Bacillus

Pest Manag Sci, 2001 May, 57(5), 413 - 21
Cross-resistance and inheritance of resistance to Bacillus thuringiensis toxin Cry1Ac in diamondback moth (Plutella xylostella L) from lowland Malaysia; Sayyed AH et al.; A field population of Plutella xylostella from Malaysia (SERD4) was divided into five sub-populations and four were selected (G2-G5) with the Bacillus thuringiensis insecticidal crystal (Cry) toxins Cry1Ac, Cry1Ab, Cry1Ca and Cry1Da . Bioassay at G6 gave resistance ratios of 88, 5, 2 and 3 for Cry1Ac, Cry1Ab, Cry1Ca and Cry1Da respectively compared with the unselected sub-population (UNSEL-SERD4) . The Cry1Ac-selected population showed little cross-resistance to Cry1Ab, Cry1Ca and Cry1Da, (3-, 2- and 3-fold compared with UNSEL-SERD4), whereas the Cry1Ab-SEL sub-population showed marked cross-resistance to Cry1Ac (40-fold), much greater than Cry1Ab itself . In contrast, the Cry1Ca- and Cry1Da-SEL sub-population showed little if any cross-resistance to Cry1Ac and Cry1Ab . The mode of inheritance of resistance to Cry1Ac was examined in Cry1Ac-selected SERD4 by standard reciprocal crosses and back-crosses using a laboratory insecticide-susceptible population (ROTH) . Logit regression analysis of F1 reciprocal crosses indicated that resistance to Cry1Ac was inherited as an incompletely dominant trait . At the highest dose of Cry1Ac tested, resistance was recessive, while at the lowest dose it was almost completely dominant . The F2 progeny from a back-cross of F1 progeny with ROTH were tested with a concentration of Cry1Ac that would kill 100% of ROTH . The mortality ranged between 50 and 95% in seven families of back-cross progeny, which indicated that more than one allele on separate loci were responsible for resistance to Cry1Ac.

Anal Biochem, 2001 Jun 1, 293(1), 67 - 70
Method for real-time detection of inorganic pyrophosphatase activity; Eriksson J et al.; A sensitive and simple method for real-time detection of inorganic pyrophosphatase (PPase) (EC 3.6.1.1) activity has been developed . The method is based on PPase-induced activation of the firefly luciferase activity in the presence of inorganic pyrophosphate (PPi) . PPi inhibits the luciferase activity, but in the presence of PPase the luciferase activity is restored and the luminescence output increases . The assay yields linear responses between 8 and 500 mU . The detection limit was found to be 8 mU PPase . The method was used to detect the hydrolytic activity of PPases from Saccharomyces cerevisiae, Escherichia coli, and Bacillus stearothermophilus . As substrate for the luciferase, adenosine 5'-phosphosulfate can replace ATP, which is an advantage for detection of PPase activity in crude extracts containing ATP-hydrolyzing activities . The method can be used for kinetic and inhibition studies as well as for detection of PPase activity during different purification procedures .

Microbiol Res, 2001, 156(1), 19 - 30
Simultaneous biosynthesis and purification of two extracellular Bacillus hydrolases in aqueous two-phase systems; Ivanova V et al.; Thermostable alpha-amylase with temperature optimum at 80 degrees C, molecular mass 58 kDa and pI point 6.9 was purified from a catabolite resistant Bacillus licheniformis strain . The enzyme was sensitive to inhibition by metal ions and N-bromosuccinimide . The partition behaviour of this enzyme in aqueous two-phase systems (ATPS) of the polymer-polymer-water type was investigated and some effects of type, molecular weight and concentration of phase components were studied . Up to 100% retention in the bottom phase of polyethylene glycol 10,000-20,000/dextran 200 system was reached . Best partition conditions were obtained in PEG 10,000-20,000/polyvinyl alcohol 200 systems, where the partition coefficient K increased 750 times to 7.5 . Simultaneous production and purification of alpha-amylase and serine proteinase in PEG-polymer-water ATPS were examined . In the system PEG 6,000/ficoll, up to 90% of the amylase was retained in the bottom phase, whereas about 95% of the total protein (K = 22.8) and 60-75% of the proteinase were in the top phase . Similar separation of the enzymes from laboratory supernatant was obtained in system PEG/Na2SO4.

J Urol, 2001 Jun, 165(6 Pt 1), 1904 - 7
Clinical experience with 5-aminolevulinic acid and photodynamic therapy for refractory superficial bladder cancer; Waidelich R et al.; PURPOSE: We determined whether photodynamic therapy after the oral administration of 5-aminolevulinic acid in patients with superficial bladder cancer that cannot be controlled by transurethral resection and intravesical bacillus Calmette-Guerin (BCG) immunotherapy would preserve the bladder, while stopping tumor progression . Side effects of treatment were also assessed . MATERIALS AND METHODS: We performed photodynamic therapy after the oral administration of 5-aminolevulinic acid in 24 patients with rapidly recurring, multifocal, BCG refractory superficial pTa-pT1 transitional cell carcinoma of the bladder and carcinoma in situ . RESULTS: At a median followup of 36 months (range 12 to 51) 3 of the 5 patients with carcinoma in situ and 4 of the 19 with papillary tumors were free of recurrence . Three patients were rendered disease-free by repeat photodynamic therapy with 5-aminolevulinic acid and 3 underwent cystectomy . Tumor progression was stopped in 20 of our 24 cases . Immediately after the oral administration of 5-aminolevulinic acid hypotension and tachycardia occurred in 19 and 10 patients, respectively, with previously known severe cardiovascular disease . No phototoxic skin reaction or decreased bladder capacity was observed . CONCLUSIONS: These initial clinical results suggest that photodynamic therapy with orally administered 5-aminolevulinic acid is effective as an organ preserving procedure for treating superficial bladder cancer even in patients with bacillus Calmette-Guerin refractory carcinoma . One should be aware of hemodynamic instability after the oral administration of 5-aminolevulinic acid, particularly in patients with cardiovascular co-morbidity.

Curr Opin Pulm Med, 2001 May, 7(3), 116 - 23
Advances in the immunopathogenesis of pulmonary tuberculosis; Rook GA et al.; Tuberculosis remains a global emergency because of our lack of understanding of the details of its pathogenesis . In the last 12 months there have been striking advances in the molecular genetics of the organism . Mutated strains of Mycobacterium tuberculosis have been used to study the genetic requirements for virulence and establishment of latency, and the biology of the interaction with host cells . Genes involved in lipid metabolism seem particularly important . The probable sites of latency within the host lungs have been identified by in situ polymerase chain reaction . The complex control by M . tuberculosis of apoptosis of T cells and macrophages has been somewhat clarified, and the data may suggest that M . tuberculosis causes death of a subset of T cells, while preserving some macrophages as hiding places with reduced microbicidal and antigen-presenting function . Similarly the demonstration of a very large relative increase in interleukin (IL)-4 and IL-13 expression, (together with IL-4delta2, the IL-4 splice variant), that correlates with lung damage, has been supported by data from flow cytometry and in situ hybridization, and indicates that a subversive T helper-2 (Th2) component in the response to M . tuberculosis may undermine the efficacy of immunity and contribute to immunopathology . Recently defined changes in metabolism of cortisol within the lesions may contribute to the development of the Th2 component . These findings underline the need to start testing vaccine candidates in models that mimic the situations in which bacille Calmette-Guerin fails, such as in the presence of latent infection, pre-existing Th2 responses to cross-reactive organisms, and stress.

Z Naturforsch {C}, 2001 Mar-Apr, 56(3-4), 245 - 8
Transformation studies of Bacillus thuringiensis cryIC gene into a nitrogen-fixing Azospirillum lipoferum; Gounder R et al.; A lepidopteran toxin gene, cryIC (pSB607) from entomopathogenic Bacillus thuringiensis subsp . aizawai was introduced into nitrogen-fixing Azospirillum lipoferum by transformation . Regeneration of spheroplasts was achieved at 99% with 39% frequency of regeneration . Transformants were screened on NB kanamycin with ampicillin plates and 4 transformants were selected after ten generations . SDS-PAGE and Western blot analysis confirmed the presence of a 68 kDa protein in the transformants . Studies on utilization of carbon sources indicate that glucose and sucrose are the most favorable carbon sources and 2% molasses is the cheap alternate carbon source for the better growth of parent A . lipoferum and transformants.

Arch Biochem Biophys, 2001 Mar 15, 387(2), 197 - 201
Differences in the specificities of the highly alkalophilic proteinases Savinase and Esperase imposed by changes in the rigidity and geometry of the substrate binding sites; Georgieva DN et al.; Savinase and Esperase are closely related highly alkalophilic proteinases produced by Bacillus lentus . They are suitable couple for investigating the structural basis of proteinase specificity due to the identity of the catalytic and the differences in the substrate binding sites . Two of the substitutions in these sites are very important: T129P and G131P . The two prolines provide an extra rigidity of the Savinase-binding site . The substitutions S166N and Q191T in the S1 recognition loop change the binding geometry of the substrate P1 residue . The geometry of S1 in Esperase is more favorable for binding and catalysis in comparison to that in Savinase . Differences in P3 specificity are probably created by the substitution V104L, which influences the conformation of S3 . Leu in position 104 is more favorable for the binding of Phe to S4 than Val . The lower affinity and catalytic efficiency as well as more narrow proteolytic specificity of Savinase in comparison to those of Esperase are explained with the extra rigidity and unfavorable changes in geometry of the substrate binding site of the first enzyme.

Int J Epidemiol, 2001 Apr, 30(2), 380 - 5
Economic evaluation of universal BCG vaccination of Japanese infants; Rahman M et al.; BACKGROUND: The international controversy surrounding the use and effectiveness of the Bacillus Calmette-Guerin (BCG) vaccine and the low incidence of tuberculosis (TB) among Japanese children prompted this study . METHODS: We compared 'universal BCG vaccination' with 'no vaccination at all' using a cost-effectiveness analysis . The study population was a hypothetical cohort comprising a total of 1.2 million infants born in 1996 at locations all over Japan . A model was developed to calculate the number of TB cases prevented by the vaccination programme . Assuming 40-80% overall vaccine efficacy (64-86% for TB-meningitis) and 10 years of protection, we calculated the cost and number of immunizations required to prevent one child from developing TB, the total number of TB cases averted by vaccination and total costs required for the programme . RESULTS: Based on an assumption of flexible vaccine efficacy (40-80%), we estimated that 111-542 TB cases including 10-27 of TB-meningitis would be prevented during the 10 years after BCG vaccination among the cohort of infants born in 1996 . About US$35 950-175 862 or 2125-10 399 immunizations would be required to prevent one child from developing TB . Sensitivity analyses covering a wide duration of protection, incidence of TB, vaccine coverage and discount rate, revealed that other than vaccine efficacy, the cost of preventing a single case of TB is highly sensitive to the duration of BCG protection and TB incidence . CONCLUSION: The cost per case of TB prevented is heavily dependent on vaccine efficacy and the duration of protection, and is high compared with the cost of treating one child who has developed TB.

Microbios, 2001, 105(410), 15 - 27
Microbial ecology of muffins baked from cassava and other nonwheat flours; Chauhan S et al.; Cassava flour was evaluated as a partial substitute for wheat flour in gluten-free muffins . Four replicate test bakes were carried out to evaluate the microbial ecology of two muffin mixes, made with different combinations of cassava, soya, sorghum, sago, and potato flour . The muffins were baked at 180 degrees C for 25 min, stored at 30 degrees C and analysed microbiologically for up to 10 days post-baking . Raw materials were similarly analysed microbiologically on the day of baking . Characterization of predominant bacterial and mould isolates from the raw materials and the muffins was carried out . The rope-free and minimum mould-free shelf life of both muffin types were approximately 1 day and approximately 3 days, respectively . The predominant bacterial isolates from the raw materials and muffins were Bacillus species (83 and 99%, respectively) . The moulds predominating in the raw materials were Gloeosporium (42%), Penicillium (13%) and Mucorales (13%), and from muffins Penicillium (37%) and Aspergillus (37%).

Pediatrics . 2001 Apr;107(4):E54.
Targeted testing of children for tuberculosis: validation of a risk assessment questionnaire; Froehlich H et al.; OBJECTIVE: Given the directive of the American Academy of Pediatrics to test children for tuberculosis (TB) only if they are at high risk for the disease, we sought to determine how well a risk assessment questionnaire can predict a positive tuberculin skin test (TST) result among children seen in a medical office setting . METHODS: In a prospective observational study, we identified 31 926 children who received well-child care in 18 pediatric offices of the Kaiser Permanente Northern California Region from August 1996 through November 1998 and who were due to receive a routine TST (Mantoux method) as part of universal screening . Parents were asked to complete a questionnaire about risk factors for TB infection that included demographic information . The TST result at 48 to 72 hours was compared with questionnaire responses to identify responses that were most highly associated with a positive TST result at both the 10-mm and 15-mm cutoffs . A concurrent study was conducted to determine whether parents can recognize induration . RESULTS: This population was diverse in age (range: 0-18 years), race/ethnicity (white: 37%; Hispanic: 26.4%; Asian: 15.0%; black: 11.8%; other: 8.4%; not stated by parent: 1.6%), and household annual income (range: $10 524-$175 282) . Overall incidence of positive TST results was 1.0% at the 10-mm cutoff and 0.5% at the 15-mm cutoff . Positive predictive value of selected individual risk factors at the 10-mm cutoff were: child born outside the United States, 10.4%; history of receiving bacille Calmette-Guerin vaccine, 5.5%; and child having lived outside the United States, 5.3% . Using multivariate analysis, we selected a subset of risk factors that were independently and significantly associated with a positive TST result >/=10 mm: history of receiving bacille Calmette-Guerin vaccine (odds ratio {OR}: 2.31; 95% confidence interval {CI}: 1.70-3.13); household member with history of positive TST result or TB disease (OR: 1.53; 95% CI: 1.14-2.04); child born outside the United States (OR: 8.63; 95% CI: 6.16-12.09); child having lived outside the United States (OR: 2.06; 95% CI: 1.49-2.85); and race/ethnicity reported by parent as Asian (OR: 2.28; 95% CI: 1.59-3.27) or Hispanic (OR: 1.57; 95% CI: 1.09-2.26) . Several factors were not statistically significant predictors of a positive TST result: age, sex, household annual income, household member infected with human immunodeficiency virus or who had stayed in a homeless shelter, and being an adopted or foster child . Overall sensitivity of the 9 main items on the questionnaire was 80.9%; when a subset of 4 of these questions plus the race/ethnicity questions were used, sensitivity of responses was 83.5% . Parents failed to recognize positive TST results at a rate of 9.9% (for the 10-mm cutoff) and 5.9% (at the 15-mm cutoff) . CONCLUSION: A 5-question risk assessment questionnaire completed by parents can be used to accurately identify risk factors associated with TB infection in children . In our population, some risk factors suggested by the American Academy of Pediatrics could not be validated . Parents cannot be relied on to read TST results accurately . Screening for TB can be enabled by using a standardized, validated questionnaire to identify children who should be given tuberculin skin testing.

Pediatrics . 2001 Apr;107(4):E48.
High levels of interferon gamma in the plasma of children with complete interferon gamma receptor deficiency; Fieschi C et al.; We have found that children with complete interferon gamma (IFNgamma) receptor deficiency, unlike patients with other genetic defects predisposing them to mycobacterial diseases, have very high levels of IFNgamma in their plasma . This unexpected observation provides a simple and accurate diagnostic method for complete IFNgamma receptor deficiency in children with clinical disease caused by bacille Calmette-Guerin vaccines or environmental nontuberculous mycobacteria.

Pediatr AIDS HIV Infect, 1996 Apr, 7(2), 98 - 102
Evidence for tuberculous infection in Romanian HIV-positive children by enzyme-linked immunosorbent assay; Ispas D et al.; The pandemic spread of tuberculosis (TB) and human immunodeficiency virus (HIV) represents a serious world problem . The diagnosis of TB in developing countries remains difficult, particularly in patients with concomitant HIV infection . Anergia to tuberculin frequently occurs in HIV-positive patients with pulmonary or extrapulmonary disease, and radiographic images are atypical or nondiagnostic . Children are often in an even more unfavorable situation: they cannot expectorate, and the biological samples required for bacteriological examination and culture are more difficult to obtain . We present in this work the correlation between the presence of serum antimycobacterial antibodies {as demonstrated by an in-house enzyme-linked immunosorbent assay (ELISA)} in 41 out of 279 HIV-infected children, and clinical, bacteriological, radiological, and pathological data that support the diagnosis of TB in these children . The prevalence of antimycobacterial antibodies in our group of HIV-positive children was 23.3% . In only 4 of the total cases investigated could the diagnosis of TB not be supported by the results of standard tests for TB . The control group showed an insignificant interference from bacillus Calmette-Guerin (BCG) vaccination.

Zhonghua Jie He He Hu Xi Za Zhi, 1998 Aug, 21(8), 504 - 6
{Epidemiological characteristics of tuberculosis patients complicated with diabetes in Shanghai}; Lin S et al.; OBJECTIVE: To understand the epidemiological trends and characteristics of tuberculosis patients complicated with diabetes in Shanghai . METHOD: All the registered tuberculosis (TB) cases from 1992 to 1997 in Shanghai were included in the analysis . The new registration rates and percentages of TB cases complicated with diabetes were calculated . RESULT: The new registration rate of the cases increased from 0.70/100,000 in 1992 to 2.10/100,000 in 1997 with an annual increase rate of 24.57% . The cases with the complication accounted for 4.86% in 1997 . Diabetes tends to be more prevalent among TB patients in urban area . The greater the age, the more diabetes were found in TB patients . A significant difference was also observed between sex . Bacillary positive rate and percentage of infiltration type were higher, and cavity was more common in cases with the complication . The percentage of cases with relapse was also higher . CONCLUSION: The complication of diabetes significantly affected the epidemiological trend of TB in Shanghai . The diagnosis of diabetes in TB patients should be enforced, and short-course chemotherapy targeting the cases should be investigated.

J Biol Chem, 2001 May 25, 276(21), 18557 - 62 Epub 2001 Feb 28.
The mechanism of aubstrate eecognition of pyroglutamyl-peptidase I from Bacillus amyloliquefaciens as determined by X-ray crystallography and site-directed mutagenesis; Ito K et al.; Pyroglutamyl-peptidase is able to specifically remove the amino-terminal pyroglutamyl residue protecting proteins or peptides from aminopeptidases . To clarify the mechanism of substrate recognition for the unique structure of the pyrrolidone ring, x-ray crystallography and site-directed mutagenesis were applied . The crystal structure of pyroglutamyl-peptidase bound to a transition state analog inhibitor (Inh), pyroglutaminal, was determined . Two hydrogen bonds were located between the main chain of the enzyme and the inhibitor (71:O.H-N:Inh and Gln71:N-H.OE:Inh), and the pyrrolidone ring of the inhibitor was inserted into the hydrophobic pocket composed of Phe-10, Phe-13, Thr-45, Ile-92, Phe-142, and Val-143 . To study in detail the hydrophobic pocket, Phe-10, Phe-13, and Phe-142 were selected for mutation experiments . The k(cat) value of the F10Y mutant decreased, but the two phenylalanine mutants F13Y and F142Y did not exhibit significant changes in kinetic parameters compared with the wild-type enzyme . The catalytic efficiencies (k(cat)/K(m)) for the F13A and F142A mutants were less than 1000-fold that of the wild-type enzyme . The x-ray crystallographic study of the F142A mutant showed no significant change except for a minor one in the hydrophobic pocket compared with the wild type . These findings indicate that the molecular recognition of pyroglutamic acid is achieved through two hydrogen bonds and an insertion in the hydrophobic pocket . In the pocket, Phe-10 is more important to the hydrophobic interaction than is Phe-142, and furthermore Phe-13 serves as an "induced fit" mechanism.

Environ Health Perspect, 2001 May, 109 Suppl 2, 337 - 42
Prospects for reducing fumonisin contamination of maize through genetic modification; Duvick J; Fumonisins (FB) are mycotoxins found in (italic)Fusarium verticillioides-infected maize grain worldwide . Attention has focused on FBs because of their widespread occurrence, acute toxicity to certain livestock, and their potential carcinogenicity . FBs are present at low levels in most field-grown maize but may spike to high levels depending on both the environment and genetics of the host plant . Among the strategies for reducing risk of FB contamination in maize supplied to the market, development and deployment of Fusarium ear mold-resistant maize germplasm is a high priority . Breeding for increased ear mold tolerance and reduced mycotoxin levels is being practiced today in both commercial and public programs, but the amount of resistance achievable may be limited due to complicated genetics and/or linkage to undesirable agronomic traits . Molecular markers can be employed to speed up the incorporation of chromosomal regions that have a quantitative effect on resistance (quantitative trait loci) . Transgenic approaches to ear mold/mycotoxin resistance are now feasible as well . These potentially include genetically enhanced resistance to insect feeding, increased fungal resistance, and detoxification/prevention of mycotoxins in the grain . An example of the first of these approaches is already on the market, namely transgenic maize expressing Bacillus thuringiensis (Bt) toxin, targeted to the European corn borer . Some Bt maize hybrids have the potential to reduce FB levels in field-harvested grain, presumably through reduced feeding of Bt-susceptible insects in ear tissues . However, improved ear mold resistance per se is still an important goal, as the plant will still be vulnerable to noninsect routes of entry to (italic)Fusarium . A second approach, transgene-mediated control of the ability of Fusarium to infect and colonize the ear, could potentially be achieved through overexpression of specific antifungal proteins and metabolites, or enhancement of the plant's own defense systems in kernel tissues . This has not yet been accomplished in maize, although promising results have been obtained recently in other monocots versus other fungal and bacterial pathogens . Achieving reproducible and stable enhanced ear mold resistance under field conditions will be immensely challenging for biotechnologists . A third approach, transgene strategies aimed at preventing mycotoxin biosynthesis, or detoxifying mycotoxins in planta, could provide further protection for the grower in environments where FBs present a risk to the crop even when the maize is relatively resistant to Fusarium mold . In one example of such a strategy, enzymes that degrade FBs have been identified in a filamentous saprophytic fungus isolated from maize, and corresponding genes have been cloned and are currently being tested in transgenic maize.

J Interferon Cytokine Res, 2001 Apr, 21(4), 209 - 17
Protein tyrosine kinase regulates FAS-mediated apoptosis in human BCG-infected monocytes; Mendez-Samperio P et al.; Apoptosis of monocytes/macrophages has emerged as a central regulatory event in the defense against mycobacterial infections . The involvement of protein tyrosine kinases (PTK) in Fas-mediated apoptosis in T cells is well established, but the possible role of PTK in Fas-dependent death of human bacillus Calmette-Guerin (BCG)-infected monocytes remains unclear . Here, we first examined the expression and function of Fas on BCG-infected human monocytes by flow cytometry . The results demonstrated that BCG-infected monocytes expressed significant Fas protein levels . In addition, engagement of the Fas antigen with its agonistic antibody (Ab) resulted in apoptosis of monocytes, as monitored by DNA analysis and fluorescence-activated cell sorter (FACS) analysis . The apoptotic action of Fas was suppressed significantly by genistein, indicating a role for PTK in this death process . Consistent with this observation, herbimycin A and tyrphostin, two selective tyrosine kinase inhibitors with different mechanisms of action, effectively inhibited Fas-mediated apoptosis of BCG-infected monocytes, as demonstrated by DNA content analysis . Moreover, we confirmed the effect of genistein, herbimycin A, and tyrphostin by examining apoptosis with the terminal transferase dUTP nick endlabeling (TUNEL) assay . Collectively, these data demonstrate that Fas-induced apoptosis may represent an important mechanism for eliminating BCG-activated human monocytes and that this apoptosis is due, at least in part, to signaling via a PTK pathway.

Eur J Biochem, 2001 May, 268(10), 3117 - 25
Protein engineering of Bacillus megaterium CYP102.The oxidation of polycyclic aromatic hydrocarbons; Carmichael AB et al.; Cytochrome P450 (CYP) enzymes are involved in activating the carcinogenicity of polycyclic aromatic hydrocarbons (PAHs) in mammals, but they are also utilized by microorganisms for the degradation of these hazardous environmental contaminants . Wild-type CYP102 (P450(BM-3)) from Bacillus megaterium has low activity for the oxidation of the PAHs phenanthrene, fluoranthene and pyrene . The double hydrophobic substitution R47L/Y51F at the entrance of the substrate access channel increased the PAH oxidation activity by up to 40-fold . Combining these mutations with the active site mutations F87A and A264G lead to order of magnitude increases in activity . Both these mutations increased the NADPH turnover rate, but the A264G mutation increased the coupling efficiency while the F87A mutation had dominant effects in product selectivity . Fast NADPH oxidation rates were observed (2250 min-1 for the R47L/Y51F/F87A mutant with phenanthrene) but the coupling efficiencies were relatively low (< 13%), resulting in a highest substrate oxidation rate of 110 min-1 for fluoranthene oxidation by the R47L/Y51F/A264G mutant . Mutation of M354 and L437 inside the substrate access channel reduced PAH oxidation activity . The PAHs were oxidized to a mixture of phenols and quinones . Notably mutants containing the A264G mutation showed some similarity to mammalian CYP enzymes in that some 9,10-phenanthrenequinone, the K-region oxidation product from phenanthrene, was formed . The results suggest that CYP102 mutants could be useful models for PAH oxidation by mammalian CYP enzymes, and also potentially for the preparation of novel PAH bioremediation systems.

Eur J Biochem, 2001 May, 268(10), 3006 - 16
Biochemical characterization and identification of catalytic residues in alpha-glucuronidase from Bacillus stearothermophilus T-6; Zaide G et al.; Alpha-D-glucuronidases cleave the alpha-1,2-glycosidic bond of the 4-O-methyl-D-glucuronic acid side chain of xylan, as a part of an array of xylan hydrolyzing enzymes . The alpha-D-glucuronidase from Bacillus stearothermophilus T-6 was overexpressed in Escherichia coli using the T7 polymerase expression system . The purification procedure included two steps, heat treatment and gel filtration chromatography, and provided over 0.3 g of pure enzyme from 1 L of overnight culture . Based on gel filtration, the native protein is comprised of two identical subunits . Kinetic constants with aldotetraouronic acid as a substrate, at 55 degrees C, were a Km of 0.2 mM, and a specific activity of 42 U x mg(-1) (kcat = 54.9 s(-1)) . The enzyme was most active at 65 degrees C, pH 5.5-6.0, in a 10-min assay, and retained 100% of its activity following incubation at 70 degrees C for 20 min . Based on differential scanning calorimetry, the protein denatured at 73.4 degrees C . Truncated forms of the enzyme, lacking either 126 amino acids from its N-terminus or 81 amino acids from its C-terminus, exhibited low residual activity, indicating that the catalytic site is located in the central region of the protein . To identify the potential catalytic residues, site-directed mutagenesis was applied on highly conserved acidic amino acids in the central region . The replacements Glu392-->Cys and Asp364-->Ala resulted in a decrease in activity of about five orders of magnitude, suggesting that these residues are the catalytic pair.

Prikl Biokhim Mikrobiol, 2001 Mar-Apr, 37(2), 160 - 3
{Depolymerization of chitosan by chinolytic complex from Bacillus sp . 739}; Il'ina AV et al.; Low-molecular-weight (3-6 kDa) water-soluble chitosan was obtained by enzymatic depolymerization . Hydrolysis of crab chitosan was induced by O-glycoside hydrolase (EC 3.2.1), an extracellular chitinolytic complex from Bacillus sp . 739 . The optimum conditions for hydrolysis were found (sodium-acetate buffer, pH 5.2; 55 degrees C; an enzyme/substrate ratio 4 U/g chitosan; 1 h).

Prikl Biokhim Mikrobiol, 2001 Mar-Apr, 37(2), 156 - 9
{Isolation of highly purified cyclodextrin glucanotransferase from Bacillus sp . 1070}; Volkova DA et al.; Two chromatographic processes for purification of cyclodextringlucanotransferase (CGTase) from Bacillus sp . 1070 was carried out . The enzyme has been purified into 9.5 times on Butyl-Toyopearl and followed immobilized metal ion chromatography on Cu(II)-Iminodiacetic (IDA)-agarose . By the application of second purification scheme (chromatography on Butyl-Toyopearl and DEAE-Sephacel) the specific activity of CGTase has folded into 13.5 times . The purity of enzyme was shown to be approximately 90% by SDS-electrophoreses data . It was shown that isolated enzyme has two isoelectric points estimated as 5.1 and 5.3.

Urology, 2001 May, 57(5), 900 - 5
Do bacteriostatic urethral lubricants affect the clinical efficacy of intravesical bacillus Calmette-Guérin therapy?
Loertzer H, Brake M, Horsch R, Keller H.
OBJECTIVES: To investigate the effect of a bacteriostatic urethral lubricant on the clinical efficacy of intravesical bacillus Calmette-Guerin (BCG) therapy . METHODS: Between July 1987 and August 1999, 389 patients with superficial urothelial carcinoma of the bladder (pTa {multilocular, size greater than 2 cm, recurrent tumor}, pT1, pTis) were treated adjuvantly with BCG in a 6-week intravesical cycle after complete transurethral tumor resection of the bladder . Within the framework of a prospective study on recurrence and progression of superficial urothelial carcinoma of the bladder after transurethral resection and BCG therapy, we retrospectively studied the clinical value of the observation that the use of bacteriostatic urethral lubricants reduces the viability and thus the efficacy of BCG . If a lubricant induces a clinically relevant reduction in the viability of BCG, instillation without lubricant should lead to a lower rate of recurrence and possibly a lower rate of progression . Lubricant (11 mL) was used during catheterization in every male patient in our population (group 1, n = 317, 81.5%); no lubricant was used in the women serving as the control group (group 2, n = 72, 18.5%) . The two groups were similar with respect to age, distribution of tumor stage and grade, multifocality, and frequency of previous bladder carcinoma (Levene test, P = 0.008) . The median follow-up was 54 months (range 4 to 143) . RESULTS: Of the 389 patients, 90 (23.1%) developed recurrence during the follow-up period: 73 (23%) in group 1 and 17 (23.6%) in group 2 (P value not significant) . Progression occurred in 14 patients in group 1 (4.4%) and in 8 patients in group 2 (11%) (P = 0.043) . In groups 1 and 2, 19.2% and 47.1%, respectively, of the recurrences were progressive (P = 0.026) . CONCLUSIONS: The use of bacteriostatic lubricants in the usual dose before BCG instillation had no detectable adverse effect on the clinical efficacy of intravesical BCG immunotherapy . To avoid traumatic catheterization with possible systemic BCG administration, we therefore recommend, especially in men, the additional use of a sufficient quantity of urethral lubricant.

Urology, 2001 May, 57(5), 883 - 8
Tolerability of bacille Calmette-Guérin maintenance therapy for superficial bladder cancer; Saint F et al.; OBJECTIVES: To study the influence of adverse reactions on adherence to an immunotherapy maintenance schedule and the recurrence rate of bladder cancer . Bacille Calmette-Guerin immunotherapy has documented efficacy in the management of high-risk superficial bladder cancer . However, the optimal duration of intravesical bacille Calmette-Guerin therapy and the risk/benefit ratio of maintenance therapy are controversial . METHODS: From April 1996 to April 2000, 72 patients with superficial bladder cancer were treated with Immucyst (six consecutive weekly instillations of 81 mg) and then received maintenance therapy consisting of three consecutive weekly instillations 3, 6, 12, 18, 24, 30, and 36 months later . Adverse reactions, studied during 518 instillations, were classified in four categories using a scale based on the World Health Organization recommendations, and their impact on the adherence to therapy was analyzed . RESULTS: After an average follow-up of 24 months, a durable disease-free response was observed in 84.9% of the patients; 12.5% of patients had a relapse and 2.6% had disease progression . The response rate was similar in patients with and without adverse reactions . Only 14 patients (19%) received all the scheduled maintenance instillations . The dose was reduced in 41 patients (57%), and treatment was stopped in 28 patients (39%) . In multivariate analysis, an adverse event score of 1.5 or greater during induction therapy was significantly associated with cessation or modification of maintenance therapy (P = 0.01) . CONCLUSIONS: The scale developed in this study to monitor the adverse reactions to bacille Calmette-Guerin and their impact on the adherence to maintenance therapy may be helpful for tailoring maintenance regimens or implementing protective measures (dose reduction or treatment postponement).

Urology, 2001 May, 57(5), 872 - 7
Efficacy of chemotherapy for prostatic tuberculosis-a clinical and histologic follow-up study; Lee Y et al.; OBJECTIVES: To characterize the clinical features of prostatic tuberculosis and to evaluate the short and long-term results of antituberculous chemotherapy . METHODS: Eighteen patients (mean age 66.7 +/- 10.2 years) with prostatic tuberculosis were included in this study . The median pretreatment prostate-specific antigen (PSA) level was 2.7 ng/mL (range 0.3 to 31) . The PSA level was greater than 4.0 ng/mL in 6 patients (33.3%) . Eight patients (44.4%) received a triple-drug regimen of rifampin, ethambutol, and isoniazid for more than 6 months . The mean duration of chemotherapy was 7.6 months (range 6 to 12) . Of the 8 patients, 3 underwent chemotherapy longer because of concurrent tuberculosis of other organs . Follow-up studies included digital rectal examination, total PSA determination, and transrectal prostate biopsy . RESULTS: Ten patients were eligible for regular follow-up . All the patients were symptom free during follow-up . The median length of follow-up was 3.4 years (range 1 to 9) . The average number of follow-up transrectal prostate biopsies was 2.4 (range 2 to 3) . The follow-up histologic findings showed nodular hyperplasia in 7 patients and chronic inflammatory cell infiltration in 3 patients . No acid-fast bacillus was found in any follow-up specimen . Similarly, subsequent transrectal biopsy showed no relapse after a median length of 3.4 years of follow-up . Of the 6 patients with elevated PSA levels, the post-treatment PSA returned to normal in 3 patients . CONCLUSIONS: Our results suggest that a triple-drug regimen of 6 months' duration can successfully control prostatic tuberculosis . Histologic follow-up is a good method for monitoring the efficacy of treatment . Transrectal prostate biopsy is an important tool for the diagnosis and follow-up of prostatic tuberculosis.

Biochem, Eng . J. . 2001 Jul, 8(1), 61 - 81
Carbon sources affect metabolic capacities of Bacillus species for the production of industrial enzymes: theoretical analyses for serine and neutral proteases and alpha-amylase; Calik P et al.; The metabolic fluxes through the central carbon pathways were calculated for the genus Bacillus separately for the enzymes serine alkaline protease (SAP), neutral protease (NP) and alpha-amylase (AMY) on five carbon sources that have different reduction degrees (gamma), to determine the theoretical ultimate limits of the production capacities of Bacillus species and to predict the selective substrate for the media design . Glucose (gamma=4.0), acetate (gamma=4.0), and the TCA cycle organic-acids succinate (gamma=3.5), malate (gamma=3.0), and citrate (gamma=3.0) were selected for the theoretical analyses and comparisons . A detailed mass flux balance-based general stoichiometric model based on the proposed metabolic reaction network starting with the alternative five carbon sources for the synthesis of each enzyme in Bacillus licheniformis that simulates the behaviour of the metabolic pathways with 107 metabolites and 150 reaction fluxes is developed . Highest and lowest specific cell growth rates (&mgr;) were calculated as 1.142 and 0.766h(-1), respectively, when glucose that has the highest degree of reduction and citrate that has the lowest degree of reduction were used as the carbon sources . Highest and lowest SAP, NP and AMY synthesis rates were also obtained, respectively, when glucose and citrate were used . Metabolic capacity analyses showed that the maximum SAP, NP, and AMY synthesis rates were, respectively, 0.0483, 0.0215 and 0.0191mmolg(-1)DWh(-1) when glucose uptake rate was 10mmolg(-1)DWh(-1) and specific growth rate was zero . The amino acid compositions and the molecular weights of the enzyme influence the production yield and selectivity . For SAP and NP oxaloacetate and pyruvate, for AMY oxaloacetate appear to be the critical main branch points . Consequently, for SAP and NP syntheses the fluxes towards the alanine group and aspartate group, and for AMY synthesis the flux towards the aspartate group amino acids need to be high . The results encourage the discussion of the potential strategies for improving productions of SAP, NP and AMY.

Biochem Soc Trans, 2001 May, 29(Pt 2), 147 - 52
Control of electron transfer in neuronal NO synthase; Daff S et al.; The nitric oxide synthases (NOSs) are dimeric flavocytochromes consisting of an oxygenase domain with cytochrome P450-like Cys-ligated haem, coupled to a diflavin reductase domain, which is related to cytochrome P450 reductase . The NOSs catalyse the sequential mono-oxygenation of arginine to N-hydroxyarginine and then to citrulline and NO . The constitutive NOS isoforms (cNOSs) are regulated by calmodulin (CaM), which binds at elevated concentrations of free Ca(2+), whereas the inducible isoform binds CaM irreversibly . One of the main structural differences between the constitutive and inducible isoforms is an insert of 40-50 amino acids in the FMN-binding domain of the cNOSs . Deletion of the insert in rat neuronal NOS (nNOS) led to a mutant enzyme which binds CaM at lower Ca(2+) concentrations and which retains activity in the absence of CaM . In order to resolve the mechanism of action of CaM activation we determined reduction potentials for the FMN and FAD cofactors of rat nNOS in the presence and absence of CaM using a recombinant form of the reductase domain . The results indicate that CaM binding does not modulate the reduction potentials of the flavins, but appears to control electron transfer primarily via a large structural rearrangement . We also report the creation of chimaeric enzymes in which the reductase domains of nNOS and flavocytochrome P450 BM3 (Bacillus megaterium III) have been exchanged . Despite its very different flavin redox potentials, the BM3 reductase domain was able to support low levels of CaM-dependent NO synthesis, whereas the NOS reductase domain did not effectively substitute for that of cytochrome P450 BM3.

J Invertebr Pathol, 2001 Apr, 77(3), 173 - 9
Mortality of Colorado potato beetle (Leptinotarsa decemlineata) after sublethal stress with the CryIIIA delta-endotoxin of Bacillus thuringiensis and subsequent exposure to Beauveria bassiana; Costa SD et al.; Acute or chronic sublethal exposure of Colorado potato beetle larvae to the CryIIIA delta-endotoxin of Bacillus thuringiensis Berliner did not significantly (P > 0.05) alter their subsequent susceptibility to Beauveria bassiana (Balsamo) Vuillemin . During the period of exposure to B . bassiana there was continued mortality from previous exposure to delta-endotoxin, and B . bassiana also caused significant mortality . Acute and chronic exposure to delta-endotoxin significantly prolonged larval development . The weights of prepupae and adults were significantly reduced by exposure to delta-endotoxin, with the greatest effect being from chronic exposure . Separation of the manifestations of stress in time (feeding vs soil stages) and space (toxin damage to the insect gut vs fungal penetration of the cuticle and activity in the hemocoel) may have precluded alteration of insect susceptibility to infection by B . bassiana . Endemic populations of B . bassiana are not expected to influence the development of resistance in the Colorado potato beetle to the delta-endotoxin of B . thuringiensis .

Semin Urol Oncol, 2001 May, 19(2), 141 - 7
Vaccine and gene therapy of renal cell carcinoma; Gitlitz BJ et al.; The concept of tumor vaccines is not new . However, advances in gene transfer technology, tumor immunology, molecular biology, and methods of monitoring antitumor response, have allowed for novel, more specific vaccine approaches . For example, first-generation tumor vaccines were composed of whole inactivated cancer cells, or tumor lysates (Tuly) given together with immune adjuvants like bacillus Calmette-Guerin (BCG) . Current strategies include tumor cells modified with genes encoding molecules necessary to stimulate a cytotoxic T cell response, such as cytokine genes, foreign HLA genes, tumor-associated antigen (TAA) genes, and even costimulatory molecules . Activation of cellular immunity requires at least three synergistic signals including presentation of specific tumor antigens, costimulatory signals (B7 molecules), and propagation of the immune response via cytokine release . In general, tumor cells often fail to demonstrate any of these immunostimulatory properties . Dendritic cell-based vaccines are gaining popularity as these cells can properly present TAA to the immune system, thus circumventing the poor antigen-presenting qualities of tumor cells . Dendritic cells can be "loaded" with TAA or other molecules either by their natural endocytotic capabilities, or by genetic modification.

