs of 32500 and 27000, respectively . Subsequent cDNA synthesis and immunological screening of the cDNA library with both antisera resulted in the isolation of six cDNA clones encoding light-harvesting subunits . Full-length precursors were 199-210 amino acids in length and had Mrs of 20000-23000 . The lengths of the putative signal peptides were 29 or 30 amino acids . Pairwise comparison revealed that the similarity between the clones ranged from 54-99% on the nucleotide level and from 36-99% at the amino acid level . In agreement with the data from the screens with the two antisera, the genes clustered into two groups . The data provide evidence that the genes constitute a heterogeneous multigene family and that the light-harvesting system of Cy . cryptica might be as complex as that of higher plants and green algae.

Immunol Lett, 1998 Dec, 64(2-3), 161 - 6
Effect of Bacillus firmus on antibody formation after mucosal and parenteral immunization in mice; Prokesova L et al.; Immunostimulatory properties of B . firmus, a nontoxic, nonpathogenic G + bacterium of external environment, were described previously . Antiinfectious and antitumor activity, macrophage activation and strong polyclonal stimulation of B lymphocytes were proved in human, mice and rats . The adjuvant effect of B . firmus on specific antibody response to ovalbumin in BALB/c mice is the topic of the present study . Against our expectation, B . firmus exerts more suppressive than stimulatory effect on specific antibody response . Formolized B . firmus decreased anti-ovalbumin response after subcutaneous immunization and only slightly increased serum antibodies after intraperitoneal immunization . After mucosal immunization, both oral and rectal, ovalbumin itself did not cause a significant systemic response but induced IgA anti-ovalbumin response in the intestine . B . firmus applied together with ovalbumin increased systemic serum response but absolutely eliminated intestinal response . The rectal route of antigen administration has been found less convenient because of less precise dosing of antigen in this mode of immunization.

FEMS Microbiol Lett, 1998 Dec 15, 169(2), 213 - 8
Similarity in moth-fly specific larvicidal activity between two serologically unrelated Bacillus thuringiensis strains; Higuchi K et al.; Parasporal inclusions of a Bacillus thuringiensis isolate designated 92-KU-105-9 (H14/19) exhibited unusual larvicidal activity, specific for the moth-fly, Telmatoscopus albipunctatus (Diptera: Psychodidae), similar to that of a previously reported B . thuringiensis serovar leesis (H33) strain . The LC50 value of the purified inclusions was 4.92 micrograms ml-1 for the moth-fly larvae, while no mortality was shown in the mosquitoes Culex pipiens molestus and Anopheles stephensi, at protein concentrations up to 10 mg ml-1 . Morphologically, the inclusion was a homogeneous globular body surrounded by an electron-dense, thick envelope . Multilamellar inner structure was evident between envelope membrane and inclusion matrix . SDS-PAGE revealed that the inclusions consist of five proteins with molecular masses of 72, 70, 68, 56 and 30 kDa . These proteins cross-reacted with the antibodies against inclusion proteins of the serovar leesis strain . High homologies existed in N-terminal amino acid sequences between the three major proteins (72, 70 and 68 kDa) and the two established protein classes, Cry4A and Cry10A.

Hunan Yi Ke Da Xue Xue Bao, 1997, 22(3), 229 - 32
{A study of anaerobic infection in maxillofacial region}; Deng F et al.; To analyse the anaerobic infection of maxillofacial surgery and estimate the efficacy of antianaerobic therapy, 45 patients were divided into two groups, tinidazole group and metronidazole group . Bacterial culture was positive before treating in all cases . There were Bacillus Melaniogenicus, Veillonella, Peptococcus and Peptostreptococus, etc . There was excellent efficacy in the treatment of maxillofacial anaerobic infection by tinidazole intravenously . After treatment, the result of bacterial examination was negative . The healing rate was 96.4% in 28 cases which used tinidazol, but 82.4% in control group which used metronidazole . The value of white blood cell and the function of liver and kidney pro- and post-treatment were not significantly different (P < 0.05) by comparision.

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