Extremophiles, 2001 Apr, 5(2), 135 - 44
Starch-hydrolyzing bacteria from Ethiopian soda lakes; Martins RF et al.; Alkaliphilic bacteria were isolated from soil and water samples obtained from Ethiopian soda lakes in the Rift Valley area--Lake Shala, Lake Abijata, and Lake Arenguadi . Starch-hydrolyzing isolates were selected on the basis of their activity on starch agar plate assay . Sixteen isolates were chosen, characterized, and subjected to 16S rRNA gene sequence analysis . All the isolates were gram positive and catalase- and beta-galactosidase positive . All isolates except one were motile endospore-forming rods and were found to be closely related to the Bacillus cluster, being grouped with Bacillus pseudofirmus, Bacillus cohnii, Bacillus vedderi, and Bacillus agaradhaerens . The one exception had nonmotile coccoid cells and was closely related to Nesterenkonia halobia . The majority of the isolates showed optimal growth at 37 degrees C and tolerated salinity up to 10% (w/v) NaCl . Both extracellular and cell-bound amylase activity was detected among the isolates . The amylase activity of two isolates, related to B . vedderi and B . cohnii, was stimulated by ethylenediaminetetraacetic acid (EDTA) and inhibited in the presence of calcium ions . Pullulanase activity was expressed by isolates grouped with B . vedderi and also most of the isolates clustered with B . cohnii; cyclodextrin glycosyltransferase was expressed by most of the B . agaradhaerens-related strains . Minor levels of alpha-glucosidase activity were detected in all the strains.

Biotechnol Bioeng, 2001 Jul 5, 74(1), 62 - 9
Production of a recombinant hybrid molecule of cholera toxin-B-subunit and proteolipid-protein-peptide for the treatment of experimental encephalomyelitis; Yuki Y et al.; Mucosal administration of experimental autoimmune encephalomyelitis (EAE)-specific autoantigens can reduce the onset of disease . To examine whether cholera toxin-B-subunit (CTB)-conjugated EAE-specific T-cell epitope can reduce development of the autoimmune disease in mice, we produced a recombinant hybrid molecule of CTB fusion protein linked with proteolipid-protein (PLP)-peptide139-151(C140S) at levels up to 0.1 gram per liter culture media in Bacillus brevis as a secretion-expression system . Amino acid sequencing and GM1-receptor binding assay showed that this expression system produced a uniformed recombinant hybrid protein . EAE was induced in SJL/J mice by systemic administration with the PLP-peptide . When nasally immunized 5 times with 70 microg rCTB PLP-peptide hybrid protein, mice showed a significantly suppressed development of ongoing EAE and an inhibition of both the PLP-peptide-specific delayed-type hypersensitivity (DTH) responses and leukocyte infiltration into the spinal cord . In contrast, all mice given the PLP-peptide alone or the PLP-peptide with the free form of CTB did not suppress the development of EAE and DTH responses . These results suggest that nasal treatment with the recombinant B . brevis-derived hybrid protein of CTB and autoantigen peptide could prove useful in the control of multiple sclerosis .

Environ Sci Technol, 2001 Mar 1, 35(5), 917 - 22
Fluoranthene-2,3- and -1,5-diones are novel products from the bacterial transformation of fluoranthene; Kazunga C et al.; Fluoranthene is one of the predominant compounds found in soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAH) . Four bacterial strains isolated from PAH-contaminated soils transformed fluoranthene to a number of products during growth on phenanthrene, including the novel metabolites fluoranthene-2,3-dione (F23Q) and fluoranthene-1,5-dione (F15Q) . Given the known toxicity and mutagenicity of F23Q, we focused on characterizing this metabolite with respect to its effects on the metabolism of other PAH . The yield of F23Q from fluoranthene ranged from 2% for Sphingomonas yanoikuyae R1 to greater than 20% for Pseudomonas stutzeri P16 and Bacillus cereus P21 . None of the strains appeared capable of metabolizing F23Q any further . F23Q strongly inhibited phenanthrene removal by strain R1 but had a negligible to minor effect on phenanthrene degradation by the other organisms . At a concentration of 6.8 microM, F23Q also substantially inhibited the mineralization of benz{a}anthracene, benzo{a}pyrene (BaP), and chrysene by strain R1 as well as BaP mineralization by Pseudomonas saccharophila P15 . Inhibition of BaP mineralization by strain P15 was still evident at an F23Q concentration of 0.68 microM . The inhibition of strain R1 by F23Q was explained in part by a cytotoxic effect, but results with strain P15 indicate that other mechanisms of inhibition occur . These findings suggest that quinones such as F23Q and F15Q have the potential to accumulate in PAH-contaminated systems and can inhibit the degradation of other PAH.

Wound Repair Regen, 2001 Mar-Apr, 9(2), 86 - 94
Exogenous phospholipase C stimulates epithelial cell migration and integrin expression in vitro; Firth JD et al.; Phospholipase C secreted by bacterial pathogens has been identified as a virulence factor in several human diseases and has been implicated in impeding wound healing . The role of phospholipase C in the intracellular signal control of epithelial growth was studied in normal human skin keratinocytes cultured in conditions simulating aspects of wound healing . Bacillus cereus phospholipase C decreased cell-cell contact and increased cell migration resulting in disruption of the advancing epithelial sheet . Phospholipase C-induced migration was blocked by inhibitor of the phosphoinositol signal transduction pathway neomycin sulfate and protein kinase C inhibitor RO-31-8220 . Induced migration was associated with elevated levels of matrix metalloproteinase-9 which, when blocked by tissue inhibitor of metalloproteinase-1, was accompanied by a loss of migration . Adhesion studies showed that phospholipase C treatment enhanced cell binding to fibronectin, vitronectin and collagen IV . Immunostained phospholipase C-stimulated cells cultured on fibronectin showed enhanced expression and relocation of the integrin subunits alpha(v), alpha5 and beta1 . Confocal microscopy showed that phospholipase C-induced levels of integrin subunit beta1 were predominantly deposited on the basal surface of the cell apparently in focal contacts and associated with actin stress fibers . These results indicate that exogenous phospholipase C signaling from a bacterial source may play an important role in perturbing normal reepithelialization via altered expression of integrins and matrix metalloproteinase-9.

RNA, 2001 May, 7(5), 692 - 701
Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding; Nakashima T et al.; Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA . The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution . The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain . The molecular shape and electrostatic representation suggest that the concave surface and loop regions are involved in 5S rRNA binding . To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurring in the beta-strands and loop regions . The mutations of Asn37 at the beta1-strand and Gln63 at the loop between helix 2 and beta3-strand as well as that of Phe77 at the tip of the loop structure between the beta2- and beta3-strands caused a significant reduction in 5S rRNA binding . In addition, the mutations of Thr90 on the beta3-strand and Ile141 and Asp144 at the loop between beta4- and beta5-strands moderately reduced the 5S rRNA-binding affinity . Comparison of these results with the more recently analyzed structure of the 50S subunit from Haloarcula marismortui suggests that there are significant differences in the structure at N- and C-terminal regions and probably in the 5S rRNA binding.

Indian J Exp Biol, 2001 Jan, 39(1), 82 - 4
Development of a self floating slow release formulation of Bacillus thuringiensis var . israelensis and its larvicidal activity; Prabakaran G et al.; Alginate encapsulated B . thuringiensis var . israelensis (B . t . i.) self floating type formulations were prepared . Its spore release rate, floating efficacy and larvicidal activity against Culex quinquefasiatus were tested in the laboratory . The larval mortality of 91-100% was induced by the floating formulation with a mean spore release of 3.04 x 10(4)/ml/day from 6th day to 27th day . From day 28 to 33 the mean number of spores released were 1.16 x 10(4)/ml/day which caused 72.2-88.2% mortality . From 34th day to 40th day the mean number of spores released were 4.97 x 10(3)/ml/day which caused 42.2-67.2% mortality . However, the self floating alginate encapsulated beads were intact and found to float upto 40 days.

Infect Immun, 2001 Jun, 69(6), 4174 - 6
Priming by DNA immunization augments protective efficacy of Mycobacterium bovis Bacille Calmette-Guerin against tuberculosis; Feng CG et al.; Sequential immunization with mycobacterial antigen Ag85B-expressing DNA and Mycobacterium bovis bacille Calmette-Guerin (BCG) was more effective than BCG immunization in protecting against Mycobacterium tuberculosis infection . Depletion of the CD8(+) T cells in the immunized mice impaired protection in their spleens, indicating that this improved efficacy was partially mediated by CD8(+) T cells.

Infect Immun, 2001 Jun, 69(6), 3697 - 702
Differential interleukin-10 and gamma interferon mRNA expression in lungs of cilium-associated respiratory bacillus-infected mice; Kendall LV et al.; The cilium-associated respiratory (CAR) bacillus is a gram-negative, extracellular bacterium that causes persistent respiratory tract infections in rodents . We have previously demonstrated that BALB/c mice are more susceptible to CAR bacillus-induced disease than resistant C57BL/6 mice, with elevations in pulmonary gamma interferon (IFN-gamma) and interleukin (IL)-4 . IL-10 is a type 2 cytokine that can increase host susceptibility to bacterial diseases through its anti-inflammatory effects, including suppression of macrophage function . The purpose of this study was to further describe the cytokine profiles associated with histologic lesions in CAR bacillus-infected mice and to assess the effects of cytokine depletion on the pathogenesis of disease . Six-week-old female BALB/c and C57BL/6 mice and mice with targeted mutations in IFN-gamma and IL-4 were inoculated intratracheally with 10(5) CAR bacillus organisms, and samples were collected at 6 to 7 weeks postinoculation . Lung samples were collected for histopathologic examination and analysis of cytokine mRNA . IFN-gamma, IL-10, and IL-4 mRNA levels in the lungs of infected mice were semiquantitatively measured using a reverse transcriptase-mediated PCR assay and compared to those in uninfected control animals of each strain . BALB/c mice infected with CAR bacillus had a median lung lesion score of 6 and IL-10 and IL-4 mRNA levels were significantly elevated . The majority of C57BL/6 mice were resistant to disease characterized by lung lesions scores of 2 or less and a dominant IFN-gamma mRNA cytokine profile . A few C57BL/6 mice with lesions scores of 5 or greater had elevations in all three cytokines and were susceptible to disease . C57BL/6 IFN-gamma knockout mice had increased disease with elevations in IL-10 and IL-4 mRNA, while BALB/c IL-4 knockout mice infected with CAR bacillus had a mild decrease in lesion severity and an attenuated IL-10 mRNA expression compared to wild-type BALB/c mice . These data indicate that IL-10 and IL-4 predominate in CAR bacillus-induced histologic lesions in mice, while IFN-gamma may play a role in resistance to disease.

Vaccine, 2001 May 14, 19(25-26), 3518 - 25
Comparison of different delivery systems of vaccination for the induction of protection against tuberculosis in mice; Lima KM et al.; The way to deliver antigens and cellular requirements for long-lasting protection against tuberculosis are not known . Immunizations with mycobacterial 65 kDa heat shock protein (hsp65) expressed from J774-hsp65 cells (antigen-presenting cells that endogenously produce hsp65 antigen) or from plasmid DNA, or with the protein entrapped in cationic liposomes, can each give protective immunity similar to that obtained from live Bacillus Calmette Guerin (BCG), whereas injecting the protein in Freund's incomplete adjuvant (FIA) has minimal effect . Protective procedures elicited high frequencies of antigen-reactive alphabeta T cells with CD4+/CD8- and CD8+/CD4- phenotypes . Protection correlated with the abundance of hsp65-dependent cytotoxic CD8+/CD4-/CD44hi cells . The frequency of these cells and the level of protection declined during 8 months after J774-hsp65 or liposome-mediated immunization with hsp65 protein but were sustained or steadily increased over this period after hsp65-DNA or BCG immunizations . IFN-gamma predominated over IL-4 among the hsp65-reactive CD8+/CD4- and CD4+/CD8- populations after J774-hsp65-, hsp65-liposome-, and hsp65-DNA-mediated immunizations, but similar levels of these cytokines prevailed after BCG vaccination.

Immunol Lett, 2001 May 1, 77(1), 39 - 45
Enhanced systemic and mucosal antibody responses to a model protein antigen after intranasal and intratracheal immunisation using Bacillus firmus as an adjuvant; Mlckova P et al.; Bacillus firmus, a non-pathogenic Gram positive (G+) bacterium of the external environment was investigated for immunomodulatory properties . It stimulated an increase in anti-ovalbumin IgG in sera, bronchoalveolar lavages and intestinal washings after both intranasal (i.n.) and intratracheal (i.t.) immunisation, and enhanced anti-ovalbumin IgA in intestinal secretions and in bronchoalveolar lavage fluid after i.n . or i.t . immunisation, respectively . The immunomodulatory effect of B . firmus on antibody formation was antigen specific.

Mol Ecol, 2001 Apr, 10(4), 1069 - 74
Diamondback moth compensatory consumption of protease inhibitor-transformed plants; Winterer J et al.; Prior study of the effect of protease inhibitors (PIs) on diamondback moths suggests that moths are resistant to them, so PIs represent an ineffective defence against moths . However, our data suggest that diamondback moths do suffer lower growth rates when they consume plants transformed with potato protease inhibitor (PI2), but that effect is hidden by compensatory consumption . Plants, instead of gaining an advantage by lowering the insect growth rate, suffer a disadvantage as moths consume more tissue to mitigate the effect . Furthermore, PI2, when used in conjunction with another transgenic pesticidal protein, Bt (from Bacillus thuringiensis) counteracts the effectiveness of Bt at protecting plant tissue . Thus, transgenic PIs are not only less effective than previously thought in protecting Brassica plants from diamondback moths, they may actually lead to increased plant damage by the moths.

J Am Mosq Control Assoc, 2001 Mar, 17(1), 8 - 12
Evaluation of liquid Bacillus thuringiensis var . israelensis products for control of Australian Aedes arbovirus vectors; Brown MD et al.; Laboratory bioassay studies were conducted in southeast Queensland, Australia, on the efficacy of Teknar, VectoBac 12AS, and Cybate (active ingredient: 1,200 international toxic units Bacillus thuringiensis var . israelensis {Bti}) against 3rd instars of the arbovirus vectors Aedes aegypti, Ae . notoscriptus, Ae . vigilax, and Ae . camptorhynchus . Probit analyses were then used to determine LD50 (median lethal dose), LD50, and lethal dose ratios (LDR) . Aedes aegypti and Ae . notoscriptus, both container-habitat species, tolerated the highest Bti concentrations compared with saltmarsh Ae . vigilax and Ae . camptorhynchus . For example, the LDR for Ae . vigilax versus Ae . notoscriptus exposed to Cybate was 0.14 (95% confidence limit {CL} 0.03-0.61) . Similarly, the Cybate LDR for Ae . camptorhynchus versus Ae . notoscriptus was 0.22 (95% CL 0.07-0.70) . Teknar produced similar results with an LDR of 0.21 (95% CL 0.04-1.10) for Aedes vigilax versus Aedes notoscriptus . Differences in product efficacy were found when tested against the 2 container-breeding species . Cybate was less effective than Teknar with LDRs of 1.55 (95% CL 0.65-3.67) and 1.87 (95% CL 0.68-5.15) for Aedes aegypti and Ae . notoscriptus, respectively . The significant differences in susceptibility between mosquito species and varying efficacy between products highlight the importance of evaluating concentration-response data prior to contracting with distributors of mosquito control products . This information is crucial to resistance management strategies.

J Am Mosq Control Assoc, 2001 Mar, 17(1), 45 - 50
Impact of a 26-month Bacillus sphaericus trial on the preimaginal density of Culex quinquefasciatus in an urban area of Recife, Brazil; Silva-Filha MH et al.; A field trial using the entomopathogen Bacillus sphaericus against Culex quinquefasciatus was conducted in a district of Recife, Brazil, an area with a high prevalence of lymphatic filariasis . In this urban area more than 2,500 Cx . quinquefasciatus breeding sites within a 1.2-km2 area were found and subsequently submitted to a long-term treatment with B . sphaericus . To evaluate the impact of treatment on the densities of Cx . quinquefasciatus preimaginal forms, 26 breeding sites, representative of the major site types found in that area, were monitored for 3 years . Parameters such as mean and maximum densities of larval population as well as the frequency of infested sites were recorded before, during, and after the trial . The level of each parameter was greatly reduced during the treatment period, particularly in the 2nd treatment year . A low density of Cx . quinquefasciatus was also maintained throughout the last year, in the absence of treatment . Analysis of the data reported here confirms B . sphaericus as a very effective larvicide against Cx . quinquefasciatus despite the optimal environmental conditions for mosquito proliferation in the urban area of Recife.

Scand J Infect Dis, 2001, 33(4), 249 - 52
Correlation between BCG genomics and protective efficacy; Behr MA; Between the derivation of bacille Calmette-Guerin (BCG) vaccine in 1921 and the lyophilization of BCG daughter strains in the 1960s, a number of clinical trials were performed looking at the protective efficacy of BCG vaccination against tuberculosis . These trials differed from one another in a number of ways: they employed different methodologies for delivering the vaccine and interpreting outcomes; they were performed on populations with different genetic backgrounds and different levels of exposure to environmental Mycobacteria; and, finally, they used different BCG vaccine strains . The results of these trials were estimates of protective efficacy against pulmonary tuberculosis ranging from about 80% to nil . Because of the differences in outcomes and confounding variables, it is difficult to conclude whether differences in interventions alone may have contributed to the remarkably variable results . Analysis of BCG vaccines used in clinical trials suggests a trend towards decreasing efficacy with increased passage in the laboratory; however, trials that used relatively "older" BCG strains were generally performed at different sites than trials which used "younger" BCG strains . Genomic analysis of BCG vaccines demonstrates that during the half-century of ongoing passage of BCG vaccines in vitro there have been numerous genetic changes, comprising single nucleotide polymorphisms, duplications and deletions . The impact of these changes in the BCG genome on the protective efficacy observed in field trials remains to be determined.

Biochim Biophys Acta, 2001 May 5, 1547(1), 57 - 63
A 28 kDa protein of the Bacillus thuringiensis serovar shandongiensis isolate 89-T-34-22 induces a human leukemic cell-specific cytotoxicity; Lee D et al.; A 28 kDa protein that exhibits cytocidal activity specific for human leukemic T (MOLT-4) cells was purified from proteinase K-digested parasporal inclusion of a Bacillus thuringiensis serovar shandongiensis isolate . The N-terminal sequence of the protein was identical with that of the 32 kDa protein, regarded as a protoxin, of the inclusion proteins . The median effective concentration of this protein was 0.23 microg/ml against MOLT-4 cells and its specific activity was 7.9 times greater than that of the whole inclusion proteins . The 28 kDa protein induced necrosis-like cytotoxicity against MOLT-4 cells and the cytopathic effect with the passage of time was characterized by cell swelling, nuclear membrane isolation and chromatin condensation.

J Urol, 2001 May, 165(5), 1488 - 91
Control group and maintenance treatment with bacillus Calmette-Guerin for carcinoma in situ and/or high grade bladder tumors; Palou J et al.; PURPOSE: Intravesical instillations of bacillus Calmette-Guerin have demonstrated satisfactory results in the treatment of vesical carcinoma in situ and high grade superficial bladder tumors . We designed a protocol to evaluate the decrease in tumor recurrence with maintenance therapy . MATERIALS AND METHODS: Between June 1989 and May 1995 an initial course of 6 intravesical instillations of Connaught strain bacillus Calmette-Guerin was administered in patients with carcinoma in situ and/or high grade superficial bladder tumors . Six months later 131 disease-free patients were randomly assigned to a control group or a maintenance therapy group that received 6 instillations every 6 months (6 x 6) for a 2-year period . RESULTS: Of the 126 evaluable patients at a mean followup of 79 months there were no significant differences in recurrence nor progression . A total of 16 patients (26.2%) in the control and 10 (15.1%) in the maintenance group had superficial relapse at a mean of 24 and 20 months, respectively (p = 0.07) . Eight patients underwent radical cystectomy due to bladder contraction in 1, high grade superficial recurrence in 4 and disease progression in 3 . Of the 65 patients on maintenance therapy 22 (33.85%) completed the planned 2-year treatment . CONCLUSIONS: Six-month maintenance therapy in patients treated initially for carcinoma in situ and/or high grade superficial bladder tumors who are disease-free at 6 months did not significantly decrease recurrence or progression.

Protein Eng, 2001 Mar, 14(3), 161 - 6
Analysis of a conserved hydrophobic pocket important for the thermostability of Bacillus pumilus chloramphenicol acetyltransferase (CAT-86); Chirakkal H et al.; Site-directed mutagenesis was carried out on Bacillus pumilus chloramphenicol acetyltransferase (CAT-86) to determine the effects of substitution at a conserved hydrophobic pocket identified earlier as important for thermostability . Mutations were introduced that would substitute residues at consensus positions 33, 191 and 203 in the enzyme, both individually and in combination . Two mutants, SDM1 (CAT-86 Y33F, A203V) and SDM5 (CAT-86 A203I), were more thermostable than wild-type and two mutants, SDM4 (CAT-86 I191V) and SDM7 (CAT-86 A203G), were less stable . Reconstruction of the residues of this hydrophobic pocket to that of a more thermostable CAT-R387 enzyme pocket (as a Y33F, I191V, A203V triple mutant) increased the thermostability of the enzyme above the wild-type, but its stability was less than that of SDM1 and SDM5 . The K(m) values of the mutant enzymes for chloramphenicol and acetyl-CoA were essentially unaltered (in the ranges 15-30 and 26-35 microM respectively) and the specific activity of purified enzyme was in the range 270-710 units/mg protein . The possible effects of the amino acid substitutions on the CAT-86 structure were determined by homology modelling . A reduction in conformational strain and optimized hydrophobic interactions are predicted to be responsible for the increased thermostability of the SDM1 and SDM5 mutants.

J Immunol, 2001 May 15, 166(10), 6367 - 75
Mycobacterium bovis strain bacillus Calmette-Guérin-induced liver granulomas contain a diverse TCR repertoire, but a monoclonal T cell population is sufficient for protective granuloma formation; Hogan LH et al.; Granuloma formation is a form of delayed-type hypersensitivity requiring CD4(+) T cells . Granulomas control the growth and dissemination of pathogens, preventing host inflammation from harming surrounding tissues . Using a murine model of Mycobacterium bovis strain bacillus Calmette-Guerin (BCG) infection we studied the extent of T cell heterogeneity present in liver granulomas . We demonstrate that the TCR repertoire of granuloma-infiltrating T cells is very diverse even at the single-granuloma level, suggesting that before granuloma closure, a large number of different T cells are recruited to the lesion . At the same time, the TCR repertoire is selected, because AND TCR transgenic T cells (Valpha11/Vbeta3 anti-pigeon cytochrome c) are preferentially excluded from granulomas of BCG-infected AND mice, and cells expressing secondary endemic Vbeta-chains are enriched among AND cells homing to granulomas . Next, we addressed whether TCR heterogeneity is required for effective granuloma formation . We infected 5CC7/recombinase-activating gene 2(-/-) mice with recombinant BCG that express pigeon cytochrome c peptide in a mycobacterial 19-kDa bacterial surface lipoprotein . A CD4(+) T cell with a single specificity in the absence of CD8(+) T cells is sufficient to form granulomas and adequately control bacteria . Our study shows that expanded monoclonal T cell populations can be protective in mycobacterial infection.

J Immunol, 2001 May 15, 166(10), 6276 - 86
ATP-mediated killing of Mycobacterium bovis bacille Calmette-Guérin within human macrophages is calcium dependent and associated with the acidification of mycobacteria-containing phagosomes; Stober CB et al.; We previously demonstrated that extracellular ATP stimulated macrophage death and mycobacterial killing within Mycobacterium bovis Bacille Calmette-Guerin (BCG)-infected human macrophages . ATP increases the cytosolic Ca(2+) concentration in macrophages by mobilizing intracellular Ca(2+) via G protein-coupled P2Y receptors, or promoting the influx of extracellular Ca(2+) via P2X purinoceptors . The relative contribution of these receptors and Ca(2+) sources to ATP-stimulated macrophage death and mycobacterial killing was investigated . We demonstrate that 1) ATP mobilizes Ca(2+) in UTP-desensitized macrophages (in Ca(2+)-free medium) and 2) UTP but not ATP fails to deplete the intracellular Ca(2+) store, suggesting that the pharmacological properties of ATP and UTP differ, and that a Ca(2+)-mobilizing P2Y purinoceptor in addition to the P2Y(2) subtype is expressed on human macrophages . ATP and the Ca(2+) ionophore, ionomycin, promoted macrophage death and BCG killing, but ionomycin-mediated macrophage death was inhibited whereas BCG killing was largely retained in Ca(2+)-free medium . Pretreatment of cells with thapsigargin (which depletes inositol (1,4,5)-trisphosphate-mobilizable intracellular stores) or 1,2-bis-(2-aminophenoxy)ethane-N, N, N',N'-tetraacetic acid acetoxymethyl ester (an intracellular Ca(2+) chelator) failed to inhibit ATP-stimulated macrophage death but blocked mycobacterial killing . Using the acidotropic molecular probe, 3-(2,4-dinitroanilino)-3'-amino-N-methyl dipropylamine, it was revealed that ATP stimulation promoted the acidification of BCG-containing phagosomes within human macrophages, and this effect was similarly dependent upon Ca(2+) mobilization from intracellular stores . We conclude that the cytotoxic and bactericidal effects of ATP can be uncoupled and that BCG killing is not the inevitable consequence of death of the host macrophage.

J Immunol, 2001 May 15, 166(10), 6227 - 35
Vaccination with the T cell antigen Mtb 8.4 protects against challenge with Mycobacterium tuberculosis; Coler RN et al.; The development of an effective vaccine against Mycobacterium tuberculosis is a research area of intense interest . Mounting evidence suggests that protective immunity to M . tuberculosis relies on both MHC class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells . By purifying polypeptides present in the culture filtrate of M . tuberculosis and evaluating these molecules for their ability to stimulate PBMC from purified protein derivative-positive healthy individuals, we previously identified a low-m.w . immunoreactive T cell Ag, Mtb 8.4, which elicited strong Th1 T cell responses in healthy purified protein derivative-positive human PBMC and in mice immunized with recombinant Mtb 8.4 . Herein we report that Mtb 8.4-specific T cells can be detected in mice immunized with the current live attenuated vaccine, Mycobacterium bovis-bacillus Calmette-Guerin as well as in mice infected i.v . with M . tuberculosis . More importantly, immunization of mice with either plasmid DNA encoding Mtb 8.4 or Mtb 8.4 recombinant protein formulated with IFA elicited strong CD4(+) T cell and CD8(+) CTL responses and induced protection on challenge with virulent M . tuberculosis . Thus, these results suggest that Mtb 8.4 is a potential candidate for inclusion in a subunit vaccine against TB.

J Immunol, 2001 May 15, 166(10), 6203 - 11
Culture at high density improves the ability of human macrophages to control mycobacterial growth; Boechat N et al.; The mechanisms through which granuloma formation helps control mycobacterial infection are poorly understood, but it is possible that the accumulation of macrophages at high density at sites of infection promotes the differentiation of macrophages into cells with improved mycobactericidal activity . To test this possibility, varying numbers of monocytes were cultured in 96-well plates for 3 days, infected with Mycobacterium bovis bacillus Calmette-Guerin, and mycobacterial number was assessed 7 days after infection based on the measurement of luciferase activity expressed by a mycobacterial reporter strain or by counting CFU . Mycobacterial growth was optimal in cultures containing 5 x 10(4) cells/well, but increasing the number of cells to 2 x 10(5) cells/well resulted in complete inhibition of mycobacterial growth . This effect could not be explained by differences in mycobacterial uptake, multiplicity of infection, acidification of the extracellular medium in high density cultures, enhanced NO production, or paracrine stimulation resulting from secretion of cytokines or other proteins . The morphology of cells cultured at high density was strikingly different from that of monocytes cultured at 5 x 10(4) cells/well, including the appearance of numerous giant cells . The bacteriostatic activity of monocyte-derived macrophages was also dependent on cell number, but fewer of these more mature cells were required to control mycobacterial growth . Thus, the ability of human macrophages to control mycobacterial infection in vitro is influenced by the density of cells present, findings that may help explain why the formation of granulomas in vivo appears to be a key event in the control of mycobacterial infections.

Biochim Biophys Acta, 2001 Feb 9, 1545(1-2), 114 - 21
Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant; Li QS et al.; The single mutant F87A of cytochrome P-450 BM-3 from Bacillus megaterium was engineered by rational evolution to achieve improved hydroxylation activity for medium chain length substrates (C8-C10) . Rational evolution combines rational design and directed evolution to overcome the drawbacks of these methods when applied individually . Based on the X-ray structure of the enzyme, eight mutation sites (P25, V26, R47, Y51, S72, A74, L188, and M354) were identified by modeling . Sublibraries created by site-specific randomization mutagenesis of each single site were screened using a spectroscopic assay based on omega-p-nitrophenoxycarboxylic acids (pNCA) . The mutants showing activity for shorter chain length substrates were combined, and these combi-libraries were screened again for mutants with even better catalytic properties . Using this approach, a P-450 BM-3 variant with five mutations (V26T, R47F, A74G, L188K, and F87A) that efficiently hydrolyzes 8-pNCA was obtained . The catalytic efficiency of this mutant towards omega-p-nitrophenoxydecanoic acid (10-pNCA) and omega-p-nitrophenoxydodecanoic acid (12-pNCA) is comparable to that of the wild-type P-450 BM-3.

Bioresour Technol, 2001 Jul, 78(3), 267 - 72
Production of a biopolymer flocculant from Bacillus licheniformis and its flocculation properties; Shih IL et al.; Bacillus licheniformis CCRC 12826 produced extracellularly an excellent biopolymer flocculant in a large amount when it was grown aerobically in a culture medium containing citric acid, glutamic acid and glycerol as carbon sources . The biopolymer flocculant was an extremely viscous material with a molecular weight over 2 x 10(6) by gel permeation chromatography . It could be easily purified from the culture medium by ethanol precipitation . It was shown to be a homopolymer of glutamic acid by amino acid analysis and thin layer chromatography and presumed to be poly-glutamic acid (PGA) . This bioflocculant efficiently flocculated various organic and inorganic suspensions . It flocculated a suspended kaolin suspension without cations, although its flocculating activity was synergistically stimulated by the addition of bivalent or trivalent cations Ca2+, Fe3+ and Al3+ . However, the synergistic effects of metal cations were most effective at neutral pH ranges . The comparison of the flocculating activity between the present biopolymer and a commercial lower molecular weight product showed that the biopolymer of the present study had much higher activity . The high productivity and versatile applications of PGA make its development as a new biodegradable, harmless, biopolymer flocculant economical and advantageous.

Clin Experiment Ophthalmol, 2001 Apr, 29(2), 92 - 3
Bacillus circulans endophthalmitis; Tandon A et al.; An 80-year-old woman presented with right endophthalmitis, characterized by chalky white deposits covering her posterior capsule . This occurred 17 months after uncomplicated right cataract surgery . A three-port pars plana vitrectomy and partial posterior capsulectomy isolated Bacillus circulans, and the patient made a rapid and full recovery on topical cephalothin and prednisolone acetate 1% . The case demonstrates that, unlike endophthalmitis due to other Bacillus spp., B . circulans endophthalmitis does not necessarily follow a fulminant course . It is the first report describing a subacute presentation, and response to posterior capsulectomy and simple antibiotic therapy It is also the first description of B . circulans causing white plaques in the posterior capsule, a finding characteristic of chronic endophthalmitis and previously considered pathognomonic of Proprionibacterium acnes endophthalmitis.

Int J Cancer, 2001 Jun 1, 92(5), 697 - 702
NK cells are essential for effective BCG immunotherapy; Brandau S et al.; Adjuvant intravesical bacillus Calmette-Guerin (BCG) therapy is a well-established and successful adjuvant immunotherapy in the treatment of superficial bladder cancer . Although the function of natural killer (NK) cells in other immunotherapeutic regimens (e.g., lymphokine-activated killer {LAK} cell or interleukin-2 {IL-2} therapy) has been established, the contribution of NK cells to effective BCG immunotherapy is not clear . We used a human in vitro system to analyze the role of NK cells in BCG-induced cellular cytotoxicity . After stimulation of mononuclear cells with BCG for 7 days, these BCG-activated killer (BAK) cells displayed substantial cytotoxicity against bladder tumor cells . Magnetic depletion experiments and fluorescence activated cell sorting revealed that NK cells were the major effector cell population . To address NK cell function in vivo, we studied a syngeneic orthotopic murine bladder cancer model and compared BCG immunotherapy in C57BL/6 wild-type mice, NK-deficient beige mice and mice treated with anti-NK1.1 monoclonal antibody . Four weekly instillations of viable BCG significantly prolonged survival in wild-type mice compared with control mice treated with solvent alone . In contrast, BCG therapy was completely ineffective in NK-deficient beige mice and in mice treated with anti-NK1.1 monoclonal antibody . These findings suggest a key role for NK cells during BCG immunotherapy .

Braz J Biol, 2001 Feb, 61(1), 159 - 70
Triceratium moreirae sp . nov . and Triceratium dubium (Triceratiaceae - Bacillariophyta) from estuarine environments of Southern Brazil, with comments on the genus Triceratium C . G . Ehrenberg; Fernandes LF et al.; A new species of Triceratiaceae is described for estuarine environments of Southern Brazil . The valve of Triceratium moreirae sp . nov . is triangular with elevations bearing ocellus and a rimoportula in their basis . The valvar surface has robust pseudoloculi, circular to polygonal, each one confining a group of areolae arranged in a typical pattern . A central larger areola is surrounded by 5-8 smaller areolae, and they are poroidal with domed cribra . A septum emerges from the valvocopula, having the base reinforced by ribs and the margins folded to the innerside of the valve . The new species is compared to the closely related diatom Triceratium dubium, which has the same pattern of areolar disposition, formation of septum and pseudoloculi . The main differences in relation to T . moreirae are: rimoportulae intercalated to ocelli and with a characteristic morphology (spinules on the edge of the external aperture), and more elongated elevations with a constriction . Some criteria used for the classification of categories into the Triceratiaceae are also discussed, and we propose that the type of areola (poroidal with cribra) be included in the circumscription of Triceratium.

BMC Microbiol . 2001;1(1):3 . Epub 2001 Apr 24.
Analysis of stress- and host cell-induced expression of the Mycobacterium tuberculosis inorganic pyrophosphatase; Triccas JA et al.; BACKGROUND: The gene encoding the inorganic pyrophosphatase (PPase) of the intracellular pathogen Legionella pneumophila is induced during intracellular infection, but is constitutively expressed in Escherichia coli . The causative agent of tuberculosis, Mycobacterium tuberculosis, contains a well conserved copy of PPase . We sought to determine if expression of the M . tuberculosis PPase is regulated by the intracellular environment . RESULTS: A strain of Mycobacterium bovis bacille Calmette-Guerin (BCG) was constructed in which the Aequoria victoria green fluorescent protein (GFP) is controlled by the promoter of the M . tuberculosis ppa gene . After prolonged exposure of the recombinant BCG strain within murine bone-marrow-derived macrophages, there was no observed increased activity of the ppa promoter . Furthermore, there was no change in promoter activity after exposure to various stress stimuli such as reduced pH, osmotic shock, nutrient limitation or oxidative stress . CONCLUSIONS: These results suggest that macrophage induction of ppa is not a general phenomenon among intracellular pathogens.

Bioelectrochemistry, 2001 Mar, 53(2), 243 - 9
Spectroelectrochemical study of cellobiose dehydrogenase and diaphorase in a thiol-modified gold capillary in the absence of mediators; Larsson T et al.; A spectroelectrochemical cell was constructed from a gold capillary with 200 microm inner diameter as a working electrode . This allowed spectroelectrochemical study of liquid samples with available volumes less than 5 microl . The optical measurements were accomplished with an optical fibre spectrometer . The optical path of the cell was about 1 cm . To facilitate electrochemistry of biomolecules, the surface of the gold capillary was modified with thiols . The formal potential, E degrees', of the heme in cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium was determined by spectroelectrochemistry in the absence of redox mediators . The number of electrons per redox conversion of heme in CDH was found to be equal to 0.98 + 0.04 corresponding well to a theoretical value representing the redox reaction Fe3+ + e-= Fe2+ . Similar spectroelectrochemical experiments with diaphorase from Bacillus stearothermophilus showed the redox conversion of the flavin mononucleotide in diaphorase in the absence of external redox mediators.

Mikrobiologiia, 2001 Jan-Feb, 70(1), 24 - 8
{Effect of thermal processing of Bacillus intermedius ribonuclease on its catalytic activity and biological properties}; Kolpakov AI et al.; Bacillus intermedius ribonuclease (binase), which is known to exert a growth-stimulating effect at low concentrations and a genotoxic effect at high concentrations, loses these abilities completely after exposure to 100 degrees C for 10 min, but retains approximately 96% of its catalytic activity and structural integrity . Other types of modification, such as photoinactivation and site-specific mutagenesis, gave rise to enzyme forms with unaltered structure but reduced (sometimes to trace amounts) catalytic activity . Genotoxicity was always proportional to the catalytic activity of the native enzyme, while a notable growth-stimulating effect may be exerted by enzymes with low activity . The loss of biological activity of thermoinactivated binase was related to the increase in the number of negatively charged groups on the enzyme surface, which led to a substantial decline in the adhesive properties of the enzyme.

Rev Med Liege, 2001 Mar, 56(3), 181 - 5
{How I investigate...difficult cases of tuberculous meningitis}; Lemaire R et al.; To formally document the presence of the bacillus of Koch in meninges still remains difficult and depends on many variables . We report two cases where the diagnosis proved difficult . The presentation of tuberculous meningitis can take several aspects . Mantoux reaction is frequently negative . The BK cultures, which demonstrate the diagnosis, depend on the volume of CSF and on the importance of BK dissemination . It, unfortunately, remains difficult to make the diagnosis of tuberculous meningitis even though this pathology can have extremely deleterious consequences.

Hereditas, 2000, 133(2), 85 - 93
Population genetics of Chrysomela tremulae: a first step towards management of transgenic Bacillus thuringiensis poplars Populus tremula x .P . tremuloides; Genissel A et al.; Many strategies have been proposed for delaying the development of insect resistance to Bacillus thuringiensis (Bt) . The current paradigm for Bt resistance management is the high dose-refuge strategy . For this strategy to be successful: (i) heterozygotes must be killed in treated areas, (ii) resistant alleles must be rare (frequency < 10-3), and (iii) there must be a high level of gene flow between populations to ensure random mating . We studied gene flow within and between populations with a view to managing the resistance of Chrysomela tremulae (Coleoptera: Chrysomelidae) to new transgenic, highly toxic poplars expressing a synthetic Bt gene . In this study, we assessed the extent of gene flow in C . tremulae within and between 16 sites in France and Belgium, using allozyme markers . We found a high level of genetic variability in C . tremulae, with a mean of 0.206 +/- 0.16 . There were no obvious limitations to gene flow between populations of C . tremulae over large geographical distances (several hundreds of kilometres) . Nevertheless, a very low level of genetic differentiation was observed between a site located in the south of France and the sampled sites from the Centre region.

J Membr Biol, 2001 Apr 1, 180(3), 195 - 203
Lipid-induced pore formation of the Bacillus thuringiensis Cry1Aa insecticidal toxin; Vie V et al.; After activation, Bacillus thuringiensis (Bt) insecticidal toxin forms pores in larval midgut epithelial cell membranes, leading to host death . Although the crystal structure of the soluble form of Cry1Aa has been determined, the conformation of the pores and the mechanism of toxin interaction with and insertion into membranes are still not clear . Here we show that Cry1Aa spontaneously inserts into lipid mono- and bilayer membranes of appropriate compositions . Fourier Transform InfraRed spectroscopy (FTIR) indicates that insertion is accompanied by conformational changes characterized mainly by an unfolding of the beta-sheet domains . Moreover, Atomic Force Microscopy (AFM) imaging strongly suggests that the pores are composed of four subunits surrounding a 1.5 nm diameter central depression.

Comp Biochem Physiol B Biochem Mol Biol, 2001 May, 129(1), 173 - 83
Lipids of brush border membrane vesicles (BBMV) from Plutella xylostella resistant and susceptible to Cry1Ac delta-endotoxin of Bacillus thuringiensis; Kumaraswami NS et al.; Plutella xylostella (PX) that were 130000-fold more resistant to Cry1Ac were selected from the susceptible strain and maintained in the laboratory . The LC50 of the susceptible strain (PXS) was 0.38 microg toxin/g diet, whereas that of the resistant strain (PXR) was 4800 microg toxin/g diet . Brush border membrane vesicles (BBMV) were prepared from both PXS and PXR . In ligand blot analysis, Cry1Ac bound to a 120-kDa protein of BBMV; however, the intensity of the band was almost equal in both strains of insect . Hence, we analyzed the lipid components of BBMV from PXS and PXR . BBMV lipids were fractionated into non-polar lipid, phospholipid, neutral glycolipid and acidic glycolipid . Neutral glycolipid content was substantially lower in the BBMV of PXR than of PXS . The same trend was observed when lipids were extracted from whole midgut instead of BBMV . Thin layer chromatography of midgut neutral glycolipids revealed the presence of more than seven components . Among the midgut neutral glycolipids, a possible hexasaccharylceramide and a possible trisaccharylceramide of PXR were less than half the level found in PXS . The other lipid fractions in PXR and PXS were similar to each other.

Bioresour Technol, 2001 Jun, 78(2), 181 - 5
Kinetics of inhibition in the biodegradation of monoaromatic hydrocarbons in presence of heavy metals; Amor L et al.; The toxicity and inhibitory effects of heavy metals such as cadmium, nickel and zinc on alkylbenzene removal were evaluated with a Bacillus strain . The kinetics of alkylbenzene biodegradation with the different heavy metals at various concentrations were modeled using the Andrews equation which yielded a good fit between model and experimental data . Additional experiments undertaken with a Pseudomonas sp . in presence of nickel confirmed a good fit between experimental data and the Andrews model for this strain as well . The heavy metals inhibition constants (Ki) were calculated for different combinations of volatile organic compounds (VOC) and heavy metals . The present approach provides a method for evaluating and quantifying the inhibition effect of heavy metals on the biodegradtion of pollutants by specific microbial strains.

J Econ Entomol, 2001 Apr, 94(2), 586 - 92
Life history attributes of Indian meal moth (Lepidoptera: Pyralidae) and Angoumois grain moth (Lepidoptera: Gelechiidae) reared on transgenic corn kernels; Sedlacek JD et al.; The Indian meal moth, Plodia interpunctella (Hubner), and Angoumois grain moth, Sitotroga cerealella (Olivier), are two globally distributed stored-grain pests . Laboratory experiments were conducted to examine the impact that corn (Zea mays L.) kernels (i.e., grain) of some Bacillus thuringiensis Berliner (Bt) corn hybrids containing CrylAb Bt delta-endotoxin have on life history attributes of Indian meal moth and Angoumois grain moth . Stored grain is at risk to damage from Indian meal moth and Angoumois grain moth; therefore, Bt corn may provide a means of protecting this commodity from damage . Thus, the objective of this research was to quantify the effects of transgenic corn seed containing CrylAb delta-endotoxin on Indian meal moth and Angoumois grain moth survival, fecundity, and duration of development . Experiments with Bt grain, non-Bt isolines, and non-Bt grain were conducted in environmental chambers at 27 +/- 1 degrees C and > or = 60% RH in continuous dark . Fifty eggs were placed in ventilated pint jars containing 170 g of cracked or whole corn for the Indian meal moth and Angoumois grain moth, respectively . Emergence and fecundity were observed for 5 wk . Emergence and fecundity of Indian meal moth and emergence of Angoumois grain moth were significantly lower for individuals reared on P33V08 and N6800Bt, MON 810 and Bt-11 transformed hybrids, respectively, than on their non-Bt transformed isolines . Longer developmental times were observed for Indian meal moth reared on P33V08 and N6800Bt than their non-Bt-transformed isolines . These results indicate that MON 810 and Bt-11 CrylAb delta-endotoxin-containing kernels reduce laboratory populations of Indian meal moth and Angoumois grain moth . Thus, storing Bt-transformed grain is a management tactic that warrants bin scale testing and may effectively reduce Indian meal moth and Angoumois grain moth populations in grain without application of synthetic chemicals or pesticides.

J Econ Entomol, 2001 Apr, 94(2), 403 - 9
Treatment thresholds for stink bugs (Hemiptera: Pentatomidae) in cotton; Greene JK et al.; The green stink bug, Acrosternum hilare (Say), the southern green stink bug, Nezara viridula (L), and the brown stink bug, Euschistus servus (Say), were predominant phytophagous Pentatomidae detected during 1995-1997 in cotton in South Carolina . These species occurred in similar numbers in conventional and transgenic cotton 'NuCOTN33B', containing the gene for expression of CryIA(c) delta-endotoxin of Bacillus thuringiensis Berliner variety kurstaki . Adult stink bugs moved into cotton from wild and cultivated alternate hosts during July, and reproducing populations usually were detected in cotton from late July into September . Applications of either methyl parathion (0.56 kg {AI}/ha) directed for stink bugs or lambda-cyhalothrin (0.037 kg {AI}/ha) or cyfluthrin (0.056 kg {AI}/ha) for control of cotton bollworm, Helicoverpa zea (Boddie), provided effective control of pentatomids in NuCOTN33B or conventional 'DP5415' and increased yields compared with untreated plots . Fiber quality did not differ among treated or untreated plots of NuCOTN33B . The ground-cloth technique was used to estimate populations of stink bugs, and data indicated that treatment at one bug per 2 m of row adequately protected cotton from yield loss due to stink bug damage . Observations on boll damage indicated that treatment might be necessary if >20-25% reveal internal symptoms of feeding injury during mid- to late season . More detailed damage thresholds should be developed to complement an approach based on population monitoring . This study validated current recommendations for management of pentatomids in cotton, demonstrated the necessity of threshold use for stink bugs in transgenic cultivars expressing endotoxin from B . thuringiensis, and provided insight into further development of management options for pentatomids in the crop.

J Econ Entomol, 2001 Apr, 94(2), 397 - 402
Establishment of Cry9C susceptibility baselines for European corn borer and southwestern corn borer (Lepidoptera: Crambidae); Reed JP et al.; In 1997 and 1998, Cry9C susceptibility baselines were established for field-collected populations of European corn borer, Osrinia nubilalis (Hubner), and southwestern corn borer, Diatraea grandiosella Dyar . Bioassay of neonate European corn borer larvae of 16 colonies collected from the midwestern United States indicated LC50 values ranging from 13.2 to 65.1 ng of Cry9C protein per square centimeter . Neonate European corn borer LC50 values ranged from 46.5 to 214 ng/cm2 . Neonate larvae of three colonies of southwestern corn borer collected from the southern and southwestern United States exhibited LC50 values from 16.9 to 39.9 ng of Cry9C protein per square centimeter . Southwestern corn borer neonate LC90 confidence limit values ranged from 40.3 to 157 ng of Cry9C protein per centimeter . The most sensitive southwestern corn borer colony was collected from the Mississippi delta exhibiting an LC50 value of 22.6 ng of Cry9C per cm2 and also displayed the widest LC0 confidence limits of 40.3-94.8 ng of Cry9C per cm2 . Geographic baseline susceptibility data establishes the natural genetic variation and provides the foundation for future testing of insect populations exposed to increased use of Bacillus thuringiensis-based crops . Insect resistance management and stewardship of Cry9C will rely upon baseline data for the validation of discriminating dose assays for European corn borer and southwestern corn borer.

J Econ Entomol, 2001 Apr, 94(2), 315 - 25
Large-scale management of insect resistance to transgenic cotton in Arizona: can transgenic insecticidal crops be sustained?
Carriere Y, Dennehy TJ, Pedersen B, Haller S, Ellers-Kirk C, Antilla L, Liu YB, Willott E, Tabashnik BE.
A major challenge for agriculture is management of insect resistance to toxins from Bacillus thuringiensis (Bt) produced by transgenic crops . Here we describe how a large-scale program is being developed in Arizona for management of resistance to Bt cotton in the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), and other insect pests of cotton . Financial support from growers makes this program possible . Collaboration between the Arizona Cotton Research and Protection Council, the University of Arizona, and government agencies has led to development of resistance management guidelines, a remedial action plan, and tools for monitoring compliance with the proposed guidelines . Direct participation in development of resistance management policies is a strong incentive for growers to invest in resistance management research . However, more research, regularly updated regulations, and increased collaboration between stakeholders are urgently needed to maintain efficacy of Bt toxins in transgenic crops.

Int J Lepr Other Mycobact Dis, 2000 Dec, 68(4), 434 - 43
Antileprosy protective vaccination of sooty mangabey monkeys with BCG or BCG plus heat-killed Mycobacterium leprae: immunologic observations; Gormus BJ et al.; Groups of sooty mangabey monkeys (SMM) were vaccinated and boosted with Mycobacterium bovis bacillus Calmette-Guerin (BCG), or BCG + low-dose (LD) or high-dose (HD) heat-killed M . leprae (HKML), or were unvaccinated . Prior to and following vaccination-boosting and subsequent M . leprae (ML) challenge, these and unvaccinated, unchallenged control monkeys were immunologically observed longitudinally for approximately 3 years . SMM {multibacillary (MB) leprosy-prone as a species} were not protected clinically by BCG or BCG + HKML, although the disease progress was slowed by vaccination with BCG alone . The longitudinal immune response profiles to BCG or BCG + HKML in SMM showed that: 1) vaccination with BCG or BCG + HKML initially stimulated significant in vitro blood mononuclear cell blastogenic responses to ML antigens, which returned to baseline post-boosting and post-live ML challenge; 2) BCG + LD HKML-vaccinated groups gave the largest blastongenic response (SI = 23) followed by the BCG + HD HKML group (SI = 14.5) and by the BCG-only vaccinated group (SI = 3.6); 3) significantly diminished numbers of blood CD4+ (helper) and CD4+CD29+ (helper-inducer) T-cell subsets were observed longitudinally in all ML-challenged groups compared to controls regardless of whether they had been vaccinated or not; 4) CD8+ (suppressor) T-cell numbers remained longitudinally constant, on average, in all ML-challenged groups (vaccinated or not) compared to controls; 5) there was a significant decrease in the CD4+:CD8+ ratio over time in all ML-challenged groups (vaccinated or not); 6) vaccination with BCG or BCG + LD or HD HKML resulted in significantly increased numbers of CD4+CD45RA+ (suppressor-inducer) T cells longitudinally compared to the unvaccinated, ML-challenged control group; and 7) over time, vaccination with BCG + HKML followed by live ML-challenge produced higher IGM:IgG antiphenolic glycolipid-I (PGL-I) serum antibody response ratios than BCG-only vaccinated, ML-challenged monkeys or unvaccinated, ML-challenged SMM, consistent with prior observations that IgG anti-PGL-I responses correlate with resistance to and protection from clinical leprosy and IgM anti-PGL-I responses correlate with increased susceptibility.

Acta Med Okayama, 2001 Apr, 55(2), 105 - 15
Protection of mice from LPS-induced shock by CD14 antisense oligonucleotide; Furusako S et al.; CD14 is a pattern recognition receptor on myeloid cells and plays a pivotal role in an innate immune system that is responsible for Gram-negative and Gram-positive bacteria infection . Lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria, can induce production of a large quantity of proinflammatory cytokines into the circulation mediated by CD14-mediated macrophages and monocytes . These cytokines eventually cause septic shock . Several in vitro and in vivo studies have shown that suppression of a CD14 function by a CD14 antibody led to an inhibition of the production of proinflammatory cytokines such as TNF-alpha, IL-1 beta, and IL-8 . In the present study, we found that CD14 antisense oligonucleotide (ODN) can prevent lethal LPS shock in D-galactosamine-sensitized mice . This ODN inhibited CD14 expression in a mouse macrophage cell line, RAW264.7, and suppressed production of TNF-alpha in LPS-stimulated RAW264.7 cells . Furthermore, we designed a consensus antisense ODN that could hybridize human and mouse CD14 RNA, and we evaluated its efficacy . The consensus antisense ODN rescued mice primed with Mycobacterium bovis bacillus Calmette-Guerin (BCG) from the LPS-induced lethal shock . In this model, the CD14 antisense ODN down-regulated LPS-elicited CD14 expression in the liver, resulting in a decrease in LPS-induced TNF-alpha production . These findings suggest that the CD14 antisense ODN is distributed in the liver and efficiently suppresses LPS-induced TNF-alpha production by reducing CD14 expression on Kupffer cells . This CD14 antisense ODN may be useful for the development of a therapeutic agent against sepsis and septic shock.

Photochem Photobiol, 2001 Apr, 73(4), 403 - 9
Interaction between photodynamic therapy and BCG immunotherapy responsible for the reduced recurrence of treated mouse tumors; Korbelik M et al.; Subcutaneous mouse EMT6 tumors were treated by individual or combined regimens of a single Bacillus Calmette-Guerin (BCG) vaccine administration and photodynamic therapy (PDT) . Six clinically relevant photosensitizers characterized by different action mechanisms were used: Photofrin, benzoporphyrin derivative, tetra(m-hydroxyphenyl)chlorin (foscan), mono-L-aspartylchlorin e6, lutetium texaphyrin or zinc phthalocyanine . Irrespective of the type of photosensitizer used, the optimized BCG protocols improved the cure rate of PDT-treated tumors . This indicates that the interaction does not take place during the early phase of tumor ablation but at later events involved in preventing tumor recurrence . Beneficial effects on tumor cure were observed even when the BCG injection was delayed to 7 days after PDT . The accumulation of activated myeloid cells that markedly increases in tumors treated by Photofrin-based PDT was not additionally affected by BCG treatment . However, the incidence of immune memory T cells in tumor-draining lymph nodes that almost doubled at 6 days after Photofrin-PDT further increased close to three-fold with adjuvant BCG . This suggests that BCG immunotherapy amplifies the T-lymphocyte-mediated immune response against PDT-treated tumors . Since both these modalities are established for the treatment of superficial bladder carcinomas, use of their combination for this condition should be clinically tested.

Biochemistry, 2001 May 8, 40(18), 5329 - 37
Kinetic analysis of three activated phenylalanyl intermediates generated by the initiation module PheATE of gramicidin S synthetase; Luo L et al.; The three-domain initiation module PheATE (GrsA) of Bacillus brevis gramicidin S synthetase catalyzes the activation, thiolation and epimerization of L-phenylalanine (L-Phe), the first amino acid incorporated into the decapeptide antibiotic gramicidin S . There are three activated intermediates in the PheATE catalyzed chemical pathway: L-phenylalanyl-adenosine-5'-monophosphate diester (L-Phe-AMP), L-Phe-S-4'-phosphopantetheine(Ppant)- and D-Phe-S-4'-Ppant-acyl enzyme . In this study, we examined PheATE in single-turnover catalysis using rapid chemical quench techniques . Kinetic modeling of the process of disappearance of the substrate L-Phe, transient appearance and disappearance of L-Phe-AMP and the ad seriatim formation and equilibration of the L- and D-Phe-S-Ppant-acyl enzyme adducts allowed evaluation of the microscopic rate constants for the three chemical reactions in the initiation module PheATE . This study provides the first transient-state kinetic analysis of a nonribosomal peptide synthetase (NRPS) module.

Biosci Biotechnol Biochem, 2001 Mar, 65(3), 698 - 701
Thermally induced changes of lipoate acetyltransferase inner core isolated from the Bacillus stearothermophilus pyruvate dehydrogenase complex; Aso Y et al.; Incubation at 70 degrees C converted the Bacillus stearothermophilus lipoate acetyltransferase inner core into an unidentified active molecular form, X, yielding an inactive aggregate . The core and X showed similar thermostabilities, but they were different in the recovery of enzyme activity after incubation with 1.2-2.0 M guanidine hydrochloride and its subsequent removal; the core was hardly recovered, but X was well recovered.

Cell Biochem Biophys, 2000, 32 Spring, 313 - 6
The human L-pipecolic acid oxidase is similar to bacterial monomeric sarcosine oxidases rather than D-amino acid oxidases; Dodt G et al.; L-Pipecolic acid oxidase activity is deficient in patients with peroxisome biogenesis disorders (PBDs) . Because its role, if any, in these disorders is unknown, the authors cloned the human gene to order to further study its functions . BLAST search of the translated sequence showed greatest homology to Bacillus sp . NS-129 monomeric sarcosine oxidase . The purified enzyme could use either L-pipecolic acid or sarcosine as a substrate . No homology was found to the peroxisomal D-amino acid oxidases . A further comparison of L-pipecolic acid oxidase to the two D-amino acid oxidases in peroxisomes showed that the proteins differed in many ways . First, both D-amino acid oxidase and L-pipecolic acid oxidase showed no enzyme activity in liver from Zellweger syndrome patients; D-aspartate oxidase activity was unchanged from control levels . Although all were targeted to peroxisomes, their targeting signals differed . No L-pipecolic acid oxidase was found in brain or other tissues outside of liver and kidney . The D-amino acid oxidases were similarly and more widely distributed . Finally, although D-amino acid degradation is limited to peroxisomes in mammals, L-pipecolic acid can be oxidized in either mitochondria or peroxisomes, or both.

Clin Diagn Lab Immunol, 2001 May, 8(3), 571 - 8
Use of synthetic peptides derived from the antigens ESAT-6 and CFP-10 for differential diagnosis of bovine tuberculosis in cattle; Vordermeier HM et al.; In Great Britain an independent scientific review for the government has concluded that the development of a cattle vaccine against Mycobacterium bovis infection holds the best long-term prospect for tuberculosis control in British herds . A precondition for vaccination is the development of a complementary diagnostic test to differentiate between vaccinated animals and those infected with M . bovis so that testing and slaughter-based control strategies can continue alongside vaccination . To date bacillus Calmette-Guerin (BCG), an attenuated strain of M . bovis, is the only available vaccine for the prevention of tuberculosis . However, tests based on tuberculin purified protein derivative cannot distinguish between M . bovis infection and BCG vaccination . Therefore, specific antigens expressed by M . bovis but absent from BCG constitute prime candidates for differential diagnostic reagents . Recently, two such antigens, ESAT-6 and CFP-10, have been reported to be promising candidates as diagnostic reagents for the detection of M . bovis infection in cattle . Here we report the identification of promiscuous peptides of CFP-10 that were recognized by M . bovis-infected cattle . Five of these peptides were formulated into a peptide cocktail together with five peptides derived from ESAT-6 . Using this peptide cocktail in T-cell assays, M . bovis-infected animals were detected, while BCG-vaccinated or Mycobacterium avium-sensitized animals did not respond . The sensitivity of the peptide cocktail as an antigen in a whole-blood gamma interferon assay was determined using naturally infected field reactor cattle, and the specificity was determined using blood from BCG-vaccinated and noninfected, nonvaccinated animals . The sensitivity of the assay in cattle with confirmed tuberculosis was found to be 77.9%, with a specificity of 100% in BCG-vaccinated or nonvaccinated animals . This compares favorably with the specificity of tuberculin when tested in noninfected or vaccinated animals . In summary, our results demonstrate that this peptide cocktail can discriminate between M . bovis infection and BCG vaccination with a high degree of sensitivity and specificity.

Lett Appl Microbiol, 2001 May, 32(5), 362 - 7
Association of PCR and feeding bioassays as a large-scale method to screen tropical Bacillus thuringiensis isolates for a cry constitution with higher insecticidal effect against Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae; Loguercio LL et al.; AIMS: To verify whether the presence of any of the cry1C, 1D, 1E and 1F genes could be associated with high toxicity against fall armyworm . METHODS AND RESULTS: A sample of 60 strains from a large collection of tropical Bacillus thuringiensis (B.t.) isolates was subjected to feeding bioassays and gene-specific PCR . Positive amplification of cry-specific fragments, so confirmed by sequencing, revealed that cry1C was ubiquitous and distributed among high and low mortality classes, cry1D was underrepresented and showed no clear association to high toxicity, and cry1F was not detected . The presence of cry1E significantly correlated to high levels of insecticidal activity, as estimated by linear regression analysis . CONCLUSION: The PCR amplification of cry1E-specific fragments alone appears to be sufficient to identify B.t . strains with high mortality levels against tropical armyworm . SIGNIFICANCE AND IMPACT OF THE STUDY: The approach presented is promising as a simple and efficient method for first-tier, marker-assisted screening of environment-specific B.t . germplasm effective in controlling a single target pest.

Immunology, 2001 Apr, 102(4), 466 - 79
Immune responses in tuberculosis: antibodies and CD4-CD8 lymphocytes with vascular adhesion molecules and cytokines (chemokines) cause a rapid antigen-specific cell infiltration at sites of bacillus Calmette-Guérin reinfection; Shigenaga T et al.; Rabbit primary dermal bacillus Calmette-Guerin (BCG) lesions were compared with reinfection BCG lesions in order to gain insight into how immune responses protect against clinical tuberculosis . As early as 3 hr, a marked infiltration of macrophages and lymphocytes occurred in the reinfection group, while very little cell infiltration occurred in the primary group . It seems that only an antigen-antibody reaction could produce such an immediate pronounced antigen-specific chemotactic effect, because very few lymphocytes are normally present in the skin . Therefore, antibodies hasten the accumulation of an expanded antigen-specific T-lymphocyte population (memory cells) at sites of bacillary lodgement . By 1-2 days, the primary and reinfection BCG lesions differed 400- to 500-fold in size . By 4-5 days, the size of the reinfection lesions had declined, while the size of the primary lesions had increased, so that, grossly, both types of lesion were similar . At 8 days in reinfection lesions and at 12 days in primary lesions, small secondary peaks in size occurred, which were probably caused by cell-mediated immune responses . In rabbits with primary BCG lesions, skin tests with Old Tuberculin were positive at 9 days, accompanied by a rise in the levels of antibodies to the secreted antigen, phosphate-specific transport protein 1, but the levels of antibodies to the constitutive antigens, purified protein derivative and heat-shock protein 65, did not increase appreciably until some time after 23 days . In tissue sections of reinfection BCG lesions, the percentage of mononuclear cells labelled, by in situ hybridization techniques, for the mRNA of monocyte chemoattractant protein 1 (MCP-1), a chemokine, peaked at 3 hr and then was down-regulated, whereas in primary lesions, this percentage was down-regulated only after 2 days . {The percentage in the tissue sections for the mRNAs of interleukins 1beta and 8, as well as the proteins of MCP-1 and tumor necrosis factor alpha (TNF-alpha), followed a somewhat similar time-course to that of MCP-1 mRNA.} A high percentage of mononuclear cells containing the MCP-1 mRNA 'factory' would favour enlargement of the lesions and a low percentage would favour their regression . At 5 days, the percentage of CD4 and CD8 lymphocytes, stained by immunohistochemical techniques, and the amount of microvasculature stained similarly for vascular cell adhesion molecule 1 were higher in the reinfection group, indicating that prior immunization caused a more rapid (antigen-dependent) up-regulation of these factors . Tuberculin reactions resembled early reinfection BCG lesions in almost every factor evaluated herein . In brief, the production of chemokines began soon after BCG reinfection, peaked within a few hours and was markedly down-regulated by 24 hr, a time at which the lesions of reinfection were of maximal size . Therefore, the amount of cell infiltration was tightly controlled, probably by the variety of mechanisms listed herein.

Immunology, 2001 Apr, 102(4), 441 - 9
Analysis of recombinant mycobacteria as T helper type 1 vaccines in an allergy challenge model; Janssen R et al.; The potential for development of mycobacteria as T helper type 1 (Th1) vaccines capable of induction of Th1 responses to recombinant antigens was explored in a model system based on an immunodominant peptide from house dust mite . Different recombinant mycobacterial preparations were compared for their ability to induce a Th1 response to the peptidea . It was found that mycobacterial viability was not a prerequisite for Th1 immunogenicity . A dominant interferon-gamma (IFN-gamma) response to peptide was observed in splenocytes from C57BL/6J mice immunized with live or heat-killed preparations of recombinant Mycobacterium vaccae or with live attenuated bacillus Calmette-Guerin (BCG) vaccine expressing the antigen . Interleukin-5 (IL-5), a marker of a Th2 response, was detected only in mice receiving live M . vaccae . A similar pattern was observed in BALB/b mice, although the magnitude of the IFN-gamma response was much lower . Control and immunized mice were subsequently exposed to allergen using a Th2-inducing challenge protocol . A significant shift from a Th2 to a Th1 response was observed in immunized mice, as judged by cytokine expression by splenocytes and by subclass of circulating antibody . The effect was seen in three inbred mouse strains differing in their innate bias towards Th1 or Th2 responses . It was dependent on the presence of specific antigen in the mycobacterial preparation and, under the immunization conditions tested, was more pronounced with dead M . vaccae than with live BCG as carrier vaccine . The results demonstrate the potency of killed mycobacteria as Th1 adjuvants and suggest a potential application for recombinant mycobacteria in antigen-specific immune modulation.

Biochemistry, 2001 Feb 27, 40(8), 2540 - 7
Role of alpha-helix seven of Bacillus thuringiensis Cry1Ab delta-endotoxin in membrane insertion, structural stability, and ion channel activity; Alcantara EP et al.; Domain I of the Cry1Ab insecticidal toxic protein has seven alpha-helices and is considered to be involved in the ion channel activity . While other alpha-helices, particularly alpha-4 and alpha-5, have been extensively explored, the remaining alpha-helices have been slightly studied . Site-directed mutagenesis was used to generate mutations throughout sequences encoding the alpha-helix 7 to test its role in ion channel function . Every amino acid residue in alpha-helix 7 was mutated to alanine . Most resultant proteins, e.g., D225A, W226A, Y229A, N230A, R233A, R234A, D242A, and F247A yielded no protoxin or were sensitive to degradation by trypsin or Manduca sexta midgut juice . Other mutant proteins, R224A, R228A, and E235A, were resistant to degradation to the above proteases but were 8, 30, and 12 times less toxic to M . sexta, respectively, than the wild-type Cry1Ab . Circular dichroism spectroscopy indicated a very small change in the R228A spectrum, while R224A and E235A display the same spectrum as the wild-type protein . These three mutant proteins showed little differences from Cry1Ab when analyzed by saturation binding and competition binding kinetics with (125)I-labeled toxin or by surface plasmon resonance to M . sexta brush border membrane vesicles . More conservative amino acid substitutions were introduced into alpha-helix 7 residues: R228K, F232Y, E235Q, and F247Y . In comparison with wild-type Cry1Ab, mutant proteins R228K, F232Y, E235A, and E235Q selectively discriminate between K+ and Rb+, while R224A and R228A had reduced inhibition of short-circuit current for both ions, when analyzed by voltage clamping of M . sexta midguts.

Biochemistry, 2001 Feb 13, 40(6), 1640 - 50
Familial mutations and zinc stoichiometry determine the rate-limiting step of nitrocefin hydrolysis by metallo-beta-lactamase from Bacteroides fragilis; Fast W et al.; The diverse members of the metallo-beta-lactamase family are a growing clinical threat evolving under considerable selective pressure . The enzyme from Bacillus cereus differs from the Bacteroides fragilis enzyme in sequence, zinc stoichiometry, and mechanism . To chart the evolution of the more reactive B . fragilis enzyme, we have made changes in an active site cysteine residue as well as in zinc content to mimic that which occurs in the B . cereus enzyme . Specifically, by introducing a C104R mutation into the B . fragilis enzyme, binding of two zinc ions is maintained, but the k(cat) value for nitrocefin hydrolysis is decreased from 226 to 14 s(-)(1) . Removal of 1 equiv of zinc from this mutant further decreases k(cat) to 4.4 s(-)(1) . In both cases, the observed k(cat) closely approximates that found in the di- and monozinc forms of the B . cereus enzyme (12 and 6 s(-)(1), respectively) . Pre-steady-state stopped-flow studies using nitrocefin as a substrate indicate that these enzyme forms share a similar mechanism featuring an anionic intermediate but that the rate-limiting step changes from protonation of that species to the C-N bond cleavage leading to the intermediate . Overall, features that contribute 3.7 kcal/mol toward the acceleration of the C-N bond cleavage step have been uncovered although some of the total acceleration is masked in the steady-state by a change in rate-limiting step . These experiments illustrate one step in the evolution of a catalytic mechanism and, in a larger perspective, one step in the evolution of antibiotic resistance mechanisms.

Biochem Biophys Res Commun, 2001 May 4, 283(2), 347 - 50
Pressure denaturation of phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus; Roitel O et al.; The effects of hydrostatic pressure on apo wild-type glyceraldehyde-3-phosphate dehydrogenase (wtGAPDH) from Bacillus stearothermophilus (B . stearothermophilus) have been studied by fluorescence spectroscopy under pressure from 0.1 to 650 MPa . Unlike yeast GAPDH {Ruan, K . C., and Weber, G . (1989) Biochemistry 28, 2144-2153}, denaturation of the tetrameric apo wtGAPDH from B . stearothermophilus is likely to precede dissociation into subunits . As expected, denaturation is accompanied by the loss of enzymatic activity . B . stearothermophilus apo wtGAPDH interfaces are less pressure sensitive than apo yeast GAPDH ones, while NAD does not protect B . stearothermophilus wtGAPDH against denaturation by pressure . The pressure effects on B . stearothermophilus GAPDH whose R and Q-axis interfaces were destabilized by disruption of interfacial hydrogen bonds are similar to that of apo wtGAPDH.

Zhonghua Jie He He Hu Xi Za Zhi, 1998 Jul, 21(7), 392 - 4
{Polymerase chain reaction technique in monitoring treatment of bacillary pulmonary tuberculosis}; An S et al.; OBJECTIVE: To explore the relationship between sputum Mycobacterium tuberculosis as well as its DNA negative conversion and relapse in bacillary pulmonary tuberculosis during chemotherapy and 2 years after completion of treatment, and to evaluate the value of polymerase chain reaction (PCR) technique in monitoring the efficacy of treatment . METHOD: Eighty-seven patients with bacillary pulmonary tuberculosis were monthly examined by PCR technique, smear and culture methods, and were followed up for 2 years after treatment . RESULT: The duration of sputum negative conversion by PCR technique was 1-3 months later than that by smear and culture methods . The more the sputum bacteria, the longer the duration of PCR positive results . Positive PCR results maintained in 10 out of 87 patients for more than 1 year, among them 3 (30%) relapsed respectively at 8, 12 and 16 months after treatment . One PCR negative conversion case relapsed at 18 months after treatment . These 4 cases who regained sputum positive results and showed deterioration in chest X-ray films were admitted to hospital again . CONCLUSION: PCR technique is more practical than smear and culture methods in monitoring efficacy of the treatment of bacillary pulmonary tuberculosis, and is useful for evaluating cases with possible relapse.

Zhonghua Jie He He Hu Xi Za Zhi, 1998 Jul, 21(7), 388 - 91
{Controlled clinical trial on efficacy of 5-month regimens and whole course intermittent 6-month regimens in treating bacillary pulmonary tuberculosis}; Chu N et al.; OBJECTIVE: To assess the therapeutic efficacy of rifapentine (L), to reduce the duration of treatment and the frequency of drug administration, and to observe the influence on efficacy and adverse reactions of using pyrazinamide (Z) through whole-course . METHOD: Two 5-month regimens respectively including rifampin (R) and L and two whole course intermittent regimens were designed as following: I: 2SHRZ/3R2H2Z2; II: 2SHRZ/3L1H2Z2; III: 2S3H3R3Z3/4L1H2Z2; IV: 2S3H3R3Z3/4L1H2E2 . A total of 366 newly-diagnosed bacillary pulmonary tuberculosis patients were admitted and randomly allocated . RESULTS: 339 cases completed the prescribed short course chemotherapy . The sputum conversion rates at the end of the treatment of groups I, II, III and IV were 97.0%, 94.1%, 100.0% and 97.2% respectively . X-ray resolution rates were 96.0%, 97.6%, 100.0% and 94.4% respectively . Cavity-close rates of the 5-month regimens and the 6-month regimens were 77% and 76% . Comparing the results among groups, there were no statistically significant differences (P > 0.05), and no obvious side-effect was found . 305 patients have been followed up for 3 years since completion of the chemotherapy . The bacteriological relapse and bacteriological relapse with deterioration on chest X-ray in groups I, II, III and IV were seen in 2,3,6 and 3 cases respectively . CONCLUSION: Domestic-made rifapentine is a long-acting, highly effective antituberculosis drug . It is unnecessary to use Z in continuation phase, and it is possible to shorten the duration to 5 months with the appropriate combination of essential drugs, which is worthwhile for further study.

Int J Tuberc Lung Dis, 2001 Mar, 5(3), 240 - 9
Survey for tuberculosis in a tribal population in North Arcot District; Datta M et al.; SETTING: Tribal villages in the jungles of the Jawadhu hills, South India . OBJECTIVE: To estimate the prevalence of tuberculosis (TB) infection and disease in a remote tribal population . DESIGN: A cross-sectional survey with two-stage screening for identification of cases . A stratified probability proportional sample with the hamlet as the unit . METHODS: Among 56 revenue divisions with a population of about 66,000, 24 revenue divisions were selected . Among 26,320 persons registered, children < 10 years were tuberculin tested and reactions were read after 72 hours . Those over 15 were X-rayed, and tuberculosis symptoms were investigated . Sputum was collected from those with abnormal X-ray or symptoms and examined for smear and culture positivity and sensitivity . RESULTS: Of the 6952 children tested and read, 5% had BCG scars and the prevalence of infection was 5% . The annual risk of infection was 1.1 . Among adults, the prevalence of bacillary cases was 8/1000 and X-ray cases 29/1000 . The prevalence of bacillary disease was higher among males, particularly with increasing age . Thirty symptomatic cases had normal X-rays and 63 X-ray cases had no symptoms . Thus prevalence would have been underestimated if either method had been used alone for screening . Isoniazid resistance was seen in 12% of patients, two of whom also had rifampicin resistance (2.6%) . CONCLUSIONS: The prevalence and pattern of tuberculosis in this tribal group is similar to that observed in non-tribal areas.

J Clin Microbiol, 2001 May, 39(5), 2033 - 4
Mycobacterium triplex infection in a liver transplant patient; Hoff E et al.; Mycobacterium triplex was first named in 1996 as an acid-fast bacillus with features that most resemble Mycobacterium simiae and Mycobacterium avium-intracellulare complex but which possesses a distinct mycolic acid pattern as well as a distinctive 16S rRNA hypervariable region . It has been isolated from lymph node, sputum, and cerebrospinal fluid specimens, but to date only rare clinical cases of this organism have been reported in the literature . The following is a case report of M . triplex that was isolated from the pericardial and peritoneal fluid of a 13-year-old female liver transplant patient.

J Clin Microbiol, 2001 May, 39(5), 2028 - 9
Capnocytophaga cynodegmi cellulitis, bacteremia, and pneumonitis in a diabetic man; Sarma PS et al.; Capnocytophaga cynodegmi (formerly "DF-2 like organism"), a commensal organism of the canine oral cavity, is a capnophilic, gram-negative, facultative bacillus . C . cynodegmi has rarely been encountered in human diseases . We report the first known case of cellulitis, bacteremia, and pneumonitis caused by C . cynodegmi in a diabetic man from central India following a dog bite.

FEBS Lett, 2001 Apr 20, 495(1-2), 115 - 9
Glutamic acid 160 is the acid-base catalyst of beta-xylosidase from Bacillus stearothermophilus T-6: a family 39 glycoside hydrolase; Bravman T et al.; A beta-xylosidase from Bacillus stearothermophilus T-6 was cloned, overexpressed in Escherichia coli and purified to homogeneity . Based on sequence alignment, the enzyme belongs to family 39 glycoside hydrolases, which itself forms part of the wider GH-A clan . The conserved Glu160 was proposed as the acid-base catalyst . An E160A mutant was constructed and subjected to steady state and pre-steady state kinetic analysis together with azide rescue and pH activity profiles . The observed results support the assignment of Glu160 as the acid-base catalytic residue.

FEBS Lett, 2001 Apr 20, 495(1-2), 39 - 43
Stereochemistry of family 52 glycosyl hydrolases: a beta-xylosidase from Bacillus stearothermophilus T-6 is a retaining enzyme; Bravman T et al.; A beta-xylosidase from Bacillus stearothermophilus T-6 assigned to the uncharacterized glycosyl hydrolase family 52 was cloned, overexpressed in Escherichia coli and purified . The enzyme showed maximum activity at 65 degrees C and pH 5.6-6.3 . The stereochemistry of the hydrolysis of p-nitrophenyl beta-D-xylopyranoside was followed by 1H-nuclear magnetic resonance . Time dependent spectrum analysis showed that the configuration of the anomeric carbon was retained, indicating that a retaining mechanism prevails in family 52 glycosyl hydrolases . Sequence alignment and site-directed mutagenesis enabled the identification of functionally important amino acid residues of which Glu337 and Glu413 are likely to be the two key catalytic residues involved in enzyme catalysis.

Eur J Biochem, 2001 May, 268(9), 2751 - 60
Identification and molecular structural prediction analysis of a toxicity determinant in the Bacillus sphaericus crystal larvicidal toxin; Yuan Z et al.; The operon containing the genes encoding the subunits of the binary crystal toxin of Bacillus sphaericus strain LP1-G, BinA and BinB (41.9 kDa and 51.4 kDa, respectively), was cloned and sequenced . Purified crystals were not toxic to Culex pipiens larvae . Comparison of the amino-acid sequences of this strain (Bin4) with those of the three other known toxin types (Bin1, Bin2 and Bin3) revealed mutations at six positions, including a serine at position 93 of BinA4, whereas all other types of BinA toxin from B . sphaericus had a leucine at this position . Reciprocal site-directed mutagenesis was performed to replace this serine in BinA4 from LP1-G with a leucine and the leucine in the BinA2 protein from strain 1593 with a serine . Native and mutated genes were cloned and overexpressed . Inclusion bodies were tested on C . pipiens larvae . Unlike the native Bin4 toxin, the mutated protein was toxic, and the reciprocal mutation in Bin2 led to a significant loss of toxicity . In vitro receptor-binding studies showed similar binding behaviour for native and mutated toxins . In the absence of any experimental data on the 3D structure of these proteins, sequence analysis and secondary-structure predictions were performed . Amino acid 93 of the BinA polypeptide probably belongs to an alpha helix that is sensitive to amino-acid modifications . Position 93 may be a key element in the formation of the BinA-BinB complex responsible for the toxicity and stability of B . sphaericus Bin toxins.

Plasmid, 2001 Mar, 45(2), 114 - 21
A new insertion sequence, IS231M, in an autoagglutinable isolate of Bacillus thuringiensis; Jung YC et al.; An insertion sequence was isolated from an autoagglutinable strain of Bacillus thuringiensis . Analysis of its DNA sequence revealed high homology to the IS231 family . The name IS231M is proposed for this new insertion sequence . IS231M is 1652 bp long and is delimited by two imperfect 20-bp inverted repeat sequences with two mismatches, which are flanked by two perfect 11-bp direct repeats (DRs) . The region upstream of the open reading frame, presumed to be able to form a stable hairpin structure, is particularly well conserved in IS231M . Based on primary nucleotide sequences, IS231M is most homologous to IS231F and IS231G and most distant from IS231V and IS231W . However, as opposed to the single transposase A ORF found in IS231A, -B, -C, -D, -F, and -G, IS231M has two overlapping open reading frames, ORF1 and ORF2, that could code for polypeptides of 334 and 143 amino acids, respectively . Whether IS231M is a functional transposable element remains to be determined .

Int J Food Microbiol, 2001 Apr 11, 65(1-2), 105 - 11
Surface characteristics of Bacillus cereus and its adhesion to stainless steel; Peng JS et al.; The ability of a Bacillus cereus strain, isolated from spoiled milk, to adhere to the surface of stainless steel chips was evaluated during its growth in diluted tryptic soy broth (DTSB) . The number of cells that adhered to the surface increased markedly as the culture reached the end of the log phase and entered stationary phase, and continued to increase with further incubation . The surface properties of cells from the log, stationary, and late stationary phases were measured by hydrophobic interaction chromatography (HIC) and electrostatic interaction chromatography (ESIC) . It was found that surface hydrophobicity of B . cereus vegetative cells from the late stationary phase was the highest followed by those from the stationary phase and the log phase cultures . While the vegetative cells prepared from stationary phase and log phase cultures, respectively, had the highest and the lowest surface charges . Adhesion of B . cereus vegetative cells to stainless steel was positively correlated with the cell surface hydrophobicity (R = 0.979) . Surface hydrophobicity and surface positive charge noted on the spores harvested from diluted tryptic soy agar (DTSA) and Mn2+-tryptone glucose extract agar were higher than those harvested from the sucrose or lactose-added DTSA . A wide variation in the surface charge values was noted on the surface of various spores prepared from cultures grown on the four different media tested, while their ability to adhere to stainless steel chips in phosphate buffered saline (PBS) showed no significant difference (p > 0.05) . Similarly, the number of spores or vegetative cells adhering to stainless steel suspended in PBS, milk or diluted milk (1000 x) did not differ significantly (p > 0.05).

Anal Chem, 2001 Apr 1, 73(7), 1549 - 59
Electric manipulation of bioparticles and macromolecules on microfabricated electrodes; Huang Y et al.; Bioparticle separation, bioparticle enrichment, and electric field-mediated immune detection were carried out on microfabricated semiconductor chips utilizing ac and dc electric fields . Microscale separation on a chip surface having an active area of approximately 16 mm2 was demonstrated for a mixture of Bacillus globigii spores and Escherichia coli bacteria . Dielectrophoretic enrichment was performed by collecting target bioparticles from a flow stream in flow cells of 47.5 microL, achieving a 20-fold increase in the concentration of E . coli bacteria from a diluted sample, a 28-fold enrichment for peripheral blood mononuclear cells from red blood cells, and a 30-fold increase in white blood cells from diluted whole blood . The ability to manipulate and collect bioparticles and macromolecules at microfabricated electrodes with ac and dc fields was further illustrated in electric field-mediated immunoassays for analyzing the biological identities of E . coli bacteria and B . globigii spores . According to these results, the electric methods for manipulating bioparticles present themselves as viable techniques for novel biomedical applications in sample preparations and biochemical assays on microelectrode arrays.

J Clin Immunol, 2001 Jan, 21(1), 51 - 9
BCG infection in allergen-presensitized rats suppresses Th2 immune response and prevents the development of allergic asthmatic reaction; Koh YI et al.; Recent investigations demonstrate that bacille Calmette-Guerin (BCG), a potent inducer of Th1 response, infection prior to allergen sensitization inhibits Th2 immune responses to the allergen . However, it is not clear whether BCG infection in allergen-presensitized rats switches off Th2 response and prevents allergic asthmatic reaction to the subsequent allergen exposure . In this study we investigate whether BCG infection in ovalbumin (OVA)-presensitized Sprague-Dawley rats suppresses airway hyperresponsiveness and eosinophilic inflammation induced by OVA and Th2 cytokine production . BCG infection in OVA-presensitized rats significantly inhibited not only the sensitivity of airway smooth muscle to electrical field stimulation and acetylcholine but also absolute eosinophil counts in bronchoalveolar lavage fluid . As a correlate, interleukin-4 (IL-4) production significantly decreased and interferon-gamma (IFN-gamma) slightly increased, resulting in a markedly decreased ratio of IL-4-IFN-gamma in OVA-presensitized rats with BCG infection . These results indicate that BCG infection in pre-sensitized rats suppresses allergic asthmatic reaction and Th2 immune response . It is possible from these findings that BCG vaccine may be used as an immunomodulating agent for the sensitized host with preestablished Th2 memory.

J Microbiol Immunol Infect, 2001 Mar, 34(1), 25 - 34
Effects of BCG on ovalbumin-induced bronchial hyperreactivity in a guinea pig asthma model; Su YC et al.; To test the effects of Bacillus Calmette-Guerin (BCG) on ovalbumin (OVA)-induced airway hyper-reactivity in guinea pigs, a total of 40 young guinea pigs was individually vaccinated subcutaneously with 0.2 mL of 2% OVA, 50 microg BCG, or a mixture of OVA and BCG (OVA+BCG) . Airways were sensitized using nebulization with 1% OVA for 3 min once a week for two applications, followed by 2% OVA nebulized challenge for 3 min 1 week after the last application . Different concentrations of methacholine were used to detect airway hyperreactivities . At the third week, the guinea pigs were nebulized with either methacholine or OVA to test airway hyperreactivity . The OVA-vaccinated group presented with severe airway hyperresponsiveness after OVA and methacholine challenges; the BCG-vaccinated group showed mild airway hyperreactivity; and the OVA+BCG group showed the least amount of airway hyperreactivity . Lung histopathology in all groups, except the OVA+BCG-vaccinated group, showed severe thickening of the alveolar walls which became firmly fibrotic, and narrowing of the alveolar spaces was also noted . The guinea pigs in the OVA+BCG-vaccinated group had similar pulmonary morphology with that of naive guinea pigs, and had mild cell infiltration in the alveolar wall . The results of the skin biopsies at 6 h (2% OVA, 0.05 mL) and 36 h (20 microg PPD, 0.05 mL) after purified protein derivative (PPD) inoculation showed that infiltration of eosinophils and activation of CD4+ T-cells occurred in the OVA-vaccinated group . In the BCG-vaccinated groups, infiltration of CD4+ T-cells, CD8+ T-cells and macrophages occurred . OVA-specific IgG2 increased in the BCG-vaccinated groups after OVA-induced airway hyperreactivity occurred . The peripheral cell subpopulation showed that there was obviously increased activation of CD4+ and CD8+ T-cells in the OVA+BCG-vaccinated group . The phagocytic activity of macrophages also increased in both BCG- and OVA+BCG-vaccinated groups . The prevention of OVA-induced airway hyperreactivities using BCG vaccination in conjugation with OVA in these young guinea pigs indicated that it might be a good approach to avoid allergic reactions in humans.

Int J Syst Evol Microbiol, 2001 Mar, 51(Pt 2), 447 - 55
Ureibacillus gen . nov., a new genus to accommodate Bacillus thermosphaericus (Andersson et al . 1995), emendation of Ureibacillus thermosphaericus and description of Ureibacillus terrenus sp . nov; Fortina MG et al.; A polyphasic taxonomic study was performed on the type strain of Bacillus thermosphaericus DSM 10633T and three related soil isolates . On the basis of phenotypic characteristics, chemotaxonomic profiles and phylogenetic data a new genus, Ureibacillus gen . nov., is proposed for the strains in the Bacillus thermosphaericus cluster . Strains of this cluster fall into two DNA-DNA similarity groups: while one group contains the type strain of Ureibacillus thermosphaericus comb . nov . and a single soil isolate, the other contains two soil isolates . The two groups differed in the composition of isoprenoid quinones and some phenotypic properties . These data support the description of a novel species of Ureibacillus for which the name Ureibacillus terrenus is proposed . The type strain of this new species is TH9AT (= DSM 12654T = LMG 19470T).

Acta Crystallogr D Biol Crystallogr, 2001 May, 57(Pt 5), 717 - 8 Epub 2001 Apr 24.
Crystallization and preliminary X-ray diffraction studies of a novel alkaline serine protease (KP-43) from alkaliphilic Bacillus sp . strain KSM-KP43; Nonaka T et al.; A novel alkaline serine protease (KP-43) which belongs to a new class of the subtilisin superfamily was crystallized by the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant . The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 43.50 (2), b = 110.4 (1), c = 168.9 (1) A . The crystals diffract X-rays beyond 1.9 A resolution using Cu Kalpha radiation from a rotating-anode generator and are suitable for high-resolution crystal structure analysis.

Proc Natl Acad Sci U S A, 2001 May 8, 98(10), 5752 - 7 Epub 2001 Apr 24.
Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-alpha /beta; Manca C et al.; To understand how virulent mycobacteria subvert host immunity and establish disease, we examined the differential response of mice to infection with various human outbreak Mycobacterium tuberculosis clinical isolates . One clinical isolate, HN878, was found to be hypervirulent, as demonstrated by unusually early death of infected immune-competent mice, compared with infection with other clinical isolates . The differential effect on survival required lymphocyte function because severe combined immunodeficiency (SCID) mice infected with HN878 or other clinical isolates all died at the same rate . The hypervirulence of HN878 was associated with failure to induce M . tuberculosis-specific proliferation and IFN-gamma production by spleen and lymph node cells from infected mice . In addition, 2- to 4-fold lower levels of tumor necrosis factor-alpha (TNF-alpha), IL-6, IL-12, and IFN-gamma mRNAs were observed in lungs of HN878-infected mice . IL-10, IL-4, and IL-5 mRNA levels were not significantly elevated in lungs of HN878 infected mice . In contrast, IFN-alpha mRNA levels were significantly higher in lungs of these mice . To further investigate the role of Type 1 IFNs, mice infected with HN878 were treated intranasally with purified IFN-alpha/beta . The treatment resulted in increased lung bacillary loads and even further reduced survival . These results suggest that the hypervirulence of HN878 may be due to failure of this strain to stimulate Th1 type immunity . In addition, the lack of development of Th1 immunity in response to HN878 appears to be associated with increased induction of Type 1 IFNs.

Microbiology, 2001 May, 147(Pt 5), 1253 - 8
Isolation of strong expression signals of Mycobacterium tuberculosis; Triccas JA et al.; The natural fluorescence of the Aequoria victoria green fluorescent protein was exploited to isolate strong expression signals of Mycobacterium tuberculosis . Mycobacterium bovis bacille Calmette-Guerin harbouring M . tuberculosis fragments driving high levels of gfp expression were isolated by fluorescence-activated cell sorting (FACS) . DNA sequencing and subsequent comparison with the M . tuberculosis genome sequence revealed that a total of nine postulated promoters had been identified . The majority of the promoters displayed activity that was greater than or equal to the Mycobacterium fortuitum beta-lactamase promoter, one of the strongest mycobacterial promoters characterized to date . Two of the promoters corresponded to proteins predicted to be involved in calcium and magnesium utilization, the importance of such functions for cell physiology suggesting why these two genes are controlled by strong transcription signals . The seven other promoters corresponded to genes encoding proteins of unknown function . Promoter activity was maintained after prolonged incubation within macrophages, implying that these promoters could be used to drive sustained foreign gene expression in vivo . The strength of these expression signals identified could be employed for the overexpression of foreign genes in mycobacteria to aid protein purification and vaccine vector development . Furthermore, this study demonstrated that FACS provides a sensitive and efficient technique to measure and select strong mycobacterial expression signals.

J Biol Chem, 2001 Jul 6, 276(27), 25404 - 10 Epub 2001 Apr 24.
An invariant threonine is involved in self-catalyzed cleavage of the precursor protein for ornithine acetyltransferase; Marc F et al.; In Bacillus stearothermophilus ornithine acetyltransferase is a bifunctional enzyme, catalyzing the first and the fifth steps of arginine biosynthesis; it follows a ping-pong kinetic mechanism . A single chain precursor protein is cleaved between the alanine and threonine residues in a highly conserved ATML sequence leading to the formation of alpha and beta subunits that assemble into a heterotetrameric 2alpha2beta molecule . The beta subunit has been shown to form an acetylated intermediate in the course of the transacetylation reaction . The present data show that the precursor protein synthesized in vitro or in vivo undergoes a self-catalyzed cleavage involving an invariant threonine (Thr-197) . Using site-directed mutagenesis T197G, T197S, and T197C derivatives have been generated . The T197G substitution abolishes both precursor protein cleavage and catalytic activity, whereas T197S and T197C substitutions reduce precursor cleavage and catalytic activity in the order Thr-197 (wild type) --> Ser-197 --> Cys-197 . A mechanism is proposed in which Thr-197 plays a crucial role in the autoproteolytic cleavage of ornithine acetyltransferase.

Ann Dermatol Venereol, 2001 Mar, 128(3 Pt 1), 217 - 9
{New cases of leprosy at the Marchoux Institute: a comparative study 1988-1997}; Keita S et al.; BACKGROUND: The prevalence of leprosy had declined greatly over the last decade . The purpose of this work was to determine whether changes in the epidemiology, clinical and bacteriological patterns occurred among patients with leprosy treated at the Marchoux Institute in 1988 and in 1997.PATIENTS AND METHODS: We conducted a descriptive cross-sectional study, reviewing retrospectively all files of patients with leprosy seen in 1988 in comparison with a prospective series of leprosy patients seen in 1997 . Only new cases of leprosy, prior to treatment and with skin and/or nervous lesions irrespective of the bacilloscopy results, were included in the two series.RESULTS: We included 93 patients among 246 patient files in 1988 . There were 119 new cases in 1997 . The following variables showed changes: mean delay to consultation (41.2 months in 1988 versus 26.1 months in 1997; patient's suspicion of having leprosy (93 patients in 1988 versus 22 in 1997); notion of contact (35 cases in 1988 versus 45 in 1997) . Multibacilli leprosy predominated over paucibacilli leprosy in 1988 (51 versus 42 cases) . This trend was inverted in 1997 (44 versus 75) (p<0.009) . Patients with grade 2 handicap were more numerous in 1988 (20 cases versus 2 cases, p<0.001).CONCLUSION: These findings demonstrate a change in the epidemiology, clinical and bacilloscopic pattern of patients with leprosy from 1988 to 1997.

Plant Foods Hum Nutr, 2001, 56(2), 183 - 94
Development of an owoh-type product from African yam beans (Sphenostylis stenocarpa) (Hoechst (ex . A . Rich.) Harms.) seeds by solid substrate fermentation; Ogbonna DN et al.; African yam beans were fermented to obtain an owoh-type product . Microorganisms associated with the fermentation were Bacillus licheniformis, B . pumilus, B . subtilis and Staphylococcus sp . Total microbial counts increased from 1.53 x 10(5) cfu/g to 1.51 x 10(9) cfu/g under aerobic conditions, and from 8.0 x 10(3) cfu/g to 1.35 x 10(7) cfu/g under conditions of reduced oxygen tension . The pH of the substrate increased throughout the fermentation, from 6.8 to 7.5 . A comparison of unfermented seeds with the fermented product showed that there were decreases in the levels of total nitrogen, crude protein, crude fiber and lipids, and that there were increases in the levels of carbohydrate and total organic matter . Enzyme activities during fermentation revealed that amylase production was erratic showing a slight increase during the first 24 h followed by a steep rise in activity in the next 24 h . By contrast, lipase activity increased rapidly throughout the first 72 h while proteinase activity followed a type of sigmoid curve with a steady increase in activity within the first 48 h and a relatively high activity until the 96th h before plunging downwards.

Clin Infect Dis, 2001 May 15, 32(10), 1456 - 62 Epub 2001 Apr 20.
Bacillus cereus bacteremia and meningitis in immunocompromised children; Gaur AH et al.; Two cases of Bacillus cereus meningitis in immunocompromised children at our hospital within a 2-month period prompted us to review B . cereus--related invasive disease . We identified 12 patients with B . cereus isolated in blood cultures from September 1988 through August 2000 at our institution . Three of these patients also had B . cereus isolated from CSF specimens; 1 additional patient had possible CNS involvement (33%, group A), whereas 8 patients had no evidence of CNS involvement (67%, group B) . Patients in group A were more likely to have neutropenia at the onset of sepsis and were more likely to have an unfavorable outcome . They were also more likely to have received intrathecal chemotherapy in the week before the onset of their illness . Two patients from group A died . One survived with severe sequelae . The fourth patient had mild sequelae at follow-up . No sequelae or deaths occurred among patients in group B . In patients with unfavorable outcomes, the interval from the time of recognition of illness to irreversible damage or death was short, which demonstrates a need for increased awareness, early diagnosis, and more-effective therapy, particularly that which addresses B . cereus toxins.

Res Microbiol, 2001 Mar, 152(2), 175 - 7
Characterisation of a Listeria monocytogenes mutant deficient in D-arabitol fermentation; Saklani-Jusforgues H et al.; We selected and analysed a Tn917-lac Listeria monocytogenes mutant deficient in D-arabitol fermentation . Comparison of the 310-aa-long translated partial sequence of the disrupted gene with known proteins showed similarity with the phosphotransferase system galactitol-specific enzyme IIC component of the alkaliphilic Bacillus halodurans (50% identity) and of Escherichia coli (36% identity) . Fermentation of 18 other carbohydrates was unimpaired, suggesting the specificity of this transmembrane permease IIC for the pentitol D-arabitol . The deficiency in D-arabitol fermentation did not alter L . monocytogenes virulence in the BALB/c mouse model after intravenous and intragastric inoculations . This fully virulent mutant is a valuable tool to study L . monocytogenes oral infection, since the antibiotic resistance marker present on the Tn917-lac transposon will efficiently select L . monocytogenes against the intestinal microflora.

J Biol Inorg Chem, 2001 Mar, 6(3), 300 - 14
Structure-based computational study of the catalytic and inhibition mechanisms of urease; Musiani F et al.; The viability of different mechanisms of catalysis and inhibition of the nickel-containing enzyme urease was explored using the available high-resolution structures of the enzyme isolated from Bacillus pasteurii in the native form and inhibited with several substrates . The structures and charge distribution of urea, its catalytic transition state, and three enzyme inhibitors were calculated using ab initio and density functional theory methods . The DOCK program suite was employed to determine families of structures of urease complexes characterized by docking energy scores indicative of their relative stability according to steric and electrostatic criteria . Adjustment of the parameters used by DOCK, in order to account for the presence of the metal ion in the active site, resulted in the calculation of best energy structures for the nickel-bound inhibitors beta-mercaptoethanol, acetohydroxamic acid, and diamidophosphoric acid . These calculated structures are in good agreement with the experimentally determined structures, and provide hints on the reactivity and mobility of the inhibitors in the active site . The same docking protocol was applied to the substrate urea and its catalytic transition state, in order to shed light onto the possible catalytic steps occurring at the binuclear nickel active site . These calculations suggest that the most viable pathway for urea hydrolysis involve a nucleophilic attack by the bridging, and not the terminal, nickel-bound hydroxide onto a urea molecule, with active site residues playing important roles in orienting and activating the substrate, and stabilizing the catalytic transition state.

Minerva Urol Nefrol, 2000 Dec, 52(4), 215 - 8
{Repeated bladder metastases from renal cell carcinoma . Report of a case with particular attention to the use of immunomodulators}; Vecchioli Scaldazza C et al.; The case of repeated metastases of the bladder from renal cell carcinoma in a 59 year old male patient is presented . Two years after nephrectomy the ureteral stump and the bladder were interested by two different metastases . Afterwards, other nine metastases developed into the bladder during the following 12 months . Biopsy with coagulation were performed in any case . Bladder metastases ranged between 2 and 4 mm . No other metastasis was discovered by MRI and skeletal scintigraphy . Thirteen months after the first bladder metastasis, intracavitary Bacillus Calmette-Guerin was used . No report was found in the literature about BCG in the treatment of superficial bladder metastases from renal cell carcinoma . However 15 months from beginning of BCG treatment no metastasis developed from the bladder that now is disease-free after a follow-up of 19 months.

Ann Surg Oncol, 2001 Apr, 8(3), 198 - 203
Serum TA90 antigen-antibody complex as a surrogate marker for the efficacy of a polyvalent allogeneic whole-cell vaccine (CancerVax) in melanoma; Tsioulias GJ et al.; INTRODUCTION: TA90 is a tumor-associated 90-kD glycoprotein antigen expressed on most melanoma cells, including those of CancerVax, a polyvalent allogeneic whole-cell vaccine . Previous studies have shown that a TA90 antigen-antibody immune complex (IC) in the serum of patients with melanoma is a marker of subclinical tumor burden and a strong prognostic factor . We hypothesized that the induction of TA90-IC during postoperative adjuvant CancerVax therapy might indicate vaccine-mediated immune destruction of subclinical melanoma cells with release of TA90, and thereby serve as a surrogate marker of vaccine efficacy . METHODS: From 1993 to 1997, 219 melanoma patients were enrolled in a prospective phase II trial of CancerVax plus bacille Calmette-Guerin (BCG) after complete tumor resection . Coded serum samples were prospectively collected and analyzed for TA90-IC before and 2, 4, 8, 12, and 16 weeks after initiation of CancerVax therapy . TA90-IC seroconverters were those patients whose negative TA90-IC values (< .410) became positive (> or = .410) after initiation of CancerVax treatment . RESULTS: Before CancerVax therapy, 51 patients had positive TA90-IC values and 168 patients had negative TA90-IC values . During CancerVax treatment, all 51 positive patients remained positive, 79 (47%) negative patients seroconverted to positive, and 89 (53%) negative patients remained negative . Seroconverters had higher 2-year rates of disease-free survival (59% vs . 32%; P < .006) and overall survival (78% vs . 63%; P < .02) than did patients whose TA90-IC values remained positive . CONCLUSIONS: CancerVax induces TA90-IC in melanoma patients with subclinical disease . TA90-IC seroconverted patients have significantly improved disease-free and overall survival compared with TA90-IC positive patients . TA90-IC is an important prognostic factor that can serve as a surrogate marker for the clinical efficacy of CancerVax.

Mikrobiologiia, 2000 Sep-Oct, 69(5), 660 - 7
{Localization of proteolytic enzymes in cells of Bacillus intermedius}; Sharipova MR et al.; The activities of proteinases in the culture fluid and cellular fractions of Bacillus intermedius 3-19 grown under various conditions were studied . Thiol-dependent serine proteinase was the prevalent enzyme in the total pool of extracellular proteinases (70%); its catalytically active form was also detected in the cell membrane and, during active enzyme production, in the cell wall . Another enzyme, glutamyl endopeptidase (10% of the total pool), was detected in the cell membrane; it was also found in the cell wall and cytoplasm during active enzyme secretion into the growth medium . Production of these enzymes was maximal on medium containing inorganic phosphate and gelatin and decreased 2- to 4-fold on medium with glucose and lactate . The level of activity of extracellular enzymes correlated with that of corresponding membrane-bound proteins . The addition of CoCl2 (2 mM) into the medium caused an essential increase in extracellular glutamyl endopeptidase activity and promoted the release of the membrane-bound enzyme into the culture fluid . Proteolytic activity towards casein was also detected in the cytoplasm . The proteinases localized in the cytoplasm were shown to differ in their properties from those secreted.

J Basic Microbiol, 2001, 41(1), 3 - 6
Specificity of lectin activity of Bacillus thuringiensis parasporal inclusion proteins; Akao T et al.; Sheep erythrocyte-agglutinating parasporal inclusion proteins from four Bacillus thuringiensis strains (FITC-20, FITC-73, FITC-76 and IBC-1456) were examined for lectin activity against erythrocytes from four mammalian (rabbit, horse, cow and guinea pig) and an avian (chicken) species . Of the five erythrocyte species, only rabbit cells were agglutinated with the protein of the human cancer cell-killing strain IBC-1456 . No haemagglutination (HA) activities were shown in other protein-erythrocyte combinations . The lectin activity of the strain IBC-1456 against rabbit cells was strongly inhibited by preincubation with D-galactose . Overall results revealed that the B . thuringiensis lectins have a preference for sheep erythrocytes.

Mem Inst Oswaldo Cruz, 2001 Apr, 96(3), 357 - 64
A theoretical model of the tridimensional structure of Bacillus thuringiensis subsp . medellin Cry 11Bb toxin deduced by homology modelling; Gutierrez P et al.; Cry11Bb is an insecticidal crystal protein produced by Bacillus thuringiensis subsp . medellin during its stationary phase; this partial differential-endotoxin is active against dipteran insects and has great potential for mosquito borne disease control . Here, we report the first theoretical model of the tridimensional structure of a Cry11 toxin . The tridimensional structure of the Cry11Bb toxin was obtained by homology modelling on the structures of the Cry1Aa and Cry3Aa toxins . In this work we give a brief description of our model and hypothesize the residues of the Cry11Bb toxin that could be important in receptor recognition and pore formation . This model will serve as a starting point for the design of mutagenesis experiments aimed to the improvement of toxicity, and to provide a new tool for the elucidation of the mechanism of action of these mosquitocidal proteins.

FEMS Microbiol Lett, 2001 Apr 13, 197(2), 223 - 8
Psychrotolerant species from the Bacillus cereus group are not necessarily Bacillus weihenstephanensis; Stenfors LP et al.; Twenty-six strains of Bacillus cereus from different sources were determined to be either mesophilic or psychrotrophic by growth at 6 and 42 degrees C . The strains were also screened by two polymerase chain reaction (PCR) methods designed to discriminate between mesophilic and psychrotrophic types . Seventeen of the 26 strains were able to grow at 6 degrees C, but only four conformed to the new psychrotolerant species Bacillus weihenstephanensis . Among the 26 strains were two which caused outbreaks of food poisoning in Norway, and three others that were isolated from food suspected of causing illness . The presence of the gene components encoding production of enterotoxins Nhe, Hbl, EntT and a recently described cytotoxin K was determined by PCR . All the strains possessed genes for at least one of these toxins, and 19 of the 26 strains were cytotoxic in a Vero cell assay . We conclude that there are psychrotrophic B . cereus strains which cannot be classified as B . weihenstephanensis, and that intermediate forms between the two species exist . No correlation between cytotoxicity and the growth temperature of the strains was found.

Biotechnol Prog, 2001 Mar-Apr, 17(2), 211 - 6
Bacillus thuringiensis cry3Ca1 protein is toxic to the Colorado potato beetle, Leptinotarsa decemlineata (Say); Haffani YZ et al.; We expressed the wild-type cry3Aa3 and cry3Ca1 Bacillus thuringiensis genes, which code for insecticidal proteins, in an Escherichia coli expression system . Highly purified preparations of the soluble delta-endotoxins were used to perform comparative bioassays with third-instar larvae of the Colorado potato beetle (CPB) . Acute mortality data showed that Cry3Ca1 (LD(50) = 320.1 ng) was 2-fold more toxic than Cry3Aa3 (LD(50) = 672.9 ng) . We also compared the chronic effects of sublethal doses of these toxins by measuring the consumption of untreated foliage and monitoring survival and development for 6 days after intoxication . No significant additional mortality was recorded, but we found that surviving larvae fed Cry3Ca1 consumed foliage at a slower rate than the larvae fed Cry3Aa3, suggesting more damage to their digestive epithelium . This study, the first assessment of the toxicity of cry3Ca1 in third-instar CPB, suggests cry3Ca1 will prove useful for the control of this important insect pest.

J Appl Microbiol, 2001 Apr, 90(4), 643 - 7
Update on the detection of beta-exotoxin in Bacillus thuringiensis strains by HPLC analysis; Hernandez CS et al.; AIMS: The current work aimed to study the presence of beta-exotoxin by high-performance liquid chromatography (HPLC) in supernatant fluids from final whole cultures of the 69 type strains and 13 subtypes of Bacillus thuringiensis strains, as well as from some insecticidal strains . METHODS AND RESULTS: Results from HPLC and bioassays with Ephestia kuhniella (Lepidoptera Pyralidae) were compared . Type I beta-exotoxin was only detected in type strains representing serotypes H1, H9 and H10a,10b . Discrepancies between HPLC and bioassays were found in H8a,8b and some insecticidal strains, which suggests the occurrence of another soluble toxin different from type I beta-exotoxin, possibly type II beta-exotoxin . CONCLUSION: This study shows the need to use bioassays to determine the presence of beta-exotoxin activity . However, HPLC is a fast and sensitive technique if only type I beta-exotoxin is to be determined . The occurrence of beta-exotoxin in a type strain does not imply production of this metabolite by other strains belonging to the same serovar . SIGNIFICANCE AND IMPACT OF THE STUDY: These results complete the characterization of type strains belonging to the International Entomopathogenic Bacillus Collection (Institut Pasteur, Paris, France).

J Appl Microbiol, 2001 Apr, 90(4), 530 - 4
Characterization and growth of Bacillus spp . in heat-treated cream with and without nisin; Nissen H et al.; AIMS: To study the germination and growth of both inoculated and naturally occurring Bacillus strains in heat-treated cream with and without nisin . METHODS AND RESULTS: In heat-treated cream (90 degrees C for 15 min) stored at 8 degrees C, growth was dominated by naturally occurring Bacillus strains such as Bacillus pumilus and B . licheniformis . Only six of the 52 isolated strains were B . cereus/thuringiensis . All of the B . cereus strains, but none of the other strains, produced enterotoxin when tested with the TECRA and reverse passive latex agglutination kits . Bacterial growth during storage of the cream at 8 or 10 degrees C was completely inhibited by low concentrations of nisin . CONCLUSION: The high number of Bacillus strains surviving the heat treatment represent a risk for heat-treated food that contains cream . The safety of the cream, for instance in "ready-to-eat" products, can be improved by the addition of low concentrations of nisin . SIGNIFICANCE AND IMPACT OF THE STUDY: Spores of several Bacillus species may survive heat treatment of cream, but low concentration of nisin with inhibit germination and growth.

Biol Chem, 2001 Feb, 382(2), 299 - 311
Synthesis of nucleotide-activated oligosaccharides by beta-galactosidase from Bacillus circulans; Zervosen A et al.; The enzymatic access to nucleotide-activated oligosaccharides by a glycosidase-catalyzed transglycosylation reaction was explored . The nucleotide sugars UDP-GlcNAc and UDP-Glc were tested as acceptor substrates for beta-galactosidase from Bacillus circulans using lactose as donor substrate . The UDP-disaccharides Gal(beta1-4)GlcNAc(alpha1-UDP) (UDP-LacNAc) and Gal(beta1-4)Glc(alpha1-UDP) (UDP-Lac) and the UDP-trisaccharides Gal(beta1-4)Gal(beta1-4)GlcNAc(alpha1-UDP and Gal(beta1-4)Gal(beta1-4)Glc(alpha1-UDP) were formed stereo- and regioselectively . Their chemical structures were characterized by 1H and 13C NMR spectroscopy and fast atom bombardment mass spectrometry . The synthesis in frozen solution at -5 degrees C instead of 30 degrees C gave significantly higher product yields with respect to the acceptor substrates . This was due to a remarkably higher product stability in the small liquid phase of the frozen reaction mixture . Under optimized conditions, at -5 degrees C and pH 4.5 with 500 mM lactose and 100 mM UDP-GlcNAc, an overall yield of 8.2% (81.8 micromol, 62.8 mg with 100% purity) for Gal(beta1-4)GlcNAc(alpha1-UDP) and 3.6% (36.1 micromol, 35 mg with 96% purity) for Gal(beta1-4)Gal(beta1-4)GlcNAc(alpha1-UDP) was obtained . UDP-Glc as acceptor gave an overall yield of 5.0% (41.3 micromol, 32.3 mg with 93% purity) for Gal(beta1-4)Glc(alpha1-UDP) and 1.6% (13.0 micromol, 12.2 mg with 95% purity) for Gal(beta1-4)Gal(beta1-4)Glc(alpha1-UDP) . The analysis of other nucleotide sugars revealed UDP-Gal, UDP-GalNAc, UDP-Xyl and dTDP-, CDP-, ADP- and GDP-Glc as further acceptor substrates for beta-galactosidase from Bacillus circulans.

J Biol Chem, 2001 Jul 6, 276(27), 24997 - 5004 Epub 2001 Apr 16.
A novel pathway of aerobic benzoate catabolism in the bacteria Azoarcus evansii and Bacillus stearothermophilus; Zaar A et al.; The aerobic catabolism of benzoate was studied in the Gram-negative proteobacterium Azoarcus evansii and in the Gram-positive bacterium Bacillus stearothermophilus . In contrast to earlier proposals, benzoate was not converted into hydroxybenzoate or gentisate . Rather, benzoyl-CoA was a product of benzoate catabolism in both microbial species under aerobic conditions in vivo . Benzoyl-CoA was converted into various CoA thioesters by cell extracts of both species in oxygen- and NADPH-dependent reactions . Using {ring-(13)C(6)}benzoyl-CoA as substrate, cis-3,4-{2,3,4,5,6-(13)C(5)}dehydroadipyl-CoA, trans-2,3-{2,3,4,5,6-(13)C(5)}dehydroadipyl-CoA, the 3,6-lactone of 3-{2,3,4,5,6-(13)C(5)}hydroxyadipyl-CoA, and 3-{2,3,4,5,6-(13)C(5)}hydroxyadipyl-CoA were identified as products by NMR spectroscopy . A protein mixture of A . evansii transformed {ring-(13)C(6)}benzoyl-CoA in an NADPH- and oxygen-dependent reaction into 6-{2,3,4,5,6-(13)C(5)}hydroxy-3-hexenoyl-CoA . The data suggest a novel aerobic pathway of benzoate catabolism via CoA intermediates leading to beta-ketoadipyl-CoA, an intermediate of the known beta-ketoadipate pathway.

Can J Urol, 1998 Oct, 5(4), 608 - 610
Stability of reconstituted freeze-dried Bacille Calmette-Guérin used for intravesical immunotherapy for bladder cancer; Conly J et al.; The product monographs for the commercially available products of Bacille Calmette-Guerin (BCG) state they must be used immediately following reconstitution . We hypothesized that the viability of the reconstituted preparations of BCG would not be altered for a period of up to 4 hours thus allowing a longer time interval between full preparation and its intravesical administration . Vials of freeze-dried ImmuCysttrade mark (Connaught Laboratories) and TICEtrade mark BCG (Organon-Teknika) were fully reconstituted in approximately 50 ml of saline and aliquots of the fully reconstituted product were serially diluted and inoculated onto freshly prepared Lowenstein-Jensen slants and colony counts were conducted at 0, 2, and 4 hours . The colony counts of the freeze-dried preparations of BCG varied between 0.1 and 0.3 x 108 and between 0.6 and 1.1 x 108 per reconstituted volume at 4 hours for the Connaught and TICE strains, respectively . There was no decrease in the colony counts between 0 and 4 hours for either product . There were no significant differences in the viability of the fully reconstituted freeze-dried preparation of BCG for up to 4 hours after complete reconstitution . These findings would suggest some latitude may be provided for institutions where the product is fully reconstituted prior to its actual administration.

Arch Insect Biochem Physiol, 2001 Apr, 46(4), 186 - 200
Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity; Valaitis AP et al.; BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis . Rabbit antibodies specific for the Bt toxin-binding molecule were raised . Attempts to label BTR-270 by protein-directed techniques were futile, but it was degraded by proteases with broad specificity indicating the presence of a peptide . Carbohydrate was detected by labeling with digoxigenin hydrazide following periodate oxidation . Mild alkaline hydrolysis destroyed toxin and antibody binding, suggesting O-linked glycans are involved in the activity . GC/MS composition analysis showed that the predominant sugars were galactose, glucose, and N-acetyl galactosamine with lesser amounts of N-acetyl glucosamine, glucuronic acid, xylose, and fucose . The carbohydrate moiety accounted for 73% of its total mass . Amino acid analysis showed a high content of aspartic/asparagine, threonine, and serine residues in the protein moiety . The purified glycoconjugate was not visualized using Coomassie or silver staining procedures, but stained "blue" using the cationic dye Stains-all . BTR-270 was labeled with biotin and used as a diagnostic probe for screening and identifying toxins that bind to the receptor . Toxin-binding kinetics obtained using a biosensor demonstrated that the receptor binds Cry1Aa and Cry1Ab toxins with high affinity, and displays a weaker affinity for Cry1Ac, in correlation with the toxicity of these toxins towards gypsy moth . Arch .

Scand J Infect Dis, 2001, 33(3), 175 - 8
Determinants of vaccine-induced resistance in animal models of pulmonary tuberculosis; McMurray DN; A more effective vaccine will be essential if the global tuberculosis (TB) pandemic is ever to be controlled . A large number of new tuberculosis vaccines have been developed, representing the whole range of modern strategies for vaccine formulation and delivery . There is currently no alternative to testing these new vaccines in experimental animals challenged with virulent Mycobacteriurn tuberculosis in order to assess their protective efficacy . Although such testing is being carried out in several animal species (mice, guinea pigs, rabbits), all rational models include pulmonary challenge with a low dose of virulent mycobacteria . The quantitative measures for TB vaccines include increased survival, amelioration of clinical signs and symptoms (e.g . prevention of weight loss), decreased lesion size, reduction in bacillary loads in the lungs, and prevention of extrapulmonary dissemination and hematogenous reseeding of the lung . Although the ultimate objective of vaccination in humans is to prevent transmission to susceptible contacts, no such measurement is being used in animal studies of new vaccines . The validation of an immunological "correlate of protection" is urgently needed . Candidates for such a correlate include antigen-specific interferon-gamma production by T cells of the memory phenotype (CD45RB(high) or mycobacterial killing by macrophages co-cultured with immune T cells . Additional animal models must be developed for vaccines designed to prevent endogenous reactivation or exogenous reinfection, or to be used as a adjunct to chemotherapy.

Clin Infect Dis, 2001 May 1, 32(9), 1331 - 7 Epub 2001 Apr 13.
Tuberculin skin testing of physicians at a midwestern teaching hospital: a 6-year prospective study; Warren DK et al.; The epidemiology of tuberculin reactivity among physicians practicing in regions of moderate tuberculosis prevalence is unknown . We prospectively assessed the epidemiology of tuberculin skin test (TST) reactivity among physicians in training in St . Louis between 1992 and 1998 . Of 1574 physicians who were tested, 267 (17%) had positive TST results . Older age, birth outside of the United States, prior bacille Calmette-Guerin (BCG) vaccination, and practice in the fields of medicine, anesthesiology, or psychiatry were associated with a positive TST result . Among physicians born in the United States, 63 (5.7%) had positive TST results . Among physicians with > or = 2 documented TSTs, 12 had conversion to a positive TST (1.6%; 1.03 conversions per 100 person-years) . Physicians in this study had a high rate of tuberculin reactivity, despite a low conversion rate . The relationship between TST conversion and birth outside of the United States and BCG vaccination suggests a booster phenomenon rather than true new TST conversions.

Biosci Biotechnol Biochem, 2001 Feb, 65(2), 389 - 95
Construction of a chimeric shuttle plasmid via a heterodimer system: secretion of an scFv protein from Bacillus brevis cells capable of inhibiting hemagglutination; Shiroza T et al.; Passive immunization is an attractive therapy for preventing oral diseases including dental caries and periodontal disease . For this purpose, we attempted to produce a single chain variable fragment, scFv, which inhibited hemagglutination using the Bacillus brevis protein-producing system . To accomplish this, a novel strategy, a heterodimer system, was used for the construction of a chimeric shuttle plasmid . Initially, a set of new plasmids, kanamycin-resistant donor and erythromycin-resistant general cloning plasmids, were constructed . p15A ori was a common replication origin in these plasmids, while the pUB110 rep and minus origin (MO) were cloned into the donor plasmid . Next, the secretion domain of the B . subtilis alpha-amylase gene and the G2-4 gene, coding for the scFv protein, were cloned into the general cloning plasmid and fused by PCR . Both the donor plasmid and the general cloning plasmid containing the fused gene were digested with NotI and them ligated, a dimeric plasmid being constructed . The key restriction sites, AscI, are arranged such that the pUB110 rep-MO moiety was switched from the donor to the general cloning plasmid following AscI digestion . The chimeric shuttle plasmid was readily constructed by simple re-circularization and a B . brevis transformant producing the scFv protein in the culture fluid was isolated.

Clin Cancer Res, 2001 Mar, 7(3 Suppl), 933s - 939s
Suppressed T-cell receptor zeta chain expression and cytokine production in pancreatic cancer patients; Schmielau J et al.; Suppression of various functions of T cells derived from cancer patients has been linked previously to changes in the T-cell receptor (TCR)-associated signal transduction molecules, in particular the zeta chain of the TCR complex . In this study, we have examined the TCRzeta chain expression and cytokine production in vivo and in vitro in T cells of patients with metastatic adenocarcinomas of the pancreas that participated in a Phase I clinical trial of the MUC1 peptide plus bacillus Calmette-Guerin cancer vaccine . A majority of the patients had reduced TCRzeta chain expression and interleukin 4 production by T cells, and all of the patients showed decreased production of IFN-gamma of their peripheral T cells when compared with healthy individuals . Peripheral blood T cells were activated with the phorbol ester phorbol myrisate acetate and ionomycin to show that although aberrant TCRzeta chain expression and decreased cytokine production were often correlated, the reduced cytokine production was not simply a consequence of an impaired TCRzeta chain expression . Rather, these are two separate but parallel defects in signal transduction in T cells, which are potentially modulated by the same mechanisms . Half of the patients showed an improvement for TCRzeta chain or IFN-gamma expression after vaccination.

Mikrobiol Z, 2000 Jan-Feb, 62(1), 40 - 63
{Unique properties of highly radioresistant bacteria}; Romanovskaia VA et al.; In connection with the Chernobyl Nuclear Power Plant (ChNPP) accident and the negative ecological after-effects for biota in this zone the interest has arisen to radioresistant bacteria, as to the most dynamic model of the given ecosystem, and to mechanisms which provide resistance of bacteria to ionizing radiation . The analysis of published data has shown that the radioresistant bacteria are not interrelated taxonomically and phylogenetically . The extreme radioresistant bacteria are represented by the Deinococcus species, which form a group phylogenetically close to the line Thermus-Meiothermus . Other radioresistant bacteria are the representatives of the genera Rubrobacter, Methylobacterium, Kocuria, Bacillus and some archebacteria . Data on natural habitats, of radioresistant bacteria are not numerous . In a number of cases it is difficult to distinguish their natural habitats, as they were isolated from the samples which were previously exposed to X-ray or gamma-irradiation, or from the ecosystems with the naturally raised radioactivity . To understand the strategy of survival of radioresistant bacteria, we briefly reviewed the mechanism of action of various species of radiation on cells and macromolecules; physiological signs of the cell damage caused by radiation; mechanisms eliminating (repairing) these damages . More details on mechanisms of the DNA repair in D . radiodurans are described . The extreme resistance of D . radiodurans to the DNA damaging factors is defined by 1) repair mechanisms which fundamentally differ from those in other procaryotes; 2) ability to increase the efficiency of a standard set of the DNA repairing proteins . Literary and own data on the effect of radiation on survival of various groups of bacteria in natural ecosystems are summarized . The ecological consequences of the ChNPP accident for soil bacteria in this region were estimated . The reduction of the number of soil bacteria and recession of microbial diversity under the effect of anthropogenic radiation was shown.

Arq Neuropsiquiatr, 2001 Mar, 59(1), 71 - 6
{Central nervous system tuberculosis in children: 1 . Clinical and laboratorial presentation}; Gusmao Filho FA et al.; Tuberculosis still occupies a remarkable place as a worldwide health problem, chiefly in emerging countries, like Brazil . The central nervous system (CNS) involvement by Mycobacterium tuberculosis is one of the most feared features of disease, because of its high morbidity and mortality . This study aimed to describe some epidemiological, clinical and laboratorial aspects of 52 children in a tertiary pediatric hospital with CNS tuberculosis . At diagnosis, the majority of patients showed low age, compromised nutritional status, previous contact with bacillary individuals, delayed or absent immunization, advanced neurological signs and compatible abnormalities in cerebrospinal fluid (CSF) analysis and in radiological findings . The etiologic agent was identified by staining methods or CSF and other fluids culturing in 40% of patients . In most cases, despite of suggestive clinical, epidemiological and laboratorial picture and feasibility of patients access to health care centres, therapy was started late.

Can J Urol, 1998 Mar, 5(1), 477 - 481
Bacille Calmette-Guerin (BCG) associated epididymitis: a case report and review; Dawson LA et al.; A case of Bacille Calmette-Guerin (BCG)-associated epididymitis that was non responsive to 6 months of antituberculous therapy, but stable after 2 years, is reported . We review the clinical and pathological features of previously reported cases of pathologically diagnosed BCG-associated epididymitis . Surgery has been the primary treatment for BCG-associated epididymitis in all previous cases.

Mol Ecol, 2001 Feb, 10(2), 525 - 33
Immunological analysis of phloem sap of Bacillus thuringiensis corn and of the nontarget herbivore Rhopalosiphum padi (Homoptera: Aphididae) for the presence of Cry1Ab; Raps A et al.; Phloem sap of transgenic Bacillus thuringiensis (Bt) corn expressing a truncated form of the B . thuringiensis delta-endotoxin Cry1Ab, sap sucking aphids feeding on Bt corn and their honeydew were analysed for presence of Cry1Ab using ELISA . Phloem sap of Bt and non-Bt corn was collected using a newly developed technique with a microcapillary being directly inserted into the phloem tubes . Using this technique, no Cry1Ab was detected in the phloem sap . In contrast, measurable concentrations of Cry1Ab in the range of 1 ppb were detected when phloem sap of pooled leaf samples was extracted using EDTA buffer . This was probably because of Cry1Ab toxin released from damaged cells . When analysing apterous adults of Rhopalosiphum padi L . and their honeydew, no Cry1Ab could be detected . In contrast, Cry1Ab was clearly detected in both larvae of the leaf chewing herbivore Spodoptera littoralis (Boisduval) and their faeces, showing that Cry1Ab is detectable after ingestion and excretion by herbivores . These results suggest that R . padi ingests or contains no or only very low concentrations of Cry1Ab in the range of the detection limit . In consequence it is hypothesized that R . padi as an important prey for beneficial insects in corn is unlikely to cause any harm to its antagonists due to mediating Bt toxin.

J Appl Microbiol, 2001 Mar, 90(3), 353 - 7
Biosynthesis of PHB tercopolymer by Bacillus cereus UW85; Labuzek S et al.; AIMS: The study was attempted to determine the ability of a Gram-positive Bacillus cereus UW85 strain to biosynthesize poly (3-hydroxybutyrate) copolymers when epsilon-caprolactone, or epsilon-caprolactone and glucose, were used as carbon sources . METHODS AND RESULTS: Bacillus cereus was grown for 24 h under nitrogen-limited conditions in a mineral salts medium . Growth was monitored by measurement of turbidity . Glucose level was determined by the colorimetric anthrone METHOD: The epsilon-caprolactone concentration was determined by gas chromatography . The bacterial biopolymers were extracted with chloroform in a Soxhlet extractor and then characterized by nuclear magnetic resonance and gel permeation chromatography . When epsilon-caprolactone was used as a carbon substrate, the bacterial strain produced tercopolymer with 3-hydroxybutyrate, 3-hydroxyvalerate and 6-hydroxyhexanoate units . However, when caprolactone and glucose were supplied together, only homopolymer of poly (3-hydroxybutyrate) was produced . CONCLUSION: All tercopolymers isolated from B . cereus UW85 cells were obtained with yields up to 9% (w/w) and low number-average molecular weight compared with the homopolymer PHB . SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus cereus UW85 produced tercopolymer with a low molecular weight from one substrate (epsilon-caprolactone) used as a carbon source . The results are significant for the potential future application of Bacillus biopolymers to bioplastics production.

FEBS Lett, 2001 Apr 6, 494(1-2), 74 - 8
Trp122 and Trp134 on the surface of the catalytic domain are essential for crystalline chitin hydrolysis by Bacillus circulans chitinase A1; Watanabe T et al.; From the 3D-structural analysis of the catalytic domain of chitinase A1, two exposed tryptophan residues (W122 and W134) are proposed to play an important role in guiding a chitin chain into the catalytic cleft during the crystalline chitin hydrolysis . Mutation of either W122 or W134 to alanine significantly reduced the hydrolyzing activity against highly crystalline beta-chitin microfibrils . Double mutation almost completely abolished the hydrolyzing activity . On the other hand, the hydrolyzing activity against either soluble or amorphous substrate was not reduced . These mutations slightly impaired the binding activity of this enzyme . These results clearly demonstrated that the two exposed aromatic residues play a critical role in hydrolyzing the chitin chain in crystalline chitin.

J Submicrosc Cytol Pathol, 2000 Oct, 32(4), 635 - 43
Immunolocalization of different tubulin epitopes in the spermatozoon of Bacillus rossius (Insecta, Phasmatodea); Taddei AR et al.; The existence of distinct tubulins in microtubules forming the sperm axoneme has been demonstrated in various species, whereas little is known about the distribution of tubulin variants in insect spermatozoa . In the present study, a panel of specific antibodies has been used to investigate the presence and localization of tubulin isotypes and post-translationally modified tubulins in the spermatozoon of the stick insect Bacillus rossius . Indirect immunofluorescence and immunogold staining showed differences in labelling in the mature sperm and that the tubulin epitopes localized differentially in the axoneme . In particular, the tyrosinated alpha-tubulin mainly occurs on doublets . These results provide an insight into the molecular composition of the microtubules forming the sperm axoneme of B . rossius and suggest that the structural specificity could reflect distinct functional roles within axonemal microtubules.

Prog Urol, 2001 Feb, 11(1), 62 - 7
{Urogenital tuberculosis . Experience in 10 years}; el Khader K et al.; OBJECTIVE: To review the clinical, imaging and therapeutic aspects of urogenital tuberculosis . MATERIAL AND METHODS: From April 1989 to April 1999, 57 patients with urogenital tuberculosis were reviewed in our department . This series consisted of 32 males and 25 females with a mean age of 40 years (range: 18 to 72 years) . RESULTS: The most frequent clinical symptoms were irritative symptoms (47.3%) . Fever, anorexia and weight loss were rare (11%) . 16% of patients had an isolated genital lesion . 14% presented with renal failure (mean serum creatinine: 18 mg/l) . Only 3 cases (5.2%) presented with bacilluria . Urography showed abnormalities in 80% of cases . The most frequent abnormality was a non-functioning silent kidney in 23 cases (40.3%) . The positive diagnosis was based on bacteriological (5 cases) and histological data (52 cases) . Treatment consisted of antituberculous chemotherapy in all patients, in combination with surgery (75%), and/or endourological procedures (26.3%) . Nephrectomy is still indicated for non-functioning tuberculous kidneys in order to prevent the development of hypertension, abscess and fistulas . CONCLUSION: The diagnosis of urogenital tuberculosis is difficult and often late . A surgical or endourological procedure is often necessary to preserve renal function and to improve quality of life.

FEMS Microbiol Ecol, 2001 Apr, 35(2), 181 - 187
Synergism between Phyllobacterium sp . (N(2)-fixer) and Bacillus licheniformis (P-solubilizer), both from a semiarid mangrove rhizosphere; Rojas A et al.; Mangrove seedlings were treated with a mixture of two bacterial species, the slow-growing, N(2)-fixing bacterium Phyllobacterium sp . and the fast-growing, phosphate-solubilizing bacterium Bacillus licheniformis, both isolated from the rhizosphere from black, white, and red mangroves of a semiarid zone . Nitrogen fixation and phosphate solubilization increased when the mixture was used compared to the effects observed when adding individual cultures, notwithstanding that there was no increase in bacterial multiplication under these conditions . Inoculation of black mangrove seedlings in artificial seawater showed the mixture performed somewhat better than inoculation of the individual bacterium; more leaves were developed and higher levels of (15)N were incorporated into the leaves, although the total nitrogen level decreased . This study demonstrates that interactions between individual components of the rhizosphere of mangroves should be considered when evaluating these bacteria as plant growth promoters.

J Biol Chem, 2001 Jun 15, 276(24), 20966 - 72 Epub 2001 Apr 09.
Structural and regulatory properties of pyruvate kinase from the Cyanobacterium synechococcus PCC 6301; Knowles VL et al.; Pyruvate kinase (PK) from the cyanobacterium Synechococcus PCC 6301 was purified 1,300-fold to electrophoretic homogeneity and a final specific activity of 222 micromol of pyruvate produced/min/mg of protein . The enzyme was shown to have a pI of 5.7 and to exist as a 280-kDa homotetramer composed of 66-kDa subunits . This PK appears to be immunologically related to Bacillus PK and a green algal chloroplast PK, but not to rabbit muscle PK, or vascular plant cytosolic and plastidic PKs . The N-terminal amino acid sequence of the Synechococcus PK exhibited maximal (67%) identity with the corresponding region of a putative PK-A sequence deduced from the genome of the cyanobacterium, Synechocystis PCC 6803 . Synechococcus PK was relatively heat-labile and displayed a broad pH optimum around pH 7.0 . Its activity was not influenced by K(+), but required high concentrations of Mg(2+), and was relatively nonspecific with respect to the nucleoside diphosphate substrate . Potent allosteric regulation by various effectors was observed (activators: hexose monophosphates, ribose 5-phosphate, glycerol 3-phosphate, and AMP; inhibitors: fructose 1,6-bisphosphate, inorganic phosphate, ATP, and several Krebs' cycle intermediates) . The enzyme exhibited marked positive cooperativity for phosphoenolpyruvate, which was eliminated or reduced by the presence of the allosteric activators . The results are discussed in terms of the phylogeny and probable central role of PK in the control of cyanobacterial glycolysis.

Fresenius J Anal Chem, 2001 Feb, 369(3-4), 280 - 5
Improving the activity of immobilized subtilisin by site-directed attachment through a genetically engineered affinity tag; Wang J et al.; An octapeptide affinity tag, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (temied FLAG), was genetically fused to the C-terminus of subtilisin BPN' (SBT) from Bacillus amyloliquefaciens . The fusion protein SBT-FLAG was immobilized to nonporous polystyrene and silica beads both in a site-directed and a random fashion . Site-directed immobilization was achieved by employing the interaction between protein A and a monoclonal antibody specific for the FLAG peptide, while random immobilization was obtained by using glutaraldehyde as a cross-linking reagent . The activity of the immobilized enzymes was compared . It was found that the site-directed subtilisin had higher catalytic efficiency, kcat/KM, which was more than 7-fold of that of the randomly immobilized enzyme . It was also noted that the site-directly immobilized enzyme had superior storage stability over the homogeneous enzyme.

Microbiol Immunol, 2001, 45(2), 111 - 7
Cytokine responses to recombinant cholera toxin B subunit produced by Bacillus brevis as a mucosal adjuvant; Maeyama J et al.; We attempted to clarify the mechanism of the mucosal adjuvanticity of recombinant cholera toxin B subunit (rCTB), which is inherently uncontaminated with the holotoxin produced by Bacillus brevis and has a powerful mucosal adjuvant activity, on cytokine responses compared with that of cholera toxin (CT) . rCTB had no ability to stimulate cyclic AMP formation in mouse peritoneal macrophages (Mphi) . Cytokine production by non-immunized Mphi cultured with rCTB or CT and by the spleen cells of mice co-immunized intranasally with ovalbumin (OVA) and rCTB or CT was examined . rCTB alone did not induce interleukin (IL)-1alpha/beta or IL-6 production by Mphi, but combination of rCTB with lipopolysaccharide (LPS) enhanced both IL-1alpha/beta production . Conversely, CT plus LPS suppressed IL-1alpha/beta production more than LPS alone . Both rCTB and CT suppressed IL-12 secretion induced by interferon gamma (IFN gamma) plus LPS . IL-2, IL-4, IL-5, and IL-10 were secreted by mouse spleen cells restimulated with OVA after intranasal co-administration of OVA together with rCTB, and in response to CT, the same cytokines were secreted . The different effect of rCTB on Mphi from that of CT may mean a difference between the mechanisms of rCTB and CT during the early stage of an immune response.

J Immunol, 2001 Apr 15, 166(8), 4922 - 30
Modulation of inhaled antigen-induced IgE tolerance by ongoing Th2 responses in the lung; Hurst SD et al.; The normal response to inhaled Ag is the absence of Ag-specific IgE and cytokine production to later Ag challenges . Although the mechanism of this aerosol-induced IgE tolerance is not completely understood, it may prevent sensitization to inhaled Ags, which could otherwise lead to allergy and asthma . We examined the consequences of ongoing Th1 and Th2 responses in the lungs of mice during OVA inhalation to mimic conditions that may subvert tolerance and lead to sensitization . We found that concurrent, secondary Th2 lung responses to keyhole limpet hemocyanin or primary responses to Nippostrongylus larvae or Asperigillus fumagatus extract prevented establishment of IgE tolerance to aerosolized OVA . Intranasal rIL-4 given before OVA aerosolization also prevented establishment of tolerance, whereas concurrent Th1 responses to influenza virus or Mycobacterium bovis bacillus Calmette-Guerin had no effect . However, once established, aerosol tolerance to OVA could not be completely broken by OVA rechallenge concurrent with a secondary Th2 response to keyhole limpet hemocyanin or A . fumagatus extract, or by intranasal rIL-4 . These data suggest that the immune status of the lung of an individual may profoundly influence the initial response to inhaled Ag, and that aerosol-induced IgE tolerance may not be appropriately established in individuals undergoing concurrent, Th2-mediated responses to Ags or pathogens.

Southeast Asian J Trop Med Public Health, 2000 Sep, 31(3), 482 - 6
Safety and immunogenicity of Bacillus Calmette-Guerin vaccine in children born to HIV-1 infected women; Thaithumyanon P et al.; This prospective cohort study was conducted to determine the complication of Bacillus Calmette-Guerin (BCG) vaccination given to newborn infants born to HIV-1 seropositive mothers and to compare the tuberculin reaction 9 months after BCG vaccination between HIV-1 infected and non infected children . Two hundred and twenty-three infants with BCG immunization at birth were examined . No BCG complication was noted . Tuberculin skin tests were performed on 126 children (56.5%) . Eleven of them were excluded because of failure to have skin tests read at 48 hours . Of the 115 infants enrolled to this study, 15 (13%) had no BCG scar and 50 (43.5%) had no tuberculin reaction . Twenty-six children were classified as group 1 or HIV-1 infected children and 89 children were group 2 or HIV-1 non infected . Group 1 children had a smaller tuberculin skin response (X+SD) than group 2 (1.15 +/- 2.82 vs 4.64 +/- 4.29 mm; p < 0.0001) . Mean weight + SD of group 1 children was also significantly less than those in group 2 (8,013 +/- 741 vs 8,540 +/- 984 g; p < 0.05) . The proportion of children with non reactivity to the tuberculin test, a negative tuberculin test and no BCG scar in group 1 was significantly higher than that in group 2 (76.9% vs 33.7%, 92.3% vs 52.8% and 36.4% vs 6.7% respectively; p < 0.0001 for all) . But, the proportion of non reactivity to the tuberculin test in children with or without BCG scar of each group was not different (p > 0.05) . Positive tuberculin tests were 7.7% and 47.2% in group 1 and 2 respectively . None of the children with positive tuberculin tests had clinical evidence of tuberculosis . The findings of this study indicate that BCG vaccine given to newborn infants of HIV-1 seropositive mothers is safe . Although tuberculin skin responses of HIV-1 infected children are less than those of HIV-1 non-infected children, it is possible that BCG vaccine might protect these children from developing severe tuberculosis.

Rev Sci Tech, 2001 Apr, 20(1), 112 - 32
Vaccination of animals against Mycobacterium bovis; Skinner MA et al.; Vaccination could potentially be used as a practical means of controlling bovine tuberculosis in countries in which a wildlife reservoir of the disease is present, and also in those countries which cannot afford conventional control strategies . An understanding of the processes involved in the protective immune response to tuberculosis is desirable for the rational development and testing of new vaccines for tuberculosis . The authors review current knowledge regarding the processes involved in protective immune responses to tuberculosis, much of which has been derived from studies in mice . This knowledge is discussed in relation to the problem of using vaccination to induce protective immunity in cattle, deer and wildlife . Challenge models have now been developed to test candidate vaccines in many domestic animals and wildlife species and these models are being used to evaluate tuberculosis vaccines . Most studies of the efficacy of tuberculosis vaccines in target animals have focused on the use of bacillus Calmette-Guerin (BCG), an attenuated strain of Mycobacterium bovis . Recent advances in immunology and the molecular biology of mycobacteria have greatly increased the options for candidate vaccines and future studies will test new types of vaccines including new attenuated strains of M . bovis, sub-unit protein vaccines and recombinant deoxyribonucleic acid vaccines . Several of these vaccines have shown promising results when tested in small animal models . Although progress has been made in the development of vaccine delivery systems for animals, the technical problems associated with vaccination of wildlife remain a challenge.

Proteins, 2001 May 15, 43(3), 327 - 35
Enzymatic circularization of a malto-octaose linear chain studied by stochastic reaction path calculations on cyclodextrin glycosyltransferase; Uitdehaag JC et al.; Cyclodextrin glycosyltransferase (CGTase) is an enzyme belonging to the alpha-amylase family that forms cyclodextrins (circularly linked oligosaccharides) from starch . X-ray work has indicated that this cyclization reaction of CGTase involves a 23-A movement of the nonreducing end of a linear malto-oligosaccharide from a remote binding position into the enzyme acceptor site . We have studied the dynamics of this sugar chain circularization through reaction path calculations . We used the new method of the stochastic path, which is based on path integral theory, to compute an approximate molecular dynamics trajectory of the large (75-kDa) CGTase from Bacillus circulans strain 251 on a millisecond time scale . The result was checked for consistency with site-directed mutagenesis data . The combined data show how aromatic residues and a hydrophobic cavity at the surface of CGTase actively catalyze the sugar chain movement . Therefore, by using approximate trajectories, reaction path calculations can give a unique insight into the dynamics of complex enzyme reactions .

J Virol, 2001 May, 75(9), 4184 - 94
Polyadenylation in rice tungro bacilliform virus: cis-acting signals and regulation; Rothnie HM et al.; The polyadenylation signal of rice tungro bacilliform virus (RTBV) was characterized by mutational and deletion analysis . The cis-acting signals required to direct polyadenylation conformed to what is known for plant poly(A) signals in general and were very similar to those of the related cauliflower mosaic virus . Processing was directed by a canonical AAUAAA poly(A) signal, an upstream UG-rich region considerably enhanced processing efficiency, and sequences downstream of the cleavage site were not required . When present at the end of a transcription unit, the cis-acting signals for 3'-end processing were highly efficient in both monocot (rice) and dicot (Nicotiana plumbaginifolia) protoplasts . In a promoter-proximal position, as in the viral genome, the signal was also efficiently processed in rice protoplasts, giving rise to an abundant "short-stop" (SS-) RNA . The proportion of SS-RNA was considerably lower in N . plumbaginifolia protoplasts . In infected plants, SS-RNA was hardly detectable, suggesting either that SS-RNA is unstable in infected plants or that read-through of the promoter-proximal poly(A) site is very efficient . SS-RNA is readily detectable in transgenic rice plants (A . Kloti, C . Henrich, S . Bieri, X . He, G . Chen, P . K . Burkhardt, J . Wunn, P . Lucca, T . Hohn, I . Potrylus, and J . Futterer, 1999 . Plant Mol . Biol . 40:249-266), thus the absence of SS-RNA in infected plants can be attributed to poly(A) site bypass in the viral context to ensure production of the full-length pregenomic viral RNA . RTBV poly(A) site suppression thus depends both on context and the expression system; our results suggest that the circular viral minichromosome directs assembly of a transcription-processing complex with specific properties to effect read-through of the promoter-proximal poly(A) signal.

FEMS Microbiol Lett, 2001 Apr 1, 197(1), 47 - 51
CytK toxin of Bacillus cereus forms pores in planar lipid bilayers and is cytotoxic to intestinal epithelia; Hardy SP et al.; CytK is a cytotoxin isolated from a strain of Bacillus cereus cultured from cases of necrotic enteritis and the amino acid sequence of the protein suggests that it may belong to the family of beta-barrel pore-forming toxins . We show here in planar lipid bilayers the toxin is able to form pores which are weakly anion selective and exhibit an open channel probability close to one . The predicted minimum pore diameter is approximately 7 A . We also show that cytK is a potent cytotoxin against human intestinal Caco-2 epithelia . CytK, like other beta-barrel pore-forming toxins, spontaneously forms oligomers which are resistant to sodium dodecyl sulphate (SDS), but not to boiling . CytK represents a pore-forming toxin linked with human cases of necrotic enteritis.

Trends Mol Med, 2001 Mar, 7(3), 135 - 7
Disease model: pulmonary tuberculosis; McMurray DN; In spite of a massive effort to apply the tools currently available for tuberculosis (TB) control, both in this country and abroad, it is clear that complicating factors {for example, HIV co-infection, drug resistance, lack of patient compliance with chemotherapy, variable efficacy of Bacille Calmette-Guerin (BCG) vaccine} will prevent disease control unless new drugs, vaccines and diagnostic tests are developed (1) . The publication of the complete genome sequence of Mycobacterium tuberculosis in 1998 (2) has facilitated a directed search for virulence genes, new drug targets, and vaccine antigens . This research effort has been made possible by the availability of highly biologically relevant animal models of pulmonary TB ((3)).

Trends Immunol, 2001 Mar, 22(3), 160 - 8
TB vaccines: progress and problems; Andersen P; Tuberculosis (TB) is the biggest killer worldwide of any infectious disease, a situation worsened by the advent of the HIV epidemic and the emergence of multi-drug resistant strains of Mycobacterium tuberculosis . The existing vaccine, Mycobacterium bovis bacille Calmette-Guerin (BCG), has proven inefficient in several recent field trials . There is currently intense research using cutting-edge vaccine technology to combat this ancient disease . However, it is necessary to understand why BCG has failed before we can rationally develop the next generation of vaccines . Several hypotheses that might explain the failure of BCG and the strategies designed to address these shortcomings are discussed.

Mol Phylogenet Evol, 2001 Apr, 19(1), 157 - 63
The mitochondrial cytochrome oxidase II gene in Bacillus stick insects: ancestry of hybrids, androgenesis, and phylogenetic relationships; Mantovani B et al.; Sequencing of a cytochrome oxidase II (COII) gene fragment in Bacillus taxa provided evidence that the bisexual B . rossius is the maternal ancestor of the hybridogenetic B . rossius-grandii strains and revealed the same ancestry for both parthenogenetic hybrids: the diploid B . whitei (B . rossius/grandii grandii) and the triploid B . lynceorum (B . rossius/grandii grandii/atticus) . Present data clearly demonstrate that all Bacillus unisexuals arose through asymmetrical hybridization events and realized a paraphyletic derivation from the B . rossius redtenbacheri subspecies . The invention of B . rossius mitochondrial DNA haplotypes in specimens with B . grandii grandii nuclear genomes revealed the occurrence of androgenesis in nature . Natural androgens represent a peculiar escape from hybridity and can help maintain the hybridogenetic system through the production of the fathering taxon via hybrid females . Results from the COII gene support the phyletic relationships among taxa suggested by previous taxonomical approaches, but also indicate a departure of B . grandii subspecies from the established taxonomy . Assuming the existence of a molecular clock, the evaluated substitution rate brings the splitting between B . rossius and B . grandii/B . atticus back to 22.79 +/- 2.65 myr before present, while the origin of hybrids appears to be much more recent (1.06 +/- 0.53 myr) .

Mem Inst Oswaldo Cruz, 2001 Feb, 96(2), 257 - 63
Expression of mosquito active toxin genes by a Colombian native strain of the gram-negative bacterium Asticcacaulis excentricus; Romero M et al.; Mosquito control with biological insecticides, such as Bacillus sp . toxins, has been used widely in many countries . However, rapid sedimentation away from the mosquito larvae feeding zone causes a low residual effect . In order to overcome this problem, it has been proposed to clone the Bacillus toxin genes in aquatic bacteria which are able to live in the upper part of the water column . Two strains of Asticcacaulis excentricus were chosen to introduce the B . sphaericus binary toxin gene and B . thuringiensis subsp . medellin cry11Bb gene cloned in suitable vectors . In feeding experiments with these aquatic bacteria, it was shown that Culex quinquefasciatus, Aedes aegypti, and Anopheles albimanus larvae were able to survive on a diet based on this wild bacterium . A . excentricus recombinant strains were able to express both genes, but the recombinant strain expressing the B . sphaericus binary toxin was toxic to mosquito larvae . Crude protease A . excentricus extracts did not degrade the Cry11Bb toxin . The flotability studies indicated that the recombinant A . excentricus strains remained in the upper part of the water column longer than the wild type Bacillus strains.

J Biol Chem, 2001 Jun 15, 276(24), 21704 - 13 Epub 2001 Apr 02.
Direct evidence for the size and conformational variability of the pyruvate dehydrogenase complex revealed by three-dimensional electron microscopy . The "breathing" core and its functional relationship to protein dynamics; Zhou ZH et al.; Structural studies by three-dimensional electron microscopy of the Saccharomyces cerevisiae truncated dihydrolipoamide acetyltransferase (tE(2)) component of the pyruvate dehydrogenase complex reveal an extraordinary example of protein dynamics . The tE(2) forms a 60-subunit core with the morphology of a pentagonal dodecahedron and consists of 20 cone-shaped trimers interconnected by 30 bridges . Frozen-hydrated and stained molecules of tE(2) in the same field vary in size approximately 20% . Analyses of the data show that the size distribution is bell-shaped, and there is an approximately 40-A difference in the diameter of the smallest and largest structures that corresponds to approximately 14 A of variation in the length of the bridge between interconnected trimers . Companion studies of mature E(2) show that the complex of the intact subunit exhibits a similar size variation . The x-ray structure of Bacillus stearothermophilus tE(2) shows that there is an approximately 10-A gap between adjacent trimers and that the trimers are interconnected by the potentially flexible C-terminal ends of two adjacent subunits . We propose that this springlike feature is involved in a thermally driven expansion and contraction of the core and, since it appears to be a common feature in the phylogeny of pyruvate dehydrogenase complexes, protein dynamics is an integral component of the function of these multienzyme complexes.

Carbohydr Res, 2001 Mar 9, 331(1), 13 - 8
Inhibition of beta-glycosidases by acarbose analogues containing cellobiose and lactose structures; Lee SB et al.; Acarbose analogues, containing cellobiose and lactose structures, were prepared by reaction of the two disaccharides with acarbose and Bacillus stearothermophilus maltogenic amylase . The kinetics for the inhibition by the two analogues was studied for beta-glucosidase, beta-galactosidase, cyclomaltodextrin glucanosyltransferase (CGTase), and alpha-glucosidase . Both analogues were potent competitive inhibitors for beta-glucosidase, with K(I) values in the range of 0.04-2.44 microM, and the lactose analogues were good uncompetitive inhibitors for beta-galactosidase, with K(I) values in the range of 159-415 microM, while acarbose was not an inhibitor for either enzyme at 10 and 5 mM, respectively . Both analogues were also potent mixed inhibitors for CGTase, with K(I) values in the range of 0.1-9.3 microM . The lactose analogue was a 6.4-fold better competitive inhibitor for alpha-glucosidase than was acarbose.

Microbiology, 2001 Apr, 147(Pt 4), 1017 - 24
Intracellular pH regulation by Mycobacterium smegmatis and Mycobacterium bovis BCG; Rao M et al.; Mycobacteria are likely to encounter acidic pH in the environments they inhabit; however intracellular pH homeostasis has not been investigated in these bacteria . In this study, Mycobacterium smegmatis and Mycobacterium bovis {Bacille Calmette--Guerin (BCG)} were used as examples of fast- and slow-growing mycobacteria, respectively, to study biochemical and physiological responses to acidic pH . M . smegmatis and M . bovis BCG were able to grow at pH values of 4.5 and 5.0, respectively, suggesting the ability to regulate internal pH . Both species of mycobacteria maintained their internal pH between pH 6.1 and 7.2 when exposed to decreasing external pH and the maximum Delta pH observed was approximately 2.1 to 2.3 units for both bacteria . The Delta pH of M . smegmatis at external pH 5.0 was dissipated by protonophores (e.g . carbonyl cyanide m-chlorophenylhydrazone), ionophores (e.g . monensin and nigericin) and N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the proton-translocating F(1)F(0)-ATPase . These results demonstrate that permeability of the cytoplasmic membrane to protons and proton extrusion by the F(1)F(0)-ATPase plays a key role in maintaining internal pH near neutral . Correlations between measured internal pH and cell viability indicated that the lethal internal pH for both strains of mycobacteria was less than pH 6.0 . Compounds that decreased internal pH caused a rapid decrease in cell survival at acidic pH, but not at neutral pH . These data indicate that both strains of mycobacteria exhibit intracellular pH homeostasis and this was crucial for the survival of these bacteria at acidic pH values.

Int Immunol, 2001 Apr, 13(4), 451 - 8
Dendritic cells infected with Mycobacterium bovis bacillus Calmette Guerin activate CD8(+) T cells with specificity for a novel mycobacterial epitope; Feng CG et al.; Although CD4(+) T cells are essential for protective immunity against Mycobacterium tuberculosis infection, recent reports indicate that CD8(+) T cells may also play a critical role in the control of this infection . However, the epitope specificity and the mechanisms of activation of mycobacteria-reactive CD8(+) T cells are poorly characterized . In order to study the CD8(+) T cell responses to the model mycobacterial antigen, MPT64, we used recombinant vaccinia virus expressing MPT64 (VVWR-64) and a panel of MPT64-derived peptides to establish that the peptide MPT64(190-198) contains an H-2D(b)-restricted CD8(+) T cell epitope . A cytotoxic T lymphocyte response to this peptide could be demonstrated in M . bovis bacillus Calmette Guerin (BCG)-infected mice following repeated in vitro stimulation . When bone marrow-derived dendritic cells (DC) were infected with BCG, the expression of MHC class I molecules by DC was up-regulated in parallel with MHC class II and B7-2, whereas CD1d expression level was not modified . Moreover, BCG-infected DC activated MPT64(190-198)-specific CD8(+) T cells to secrete IFN-gamma, although with a lower efficacy than VVWR-64-infected DC . The production of IFN-gamma by MPT64(190-198)-specific CD8(+) T cells was inhibited by antibodies to MHC class I, but not to CD1d . These data suggest that mycobacteria-specific CD8(+) T cells are primed during infection . Therefore, anti-mycobacterial vaccine strategies targeting the activation of specific CD8(+) T cells by DC may have improved protective efficacy.

Appl Environ Microbiol, 2001 Apr, 67(4), 1956 - 8
Lack of cross-resistance to Cry19A from Bacillus thuringiensis subsp . jegathesan in Culex quinquefasciatus (Diptera: Culicidae) resistant to cry toxins from Bacillus thuringiensis subsp . israelensis; Wirth MC et al.; Culex quinquefasciatus mosquitoes with high levels of resistance to single or multiple toxins from Bacillus thuringiensis subsp . israelensis were tested for cross-resistance to the Bacillus thuringiensis subsp . jegathesan polypeptide Cry19A . No cross-resistance was detected in mosquitoes that had been selected with the Cry11A, Cry4A and Cry4B, or Cry4A, Cry4B, Cry11A, and CytA toxins . A low but statistically significant level of cross-resistance, three to fourfold, was detected in the colony selected with Cry4A, Cry4B, and Cry11A . This cross-resistance was similar to that previously detected with B . thuringiensis subsp . jegathesan in the same colony . These data help explain the toxicity of B . thuringiensis subsp . jegathesan against the resistant colonies and indicate that the Cry19A polypeptide might be useful in managing resistance and/or as a component of synthetic combinations of mosquitocidal toxins.

Appl Environ Microbiol, 2001 Apr, 67(4), 1744 - 50
Novel alpha-amylase that is highly resistant to chelating reagents and chemical oxidants from the alkaliphilic Bacillus isolate KSM-K38; Hagihara H et al.; A novel alpha-amylase (AmyK38) was found in cultures of an alkaliphilic Bacillus isolate designated KSM-K38 . Based on the morphological and physiological characteristics and phylogenetic position as determined by 16S ribosomal DNA gene sequencing and DNA-DNA reassociation analysis, it was suggested that the isolate was a new species of the genus Bacillus . The enzyme had an optimal pH of 8.0 to 9.5 and displayed maximum catalytic activity at 55 to 60 degrees C . The apparent molecular mass was approximately 55 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was around pH 4.2 . This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltohexaose, maltoheptaose, and, in addition, maltose as major end products after completion of the reaction . The activity was not prevented at all by EDTA and EGTA at concentrations as high as 100 mM . Moreover, AmyK38 was highly resistant to chemical oxidation and maintained more than 80% of its original activity even after incubation for 1 h in the presence of excess H2O2 (1.8 M).

Appl Environ Microbiol, 2001 Apr, 67(4), 1693 - 9
Sensitivities of germinating spores and carvacrol-adapted vegetative cells and spores of Bacillus cereus to nisin and pulsed-electric-field treatment; Pol IE et al.; Treatment of Bacillus cereus spores with nisin and/or pulsed-electric-field (PEF) treatment did not lead to direct inactivation of the spores or increased heat sensitivity as a result of sublethal damage . In contrast, germinating spores were found to be sensitive to PEF treatment . Nisin treatment was more efficient than PEF treatment for inactivating germinating spores . PEF resistance was lost after 50 min of germination, and not all germinated spores could be inactivated . Nisin, however, was able to inactivate the germinating spores to the same extent as heat treatment . Resistance to nisin was lost immediately when the germination process started . A decrease in the membrane fluidity of vegetative cells caused by incubation in the presence of carvacrol resulted in a dramatic increase in the sensitivity to nisin . On the other hand, inactivation by PEF treatment or by a combination of nisin and PEF treatments did not change after adaptation to carvacrol . Spores grown in the presence of carvacrol were not susceptible to nisin and/or PEF treatment in any way.

Appl Environ Microbiol, 2001 Apr, 67(4), 1453 - 60
Comparative study of the cyclization reactions of three bacterial cyclomaltodextrin glucanotransferases; Terada Y et al.; The actions of cyclomaltodextrin glucanotransferases (CGTase; EC 2.4.1.19) from alkalophilic Bacillus sp . strain A2-5a (A2-5a CGTase), Bacillus macerans (Bmac CGTase), and Bacillus stearothermophilus (Bste CGTase) on amylose were investigated . All three enzymes produced large cyclic alpha-1,4-glucans (cycloamyloses) at the early stage of the reaction, but these were subsequently converted into smaller cycloamyloses . However, the rates of this conversion differed among the three enzymes . The product specificity of each CGTase in the cyclization reaction was determined by measuring the amount of each cycloamylose from CD6 to CD31 (CDn, a cycloamylose with a degree of polymerization of n) . A2-5a CGTase produced 10 times more CD7, while Bmac CGTase produced 34 times more CD6 than other cycloamyloses . Bste CGTase produced 12 and 3 times more CD6 and CD7 than other cycloamyloses, respectively . The substrate specificities of the linearization reactions of CD6, CD7, CD8, and larger cycloamyloses (a mixture of CD22 to CD50) were investigated, and we found that CD7 and CD8 are extremely poor substrates for both hydrolytic and transglycosidic linearization (coupling) reactions while larger cycloamyloses are linearized at a much higher rate . By repeating these cyclization and linearization reactions, the larger cycloamyloses initially produced are converted into smaller cycloamyloses and finally into mainly CD6, CD7, and CD8 . These three enzymes also differ in their hydrolytic activities, which seem to accelerate the conversion of larger cycloamyloses into smaller cycloamyloses.

Eur Urol, 2001 Mar, 39(3), 349 - 56
Nitric oxide synthase gene and protein expression are upregulated by Bacille Calmette-Guérin in the rat bladder; Oh BR et al.; OBJECTIVE: We hypothesize that Bacille Calmette-Guerin (BCG) may act through the regulation of various isoforms of nitric oxide synthase (NOS) {inducible (iNOS), endothelial (eNOS), and neuronal (nNOS)} genes and protein expression in rat bladder . METHODS: Adult female Sprague-Dawley rats (200-250 g) were injected transurethrally with BCG (22 rats) and phosphate-buffered saline (22 control rats), and after 2, 4, 6, and 12 h, and 1, 2, 3, 5, 7, 10, and 14 days, the bladders were harvested . Normal and BCG-treated rat bladders were analyzed for mRNA expression for iNOS, eNOS, and nNOS by reverse-transcriptase polymerase chain reaction . Protein expression was determined by Western blotting and immunohistochemistry . RESULTS: mRNA expression for iNOS was induced after 2 h of BCG injection in the rat bladder . Gene expression for iNOS was highest at 6 h to 1 day followed by decreased expression, reaching its lowest level at 5 days . eNOS mRNA expression was detected in control bladders, but its level was higher in the BCG-treated animals . nNOS mRNA expression was present in all samples, but did not change after BCG treatment . Western blotting confirmed these findings . Immunohistochemistry analysis demonstrated that eNOS was present mainly in endothelium, while iNOS was detected in stroma and nNOS in epithelium and smooth muscle of the rat bladder . CONCLUSION: The present study demonstrates, for the first time, that BCG treatment up-regulates gene and protein expression of iNOS and eNOS in normal rat bladders, suggesting that BCG action may be mediated through NOS pathways.

J Biochem (Tokyo), 2001 Apr, 129(4), 593 - 8
Crystal structure of cyclodextrin glucanotransferase from alkalophilic Bacillus sp . 1011 complexed with 1-deoxynojirimycin at 2.0 A resolution; Kanai R et al.; 1-Deoxynojirimycin, a pseudo-monosaccharide, is a strong inhibitor of glucoamylase but a relatively weak inhibitor of cyclodextrin glucanotransferase (CGTase) . To elucidate this difference, the crystal structure of the CGTase from alkalophilic Bacillus sp . 1011 complexed with 1-deoxynojirimycin was determined at 2.0 A resolution with the crystallographic R value of 0.154 (R(free) = 0.214) . The asymmetric unit of the crystal contains two CGTase molecules and each molecule binds two 1-deoxynojirimycins . One 1-deoxynojirimycin molecule is bound to the active center by hydrogen bonds with catalytic residues and water molecules, but its binding mode differs from that expected in the substrate binding . Another 1-deoxynojirimycin found at the maltose-binding site 1 is bound to Asn-667 with a hydrogen bond and by stacking interaction with the indole moiety of Trp-662 of molecule 1 or Trp-616 of molecule 2 . Comparison of this structure with that of the acarbose-CGTase complex suggested that the lack of stacking interaction with the aromatic side chain of Tyr-100 is responsible for the weak inhibition by 1-deoxynojirimycin of the enzymatic action of CGTase.

Trends Genet, 2001 Apr, 17(4), 193 - 9
How Bacillus thuringiensis has evolved specific toxins to colonize the insect world; de Maagd RA et al.; Bacillus thuringiensis is a bacterium of great agronomic and scientific interest . Together the subspecies of this bacterium colonize and kill a large variety of host insects and even nematodes, but each strain does so with a high degree of specificity . This is mainly determined by the arsenal of crystal proteins that the bacterium produces during sporulation . Here we describe the properties of these toxin proteins and the current knowledge of the basis for their specificity . Assessment of phylogenetic relationships of the three domains of the active toxin and experimental results indicate how sequence divergence in combination with domain swapping by homologous recombination might have caused this extensive range of specificities.

Clin Imaging, 2000 Jul-Aug, 24(4), 197 - 9
Lemierre's syndrome presenting as multiple lung abscesses; Shaham D et al.; Lemierre's syndrome is thrombophlebitis of the internal jugular vein (IJV), complicating an oropharingeal infection . The causative organism is Fusobacterium, an anaerobic bacillus, and the syndrome typically occurs in previously healthy teenagers and young adults . Thromboembolic metastases are a common sequela, and the lungs are most frequently affected . We present a case of a 25-year-old woman, who presented with multiple lung abscesses, in whom IJV thrombophlebitis was subsequently noted.

Immunol Lett, 2001 Mar 1, 76(2), 79 - 88
The response of human dendritic cells to recombinant adenovirus, recombinant Mycobacterium bovis Bacillus Calmette Guerin and biolistic methods of antigen delivery: different induction of contact-dependant and soluble signals; Smith SG et al.; Dendritic cells (DCs) are the most potent antigen presenting cells for inducing T-cell immune responses . The ability to grow human DCs from monocyte precursors provides an abundant source of these cells, which can be modified in vitro to present antigens . Re-administration of modified DCs to patients as vaccines has been shown in some cases to induce immune responses against cancer and infectious disease . Gene delivery to DCs provides an intracellular source of antigen for efficient and persistent loading of major histocompatibility complex (MHC) class I molecules . The aim of this study was to use monocyte-derived DCs (MD-DCs) from healthy donors to compare in vitro gene transfer, mediated by adenovirus, M . bovis Bacillus Calmette Guerin (BCG) and biolistic delivery . Efficiency of transfection and effect on DC phenotype, allostimulatory capacity and cytokine secretion was investigated . Adenovirus and BCG both showed a comparable ability to transfect MD-DCs, whereas the biolistic delivery by gene gun was unsuccessful in the reporter gene delivery . BCG transfection promoted MD-DC maturation as is apparent in the surface phenotype, allostimulatory capacity and cytokine secretion from cells . In comparison, adenovirus and biolistic delivery had a reduced effect on MD-DCs although enhancement of co-stimulatory and MHC molecule expression occurred in the cells of some donors . Both BCG and adenovirus represent useful vectors for gene transfer to human DCs . The effect of BCG on DC maturation may provide additional signals for the induction of antigen-specific T-cell responses.

J Biol Chem, 2001 Jun 22, 276(25), 23065 - 76 Epub 2001 Mar 26.
Mycobacterium bovis Bacillus Calmette-Guerin and its cell wall complex induce a novel lysosomal membrane protein, SIMPLE, that bridges the missing link between lipopolysaccharide and p53-inducible gene, LITAF(PIG7), and estrogen-inducible gene, EET-1; Moriwaki Y et al.; LITAF and PIG7 encode an identical protein, and they have recently been reported as lipopolysaccharide and p53-inducible genes, respectively . By using the differential display approach, we identified a Mycobacterium bovis BCG cell wall skeleton (BCG-CWS)-inducible gene fragment from human monocytes, showing no homology to any reported gene . Full-length cloning of this fragment reveals the following . 1) The differential display product represents the incomplete 3'-untranslated region of LITAF/PIG7 . 2) The coding region of the transcript differs from LITAF/PIG7 due to an absence of a single guanine residue, resulting in a potential translational frameshift . 3) The newly coded protein turns out to be 86% identical and 90% similar to an estrogen-inducible rat gene, EET-1 . Repeated analysis, expressed sequence tag search, comparison with homologues, and genome sequence analysis confirmed the absence of the single guanine residue . One interesting feature of this protein is that it possesses the RING domain signature and is predicted to be localized in the nucleus . However, detailed analysis together with experimental evidence suggests it is neither a RING family member nor a nuclear protein . Comparison of a total collection of 18 proteins from various species indicates that proteins of this family are small in size and mainly conserved at the C-terminal domain with a unique motif . We characterize this novel protein as an unglycosylated small integral membrane protein of the lysosome/late endosome (SIMPLE) whose expression is elicited in monocytes by live and heat-killed BCG, BCG cell wall complex, lipopolysaccharide, and tumor necrosis factor-alpha . To our knowledge this is the first report of pathogen-associated molecular pattern (PAMP)-induced differential expression of a lysosomal membrane protein presumably involved in apoptosis.

Microb Pathog, 2001 Mar, 30(3), 129 - 38
Identification of the mycobacterial DNA-binding protein 1 region which suppresses transcription in vitro; Furugen M et al.; We recently identified a novel DNA-binding protein from Mycobacterium bovis bacillus Calmette-Guerin (BCG), termed mycobacterial DNA-binding protein 1 (MDP1) . MDP1 inhibited the in vitro syntheses of DNA, RNA and protein, and reduced growth rates of bacteria transformed with MDP1 genes . In this study, we examined the DNA binding regions of MDP1 by using a set of synthetic peptides . One dominant region was determined on peptide 4 composed of amino acids, at positions 31-50 . The peptide 4 inhibited syntheses of both DNA and RNA in vitro . The critical amino acids residues for these functions were analysed utilizing synthetic peptides substituted with Ala . This domain was perfectly conserved in MDP1 homologues in mycobacteria, but not observed in other known DNA binding proteins . These results indicate mycobacteria possess a unique nuclear protein, which might be involved in growth regulation of these organisms .

J Invertebr Pathol, 2001 Feb, 77(2), 92 - 8
The pathogenicity of Bacillus thuringiensis ssp . kurstaki to gamma-irradiated Cadra cautella (Walker) (Lepidoptera: Phycitidae); Faruki SI et al.; The present investigation deals with the effects of Bacillus thuringiensis ssp . kurstaki on various biological parameters of gamma-irradiated Cadra cautella . The pathogen, irradiation, and their combinations significantly affected the insects by increasing their mortality and developmental periods, reducing the pupal and adult survival (%), and the adult longevity and reproductive potential were also significantly reduced . It was observed that irradiation-pathogen combinations produced additive to synergistic effects on C . cautella .

Semin Oncol, 2001 Feb, 28(1 Suppl 1), 9 - 13
National Surgical Adjuvant Breast and Bowel Project trials in colon cancer; Wolmark N et al.; During the last decade, the National Surgical Adjuvant Breast and Bowel Project (NSABP) has completed six adjuvant chemotherapy trials comparing different adjuvant therapy regimens or adjuvant therapy versus surgery alone . A seventh trial is ongoing . These trials have contributed to defining the role of adjuvant therapy in colon cancer . Patients eligible for inclusion in NSABP trials had been diagnosed as having stage II or III colon cancer with no evidence of gross residual or metastatic disease . The follow-up strategies were similar in the reported trials with follow-up every 3 months for the first 2 years, then every 6 months for the next 3 to 5 years, and annually thereafter . The NSABP C-01 protocol was a three-arm trial comparing an adjuvant semustine/vincristine/5-fluorouracil (5-FU) regimen (MOF) to a Bacille Calmette-Guerin treatment, and to surgery alone . The C-02 protocol investigated whether portal vein infusion of 5-FU improved survival outcome compared with surgery alone . Protocol C-03 compared a semustine/vincristine/5-FU regimen to a 5-FU plus leucovorin (LV) (5-FU/LV) regimen . The NSABP C-04 protocol was a three-arm trial comparing 5-FU/LV, 5-FU plus levamisole, and 5-FU/LV plus levamisole . The NSABP C-05 trial compared 5-FU/LV to 5-FU/LV plus alpha-interferon . Results of NSABP C-01, C-02, C-03, C-04, and C-05 trials are summarized in this report . Patient accrual has completed in the NSABP C-06 trial comparing 5-FU/LV with oral tegafur and plus uracil leucovorin . The NSABP is currently conducting another trial (C-07) comparing 5-FU/LV with 5-FU/LV plus oxaliplatin . The role of adjuvant chemotherapy in stage II colon cancer is also discussed in this report . A recent pooled analysis of studies C-01, C-02, C-03, and C-04 has indicated that the relative treatment benefit in stage II disease is at least equal to the benefit in stage III colon cancers, and concluded that adjuvant chemotherapy also should be considered as the standard of care for stage II colon cancer patients.

J Assoc Physicians India, 1998 Apr, 46(4), 345 - 50
Association of HLA B27 antigen in Indian patients of ankylosing spondylitis and other autoimmune diseases; Kankonkar SR et al.; One thousand three hundred and forty clinically suspected patients of Ankylosing Spondylitis (AS) and other autoimmune diseases and 5000 controls were studied to detect the association of HLA B27 antigen amongst them . Other alleles studied include HLA B7, B40 (B60), B22(B55), B13, etc . Our findings show a considerable and consistent association of HLA B27 with AS irrespective of the community to which the patient, belonged his hygiene or socio-economic conditions . We also found that people in the age group of 21-39 were the most vulnerable, when number of affected individuals or severity of the disease were taken into consideration . Male members showed a preponderance over females in HLA B27 positivity . Detection of HLA B27 could help in the diagnosis of AS . Patients suffering from other autoimmune diseases such as rheumatoid arthritis, psoriasis, Reiter's syndrome and uveitis and patients with inflammatory bowel disease, colitis, eczema, bacillary or fungal infection were also found to be HLA B27 positive . A study of other alleles shows that even they sometimes associate AS and other autoimmune diseases.

Chemosphere, 2001 Mar, 42(8), 965 - 74
Risk assessment of etofenprox (Vectron) on non-target aquatic fauna compared with other pesticides used as Simulium larvicide in a tropical environment; Yameogo L et al.; Within the rotational scheme developed by the Programme to fight the resistance of Simulium damnosum to chemical larvicides, there was an operational gap at discharges between 5 and 70 m3 s(-1) for the treatment of rivers where resistance to organophosphates was present . The use of permethrin and carbosulfan was precluded because of risk of environmental impact and, Bacillus thuringiensis ser . H-14 treatments were not envisageable due to cost and logistics constraints . Among the possible complementary groups of larvicides tested, the pseudo-pyrethroids, held promise, because of a mode of action similar to that of pyrethroids, but along with a usually lower toxicity for fish . Etofenprox, one of the pseudo-pyrethroids tested, shows a global detachment of non-target insects in 24 h close to that of pyraclofos, an organo-phosphorus compound (27 against 23%) . In laboratory conditions, six times the operational dose which is 0.03 mg l(-1) 10 min, is needed to cause 50% mortality of Caridina sp . (a small shrimps species) and 30 times this same dose for 95% mortality . For fish species, a safety margin of 400-800 times the operational dose is observed for Oreochromis niloticus and 200-400 times for Tilapia zillii.

Biosci Biotechnol Biochem, 2001 Jan, 65(1), 226 - 8
A cryptic plasmid, pAO1, from a compost bacterium, Bacillus sp; Karita S et al.; The complete nucleotide sequence of a new cryptic plasmid, pAO1 isolated from a compost bacterium Bacillus sp., has been analyzed . Analysis of the PCR-based 16S rRNA sequence showed the bacterium harboring pAO1 was closely related to Bacillus pallidus . The plasmid pAO1 was 3,325 bp in size . Two open reading frames, ORF1 and ORF2, encoding putative polypeptides of 248 and 290 amino acids, respectively, were identified within the sequence . The ORF1 has a limited sequence similarity to an integrase/recombinase, while the ORF2 has high similarity with the replication protein of pBC1 from Bacillus coagulans . A putative origin sequence for a plus-strand was located between ORFs . Southern blot analysis indicates this plasmid replicates via a rolling circle-type mechanism.

Oncology (Huntingt), 2001 Jan, 15(1), 85 - 8; discussion 88-91
Maintenance therapy for superficial bladder cancer; Baselli EC et al.; Transurethral resection remains the standard for first-line treatment of transitional cell carcinoma of the bladder . This technique clearly defines the pathologic grade and is essential in determining the clinical stage of the bladder tumor . Intravesical therapy is an important adjunct to transurethral resection in the management of patients with superficial bladder cancer, many of whom are at risk for disease recurrence and progression . Pharmacotherapy consisting of cytotoxic and immunomodulating agents has demonstrated utility against superficial transitional cell carcinoma . Bacillus Calmette-Guerin and mitomycin (Mutamycin) remain the more commonly used and most effective agents in the prophylaxis against recurrence and progression of superficial bladder transitional cell carcinoma . Many studies have examined their efficacy at different schedules . This article reviews the traditional intravesical agents that are useful in the therapy and prophylaxis of superficial transitional cell carcinoma of the bladder . It also addresses their long-term efficacy when used as maintenance therapy in higher-risk patients.

Folia Microbiol (Praha), 2000, 45(3), 207 - 10
Purification and characterization of a thermostable alpha-amylase from Bacillus stearothermophilus; Chakraborty K et al.; A soil isolate of Bacillus stearothermophilus was found to synthesize thermostable alpha-amylase . The enzyme was purified to homogeneity by ammonium sulfate fractionation and IECC on DEAE-cellulose column . The purified enzyme was considered to be a monomeric protein with a molar mass of 64 kDa, as determined by SDS-PAGE . The enzyme showed a wide range of pH tolerance and maximum activity at pH 7.0 . The temperature tolerance was up to 100 degrees C with more than 90% catalytic activity; the maximum activity was observed at 50 degrees C . Divalent metal ions exhibited inhibitory effect on the enzyme activity . However, proteinase inhibitor did not react positively.

Asian Pac J Allergy Immunol, 2000 Sep, 18(3), 165 - 8
X-linked hyper IgM syndrome: a report of the first case in Thailand with a confirmed mutation of CD40 ligand gene; Santadusit S et al.; X-linked hyper IgM (XHIM) syndrome is a rare congenital immunodeficiency disease caused by failure of B cell to isotype switch from IgM to other classes of immunoglobulins in response to infections . Recently, a molecular cloning of the gene responsible for the syndrome, the CD40L gene has been accomplished and the gene was successfully mapped to the long arm of X chromosome at the position Xq26 . We, herein, report the first case of molecular proven XHIM in a Thai boy with a classic presentation and with a confirmed mutation of the CD40L gene . Case Report: A.S . was a 1 year 7 month old boy referred from Buriram Provincial Hospital for a work up and treatment for his recurrent infections consisted of chronic respiratory tract infections with otitis media (since 6 months of age), chronic diarrhea (since 9 months of age) and malnutrition (marasmus) secondary to his longstanding illnesses . He was a product of a consanguineous marriage but without history of similar illness observed in his pedigree . Abnormal laboratory works up included IgG of 300 mg/dl, IgA 10 mg/dl, IgM 1,635 mg/dl, positive stool examinations for Cryptosporidium, chronic colitis on radiographic gastrointestinal follow through study, a positive acid fast bacillus (AFB) stain of gastric aspirate and multiple positive bacterial cultures from various body sources . His anti-HIV serology was negative . His hospital course was significant for several bouts of infections of gastrointestinal, respiratory, and genitourinary systems . His treatment consisted of multiple courses of antibiotics, antituberculous drugs and IVIG administrations . His hospital course was complicated with feeding problem from an esophageal stricture requiring several esophageal dilatations . The analysis of CD40L gene revealed a point mutation of exon 5 (A619T) of the CD40L gene resulting in a stop codon confirming that indeed he had XHIM . He died with Pseudomonas septicemia during the waiting period for a bone marrow transplantation from a cord-blood stem cell.

Carbohydr Res, 2001 Feb 28, 330(4), 487 - 93
Enzymatic syntheses of T antigen-containing glycolipid mimicry using the transglycosylation activity of endo-alpha-N-acetylgalactosaminidase; Ashida H et al.; Thomsen-Friedenreich antigen (T antigen) disaccharide, beta-D-galactose-(1-->3)-alpha-N-acetyl-D-galactosamine (beta-D-Gal-(1-->3)-alpha-D-GalNAc), containing glycolipid mimicry was synthesized using the transglycosylation activity of endo-alpha-N-acetylgalactosaminidase from Bacillus sp . This enzyme could transfer the disaccharide from a p-nitrophenyl substrate to water-soluble 1-alkanols and other alcohols at a transfer ratio of 70% or more . Although the transfer ratios were lower for water-insoluble than water-soluble alcohols, they were shown to increase by adding sodium cholate to the reaction mixtures . The enzyme also transferred the disaccharide directly from asialofetuin to 1-alkanols . The anomeric bond between the disaccharide and 1-alkanols of the transglycosylation product is in the alpha configuration as determined by sequential digestion of jack bean beta-galactosidase and Acremonium alpha-N-acetylgalactosaminidase . Since the transglycosylation product, beta-D-Gal-(1-->3)-alpha-D-GalNAc-(1-->O)-hexyl, efficiently inhibits the binding of anti-T antigen monoclonal antibody to asialofetuin, it has potential as an agent for blocking T antigen-mediated cancer metastasis.

FEMS Microbiol Lett, 2001 Mar 15, 196(2), 245 - 9
Identification of genes regulated by prolonged acid exposure in Helicobacter pylori; Dong Q et al.; To investigate the influence of prolonged acid exposure on the gene expression, transcripts of Helicobacter pylori, grown under pH 5.5 and pH 7.4 for five successive passages, were analysed by differential display PCR . Eight genes were regulated by prolonged acid exposure . These genes included topA, tufB, ureB, flaA, atoE in the H . pylori genome and a cDNA fragment with 54% identity of the predicted amino acid sequence to a Bacillus cereus YkoW protein . The remaining two cDNA fragments had no significant homology to known sequences . Our data suggest that most of these genes might be required for the resistance of H . pylori to prolonged acid exposure.

FEMS Microbiol Lett, 2001 Mar 15, 196(2), 201 - 5
Initial degradation of dimethylphthalate by esterases from Bacillus species; Niazi JH et al.; Dimethylphthalate (DMP), one of the phthalate esters, is used in the manufacture of plasticizers, insect repellents, and synthetic fibers, and contributes to environmental pollution . In the present study, we report a novel bacterium belonging to the Bacillus sp., which has the ability to utilize DMP as the sole source of carbon . The esterases from the cell-free extract of the Bacillus de-esterified DMP . Native polyacrylamide gel electrophoresis showed the presence of four isoesterases designated Et1--4 . The isoesterases Et-4 and Et-1 showed a higher preference towards DMP hydrolysis as compared with Et-2 and 3 . A megaplasmid of about 60 kb was detected in this bacterium . The ability of this bacterium to utilize DMP as the sole source of carbon was lost upon plasmid curing . The isoesterases Et-1--4 were absent in the cell-free extracts of the cured bacterium . The results from our studies clearly demonstrate that de-esterification is the initial step in the degradation of DMP and the genes for these esterases seem to be harbored on the plasmid in this bacterium.

Enzyme Microb Technol, 2001 Apr 5, 28(6), 492 - 498
Purification and characterization of a Bacillus cereus exochitinase; Wang S et al.; Five extracellular chitinases of Bacillus cereus 6E1 were detected by a novel in-gel chitinase assay using carboxymethyl-chitin-remazol brilliant violet 5R (CM-chitin-RBV) as a substrate . The major chitinase activity was associated with a 36-kDa (Chi36) gel band . Chi36 was purified by a one-step, native gel purification procedure derived from the new in-gel chitinase assay . The purified Chi36 has optimal activity at pH 5.8 and retains some enzymatic activity between pH 2.5-8 . The temperature optimum for Chi36 was 35 degrees C, but the enzyme was active between 4-70 degrees C . Based on its ability to hydrolyze mainly p-nitrophenyl-(N-acetyl-beta-D-glucosaminide)(2), Chi36 is characterized as a chitobiosidase, a type of exochitinase . The N-terminal amino acid sequence of mature Chi36 was determined (25 amino acids) . Alanine is the first N-terminal amino acid residue indicating the cleavage of a signal peptide from a Chi36 precursor to form the mature extracellular Chi36 . The N-terminal sequence of Chi36 demonstrated highest similarity with Bacillus circulans WL-12 chitinase D and significant similarity with several other bacterial chitinases.

Bioresour Technol, 2001 May, 78(1), 89 - 94
Degumming of ramie fibers by alkalophilic bacteria and their polysaccharide-degrading enzymes; Zheng L et al.; Three strains of alkalophilic bacteria, Bacillus sp . NT-39, NT-53 and NT-76, were selected for the degumming of ramie fibers and production of polysaccharide-degrading enzymes . After 48 h of incubation with the strains, the loss of the gum might amount to 5.0% or more of the fibers and a number of polysaccharide-degrading enzymes were secreted to the culture supernatants . The residual gum of the fibers decreased to 9.4% after 5 h of enzymatic degumming . Analysis of gum contents and enzyme activities revealed that pectate lyase and xylanase played an important role in the degradation of residual gum . Enzymatic degumming resulted in an increment of 5.4 ISO units in fiber brightness, whereas the reduction in bundle breaking tenacity of the fibers was less than 5.% . The results confirmed that degumming of ramie fibers by alkalophilic bacteria and their enzymes had substantial advantages.

Anal Biochem, 2001 Apr 1, 291(1), 48 - 61
Mass spectrometry as a novel approach to probe cooperativity in multimeric enzymatic systems; Rogniaux H et al.; Investigating cooperativity in multimeric enzymes is of utmost interest to improve our understanding of the mechanism of enzymatic regulation . In the present article, we propose a novel approach based on mass spectrometry to probe cooperativity in the binding of a ligand to a multisubunit enzyme . This approach presents the selective advantage of giving a direct insight into all the subsequent ligation states that are formed in solution as the ligand is added to the enzyme . A quantitative interpretation of the electrospray ionization (ESI) mass spectra gives the relative abundance of all the distinct enzymatic species, which allows one to directly deduce the cooperativity of the system . The overall method is described for the addition of the oxidized cofactor nicotinamide adenine dinucleotide (NAD(+)) to a dimeric mutant of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase (GPDH) . It is then applied to four tetrameric enzymes: sturgeon muscle GPDH, wild type and S48G mutant of GPDH from B . stearothermophilus, and alcohol dehydrogenase (ADH) from Bakers yeast . The results illustrate the possibilities offered by this new technique . First, mass spectrometry allows a control of the enzymes before the addition of NAD(+) . Second, the cooperative behavior can be drawn from one single ESI mass spectrum, which makes the method highly attractive in terms of the amount of biological material required . Above all, the major benefit lies in the direct visualization of all the enzymatic species that are in equilibrium in solution . The direct measurement of cooperativity readily resolve the inconvenience of the classical approaches employed in this field, which all need to model the experimental data in order to get the cooperative behavior of the system .

Bull Acad Natl Med, 2000, 184(7), 1409 - 15; discussion 1415-6
{Genetic predisposition to infective diseases in humans . Mendelian predisposition to mycobacterial infections}; Casanova JL; Selective susceptibility to poorly pathogenic mycobacteria, such as bacille Calmette-Guerin (BCG) vaccine and environmental non-tuberculous mycobacteria (NTM), has long been suspected to be a mendelian disorder but its molecular basis has remained elusive . Recently, recessive mutations in the interferon gamma receptor ligand-binding chain (IFNgR1), interferon gamma receptor signalling chain (IFNgR2), interleukin 12 p40 subunit (IL-12p40), and interleukin 12 receptor beta 1 chain (IL-12Rb1) genes have been identified in a number of patients with disseminated BCG or NTM infection . Although genetically distinct, these conditions are immunologically related and highlight the essential role of interferon gamma-mediated immunity in the control of mycobacteria in man.

Kekkaku, 2001 Feb, 76(2), 59 - 69
{Problems and their transition of long-term hospitalized patients with tuberculosis}; Tuberculosis Research Committee (Ryoken).; In 1975, the Tuberculosis Research Committee (Ryoken) conducted its first study on long-term hospitalized TB patients who had been staying in a hospital for more than five years . Similar studies were repeated in 1981 (hospitalized for more than three years), 1988 (hospitalized for more than two years), 1993 and 1998 (hospitalized for more than one year) . The same patient cohorts in each study were followed up, each after a different time period, i.e., after 68 months for the 1975 study cohort, 36 months for the 1981 study cohort, 26 months for the 1988 study cohort, and 74 months for the 1993 study cohort . Based on the results of these series of studies, changes in the patients' characteristics and factors related to long-term hospitalization during the last 23 years were analyzed . The main findings are summarized below . 1) The proportion of patients who stayed in a hospital for more than one year in the study decreased from 41.6% in 1975 to 9.5% in 1998 . 2) The long-term hospitalized patients have become older, are more likely to be previously untreated cases, and initially bacillary cases . 3) The major cause of the treatment failure among the long-term hospitalized cases as reported by the attending doctors was "too chronic disease" in the earlier years, but the causes have changed to adverse reactions to anti-TB drugs, initial drug-resistance, and patient's poor compliance or non-adherence to treatment in recent years . 4) The cause of the long-term hospital stay after bacteriological negative conversion was chronic respiratory failure in more than one third of the patients in every study . Among these patients, an increasing proportion of cases has non-medical problems, such as poor family acceptance and reluctance to be discharged . 5) The mortality of the long-term hospitalized cases was generally high . Among bacteriologically positive cases in the study, it was 14% to 19% annually, and tuberculosis deaths occupied 60% to 80% of all the deaths . 6) The outcomes of those patients who were eventually discharged has become less favorable . Only 3% of them were returned to light work in the 2000 study, while 15% did in the 1981 study.

Immunology, 2001 Feb, 102(2), 146 - 56
The human macrophage cell line U937 as an in vitro model for selective evaluation of mycobacterial antigen-specific cytotoxic T-cell function; Passmore JS et al.; Despite strong evidence for CD8+ T-cell function in murine mycobacterial infections, their corresponding role in human tuberculosis has proven more difficult to demonstrate . We have evaluated the human macrophage (Mphi) cell line U937 as an in vitro model for human leucocyte antigen (HLA) class I-restricted presentation of mycobacterial antigens, as HLA class I is constitutively expressed at high levels by U937 cells in the absence of detectable HLA class II or CD1 molecules . U937 cells were evaluated for their ability to phagocytose Mycobacterium tuberculosis and for their ability to present mycobacterial antigens to human HLA class I-matched cytotoxic T lymphocytes (CTLs) . Differentiated U937 cells were capable of efficient phagocytosis of M . tuberculosis but did not generate a subsequent respiratory burst response, and were permissive for intracellular growth of both bacillus Calmette-Guerin (BCG) and the virulent M . tuberculosis H37Rv strain . CTL activity was restricted to live mycobacterial organisms and was shown to be mediated by M . tuberculosis-specific, HLA class I-matched, purified CD8+ CTL lines and CD8+ T-cell clones . Furthermore, M . tuberculosis-infected U937 targets were more rapidly and strongly lysed by CD8+ CTLs than were infected autologous Mphi . Finally, M . tuberculosis-infected U937 cells simultaneously provided a sensitive indicator for detection of mycobacterial-specific, HLA-unrestricted gammadelta+ CTL activity.

Cell Microbiol, 2001 Mar, 3(3), 169 - 80
Formation of stress fibres in human endothelial cells infected with Bartonella bacilliformis is associated with altered morphology, impaired migration and defects in cell morphogenesis; Verma A et al.; Bartonella bacilliformis, a Gram-negative, flagellated bacterium, infects human erythrocytes (haematic phase) and endothelial cells (tissue phase), resulting in a biphasic disease . In the tissue phase of disease (verruga peruana), infection leads to infection of endothelial cells and a pronounced proliferation of these cells, resulting in characteristic skin eruptions of papules and nodules . We have studied the properties of endothelial cells infected in vitro . Extensive cytoskeletal remodelling of endothelial cells occurred after infection in vitro with B . bacilliformis . The cells became spindle shaped and contained arrays of actin stress fibres orientated parallel to the long axis of the cell . Cell-cell contacts were disrupted, along with the distribution of the plasma membrane marker protein, PECAM-1, which participates in cell-cell junctions . The prominent stress fibres terminated in an increased number of focal contacts, which were studied using immunofluorescent staining for paxillin, a cytoplasmic protein that localizes in the focal adhesions . These morphological changes are consistent with activation of intracellular Rho by B . bacilliformis . Formation of stress fibres and the increased number of focal adhesions could be prevented by preincubation of the endothelial cells with C3 exoenzyme, which inactivates intracellular Rho by ADP ribosylation . Endothelial cell motility was greatly diminished in infected cells and the cells did not respond effectively to a stimulus that would evoke motility . In addition, infection of endothelial cells interfered with their ability to form networks of capillary tubes when suspended within three-dimensional collagen matrices . If the properties of infected endothelial cells in vivo are similar, the infected cells will probably not participate effectively in angiogenesis.

Biochemistry, 2001 Mar 6, 40(9), 3002 - 8
Equilibrium binding studies of a tryptophan-shifted mutant of phosphofructokinase from Bacillus stearothermophilus; Riley-Lovingshimer MR et al.; A tryptophan-shifted mutant of phosphofructokinase (PFK) from Bacillus stearothermophilus has been constructed . This mutant, which is functionally similar to wild-type, provides the opportunity to examine the allosteric properties of PFK under equilibrium conditions . The unique fluorescence properties of the tryptophan-shifted mutant enzyme, W179F/F230W, have been utilized to deduce the thermodynamics of ligand binding and the allosteric perturbations in the absence of catalytic turnover . Specifically, phospho(enol)pyruvate (PEP) and MgADP binding to the mutant PFK can be directly observed using tryptophan fluorescence, and dissociation constants for these ligands have been measured to be equal to 2.71 +/- 0.04 and 90.4 +/- 3.5 microM, respectively . In addition, the homotropic couplings for the allosteric ligands have been assessed for the first time . PEP binds cooperatively with a Hill number of 2.9 +/- 0.3, while MgADP binding is not cooperative . The equilibrium couplings between these ligands and the substrate fructose 6-phosphate (Fru-6-P) have also been determined and follow the same trends with temperature observed under steady-state kinetic assay conditions using wild-type PFK, indicating that the presence of bound MgATP has little influence on the allosteric interactions . Like wild-type PFK, the coupling free energies for the mutant result from largely compensating enthalpy and entropy components at 25 degrees C . Furthermore, the sign of each coupling free energy, which signifies the nature of the allosteric effect, is opposite that of the enthalpy contribution and is therefore due to the larger absolute value of the associated entropy change . This characteristic stands in direct contrast to the thermodynamic basis of the allosteric response in the homologous PFK from E . coli in which the sign of the coupling free energy is established by the sign of the coupling enthalpy.

Biochim Biophys Acta, 2001 Mar 9, 1546(1), 234 - 41
Synthesis of (di)adenosine polyphosphates by non-ribosomal peptide synthetases (NRPS); Dieckmann R et al.; In response to nutritional stress conditions, Bacillus brevis produces the cyclodecapeptide antibiotic tyrocidine via tyrocidine synthetase, a multifunctional non-ribosomal peptide synthetase . The apo-form of tyrocidine synthetase 1 forms adenosine (5')tetraphospho(5')adenosine, when incubated with MgATP(2-), amino acid and inorganic pyrophosphatase . The synthesis is an intrinsic property of the adenylation domain, is strictly dependent upon the amino acid, and proceeds from a reverse reaction of adenylate formation involving a second ATP molecule . In the presence of tri- or tetrapolyphosphate preferential synthesis of adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate occurs, respectively . A potential involvement of adenosine (5')-n-phospho(5')adenosine in the regulation of the biosynthetic process has been suggested.

Biochim Biophys Acta, 2001 Mar 9, 1546(1), 122 - 31
Structural and functional studies of alpha-helix 5 region from Bacillus thuringiensis Cry1Ab delta-endotoxin; Nunez-Valdez M et al.; The crystal insecticidal proteins from Bacillus thuringiensis are modular proteins comprised of three domains connected by single linkers . Domain I is a seven alpha-helix bundle, which has been involved in membrane insertion and pore formation activity . Site-directed mutagenesis has contributed to identify regions that might play an important role in the structure of the pore-forming domain within the membrane . There are several evidences that support that the hairpin alpha4-alpha5 inserts into the membrane in an antiparallel manner, while other helices lie on the membrane surface . We hypothesized that highly conserved residues of alpha5 could play an important role in toxin insertion, oligomerization and/or pore formation . A total of 15 Cry1Ab mutants located in six conserved residues of Cry1Ab, Y153, Y161, H168, R173, W182 and G183, were isolated . Eleven mutants were located within helix alpha5, one mutant was located in the loop alpha4-alpha5 and three mutants, W182P, W182I and G183C, were located in the loop alpha5-alpha6 . Their effect on binding, K(+) permeability and toxicity against Manduca sexta larvae was analyzed and compared . The results provide direct evidence that some residues located within alpha5 have an important role in stability of the toxin within the insect gut, while some others also have an important role in pore formation . The results also provide evidence that conserved residues within helix alpha5 are not involved in oligomer formation since mutations in these residues are able to make pores in vitro.

Biochem J, 2001 Apr 1, 355(Pt 1), 167 - 77
A new family of rhamnogalacturonan lyases contains an enzyme that binds to cellulose; McKie VA et al.; Pseudomonas cellulosa is an aerobic bacterium that synthesizes an extensive array of modular cellulases and hemicellulases, which have a modular architecture consisting of catalytic domains and distinct non-catalytic carbohydrate-binding modules (CBMs) . To investigate whether the main-chain-cleaving pectinases from this bacterium also have a modular structure, a library of P . cellulosa genomic DNA, constructed in lambdaZAPII, was screened for pectinase-encoding sequences . A recombinant phage that attacked arabinan, galactan and rhamnogalacturonan was isolated . The encoded enzyme, designated Rgl11A, had a modular structure comprising an N-terminal domain that exhibited homology to Bacillus and Streptomyces proteins of unknown function, a middle domain that exhibited sequence identity to fibronectin-3 domains, and a C-terminal domain that was homologous to family 2a CBMs . Expression of the three modules of the Pseudomonas protein in Escherichia coli showed that its C-terminal module was a functional cellulose-binding domain, and the N-terminal module consisted of a catalytic domain that hydrolysed rhamnogalacturonan-containing substrates . The activity of Rgl11A against apple- and potato-derived rhamnogalacturonan substrates indicated that the enzyme had a strong preference for rhamnogalacturonans that contained galactose side chains, and which were not esterified . The enzyme had an absolute requirement for calcium, a high optimum pH, and catalysis was associated with an increase in absorbance at 235 nm, indicating that glycosidic bond cleavage was mediated via a beta-elimination mechanism . These data indicate that Rgl11A is a rhamnogalacturonan lyase and, together with the homologous Bacillus and Streptomyces proteins, comprise a new family of polysaccharide lyases . The presence of a family 2a CBM in Rgl11A, and in a P . cellulosa pectate lyase described in the accompanying paper {Brown, Mallen, Charnock, Davies and Black (2001) Biochem . J . 355, 155-165} suggests that the capacity to bind cellulose plays an important role in the activity of main-chain-cleaving Pseudomonas pectinases, in addition to cellulases and hemicellulases.

Biochem J, 2001 Apr 1, 355(Pt 1), 155 - 65
Pectate lyase 10A from Pseudomonas cellulosa is a modular enzyme containing a family 2a carbohydrate-binding module; Brown IE et al.; Pectate lyase 10A (Pel10A) enzyme from Pseudomonas cellulosa is composed of 649 residues and has a molecular mass of 68.5 kDa . Sequence analysis revealed that Pel10A contained a signal peptide and two serine-rich linker sequences that separate three modules . Sequence similarity was seen between the 9.2 kDa N-terminal module of Pel10A and family 2a carbohydrate-binding modules (CBMs) . This N-terminal module of Pel10A was shown to encode an independently functional module with affinity to crystalline cellulose . A high sequence identity of 66% was seen between the 14.2 kDa central module of Pel10A and the functionally uncharacterized central modules of the xylan-degrading enzymes endoxylanase 10B, arabinofuranosidase 62C and esterase 1D, also from P . cellulosa . The 35.8 kDa C-terminal module of Pel10A was shown to have 30 and 36% identities with the family 10 pectate lyases from Azospirillum irakense and an alkaliphilic strain of Bacillus sp . strain KSM-P15, respectively . This His-tagged C-terminal module of the Pel10A was shown to encode an independent catalytic module (Pel10Acm) . Pel10Acm was shown to cleave pectate and pectin in an endo-fashion and to have optimal activity at pH 10 and in the presence of 2 mM Ca2+ . Highest enzyme activity was detected at 62 degrees C . Pel10Acm was shown to be most active against pectate (i.e . polygalacturonic acid) with progressively less activity against 31, 67 and 89% esterified citrus pectins . These data suggest that Pel10A has a preference for sequences of non-esterified galacturonic acid residues . Significantly, Pel10A and the P . cellulosa rhamnogalacturonan lyase 11A, in the accompanying article {McKie, Vincken, Voragen, van den Broek, Stimson and Gilbert (2001) Biochem . J . 355, 167-177}, are the first CBM-containing pectinases described to date.

Biochem J, 2001 Apr 1, 355(Pt 1), 79 - 86
Long-lived glycosyl-enzyme intermediate mimic produced by formate re-activation of a mutant endoglucanase lacking its catalytic nucleophile; Viladot JL et al.; The mutant E134A 1,3-1,4-beta-glucanase from Bacillus licheniformis, in which the catalytic nucleophilic residue has been removed by mutation to alanine, has its hydrolytic activity rescued by exogenous formate in a concentration-dependent manner . A long-lived alpha-glycosyl formate is detected and identified by (1)H-NMR and matrix-assisted laser desorption ionization-time-of-flight-MS . The intermediate is kinetically competent, since it is, at least partially, enzymically hydrolysed, and able to act as a glycosyl donor in transglycosylation reactions . This transient compound represents a true covalent glycosyl-enzyme intermediate mimic of the proposed covalent intermediate in the reaction mechanism of retaining glycosidases.

Biochimie, 2001 Jan, 83(1), 121 - 4
Perpendicular planes of FtsZ arcs in spheroidal Escherichia coli cells; Pas E et al.; Division planes in Escherichia coli, usually restricted to one dimension of the rod-shaped cell, were induced at all possible planes by transforming the cells to spheroids with mecillinam (inactivating PbpA) . Such cells displayed many nucleoids and arcs of FtsZ, genetically tagged to green fluorescent protein, that developed to rings at constriction sites all around their surface . These observations are consistent with the view (Woldringh et al., J . Bacteriol . 176 (1994) 6030-6038) that nucleoids, forced during replication to segregate in the length axis of the cell by the rigid bacillary envelope, induce assembly of FtsZ to division rings in between them.

J Immunol, 2001 Apr 1, 166(7), 4721 - 7
Requirement of A1-a for bacillus Calmette-Guérin-mediated protection of macrophages against nitric oxide-induced apoptosis; Kausalya S et al.; The role of apoptosis in regulating the course of intracellular microbial infection is not well understood . We studied the relationship between apoptotic regulation and bacillus Calmette-Guerin (BCG) treatment in murine peritoneal exudate macrophages (PEM) and the J774 macrophage cell line . In both PEM and J774 cells, mRNA expression of the anti-apoptotic gene, A1, was selectively induced by BCG treatment as compared with other bcl2 family members (bcl-w, bcl-2, bcl-xl, bcl-xs, bax, bak, bad) . In PEM, A1 expression was maximal by 8 h postinfection and was abrogated by the proteasomal inhibitor MG-132 . The induction was independent of protein synthesis as well as the p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways and did not require live organism . Three genes encoding closely related isoforms of A1 were all expressed; however, the A1-a isoform displayed the greatest fold induction in PEM . BCG-induced A1 expression was associated with protection of host macrophages from NO-mediated apoptosis in both PEM and J774 cells . BCG-mediated protection was abrogated in PEM derived from A1-a(-/-) mice, indicating a requirement of A1-a for survival of inflammatory macrophages.

Infect Immun, 2001 Apr, 69(4), 2456 - 61
Stimulation of dendritic cells via CD40 enhances immune responses to Mycobacterium tuberculosis infection; Demangel C et al.; The resolution of pulmonary tuberculosis (TB) critically depends on the development of the Th1 type of immune responses, as exemplified by the exacerbation of TB in IL-12-deficient mice . Therefore, vaccination strategies optimizing IL-12 production by antigen-presenting cells (APC) in response to mycobacteria may have enhanced protective efficacy . Since dendritic cells (DC) are the critical APC for activation of CD4+ and CD8+ T cells, we examined whether stimulation of Mycobacterium bovis bacillus Calmette Guerin (BCG)-infected DC via CD40 increased their ability to generate Th1-oriented cellular immune responses . Incubation of DC with an agonistic anti-CD40 antibody activated CD40 signaling in DC, as shown by increased expression of major histocompatibility complex class II and costimulatory molecules, mRNA production for proinflammatory cytokines and interleukin 12 (IL-12) p40 . This activation pattern was maintained when DC were stimulated with anti-CD40 antibody and infected with BCG . Importantly, CD40-stimulated BCG-infected DC displayed increased capacity to release bioactive IL-12 and to activate gamma interferon (IFN-gamma) producing T cells in vitro . Moreover, when C57BL/6 mice were immunized with these DC and challenged with aerosol Mycobacterium tuberculosis, increased levels of mRNA for IL-12 p40, IL-18, and IFN-gamma were present in the draining mediastinal lymph nodes . However, the mycobacterial burden in the lungs was not reduced compared to that in mice immunized with BCG-infected non-CD40-stimulated DC . Therefore, although the manipulation of DC via CD40 is effective for enhancing immune responses to mycobacteria in vivo, additional strategies are required to increase protection against virulent M . tuberculosis infection.

J Colloid Interface Sci, 2001 Mar 15, 235(2), 298 - 309
Surface Chemical Studies on Sphalerite and Galena Using Bacillus polymyxa; Santhiya D et al.; Biodissolution tests reveal the release of lead/zinc species from galena/sphalerite, respectively, while biosorption experiments confirm interaction of cells of Bacillus polymyxa (B . polymyxa) with the metal ions of interest . The amount of exo-polysaccharides is found to be the highest in the case of galena-interacted cells, followed by the Bromfield medium-grown cells while the sphalerite-interacted cells have the least, based on ruthenium red adsorption studies . In contrast, the sphalerite-interacted cells assay the highest amount of protein while the galena-interacted cells have the lowest amount, on a comparative basis . The adsorption of xanthate onto galena is found to be diminished in the presence of the cells whereas the xanthate adsorption density for activated sphalerite is unaffected in the pH range 9-11 . Additionally, the cell surface hydrophobicity tests confirm that the sphalerite-interacted cells are more hydrophobic relative to the galena-interacted cells . FTIR spectroscopic data lend support to the higher adsorption density of the cells onto galena vis-a-vis sphalerite . The higher exo-polysaccharide and lower protein contents together with the hydrophilic nature of the galena-interacted cells could be the contributing factors to the selective flocculation and depression of galena . In a similar manner, the higher protein and lower exo-polysaccharide contents as well as the greater hydrophobicity of the sphalerite-interacted cells favor its floatability and dispersion .

J Colloid Interface Sci, 2001 Mar 15, 235(2), 289 - 297
Surface Chemical Studies on Sphalerite and Galena Using Bacillus polymyxa; Santhiya D et al.; The interaction of sphalerite and galena with cells of Bacillus polymyxa was investigated through adsorption, electrokinetic, flotation, and flocculation studies . Adsorption experiments indicated that a higher amount of the cells was adsorbed onto galena compared to sphalerite . The adsorption density of the cells onto galena was almost independent of pH while that onto sphalerite was found to continuously decrease with increasing pH . The adsorption isotherms of the bacterial cells on galena and sphalerite exhibited Langmuirian behavior . Electrokinetic measurements showed that the negative electrophoretic mobilities of the cells were reduced in magnitude in proportion to the time of interaction with either sphalerite or galena . Similar trends were observed in the cases of sphalerite and galena after interaction with the cells . However, the magnitude of the reduction in the electrophoretic mobilities was found to be greater for galena than for sphalerite . Flotation tests revealed that galena was almost completely depressed after interaction with the cells both in the absence and in the presence of the collector . In contrast, the addition of collector and activator to sphalerite, which was initially interacted with the cells, restored the floatability at and beyond pH 8.5 . Selective flotation tests on a synthetic mixture of galena and sphalerite confirmed that sphalerite could be preferentially floated from galena, which was depressed by the bacterial cells . Selective flocculation tests further demonstrated that galena could be flocculated from sphalerite, which was dispersed in the presence of cells of B . polymyxa at pH 9-9.5 .

Can J Urol, 1999 Apr, 6(2), 749 - 750
Metastatic renal cell carcinoma regression after nephrectomy and BCG immunotherapy; Dagnone AJ et al.; Patients with renal cell carcinoma (RCC) often remain asymptomatic until the disease has progressed beyond the confines of the kidney, as almost one third of patients have metastatic disease at the time of diagnosis.1 The average survival for a patient presenting with metastatic renal cell carcinoma is 4 months and only 10% will be alive at 1 year.2 Many different treatment combinations have tried for metastatic disease, but with limited results . Some success has been shown in smaller studies using nephrectomy and Bacillus Calmette-Guerin (BCG) immunotherapy but these results have not been followed by larger randomized control trials.3 We report a case metastatic renal cell carcinoma with an unusually long survival and regression of metastases from 2 uncommon sites.

Curr Oncol Rep, 2001 Mar, 3(2), 116 - 26
Immunotherapy for colorectal cancer; Foon KA; Immunologic approaches to therapy for colorectal cancer have evolved substantially . In the past, patients were treated with nonspecific immune stimulants such as bacillus Calmette-Guerin (BCG) . The current focus lies in targeting tumor-associated antigens . This is done either through passive immune therapy, with antibodies targeted directly to tumor cells, or by active immune therapy through vaccination with tumor cells, tumor cell lysates, peptides, carbohydrates, gene constructs encoding proteins, or anti-idiotype antibodies that mimic tumor-associated antigens . These different approaches to immunotherapy are reviewed.

Int J Food Microbiol, 2001 Feb 28, 64(1-2), 81 - 8
Influence of agar content on the growth parameters of Bacillus cereus; Stecchini ML et al.; Factors governing Bacillus cereus colony growth on agar media as modified by the agar content (1-7%, w/v) were studied . Agar had a significant effect on the radial growth rate which diminished as the agar content increased . Cell density in colonies (colony density) was found to decrease during the incubation time, with lower values occurring in the presence of 1% agar . Size and DNA content of the cells grown on 1 and 7% agar were similar . An increasing proportion of cell population growing on 7% agar produced spores during the 24-h incubation period . It was shown that 'water condition' on the agar surface could be associated with colony density, with 7% agar media presenting the thinnest nominal thickness of the liquid film . On the other hand, the partial drying phenomena of the agar media which occur during preparation and incubation, could not account for the observed differences in colony growth.

Int J Food Microbiol, 2001 Feb 28, 64(1-2), 63 - 70
Detection of genes encoding for enterotoxins and determination of the production of enterotoxins by HBL blood plates and immunoassays of psychrotrophic strains of Bacillus cereus isolated from pasteurised milk; in't Veld PH et al.; The presence of genes for the production of the three components of the HBL enterotoxin complex and enterotoxin-T in Bacillus cereus was evaluated by PCR tests for strains isolated from milk . In addition enterotoxin production of B . cereus was evaluated by means of the HBL blood agar plate and two commercially available toxin tests . All three genes for the HBL enterotoxin complex were detected in 55% of the 86 strains tested, the enterotoxin-T gene was detected in 62% of the strains . A few strains showed a weak reaction in the PCR tests for the L1 or L2 components of the HBL enterotoxin complex . Many strains that were found to contain the genes for the HBL complex gave negative or doubtful results in the HBL blood agar plate test . All strains that contain the L2 part of the HBL complex showed a titer of at least 8 in the Oxoid RPLA test . Two strains that did not contain the L2 part of the HBL enterotoxin complex gave high titers (= 64) in the RPLA test.

Mol Microbiol, 2001 Mar, 39(5), 1174 - 85
Mycobacterial FurA is a negative regulator of catalase-peroxidase gene katG; Zahrt TC et al.; In several bacteria, the catalase-peroxidase gene katG is under positive control by oxyR, a transcriptional regulator of the peroxide stress response . The Mycobacterium tuberculosis genome also contains sequences corresponding to oxyR, but this gene has been inactivated in the tubercle bacillus because of the presence of multiple mutations and deletions . Thus, M . tuberculosis katG and possibly other parts of the oxidative stress response in this organism are either not regulated or are controlled by a factor different from OxyR . The mycobacterial FurA is a homologue of the ferric uptake regulator Fur and is encoded by a gene located immediately upstream of katG . Here, we examine the possibility that FurA regulates katG expression . Inactivation of furA on the Mycobacterium smegmatis chromosome, a mycobacterial species that also lacks an oxyR homologue, resulted in derepression of katG, concomitant with increased resistance of the furA mutant to H2O2 . In addition, M . smegmatis furA::Km(r) was more sensitive to the front-line antituberculosis agent isonicotinic acid hydrazide (INH) compared with the parental furA+ strain . The phenotypic manifestations were specific, as the mutant strain did not show altered sensitivity to organic peroxides, and both H2O2 and INH susceptibility profiles were complemented by the wild-type furA+ gene . We conclude that FurA is a second regulator of oxidative stress response in mycobacteria and that it negatively controls katG . In species lacking a functional oxyR, such as M . tuberculosis and M . smegmatis, FurA appears to be a dominant regulator affecting mycobacterial physiology and intracellular survival.

Mol Microbiol, 2001 Feb, 39(4), 1010 - 21
Analysis of the replicon region and identification of an rRNA operon on pBM400 of Bacillus megaterium QM B1551; Kunnimalaiyaan M et al.; An 18 633 bp region containing the replicon from the approximately 53 kb pBM400 plasmid of Bacillus megaterium QM B1551 has been sequenced and characterized . This region contained a complete rRNA operon plus 10 other potential open reading frames (ORFs) . The replicon consisted of an upstream promoter and three contiguous genes (repM400, orfB and orfC) that could encode putative proteins of 428, 251 and 289 amino acids respectively . A 1.6 kb minimal replicon was defined and contained most of repM400 . OrfB was shown to be required for stability . Three 12 bp identical tandem repeats were located within the coding region of repM400, and their presence on another plasmid caused incompatibility with their own cognate replicon . Nonsense, frameshift and deletion mutations in repM400 prevented replication, but each mutation could be complemented in trans . RepM400 had no significant similarity to sequences in the GenBank database, whereas five other ORFs had some similarity to gene products from other plasmids and the Bacillus genome . An rRNA operon was located upstream of the replication region and is the first rRNA operon to be sequenced from B . megaterium . Its unusual location on non-essential plasmid DNA has implications for systematics and evolutionary biology.

J Insect Physiol, 2001 Jun, 47(6), 599 - 606
Regeneration of cultured midgut cells after exposure to sublethal doses of toxin from two strains of Bacillus thuringiensis; Loeb MJ et al.; Toxin from two strains of Bacillus thuringiensis (Bt), AA 1-9 and HD-73, caused dose-dependent destruction of cultured midgut cells from Heliothis virescens larvae . HD-73 toxin was more effective although, at the doses used, not all cells were killed . After 2 days of exposure to 0.8 pg/microl AA 1-9 or 0.06 pg/microl HD-73, columnar and goblet cell numbers declined to ca 20% of controls . In contrast, stem and differentiating cells increased to 140-200% of controls . The dynamic of depletion and replacement depended on toxin type and concentration . Two days after toxin was washed out, ratios of cell types returned to approximate control levels, suggesting rapid population corrections in vitro . Regulation of the ratio of cell types in each population, and the rate of proliferation and differentiation of stem cells was induced by the cultured midgut cells themselves . Controls and cells treated with toxin from Bt strain AA 1-9 were stained using a polyclonal antibody to Lepidopteran midgut differentiation factor 1 (MDF1) . With Bt toxin, 1.5 times more cells stained for MDF1, suggesting increased synthesis of this differentiation factor during increased stem cell differentiation . The response of cultured midgut cells to Bt toxin injury is similar to injured vertebrate tissues dependent on stem cells for replacement and healing.

Bioorg Med Chem, 2001 Feb, 9(2), 503 - 10
Cysteinyl peptide inhibitors of Bacillus cereus zinc beta-lactamase; Bounaga S et al.; Several cysteinyl peptides have been synthesised and shown to be reversible competitive inhibitors of the Bacillus cereus metallo-beta-lactamase . The pH dependence of pKi indicates that the thiol anion displaces hydroxide ion from the active site zinc(II) . D,D-Peptides bind to the enzyme better than other diastereoisomers, which is compatible with the predicted stereochemistry of the active site.

FEMS Microbiol Ecol, 2001 Mar, 35(1), 37 - 48
Impact of cultivation on characterisation of species composition of soil bacterial communities; McCaig AE et al.; The species composition of culturable bacteria in Scottish grassland soils was investigated using a combination of Biolog and 16S rDNA analysis for characterisation of isolates . The inclusion of a molecular approach allowed direct comparison of sequences from culturable bacteria with sequences obtained during analysis of DNA extracted directly from the same soil samples . Bacterial strains were isolated on Pseudomonas isolation agar (PIA), a selective medium, and on tryptone soya agar (TSA), a general laboratory medium . In total, 12 and 21 morphologically different bacterial cultures were isolated on PIA and TSA, respectively . Biolog and sequencing placed PIA isolates in the same taxonomic groups, the majority of cultures belonging to the Pseudomonas (sensu stricto) group . However, analysis of 16S rDNA sequences proved more efficient than Biolog for characterising TSA isolates due to limitations of the Microlog database for identifying environmental bacteria . In general, 16S rDNA sequences from TSA isolates showed high similarities to cultured species represented in sequence databases, although TSA-8 showed only 92.5% similarity to the nearest relative, Bacillus insolitus . In general, there was very little overlap between the culturable and uncultured bacterial communities, although two sequences, PIA-2 and TSA-13, showed >99% similarity to soil clones . A cloning step was included prior to sequence analysis of two isolates, TSA-5 and TSA-14, and analysis of several clones confirmed that these cultures comprised at least four and three sequence types, respectively . All isolate clones were most closely related to uncultured bacteria, with clone TSA-5.1 showing 99.8% similarity to a sequence amplified directly from the same soil sample . Interestingly, one clone, TSA-5.4, clustered within a novel group comprising only uncultured sequences . This group, which is associated with the novel, deep-branching Acidobacterium capsulatum lineage, also included clones isolated during direct analysis of the same soil and from a wide range of other sample types studied elsewhere . The study demonstrates the value of fine-scale molecular analysis for identification of laboratory isolates and indicates the culturability of approximately 1% of the total population but under a restricted range of media and cultivation conditions.

Biochim Biophys Acta, 2001 May 1, 1505(1), 158 - 68
The Na(+)-dependence of alkaliphily in Bacillus; Krulwich TA et al.; A Na(+) cycle plays a central role in the remarkable capacity of aerobic, extremely alkaliphilic Bacillus species for pH homeostasis . The capacity for pH homeostasis, in turn, appears to set the upper pH limit for growth . One limb of the alkaliphile Na(+) cycle consists of Na(+)/H(+) antiporters that achieve net H(+) accumulation that is coupled to Na(+) efflux . The major antiporter on which pH homeostasis depends is thought to be the Mrp(Sha)-encoded antiporter, first identified from a partial clone in Bacillus halodurans C-125 . Mrp(Sha) may function as a complex . While this antiporter is capable of secondary antiport energized by an imposed or respiration-generated protonmotive force, the possibility of a primary mode has not been excluded . In Bacillus pseudofirmus OF4, at least two additional antiporters, including NhaC, have supporting roles in pH homeostasis . Some of these additional antiporters may be especially important for antiport at low {Na(+)} or at near-neutral pH . The second limb of the Na(+) cycle facilitates Na(+) re-entry via Na(+)/solute symporters and, perhaps, the ion channel associated with the Na(+)-dependent flagellar motor . The process of pH homeostasis is also enhanced, perhaps especially during transitions to high pH, by different arrays of secondary cell wall polymers in the two alkaliphilic Bacillus species studied most intensively . The mechanisms whereby alkaliphiles handle the challenge of Na(+) stress at very elevated {Na(+)} are just beginning to be identified, and a hypothesis has been advanced to explain the finding that B . pseudofirmus OF4 requires a higher {Na(+)} for growth at near-neutral pH than at very alkaline pH values.

Lancet, 2001 Mar 3, 357(9257), 675 - 9
Cell-wall-deficient bacteria and culture-negative febrile episodes in bone-marrow-transplant recipients; Woo PC et al.; BACKGROUND: Although about 75-80% of neutropenic fevers are thought to be caused by infections, a causal organism can be confirmed microbiologically or suspected clinically in only 30-50%, and even fewer of these cases (16%) have a documented bacteraemia . The cause of neutropenic fever in the remaining cases remains elusive . We investigate the role of cell-wall-deficient bacteria in bone-marrow transplantation (BMT) . METHODS: Blood cultures were obtained from bone-marrow-transplant recipients and were inoculated into an aerobic bottle with resin, an anaerobic bottle, and a bottle to isolate cell-wall deficient bacteria . When growth was detected in the blood-culture bottle for cell-wall-deficient bacteria, 20 microL of the broth was subcultured and the isolate identified by standard biochemical methods . The microbiological findings were correlated with the clinical characteristics of the patients . FINDINGS: From the 86 BMT recipients enrolled into the study, 798 sets of blood cultures were sent for laboratory analysis . 55 blood cultures were positive in the aerobic bottle, and the same isolates were also detected in 52 blood-culture bottles for cell-wall-deficient forms . For 20 sets of blood cultures, the isolates were detected only in the bottle for cell-wall-deficient forms . Blood for 13 (65%) of these 20 sets were sampled in the pre-engraftment neutropenic period, three (15%) during conditioning when the absolute neutrophil count was above 0.5 x 10(3)/L, and four (20%) in the post-engraftment period . 17 (85%) of the isolates were gram positive: four (20%) were Staphylococcus spp and ten (50%) were Bacillus spp . Antibiotic treatment was successful in 19 (95%) of 20 episodes . INTERPRETATION: Bacteraemia due to cell-wall-deficient forms causes a significant proportion of so-called culture-negative febrile episodes in BMT recipients.

Biometals, 2000 Dec, 13(4), 273 - 80
Cadmium biosorption by a cadmium resistant strain of Bacillus thuringiensis: regulation and optimization of cell surface affinity for metal cations; El-Helow ER et al.; A marine bacterial strain putatively identified as Bacillus thuringiensis strain DM55, showed multiple heavy metal resistance and biosorption phenotypes . Electron microscopic studies revealed that DM55 cells are encased in anionic cell wall polymers that can immobilize discrete aggregates of cations . Factors affecting cell surface affinity for metal cations, monitored by means of Cd2+ binding capability, are investigated . The mechanisms of cadmium resistance and Cd2+ biosorption by the bacterium appeared to be inducible and coincident . Medium components affecting metal removal under cadmium-stressed growth conditions were explored based on the application of two sequential multi-factorial statistical designs . Concentrations of potassium phosphates and peptone were the most significant variables . Optimized culture conditions allowed DM55 cells grown in the presence of 0.25 mM CdCl2 to remove about 79% of the metal ions within 24 h with a specific biosorption capacity of 21.57 mg g(-1) of biomass . Both fresh and dry cells of DM55 prepared under cadmium-free optimal nutrient condition were also able to biosorb Cd2+ . In addition to the concentration of phosphate in the medium, KinA, a major phosphate provider in the phosphorelay of Bacillus cells, was also demonstrated to regulate the magnitude of cell surface affinity for cadmium ions.

Int J Food Microbiol, 2001 Feb 15, 63(3), 257 - 64
Effect of heat activation and inactivation conditions on germination and thermal resistance parameters of Bacillus cereus spores; Fernandez A et al.; The effect of isothermal and non-isothermal heat activation on germination and thermoresistance of two strains of Bacillus cereus spores was studied . Results indicated that the germination after isothermal activation was lower than after non-isothermal heating . The activation rate affected the z value, which increased with faster heating rates . For each temperature and inactivation rate, the non-isothermal activation at rate of 2 degrees C/min resulted in larger D values (D90 = 4.70 min) than isothermal activation (D90 = 4.04 min) . The two mathematical equations used to analyse non-isothermal data produced similar predicted D and z values, nevertheless the Hayakawa equation modified in this work for non-linear regression analysis, requires less computational effort.

Int J Food Microbiol, 2001 Feb 15, 63(3), 199 - 207
Prevalence and behaviour of Bacillus cereus in a REPFED of Italian origin; Del Torre M et al.; This paper reports on the prevalence and behaviour of Bacillus cereus in gnocchi, a REPFED of Italian origin . A survey of gnocchi under varying storage conditions, revealed that, although B . cereus was found in 33% of the samples, the contamination level was lower than 10(2) CFU/g for the unstored and the refrigerated (8 degrees C) samples . Counts increased with increasing storage temperatures and prolonged storage times in samples prepared without sorbic acid . The effect of different formulations (sorbic, citric and lactic acid) and storage conditions (8, 12 and 20 degrees C) have been evaluated in a challenge testing with spores of B . cereus . Results indicate that the use of sorbic acid in association with citric or lactic acid to pH 5.0 is effective in inhibiting growth of B . cereus and the anticipated shelf life of the product is safe even if temperature abuse occurs . If sorbic acid is omitted, lactic acid can inhibit B . cereus growth during storage at 8 degrees C . On the contrary, when temperature abuse occurs (12 and 20 degrees C), lactic or citric acid are not able to prevent growth of B . cereus.

Rev Inst Med Trop Sao Paulo, 2001 Jan-Feb, 43(1), 1 - 6
Bacillary angiomatosis: description of 13 cases reported in five reference centers for AIDS treatment in Rio de Janeiro, Brazil; Gazineo JL et al.; The aim of this case series was to describe the clinical, laboratory and epidemiological characteristics and the presentation of bacillary angiomatosis cases (and/or parenchymal bacillary peliosis) that were identified in five public hospitals of Rio de Janeiro state between 1990 and 1997; these cases were compared with those previously described in the medical literature . Thirteen case-patients were enrolled in the study; the median age was 39 years and all patients were male . All patients were human immunodeficiency virus type 1 (HIV-1) infected and they had previous or concomitant HIV-associated opportunistic infections or malignancies diagnosed at the time bacillary angiomatosis was diagnosed . Median T4 helper lymphocyte counts of patients was 96 cells per mm(3) . Cutaneous involvement was the most common clinical manifestation of bacillary angiomatosis in this study . Clinical remission following appropriate treatment was more common in our case series than that reported in the medical literature, while the incidence of relapse was similar . The frequency of bacillary angiomatosis in HIV patients calculated from two of the hospitals included in our study was 1.42 cases per 1000 patients, similar to the frequencies reported in the medical literature . Bacillary angiomatosis is an unusual opportunistic pathogen in our setting.

J Biochem Biophys Methods, 2001 Feb 26, 47(3), 177 - 88
Screening of the Bacillus thuringiensis Cry1Ac delta-endotoxin on the artificial phospholipid monolayer incorporated with brush border membrane vesicles of Plutella xylostella by optical biosensor technology; Okumura S et al.; The binding of Cry1Ac, an insecticidal protein of Bacillus thuringiensis, to a brush border membrane (BBM) isolated from midguts of the diamondback moth Plutella xylostella was examined by surface plasmon resonance (SPR)-based biosensor . BBM was mixed with 1,3-ditetradecylglycero-2-phosphocholine (PC14), a neutral charged artificial lipid, and was reconstructed to a monolayer on a hydrophobic chip for the biosensor . The binding of Cry1Ac to the reconstructed monolayer was analyzed by a two-state binding model, and it was shown that Cry1Ac bound to the monolayer in the first step with an affinity constant (K(1)) of 508 nM, followed by the second uni-molecular step with an equilibrium constant (K(2)) of 0.472 . The overall affinity constant K(d) was determined to be 240 nM . The binding was markedly inhibited by N-acetyl-D-galactosamine (K(i)=8 mM) . The monolayer was shown to retain a high affinity to Cry1Ac, providing an insect-free system for rapid and large-scale screening of B . thuringiensis insecticidal proteins by the SPR-based biosensor technology.

J Mol Biol, 2001 Mar 16, 307(1), 75 - 92
Function of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase in RNA splicing . Role of the idiosyncratic N-terminal extension and different modes of interaction with different group I introns; Mohr G et al.; The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) promotes the splicing of group I introns by helping the intron RNA fold into the catalytically active structure . The regions required for splicing include an idiosyncratic N-terminal extension, the nucleotide-binding fold domain, and the C-terminal RNA-binding domain . Here, we show that the idiosyncratic N-terminal region is in fact comprised of two functionally distinct parts: an upstream region consisting predominantly of a predicted amphipathic alpha-helix (H0), which is absent from bacterial tyrosyl-tRNA synthetases (TyrRSs), and a downstream region, which contains predicted alpha-helices H1 and H2, corresponding to features in the X-ray crystal structure of the Bacillus stearothermophilus TyrRS . Bacterial genetic assays with libraries of CYT-18 mutants having random mutations in the N-terminal region identified functionally important amino acid residues and supported the predicted structures of the H0 and H1 alpha-helices . The function of N and C-terminal domains of CYT-18 was investigated by detailed biochemical analysis of deletion mutants . The results confirmed that the N-terminal extension is required only for splicing activity, but surprisingly, at least in the case of the N . crassa mitochondrial (mt) large ribosomal subunit (LSU) intron, it appears to act primarily by stabilizing the structure of another region that interacts directly with the intron RNA . The H1/H2 region is required for splicing activity and TyrRS activity with the N . crassa mt tRNA(Tyr), but not for TyrRS activity with Escherichia coli tRNA(Tyr), implying a somewhat different mode of recognition of the two tyrosyl-tRNAs . Finally, a CYT-18 mutant lacking the N-terminal H0 region is totally defective in binding or splicing the N . crassa ND1 intron, but retains substantial residual activity with the mt LSU intron, and conversely, a CYT-18 mutant lacking the C-terminal RNA-binding domain is totally defective in binding or splicing the mt LSU intron, but retains substantial residual activity with the ND1 intron . These findings lead to the surprising conclusion that CYT-18 promotes splicing via different sets of interactions with different group I introns . We suggest that these different modes of promoting splicing evolved from an initial interaction based on the recognition of conserved tRNA-like structural features of the group I intron catalytic core .

Indian J Public Health, 1999 Oct-Dec, 43(4), 152 - 5
Differential efficacy evaluation of formulations of Bacillus thuringiensis Var israelensis against mosquitoes: a laboratory investigation; Srivastava R et al.; The efficacy evaluation of three formulations; wettable powder (W.P.) floating pellet and beads of Bacillus thuringiensis Var israelensis (Bti), revealed a greater susceptibility of the early larval instars of mosquitoes to Bti, sensitivity of anophelines to floating pellet, culicines to bead and equal efficacy and faster kill of W.P . to all the mosquitoes tested . A greater persistence of the slow release formulations, floating pellet and beads for 49 and 28 days against anophelines and culicines respectively was observed in contrast to a maximum persistence for 21 days in case of W.P . formulation.

Enzyme Microb Technol, 2001 Mar 8, 28(4-5), 404 - 409
Calcite precipitation induced by polyurethane-immobilized Bacillus pasteurii; Bang SS et al.; Polyurethane (PU) foam was used to immobilize the whole cell of Bacillus pasteurii . The immobilized cells exhibited the rates of calcite precipitation and ammonia production as high as those of the free cells . Scanning electron micrographs identified the cells embedded in calcite crystals throughout PU matrices . Calcite in PU showed little effect on the elastic modulus and tensile strength of the polymer, but increased the compressive strengths of concrete cubes, whose cracks were remediated with PU-immobilized cells . These observations led us to believe that the calcite might remain as a form of precipitation, not as a bonding material within the matrices.

Enzyme Microb Technol, 2001 Mar 8, 28(4-5), 376 - 382
Microbial reclamation of shellfish wastes for the production of chitinases; Wang S et al.; Shrimp and crab shell powder (SCSP), prepared by treating shellfish processing waste with boiling and crushing, was used as a substrate for isolating chitinolytic microorganisms . Three potential strains (E1, J1, and J1-1) were isolated and identified as Bacillus cereus, B . alvei, and B . sphaericus, respectively . Three extracellular chitinases (FB1, FB2, and FB3) were purified from the culture supernatants of Bacillus cereus E1, B . alvei J1, and B . sphaericus J1-1, respectively . The molecular weights of FB1, FB2, and FB3 were 71,000, 71,000, and 65,000, respectively, by SDS-PAGE . The pIs for FB1, FB2, and FB3 were 7.1, 7.2, and 7.4, respectively . The optimum pH, optimum temperature, pH stability, and thermal stability of FB1 were pH 9, 50 degrees C, pH 7 to 10, and 70 degrees C; those of FB2 were pH 9, 60 degrees C, pH 5 to 9, and 70 degrees C; and those of FB3 were pH 7, 50 degrees C, pH 5 to 9, and 60 degrees C . The activities of all enzymes were strongly inhibited by Hg(2+) and completely inhibited by glutathione, dithiothreitol, and 2-mercaptoethanol.

FEBS Lett, 2001 Mar 2, 491(3), 257 - 60
Conserved amino acids near the carboxy terminus of bacterial tyrosyl-tRNA synthetase are involved in tRNA and Tyr-AMP binding; Salazar JC et al.; Bacterial tyrosyl-tRNA synthetases occur in two large subfamilies, TyrRS and TyrRZ, that possess about 25% amino acid identity . Their amino-terminal region, the active site domain, is more conserved (>36% identity) . The carboxy-terminal segment of these enzymes includes the tRNA binding domain and contains only few conserved residues . Replacement of three of these residues in Acidithiobacillus ferrooxidans TyrRZ revealed that S356 and K395 play roles in tRNA binding, while H306, a residue at the junction of the catalytic and tRNA binding domains, stabilizes the Tyr-AMP:TyrRZ complex . The replacement data suggest that conserved amino acids in A . ferrooxidans TyrRZ and Bacillus stearothermophilus TyrRS play equivalent roles in enzyme function.

J Microbiol Methods, 2001 Apr, 44(3), 205 - 8
Rapid detection of Bacillus stearothermophilus using impedance-splitting; Flint SH et al.; An impedance splitting method was used to detect Bacillus stearothermophilus in suspension and attached to stainless steel surfaces . The effects of bacterial metabolism on the impedance of the culture medium and the ionic layers of the measuring electrodes were recorded using the BacTrac 4000 microorganism growth analyser . Impedance changes were measured at 55 degrees C . Seven of the eight media produced changes in the electrode impedance (E-value) and all media produced negligible changes in the impedance of the culture medium (M-value) . Good correlations were obtained between cell numbers and the E-value measured over 18 h (r > 0.9) for the two strains of B . stearothermophilus tested in trypticase soy broth . The E-value correlations were used to estimate the numbers of both vegetative and spore forms of B . stearothermophilus as either planktonic or adhered cells . For the detection of B . stearothermophilus using impedance, only methods where the E-value impedance is recorded, can be used.

J Nutr, 2001 Mar, 131(3s), 1067S - 70S
Enhanced immunocompetence by garlic: role in bladder cancer and other malignancies; Lamm DL et al.; Of the many beneficial actions of garlic, inhibition of the growth of cancer is perhaps the most remarkable . Our previous animal studies demonstrated that aged garlic extract was highly effective, and unlike the approved immunotherapy for human bladder cancer, bacillus Calmette--Guerin (BCG), garlic was effective when added to the diet . To elucidate the mechanism of this antitumor effect, the literature describing antitumor and immune-enhancing effects of garlic is reviewed . Garlic can detoxify carcinogens by stimulation of cytochrome P(450) enzymes, antioxidant activity or sulfur compound binding . Studies demonstrate a direct toxic effect of garlic to sarcoma and gastric, colon, bladder and prostate cancer cells in tissue culture, but these effects cannot explain the inhibition of growth of transplanted cancer in animal models . The most likely explanation of this effect is immune stimulation . Comparison of the effects of garlic to BCG immunotherapy reveals many similarities . Both stimulate proliferation of lymphocytes and macrophage phagocytosis, induce the infiltration of macrophages and lymphocytes in transplanted tumors, induce splenic hypertrophy, stimulate release of interleukin-2, tumor necrosis factor-alpha and interferon-gamma, enhance natural killer cell, killer cell and lymphokine-activated killer cell activity . These activities represent effective stimulation of the immune response . Studies suggest that garlic may be useful in preventing the suppression of immune response that is associated with increased risk of malignancy . Data suggest that maintenance of immune stimulation can significantly reduce the risk of cancer . Clinical trials should be initiated to test the hypothesis that the immune stimulation and other beneficial effects of garlic are able to reduce the incidence of cancer.

J Infect Dis, 2001 Mar 15, 183(6), 928 - 34 Epub 2001 Feb 21.
HLA-B*35-restricted CD8 T cell epitopes in the antigen 85 complex of Mycobacterium tuberculosis; Klein MR et al.; Few target epitopes have been described for human CD8 T lymphocytes in antigens of Mycobacterium tuberculosis . By use of a reverse immunogenetics approach, 23 motif-bearing peptides of the Ag85 complex were tested for binding to HLA-B*35, one of the common B-types in West Africa . Three 9-mer peptides bound with high affinity to HLA-B*3501 and displayed low dissociation rates of peptide-major histocompatibility complexes (MHCs) . IC(50) and half-life values of peptide-MHC class I complexes were in the same range as reported earlier for other immunogenic peptides . Immune responses against peptide Ag85C (aa 204-212) WPTLIGLAM were characterized in detail . Peptide-stimulated effector cells were able to kill macrophages infected with M . tuberculosis or bacille Calmette-Guerin . Peptide-specific CD8 T cells could be visualized by using HLA-B*3501 tetramers and were shown to produce interferon-gamma and tumor necrosis factor-alpha . Together with other published epitopes, these peptides can be used to study more closely the role of CD8 T cells in mycobacterial infection and tuberculosis.

Hinyokika Kiyo, 2001 Jan, 47(1), 1 - 4
{The sensitivity and clinical implications of periodical bladder biopsy following transurethral resection of superficial bladder transitional cell carcinoma}; Kanematsu A et al.; The role of the periodical bladder biopsy after transurethral resection (TUR-Bt) of superficial bladder cancer (sBT) was evaluated . Sixty-four patients (85 TURs) with sBT who underwent TUR-Bt between 1993 and 1998 were divided into 14 (22 TURs) who had carcinoma in situ (CIS) at the first TUR (group A), and 50 (64 TURs) who had papillary tumors without concomitant CIS (group B) . Post-TUR intravesical instillation was performed with bacillus Calmette-Guerin for the majority of group A, and mitomycin C for the majority of group B . The first biopsy was performed at 3 months postoperatively, and the second biopsy was done at 8 to 12 months postoperatively . The mean observation time was 4 years and 6 months . Residual cancer was detected in 7 out of 34 biopsies (20.6%) in group A, and 19 out of 94 (20.2%) in group B . Every residual lesion in group A was CIS with negative cytology . In group B, with exclusion of 11 recurrent papillary tumors, the detection rate was only 8/83 (9.6%) . In both groups, even in the cases with no sign of disease in biopsies, the recurrence immediately after the termination of the biopsy protocol was common . The progression of the cancer was more frequent in group A (4 patients), than in group B (2 patients) (p < 0.01, log-rank test), and no case in group B showed local progression . The periodical biopsy may have a certain, but limited advantage over conventional examinations . A less invasive and more sensitive method in awaited.

Prikl Biokhim Mikrobiol, 2001 Jan-Feb, 37(1), 86 - 9
{Use of celluloselytic nitrogen-fixing bacteria in the enrichment of roughage with protein}; Smirnova IE et al.; A new strain of acid-tolerant facultative anaerobic cellulose-degrading bacteria Bacillus cytaseus 21 (Mc Bethe ef Scales, 1912), which are capable to fixing atmospheric nitrogen, was isolated . This strain is intended for solid-phase fermentation and enrichment with protein of cellulose-containing waste of plant cultivation.

Mol Biol (Mosk), 2001 Jan-Feb, 35(1), 42 - 51
{DNA-(N4-cytosine)-methyltransferase from Bacillus amyloliquefaciens: kinetic and substrate binding properties}; Malygin EG et al.; Interaction of DNA-(N4-cytosine)-methyltransferase from the Bacillus amyloliquefaciens (BamHI MTase, 49 kDa) with a 20-mer oligonucleotide duplex containing the palindrome recognition site GGATCC was studied by methods of steady-state and presteady-state kinetics of the methyl group transfer, gel retardation, and crosslinking of the enzyme subunits with glutaric aldehyde . In steady-state conditions, BamHI MTase displays a simple kinetic behavior toward a 20-mer oligonucleotide substrate . A linear dependence was observed for the reaction rate on the enzyme concentration and a Michaelis dependence of the reaction rate on the concentration of both substrates: S-adenosyl-L-methionine (SAM), the methyl group donor, and DNA, the methyl group acceptor . In independent experiments, the concentration of the 20-mer duplex or SAM was changed, the enzyme concentration being substantially lower then the concentrations of substrates . The kcat values determined in these conditions are in good agreement with one another and approximately equal to 0.05 s-1 . The Km values for the duplex and SAM are 0.35 and 1.6 microM, respectively . An analysis of single turnover kinetics (at limiting concentration of the 20-mer oligonucleotide duplex) revealed the following characteristics of the BamHI MTase-dependent methylation of DNA . The value of rate constant of the DNA methylation step at the enzyme saturating concentration is on average 0.085 s-1, which is only 1.6 times higher than the value determined in steady-state conditions . Only one of two target cytidine residues was methylated in the course of the enzyme single turnover, which coincides with the earlier data on EcoRI MTase . Regardless of the order of the enzyme preincubation with SAM and DNA, both curves for the single turnover methylation are comparable . These results are consistent with the model of the random order of the productive ternary enzyme-substrate complex formation . In contrast to the relatively simple kinetic behavior of BamHI MTase in the steady-state reaction are the data on the enzyme binding of DNA . In gel retardation experiments, there was no stoichiometrically simple complexes with the oligonucleotide duplex even at low enzyme concentrations . The molecular mass of the complexes was so high that they did not enter 12% PAG . In experiments on crosslinking of the BamHI MTase subunits, it was shown that the enzyme in a free state exists as a dimer . Introduction of substoichiometric amounts of DNA into the reaction mixture results in pronounced multimerization of the enzyme . However, addition of SAM in saturating concentration at an excess of the oligonucleotide duplex over BamHI MTase converts most of the enzyme into a monomeric state.

Nature, 2001 Feb 22, 409(6823), 1007 - 11
Massive gene decay in the leprosy bacillus; Cole ST et al.; Leprosy, a chronic human neurological disease, results from infection with the obligate intracellular pathogen Mycobacterium leprae, a close relative of the tubercle bacillus . Mycobacterium leprae has the longest doubling time of all known bacteria and has thwarted every effort at culture in the laboratory . Comparing the 3.27-megabase (Mb) genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus with that of Mycobacterium tuberculosis (4.41 Mb) provides clear explanations for these properties and reveals an extreme case of reductive evolution . Less than half of the genome contains functional genes but pseudogenes, with intact counterparts in M . tuberculosis, abound . Genome downsizing and the current mosaic arrangement appear to have resulted from extensive recombination events between dispersed repetitive sequences . Gene deletion and decay have eliminated many important metabolic activities including siderophore production, part of the oxidative and most of the microaerophilic and anaerobic respiratory chains, and numerous catabolic systems and their regulatory circuits.

J Chemother, 2001 Feb, 13(1), 52 - 8
Bacillus Calmette-Guerin down-regulates CD1b induction by granulocyte-macrophage colony stimulating factor in human peripheral blood monocytes; Prete SP et al.; Non-peptide antigens (e.g . glycolipids of microbial origin) presented by monocyte-associated CD1 molecules to T cells appear to play an important role in host immunity against tuberculosis and other pathogenic bacteria . Since vaccination with Bacillus Calmette-Guerin (BCG) has limited efficacy, the influence of viable BCG organisms on the induction of CD1b antigen by granulocyte macrophage-colony stimulating factor (GM-CSF) has been tested in adherent mononuclear cells obtained from peripheral blood of healthy donors . The results indicate that the vaccine reduces substantially CD1b induction by GM-CSF . On the other hand, BCG was found to promote a slight increase in the expression of this molecule on target cells not exposed to GM-CSF . Attempts to reverse the antagonistic effects of BCG on GM-CSF with high concentrations of GM-CSF, alone, or associated with IL-4, were unsuccessful . Moreover, mycobacteria suppression by 10 microg/ml of rifampin, did not affect BCG influence on CD1b induction . The present results suggest that mycobacterium-induced impairment of the CD1 system could play a role in the unsatisfactory results obtained with BCG vaccination.

J Protein Chem, 2000 Oct, 19(7), 621 - 30
Analysis of essential leucine residue for catalytic activity of novel thermostable chitosanase by site-directed mutagenesis; Yoon HG et al.; Bacterial chitosanases share weak amino acid sequence similarities at certain regions of each enzyme . These regions have been assumed to be important for catalytic activities of the enzyme . To verify this assumption, the functional importance of the conserved region in a novel thermostable chitosanase (TCH-2) from Bacillus coagulans CK108 was investigated . Each of the conserved amino acid residues (Leu64, Glu80, Glu94, Asp98, and Gly108) was changed to aspartate and glutamine or asparagine and glutamate by site-directed mutagenesis, respectively . Kinetic parameters for colloidal chitosan hydrolysis were determined with wild-type and 10 mutant chitosanases . The Leu64 --> Arg and Leu64 --> Gln mutations were essentially inactive and kinetic parameters such as Vmax and kcat were approximately 1/10(7) of those of the wild-type enzyme . The Asp98 --> Asn mutation did not affect the Km value significantly, but decreased kcat to 15% of that of wild-type chitosanase . On the other hand, the Asp98 --> Glu mutation affected neither Km nor kcat . The observation that approximately 15% of activity remained after the substitution of Asp98 by Asn indicated that the carboxyl side chain of Asp98 is not absolutely required for catalytic activity . These results indicate that the Leu64 residue is directly involved in the catalytic activity of TCH-2.

J Protein Chem, 2000 Oct, 19(7), 563 - 8
Enzyme-induced covalent modification of methionyl-tRNA synthetase from Bacillus stearothermophilus by methionyl-adenylate: identification of the labeled amino acid residues by matrix-assisted laser desorption-ionization mass spectrometry; Hountondji C et al.; Methionyl-tRNA synthetase (MetRS) from Bacillus stearothermophilus was shown to undergo covalent methionylation by a donor methionyl-adenylate, the mixed carboxylic-phosphoric acid anhydride synthesized by the enzyme itself . Covalent reaction of methionyl-adenylate with the synthetase or other proteins proceeds through the formation of an isopeptide bond between the carboxylate of the amino acid and the epsilon-NH2 group of lysyl residues . The stoichiometries of labeling, as followed by TCA precipitation, were 2.2 +/- 0.1 and 4.3 +/- 0.1 mol of {14C}Met incorporated by 1 mol of the monomeric MS534 and the native dimeric species of B . stearo methionyl-tRNA synthetase, respectively . Matrix-assisted laser desorption-ionization mass spectrometry designated lysines-261, -295, -301 and -528 (or -534) of truncated methionyl-tRNA synthetase as the target residues for covalent binding of methionine . By analogy with the 3D structure of the monomeric M547 species of E . coli methionyl-tRNA synthetase, lysines-261, -295, and -301 would be located in the catalytic crevice of the thermostable enzyme where methionine activation and transfer take place . It is proposed that, once activated by ATP, most of the methionine molecules react with the closest reactive lysyl residues.

J Econ Entomol, 2001 Feb, 94(1), 86 - 92
Efficacy of insecticides of different chemistries against Helicoverpa zea (Lepidoptera: Noctuidae) in transgenic Bacillus thuringiensis and conventional cotton; Brickle DS et al.; Six insecticides of different chemistries were evaluated against the cotton bollworm, Helicoverpa zea (Boddie), in non-B.t . (Deltapine 'DP 5415', Deltapine 'DP 5415RR') and transgenic Bacillus thuringiensis (Berliner) (B.t.) (Deltapine 'NuCOTN 33B', Deltapine 'DP 458 B/RR') cotton . In 1998, treatments consisted of three rates each of a pyrethroid (lambda-cyhalothrin), spinosyn (spinosad), carbamate (thiodicarb), pyrrole (chlorfenapyr), oxadiazine (indoxacarb), and avermectin (emamectin benzoate) in a nonirrigated field . In 1999, treatments consisted of three rates each of lambda-cyhalothrin, spinosad, thiodicarb, and indoxacarb in an irrigated and a nonirrigated (dryland) field . The highest rate of each insecticide corresponded to normal grower-use rates . Spinosad and thiodicarb controlled H . zea in non-B.t . cotton, whereas other materials were less effective . Even though H . zea is becoming increasingly resistant to pyrethroid insecticides, lambda-cyhalothrin was highly effective in dryland B . thuringiensis cotton . Spinosad and thiodicarb were equally effective . Data indicated that reduced rates of lambda-cyhalothrin, spinosad, and thiodicarb could be used for control of H . zea in dryland B.t . cotton systems . However, reduced rates of these insecticides in a heavily irrigated B.t . cotton system did not provide adequate control.

J Econ Entomol, 2001 Feb, 94(1), 68 - 75
Spider fauna in apple ecosystem of western Oregon and its field susceptibility to chemical and microbial insecticides; Bajwa WI et al.; Twelve families, 26 genera, and 30 identifiable spider species were found in surveys conducted in apple orchards of western Oregon . The Salticidae, Linyphiidae, Clubionidae, Philodromidae, and Theridiidae comprised 85.56% ofthe total spiders collected . The most common species in order of abundance were Metaphidippus aeneolus Curtis, Spirembolus mundus Chamberlin & Ivie, Cheiracanthium inclusum (Hentz), Philodromus spectabilis Keyserling, Eris marginata (Walckenaer), and Theridion lawrencei Gertsch & Archer . Individuals of these species were collected in 50-60% of the samples and were most abundant in the month of August . The Bacillus thuringiensis-based insecticides, DiPel (100 Million International Units/100 liters) and MVP (250 ml/100 liters), summer oil (0.5-1.0 liter/100 liters), the insect growth regulator (IGR) diflubenzuron (3-12 g/100 liters), and organophosphate Phosmet (6-60 g/100 liters) were generally harmless (P > 0.05) to these spider species . Full field rates of organophosphate azinphosmethyl (25 g/100 liters) and carbamate carbaryl (60 g/100 liters) were slightly to moderately harmful (25-75% mortality) . These insecticides at reduced rates (azinphosmethyl 2.5-5.0 g and carbaryl 12 g/100 liters) applied alone or in combination with DiPel and MVP, had a negligible effect . Full rates of pyrethroids esfenvalerate (2.5 g/100 liters) and permethrin (4.0 g/100 liters) were moderately to highly harmful (50-75% mortality) and their reduced rates (esfenvalerate 0.25-0.50 g and permethrin 0.4-0.8 g/100 liters) were selective to the spiders.

J Econ Entomol, 2001 Feb, 94(1), 284 - 90
Correlating differences in larval survival and development of bollworm (Lepidoptera: Noctuidae) and fall armyworm (Lepidoptera: Noctuidae) to differential expression of Cry1A(c) delta-endotoxin in various plant parts among commercial cultivars of transgenic Bacillus thuringiensis cotton; Adamczyk JJ et al.; Differences in larval survival and development of bollworm, Helicoverpa zea (Boddie), and fall armyworm, Spodoptera frugiperda (J . E . Smith), respectively, were found to exist among commercially available Cry1A(c) transgenic Bacillus thuringiensis Berliner (Bt) varieties . Using a quantification assay (ELISA) to measure the levels of delta-endotoxin in two of these varieties ('DP 451B/RR' and 'NuCOTN 33B'), differences in the amount of delta-endotoxin present in various plant parts was correlated with larval survival of bollworms and larval development of fall armyworms throughout the growing season . Larvae that were fed on DP 451B/RR completed development faster and exhibited better survivorship than those larvae fed NuCOTN 33B, whereas lower levels of delta-endotoxin were generally detected in plant parts from DP 451B/RR compared with NuCOTN 33B . These differences may impact population dynamics of these pests which may be a critical factor in managing resistance to Bt . Furthermore, the utility of using this system for providing information to the grower concerning varietal choices may be more common in the future.

J Econ Entomol, 2001 Feb, 94(1), 271 - 6
Field evaluation of resistance of transgenic rice containing a synthetic cry1Ab gene from Bacillus thuringiensis Berliner to two stem borers; Ye GY et al.; Two transgenic rice (Oryza sativa L.) lines, KMD1 and KMD2 at the R4 generation, transformed with a synthetic cry1Ab gene from Bacillus thuringiensis Berliner, were first evaluated for stem borer resistance in the field during the rice growing season of 1998 in two areas of Zhejiang Province, China . Both KMD1 and KMD2 were highly resistant to the stem borers Chilo suppressalis (Walker) and Scirpophaga incertulas (Walker), and were completely undamaged during the whole rice growing season . In contrast, damage to the plants of the untransformed parental control (Xiushui 11) was in the form of deadhearts or whiteheads . Under natural infestation by the C . suppressalis, the damage to control plants reached a peak of 88.7% of plants and 20.1% of tillers encountered with deadhearts . Under artificial and natural infestation of neonate striped stem borers at the vegetative stage and booting stage, 100% of plants and 25.6% of tillers, 78.9% of plants and 15.6% of productive tillers among artificially infested control plants were observed with the symptom of deadhearts and whiteheads, respectively . Damage to the control plants from artificial infestation by the S . incertulas reached a peak of 97.0% of plants and 22.9% of tillers damaged . The field research indicated that both KMD1 and KMD2 show great potential for protecting rice from attack by these two stem borers.

J Econ Entomol, 2001 Feb, 94(1), 248 - 52
Genetics of pink bollworm resistance to Bacillus thuringiensis toxin Cry1Ac; Liu YB et al.; Laboratory selection increased resistance of pink bollworm (Pectinophora gossypiella) to the Bacillus thuringiensis toxin Cry1Ac . Three selections with Cry1Ac in artificial diet increased resistance from a low level to >100-fold relative to a susceptible strain . We used artificial diet bioassays to test F1 hybrid progeny from reciprocal crosses between resistant and susceptible strains . The similarity between F1 progeny from the two reciprocal crosses indicates autosomal inheritance of resistance . The dominance of resistance to Cry1Ac depended on the concentration . Resistance was codominant at a low concentration of Cry1Ac, partially recessive at an intermediate concentration, and completely recessive at a high concentration . Comparison of the artificial diet results with previously reported results from greenhouse bioassays shows that the high concentration of Cry1Ac in bolls of transgenic cotton is essential for achieving functionally recessive inheritance of resistance.

J Econ Entomol, 2001 Feb, 94(1), 240 - 7
Greenhouse tests on resistance management of Bt transgenic plants using refuge strategies; Tang JD et al.; Experimental evaluation of the effectiveness of resistance management tactics is vital to help provide guidelines for the deployment of transgenic insecticidal crops . Transgenic broccoli expressing a Cry1Ac gene of Bacillus thuringiensis (Bt) and the diamondback moth, Plutella xylostella (L.), were used in greenhouse tests to evaluate the influence of size and placement of nontransgenic refuge plants on changes in resistance allele frequency and pest population growth . In the first test with an initial Cry1Ac-resistance (R) allele frequency of 0.007, P . xylostella were introduced into cages with the following treatments: 0, 3.3, 10, 20, and 100% refuge plants . Results after four generations showed that resistance could be delayed by increasing the proportion of refuge plants in the cage . Population growth was also influenced by refuge size with the highest populations occurring in treatments that had either no refuge plants or all refuge plants . In the second test, we evaluated the effect of refuge placement by comparing 20% separate and 20% mixed refuges . P . xylostella with an initial frequency of resistant alleles at 0.0125 were introduced into cages and allowed to cycle; later generations were evaluated for resistance and population growth . Separating the refuge had a pronounced effect on delaying resistance and slowing establishment of resistant larvae on Bt plants . Combining information from both trials, we found a strong negative correlation between the number of larvae on Bt plants and the mortality of the population in leaf dip bioassays . Results from larval movement studies showed that separate refuges delayed resistance better than mixed refuges because they conserved relatively more susceptible alleles than R alleles and did not increase the effective dominance of resistance.

J Econ Entomol, 2001 Feb, 94(1), 197 - 203
Field efficacy of sweet corn hybrids expressing a Bacillus thuringiensis toxin for management of Ostrinia nubilalis (Lepidoptera: Crambidae) and Helicoverpa zea (Lepidoptera: Noctuidae); Burkness EC et al.; Field studies were done in 1995-1996 to assess the efficacy of three sweet corn hybrids that express the Bacillus thuringiensis (Bt) toxin, CrylAb, against two lepidopteran pests, Ostrinia nubilalis (Hubner) and Helicoverpa zea (Boddie) . The Bt hybrids tested were developed by Novartis Seeds, using the event BT-11, which expresses Bt toxin in green tissue as well as reproductive tissues including the tassel, silk, and kernel . Bt hybrids were compared with a standard non-Bt control or the non-Bt isoline for each hybrid; none of the hybrids were treated with insecticides during the study . Hybrid efficacy was based on larval control of each pest, as well as plant or ear damage associated with each pest . In both years, control of O . nubilalis larvae in primary ears of all Bt hybrids was 99-100% compared with the appropriate non-Bt check . Plant damage was also significantly reduced in all Bt hybrids . In 1996, control of H . zea in Bt hybrids ranged from 85 to 88% when compared with the appropriate non-Bt control . In 1996, a University of Minnesota experimental non-Bt hybrid (MN2 x MN3) performed as well as the Bt hybrids for control of O . nubilalis . Also, in 1996, two additional University of Minnesota experimental non-Bt hybrids (A684su X MN94 and MN2 X MN3) performed as well as Bt hybrids for percent marketable ears (ears with no damage or larvae) . In addition, compared with the non-Bt hybrids, percent marketable ears were significantly higher for all Bt hybrids and in most cases ranged from 98 to 100% . By comparison, percent marketable ears for the non-Bt hybrids averaged 45.5 and 37.4% in 1995 and 1996, respectively . Results from the 2-yr study strongly suggest that Bt sweet corn hybrids will provide high levels of larval control for growers in both fresh and processing markets . Specifically, Bt sweet corn hybrids, in the absence of conventional insecticide use, provided excellent control of O . nubilalis, and very good control of H . zea . However, depending on location of specific production regions, and the associated insect pests of sweet corn in each area, some insecticide applications may still be necessary.

J Econ Entomol, 2001 Feb, 94(1), 112 - 5
Effects of a broad spectrum and biorational insecticides on parasitoids of the Nantucket pine tip moth (Lepidoptera: Tortricidae); McCravy KW et al.; We examined effects of aerial application of acephate (Orthene), Bacillus thuringiensis variety kurstaki Berliner (Foray), and tebufenozide (Mimic) on larval/pupal parasitoids of the Nantucket pine tip moth, Rhyacionia frustrana (Comstock), in the southwestern Georgia coastal plain . Parasitism of tip moths in acephate-treated plots was significantly lower than in untreated plots . Bacillus thuringiensis and tebufenozide showed no significant effects on parasitism . A tachinid, Lixophaga mediocris Aldrich, comprised a significantly greater proportion of emerging parasitoids in acephate-treated than in untreated control plots, whereas a chalcidid, Haltichella rhyacioniae Gahan, was less abundant in the acephate-treated plots . Acephate has a negative, but somewhat species-specific, impact on tip moth parasitism.

Infect Control Hosp Epidemiol, 2001 Feb, 22(2), 88 - 93
Factors associated with tuberculin reactivity in two general hospitals in Mexico; Garcia-Garcia ML et al.; OBJECTIVE: To identify risk factors associated with tuberculin reactivity in healthcare workers (HCWs) . DESIGN: Cross-sectional survey of tuberculin reactivity (2 TU of purified protein derivative (PPD) RT23, using the Mantoux two-step test) . SETTING: Two general hospitals located in a region with a high prevalence of tuberculosis and high bacille Calmette-Guerin (BCG) coverage . PARTICIPANTS: Volunteer sample of HCWs . RESULTS: 605 HCWs were recruited: 71.2% female; mean age, 36.4 (standard deviation {SD}, 8.2) years; 48.9% nurses, 10.4% physicians, 26.8% administrative personnel; mean time of employment, 10.9 (SD, 6.7) years . PPD reactivity (> or =10 mm) was found in 390 (64.5%) . Multivariate analysis revealed an association of tuberculin reactivity with occupational exposure in the hospital: participation in autopsies (odds ratio {OR}, 9.3; 95% confidence interval {CI95}, 2.1-40.5; P=.003.), more than 1 year of employment (OR, 2.4; CI95, 1.1-5.0; P=.02), work in the emergency or radiology departments (OR, 2.0; CI95, 1.03-3.81; P=.04), being physicians or nurses (OR, 1.5; CI95, 1.04-2.11; P=.03), age (OR, 1.04; CI95, 1.02-1.07 per year of age; P<.001), and BCG scar (OR, 2.1; CI95, 1.2-3.4; P=.005) . CONCLUSIONS: Although the studied population has a high baseline prevalence of tuberculosis infection and high coverage of BCG vaccination, nosocomial risk factors associated with PPD reactivity were identified as professional risks; strict early preventive measures must be implemented accordingly.

J Chromatogr B Biomed Sci Appl, 2001 Feb 10, 751(1), 143 - 51
Electron-capture gas chromatographic-chemical ionization mass spectrometric study of sera from people vaccinated with bacille Calmette-Guerin for characteristic metabolites; Syriopoulou V et al.; Serum samples from 26 individuals vaccinated with bacille Calmette-Guerin (BCG) and from 26 controls (10 patients with pulmonary tuberculosis and 16 non BCG-vaccinated healthy individuals) were analyzed by frequency-pulsed electron-capture gas chromatography (FPEC-GC) and chemical ionization gas chromatography-mass spectrometry (CIGC-MS) for the presence of characteristic metabolites . A distinct pattern consisted of tuberculostearic acid (TSA) and a peak, labeled peak 1, was observed in all BCG-vaccinated individuals, whereas only three of 26 controls generated this chromatography profile . TSA was detected in all patients with pulmonary tuberculosis but peak 1 was absent . Sera drawn from 12 individuals 11 to 14 days after BCG vaccination yielded three transitional FPEC-GC profiles . A permanent FPEC-GC profile consisting of TSA and of a full scale peak 1 appeared 28 days to a few months after BCG vaccination . Peak 1 was tentatively identified by CIGC-MS as 9-methyl-hexacosanol . The findings suggest that peak 1 may serve as a marker to detect Mycobacterium bovis BCG and to distinguish individuals infected with M . tuberculosis from individuals vaccinated with BCG.

Pediatrics . 2001 Mar;107(3):E36.
Delayed hypersensitivity to tuberculin, total immunoglobulin E, specific sensitization, and atopic manifestation in longitudinally followed early Bacille Calmette-Guérin-vaccinated and nonvaccinated children; Gruber C et al.; BACKGROUND: Bacille Calmette-Guerin (BCG) is a strong T helper 1 incentive and, thus, may contribute to a decreased risk of T helper 2-dependent atopic disease . OBJECTIVE: To investigate the natural course of specific immunoglobulin E (IgE) responses and atopic disease in BCG-vaccinated and nonvaccinated children . PARTICIPANTS: Seven hundred seventy-four children from a prospectively followed birth cohort . OUTCOME MEASURES: Physical examination and case history were performed at 3, 6, 12, 18, 24, 36, 48, 60, 72, and 84 months of age . Total and specific serum IgE levels to 9 common inhalant and food allergens were determined (CAP; Pharmacia, Freiburg, Germany) at 12, 24, 36, 60, 72, and 84 months of age . Purified protein derivative (PPD) skin testing was performed at 84 months . RESULTS: Period and lifetime prevalences of atopic dermatitis and recurrent wheezing tended to be lower in the BCG-vaccinated group early in life, whereas no such trend was found after the second birthday or for allergic rhinitis . The proportion of children remaining free of clinical manifestations tended to be higher in the BCG-vaccinated group but differences decreased over time . No statistically significant differences were found for total IgE levels (median) . Atopic sensitization tended to be lower among BCG-vaccinated children during the first 2 years of life . The diameter of the skin reaction to PPD did not correlate with total serum IgE . Clinical and serologic correlates of atopy were not significantly different between children with a skin test diameter of >/=5 mm and those with a smaller diameter . CONCLUSION: These results do not support the hypothesis that BCG vaccination in early infancy is associated with a subsequently markedly decreased risk of atopic sensitization or allergy . In addition, PPD skin test reactivity was not impaired in atopic individuals.

J Clin Microbiol, 2001 Mar, 39(3), 1032 - 5
Evaluation of a fluorescence in situ hybridization assay for differentiation between tuberculous and nontuberculous Mycobacterium species in smears of Lowenstein-Jensen and Mycobacteria Growth Indicator Tube cultures using peptide nucleic acid probes; Hongmanee P et al.; A new fluorescence in situ hybridization assay based on peptide nucleic acid probes (MTB and NTM probes targeting tuberculous and nontuberculous species, respectively) for the identification of Mycobacterium tuberculosis complex and differentiation between tuberculous and nontuberculous mycobacteria (NTM) was evaluated using Lowenstein-Jensen (LJ) solid cultures from 100 consecutive sputum samples and 50 acid-fast bacillus (AFB)-positive sputum samples as well as Mycobacteria Growth Indicator Tube (MGIT) liquid cultures from 80 AFB-positive sputum samples . Mycobacterium species could be identified from a total of 53 LJ cultures and 77 MGIT cultures . The diagnostic specificities of the MTB and NTM probes were 100% for both cultures . The diagnostic sensitivities of the MTB probe for the LJ and MGIT cultures were 98 and 99%, respectively, whereas the sensitivities of the NTM probe were 57 and 100%, respectively . The relatively low sensitivity of the NTM probe was due to a high proportion of M . fortuitum, which is not identified by the probe.

Appl Environ Microbiol, 2001 Mar, 67(3), 1300 - 7
Solid-phase capture of proteins, spores, and bacteria; Weimer BC et al.; Current methods for the detection of pathogens in food and water samples generally require a preenrichment step that allows selective enrichment of the test organism . The objective of this research was to eliminate an enrichment step to allow detection of bacteria directly in food and water samples in 30 min . A high-flow-rate, fluidized bed to capture and concentrate large (bacteria and spores) and small (protein) molecules was developed . This format, ImmunoFlow, is volume independent and uses large beads (greater than 3 mm in diameter) when capturing bacteria to prevent sample clogging when testing food samples . Detection of bound targets was done using existing enzyme-linked immunosorbent assay (ELISA) protocols . Four antibodies (anti-Escherichia coli O157:H7, -Bacillus globigii, -bovine serum albumin {BSA}, and -ovalbumin {OVA}) were covalently coupled to various glass and ceramic beads . Very small amounts of BSA (<1 ng) and OVA (0.2 to 4.0 microg) were detected . Various industrial and environmental samples were used to observe the effect of the sample composition on the capture of anti-B . globigii and anti-E . coli O157:H7 modified beads . The lower limit of detection for both E . coli O157:H7 and B . globigii was 1 spore/cell independent of the sample size . The activity of anti-B . globigii modified beads declined after 3 days . Anti-E . coli O157:H7 modified beads declined in their capture ability after 2 days in various storage buffers . Storage temperature (4 and 25 degrees C) did not influence the stability . The ImmunoFlow technology is capable of capturing bacteria and spores directly from samples, with subsequent detection in an ELISA format in 30 min.

Appl Environ Microbiol, 2001 Mar, 67(3), 1090 - 6
The Bacillus thuringiensis cyt genes for hemolytic endotoxins constitute a gene family; Guerchicoff A et al.; In the same way that cry genes, coding for larvicidal delta endotoxins, constitute a large and diverse gene family, the cyt genes for hemolytic toxins seem to compose another set of highly related genes in Bacillus thuringiensis . Although the occurrence of Cyt hemolytic factors in B . thuringiensis has been typically associated with mosquitocidal strains, we have recently shown that cyt genes are also present in strains with different pathotypes; this is the case for the morrisoni subspecies, which includes strains biologically active against dipteran, lepidopteran, and coleopteran larvae . In addition, while one Cyt type of protein has been described in all of the mosquitocidal strains studied so far, the present study confirms that at least two Cyt toxins coexist in the more toxic antidipteran strains, such as B . thuringiensis subsp . israelensis and subsp . morrisoni PG14, and that this could also be the case for many others . In fact, PCR screening and Western blot analysis of 50 B . thuringiensis strains revealed that cyt2-related genes are present in all strains with known antidipteran activity, as well as in some others with different or unknown host ranges . Partial DNA sequences for several of these genes were determined, and protein sequence alignments revealed a high degree of conservation of the structural domains . These findings point to an important biological role for Cyt toxins in the final in vivo toxic activity of many B . thuringiensis strains.

Appl Environ Microbiol, 2001 Mar, 67(3), 1085 - 9
Different mechanisms of resistance to Bacillus thuringiensis toxins in the indianmeal moth; Herrero S et al.; Susceptibility to protoxin and toxin forms of Cry1Ab and the binding of (125)I-labeled Cry1Ab and Cry1Ac has been examined in three Plodia interpunctella colonies, one susceptible (688(s)) and two resistant (198(r) and Dpl(r)) to Bacillus thuringiensis . Toxicological studies showed that the 198(r) colony was 11-fold more resistant to Cry1Ab protoxin than to Cry1Ab activated toxin, whereas the Dpl(r) colony was 4-fold more resistant to protoxin versus toxin . Binding results with (125)I-labeled toxins indicated the occurrence of two different binding sites for Cry1Ab in the susceptible insects, one of them shared with Cry1Ac . Cry1Ab binding was found to be altered in insects from both resistant colonies, though in different ways . Compared with the susceptible colony, insects from the Dpl(r) colony showed a drastic reduction in binding affinity (60-fold higher K(d)), although they had similar concentrations of binding sites . Insects from the 198(r) colony showed a slight reduction in both binding affinity and binding site concentration (five-fold-higher K(d) and ca . three-fold-lower R(t) compared with the 688(s) colony) . No major difference in Cry1Ac binding was found among the three colonies . The fact that the 198(r) colony also has a protease-mediated mechanism of resistance (B . Oppert, R . Hammel, J . E . Throne, and K . J . Kramer, J . Biol . Chem . 272:23473-23476, 1997) is in agreement with our toxicological data in which this colony has a different susceptibility to the protoxin and toxin forms of Cry1Ab . It is noteworthy that the three colonies used in this work derived originally from ca . 100 insects, which reflects the high variability and high frequency of B . thuringiensis resistance genes occurring in natural populations.

Appl Environ Microbiol, 2001 Mar, 67(3), 1035 - 43
Identification of Bacillus thuringiensis subsp . kurstaki strain HD1-Like bacteria from environmental and human samples after aerial spraying of Victoria, British Columbia, Canada, with Foray 48B; Valadares De Amorim G et al.; Aerial applications of Foray 48B, which contains Bacillus thuringiensis strain HD1, were carried out on 9 to 10 May, 19 to 21 May, and 8 to 9 June 1999 to control European gypsy moth (Lymantria dispar) populations in Victoria, British Columbia, Canada . A major assessment of the health impact of B . thuringiensis subsp . kurstaki was conducted by the Office of the Medical Health Officer of the Capital Health Region during this period . Environmental (air and water) and human (nasal swab) samples, collected before and after aerial applications of Foray 48B, both in the spray zone and outside of the spray zone, were analyzed for the presence of strain HD1-like bacteria . Random amplified polymorphic DNA analysis, cry gene-specific PCR, and dot blot DNA hybridization techniques were used to screen over 11,000 isolates of bacteria . We identified bacteria with genetic patterns consistent with those of B . thuringiensis subsp . kurstaki HD1 in 9,102 of 10,659 (85.4%) isolates obtained from the air samples, 13 of 440 (2.9%) isolates obtained from the water samples, and 131 of 171 (76.6%) isolates from the nasal swab samples . These analyses suggest that B . thuringiensis subsp . kurstaki HD1-like bacteria were present both in the environment and in the human population of Victoria prior to aerial applications of Foray 48B . The presence of B . thuringiensis subsp . kurstaki HD1-like bacteria in human nasal passages increased significantly after the application of Foray 48B, both inside and outside the spray zone.

Vaccine, 2001 Feb 28, 19(15-16), 2061 - 70
Nasal immunization with E . coli verotoxin 1 (VT1)-B subunit and a nontoxic mutant of cholera toxin elicits serum neutralizing antibodies; Byun Y et al.; Escherichia coli O157:H7 produces two forms of verotoxin (VT), VT1 and VT2, which cause hemorrhagic colitis with development, in some cases, of hemolytic uremic syndrome . These toxins consist of an enzymatically active A subunit and pentamers of B subunit responsible for their binding to host cells . We used the secretion-expression system of Bacillus brevis to produce recombinant VT1B and VT2B . The secreted B subunits were purified and sequenced to verify their structure . Receptor-binding showed that rVT1B but not rVT2B bound to Gb3-receptor . When mice were nasally immunized with rVT1B or rVT2B together with a nontoxic mutant of cholera toxin (mCT) or native cholera toxin (nCT) as adjuvants, serum IgG and mucosal IgA antibody responses to VT1B were induced . The VT1B-specific antibodies prevented VT1B binding to its Gb3 receptor . In contrast, poor serum and no mucosal VT2B-specific antibodies but brisk CTB-specific antibody responses were induced by nasal immunization with rVT2B in the presence of mCT or nCT . These results show that nasal immunization with rVTB and mCT as a nontoxic mucosal adjuvant is an effective regimen for the induction of VT1B but not VT2B antibody responses which inhibit VT1B binding to Gb3 receptor.

Vaccine, 2001 Feb 28, 19(15-16), 1968 - 77
Immune reactions of CD4- and CD8-positive T cell subpopulations in spleen and lymph nodes of guinea pigs after vaccination with Bacillus Calmette Guerin; Klunner T et al.; Vaccination of guinea pigs with Mycobacterium bovis BCG confers partial resistance against infection with Mycobacterium tuberculosis . Induction of immunity is associated with a strong T cell response . The reactions of the cytotoxic and helper T lymphocyte subsets after BCG vaccination were analyzed by cytofluorometry and in functional tests . The relative number of CD8(+) T cells in the spleen increased substantially after injection of BCG . In vitro restimulation after immunization induced a strong proliferative response but no cytotoxic reactions of CD8(+) T cells against BCG-infected macrophages . A specific induction of IFN-gamma and RANTES mRNA was observed after vaccination particularly in CD8(+) but not in CD4(+) T cells of the lymph nodes.

Vaccine, 2001 Feb 28, 19(15-16), 1906 - 10
Protective responses against experimental Mycobacterium leprae infection in mice induced by recombinant Bacillus Calmette-Guérin over-producing three putative protective antigen candidates; Ohara N et al.; The components of Ag85 (Ag85A, Ag85B, and Ag85C) are putative protective antigen candidates against mycobacterial infection . A recombinant Mycobacterium bovis Bacillus Calmette-Guerin (rBCG) over-producing Ag85A, Ag85B, and MPB51 (rBCG/BA51) was constructed . rBCG/BA51 could secrete these antigens at levels more than five times higher than parental BCG . Immunization of C57BL/6 and BALB/c mice with this rBCG reduced the multiplication of Mycobacterium leprae in the foot pads of both strains of mice . The inhibition by rBCG/BA51 was more evident than that by parental BCG.

J Biochem (Tokyo), 2001 Mar, 129(3), 461 - 8
Crystal structure of Bacillus stearothermophilus alpha-amylase: possible factors determining the thermostability; Suvd D et al.; The crystal structure of a thermostable alpha-amylase from Bacillus stearothermophilus (BSTA) has been determined at 2.0 A resolution . The main-chain fold is almost identical to that of the known crystal structure of Bacillus licheniformis alpha-amylase (BLA) . BLA is known to be more stable than BSTA . A structural comparison between the crystal structures of BSTA and BLA showed significant differences that may account for the difference in their thermostabilities, as follows . (i) The two-residue insertion in BSTA, Ile181-Gly182, pushes away the spatially contacting region including Asp207, which corresponds to Ca(2+)-coordinating Asp204 in BLA . As a result, Asp207 cannot coordinate the Ca(2+) . (ii) BSTA contains nine fewer hydrogen bonds than BLA, which costs about 12 kcal/mol . This tendency is prominent in the (beta/alpha)(8)-barrel, where 10 fewer hydrogen bonds were observed in BSTA . BLA forms a denser hydrogen bond network in the inter-helical region, which may stabilize alpha-helices in the barrel . (iii) A few small voids observed in the alpha-helical region of the (beta/alpha)(8)-barrel in BSTA decrease inter-helical compactness and hydrophobic interactions . (iv) The solvent-accessible surface area of charged residues in BLA is about two times larger than that in BSTA.

J Steroid Biochem Mol Biol, 2000 Dec 15, 75(2-3), 187 - 99
Transformation of steroids by Bacillus strains isolated from the foregut of water beetles (Coleoptera: Dytiscidae): II . Metabolism of 3 beta-hydroxypregn-5-en-20-one (pregnenolone); Schaaf O et al.; The in vitro metabolism of pregnenolone by two Bacillus strains (HA-V6-3 and HA-V6-11) isolated from the foregut of the water beetle Agabus affinis (Payk.) was examined in the course of our studies about a possible participation of gut micro-organisms in the biosynthesis of prothoracic defensive steroids of dytiscids . The transformation products were identified by EI GC--MS of culture extracts after derivatization . The dominating reactions were hydroxylations, with 7 alpha-hydroxypregnenolone as the major product . With considerably lower yields, 7 beta- and 15xi-hydroxypregenolone were formed by both strains, while 11, 17 and 16 alpha-hydroxypregnenolone were produced only by HA-V6-3 . The occurrence of 7, 11 alpha- and 7 beta, 11 alpha-dihydroxypregnenolone as well as several minor products containing a 17 alpha-OH group proved the capability of HA-V6-11 to hydroxylate pregenenolone at C(11) and C(17) as well . The monohydroxylated 7-OH-pregnenolones were partly oxidized to 7-oxopregnenolone by both strains . In trace amounts, HA-V6-3 performed 3 beta-acetylation of pregnenolone.

J Virol Methods, 2001 Apr, 92(2), 193 - 7
Detection of prawn white spot bacilliform virus by immunoassay with recombinant antigen; Zhang X et al.; Prawn white spot bacilliform virus (WSBV) is the major pathogen of prawn disease . To develop a sensitive assay for the early detection of this virus, we generated an antibody against a WSBV-specific protein, P204 . The p204 gene was cloned from a WSBV cDNA library and expressed in Escherichia coli . The peptide (P204) encoded by p204 was purified, and its antibody raised in mice . IgG fraction of the anti-P204 serum was purified using a Sepharose column and the Fab fragment was obtained by pepsin digestion . An enzyme-linked immunosorbent assay (ELISA) using this Fab fragment was developed to detect the WSBV in prawn tissues . The experiments showed that Fab of anti-P204 antibody is highly specific for WSBV, and displays a sensitivity of 10(5) viral particles/mg prawn tissues . This is the first report using the Fab fragment prepared from a recombinant antigen to detect prawn viruses by ELISA.

Klin Padiatr, 2001 Jan-Feb, 213(1), 39 - 42
{Tufted angioma}; Michel S et al.; We present a 2-year-old boy with a red, cutaneous-subcutaneous, nodule on the right elbow and a 2.5 year-old girl with an red-brown, indurated plaque on the left knee . Colour-coded doppler sonography of the boy's lesion showed vascular structures . A biopsy established the diagnosis of tufted angioma in both patients . Tufted angioma is clinically characterized by slowly spreading erythematous macules and plaques preferentially located on the upper trunk and neck in children . It is a benign tumor, malignant transformation has not been reported . The case history, clinical and histological findings contribute to the diagnosis . Tufted angioma has to be distinguished from Kaposi's sarcoma, angiosarcoma, hemangioma of infancy, sometimes bacillary angiomatosis and other cutaneous capillary malformations . Treatment of tufted angioma is difficult, various modalities like glucocorticosteroids, Interferon-alpha, flashlamp-pumped pulsed dye laser, excision and spontaneous regression have been described with varying results.

Int J Lepr Other Mycobact Dis, 2000 Sep, 68(3), 247 - 57
Single lesion paucibacillary leprosy: baseline profile of the Brazilian Multicenter Cohort Study; Martelli CM et al.; In Brazil, there is little information about the clinical and epidemiological characteristics of paucibacillary, single skin lesion leprosy patients (SSL-PB) . Only recently has the official notification system distinguished leprosy patients with a single lesion as a clinical entity, for whom the single-dose ROM (rifampin, ofloxacin and minocycline) regimen has been recommended . In this paper, we describe the baseline clinical features and the immunological background of a multicenter cohort of SSL-PB leprosy cases enrolled between December 1997-1998 . Patients were recruited at health centers located in the following regions: Southeast = Rio de Janeiro; North = Amazon and Rondonia states and Center-West = Goias state . Eligible cases were newly detected, untreated single-lesion leprosy patients without thickened nerve involvement, and were assessed by clinical, bacilloscopic and histopathological exams . The Mitsuda skin test and anti-PGL-I serology (ELISA) were also performed . Of the 299 SSL-PB leprosy patients, 259 (86.6%) fulfilled the criteria for single-dose ROM intervention . Our results showed that patients recruited from different sites had similar features, considering the clinical and immunological profiles . There was a predominance of adults (mean age 32.4; S.D . = 16.0), and a BCG scar was detected in 76.7% of the children (< or = 15 years old) . Only 7 cases were diagnosed as the multibacillary type, representing less than 3% of the patients being misclassified . Our data indicate that in Brazil SSL-PB case ascertainment based on clinical and bacilloscopic criteria can be accurately defined under a routine control program; 75.0% of SSL-PB cases were Mitsuda positive (> or = 5 mm) and seropositivity for anti-PGL-I was detected in 17.3% of the patients . These data are compatible with effective cell-mediated immunity and low bacillary load, suggesting favorable clinical outcomes for most SSL-PB participants of this cohort.

Med Arh, 2000, 54(5-6), 321 - 2
{Cat-scratch disease}; Saracevic E et al.; We report a case of fourteen years old boy with cat scratch disease . He was treated at Pediatric Clinic in January and February 2000 . Cat scratch disease is benign infectious chronic disease which appears in all ages . It begins after contact with cat and its scratch . Yearly incidence of this disease for whole population is 0.77-0.86/100.000 . The cause is bacillus Bartonella which is also called genus Rochalimaea . It is Gram negative, mobile, aerobe bacillus with ten flagellas on one pole . Our patient was admitted at hospital because of high temperature, paleness, fatigue, bad appetite and pains in his joint . Ultrasound and magnet resonance scans of liver and spleen showed enlarged but regulary contured liver and two focal changes with diameter 16 mm, 12 mm and 7 mm in spleen . All those focal changes were described as abscesses . Diagnose was set up according increasing titre of IF-Bartonella hensalae IgG 1:256 (positive) . This diagnostic test was performed at Institute for microbiology and Immunology at Medical Faculty in Ljubljana . Disease had long lasting course, and was cured by antibiotics without surgical intervention.

J Antibiot (Tokyo), 2000 Dec, 53(12), 1363 - 72
Molecular cloning and sequencing of the edeine B1 amidinohydrolase gene of Bacillus brevis TT02-8 and its expression in Escherichia coli; Shimotohno KW et al.; The gene encoding edeine B1 amidinohydrolase from Bacillus brevis TT02-8 was cloned into Escherichia coli and its nucleotide sequence was determined . An open reading frame was identified and was found to encode a polypeptide of 289 amino acid residues with a predicted molecular weight of 32,455, which was consistent with that previously calculated for edeine B1 amidinohydrolase purified from this bacterium . Comparison of the deduced amino acid sequence of this enzyme with other amidinohydrolases revealed the highest homology to B . subtilis agmatine ureohydolase . The enzymatic activity of the protein produced in Escherichia coli was analyzed . Three histidine residues, H-112, H-137 and H-151 in the edeine B1 amidinohydrolase, which are highly conserved in amidinohydrolases, were changed to alanine by site-directed mutagenesis . Analysis of each of these mutants revealed that three histidine residues are important but not essential for the enzyme activity.

Chem Pharm Bull (Tokyo), 2001 Feb, 49(2), 129 - 33
Formulation design of ointment base suitable for healing of lesions in treatment of bedsores; Shigeyama M et al.; We intended to develop a desired ointment base suitable for treatment of bedsores including the proliferation of granulation and epidermis . The main bedsore bacteria detected in our hospital were S . aureus in gram-positive coccus and P . aeruginosa in gram-negative bacillus . As the macrogol ointment (MO) was found to have bactericidal effects on these bacteria, MO was adopted as the base for the objective ointment . To improve the properties of the ointment base such as regulating the humidity of the exudation and controlling the release of antibiotics formulated in the ointment, co-formulating effects of various additives to MO were evaluated . The sustained release function of the ointment base was obtained by adding hydrophilic petrolatum (HP) to MO . However, the resultant ointment was found to have a poor humidity regulating property . On the other hand, MO containing 5% of hydroxypropyl cellulose (HPC) showed both the humidity regulating and the controlled drug releasing properties . It was considered that HPC particles dispersed in the ointment could be swelled by absorbing water to form a gel network . The curd tension meter tests for the ointments prepared with the various polymers showed that the MO-HPC base, which showed the highest sustained drug releasing property, was found to have the highest hardness . This result means that HPC formulated into the base forms the most rigid gel structure to resist the erosion of the ointment and to control the drug release.

Tidsskr Nor Laegeforen, 2000 Dec 10, 120(30), 3714 - 8
{From The Origin of Species to Artenes Oprindelse}; Lie T; The Origin of Species, Charles Darwin's most important work, was published in London in 1859 . The first presentation in Norway, by P . Chr . Asbjornsen, appeared in the journal Budstikken . About 30 years passed before it was translated into Norwegian, and it took some time before Darwin's theories were debated in Norway . The zoologist Michael Sars introduced them in the Scientific Society in Christiania (i.e., Oslo) in 1869, but he was not met with a great deal of interest . However, a new generation of scientist saw this differently, mainly the botanist Axel Blytt, the zoologist G.O . Sars and the geologist W.C . Brogger . Two prominent professors of medicine were also involved in the debate, on different sides . The Darwinist Gerhard Henrik Armauer Hansen, who discovered the lepra bacillus, wrote several books and articles about Darwinism, while Professor Ernst Ferdinand Lochmann, though admiring Darwin as a prominent naturalist, strongly rejected Darwinism as a scientific theory.

Arch Esp Urol, 2000 Dec, 53(10), 879 - 92
{Immunotherapy in superficial bladder carcinoma}; Rios Gonzalez E et al.; OBJECTIVE: To review the current status of superficial bladder cancer treatment with several immune response modifiers . METHODS: A review of the advances in the treatment of superficial bladder cancer with BCG, interferon, interleukin-2, bropirimine and keyhole-limpet hemocyanin was performed . RESULTS: Treatment with BCG has been demonstrated to be superior to intravesical chemotherapy and other immune response modifiers in the trials reviewed . BCG therapy, however, carries a higher toxicity . Several trials have demonstrated that in low and medium risk patients, it is better to administer low BCG doses to reduce the toxicity . Furthermore, the trials confirm the utility of maintenance schedules with BCG . CONCLUSION: The Calmette-Guerin Bacillus is the most effective adjuvant treatment in superficial bladder cancer, especially in the high risk patients . The other immune response modifiers are an alternative to this treatment.

Indian J Lepr, 2000 Oct-Dec, 72(4), 437 - 42
Assessment of viability by normal mouse foot-pad and bacillary ATP bioluminescence assay in multibacillary cases treated with an MDT regimen using conventional as well as newer drugs like minocycline and ofloxacin; Gupta UD et al.; The therapeutic effect of a drug regimen of conventional drugs as well as newer drugs like ofloxacin and minocycline in smear-positive multibacillary (MB) leprosy cases was assessed by mouse foot-pad and ATP bioluminiscence methods . Biopsies were taken before starting treatment and after one year of treatment . They were processed for viability assessment by normal mouse foot-pad inoculation and bacillary ATP assay techniques . The test regimen was quite effective in its anti-bacterial effect as it was found to result in loss of bacillary viability in all the cases, as assessed by both methods.

Med Parazitol (Mosk), 2000 Oct-Dec, (4), 25 - 9
{Duration of the larvicidal effect of spore crystalline mass of bacteria Bacillus thuringiensis spp . israelensis and Bacillus sphaericus in the laboratory setting}; Ganushkina LA et al.; The duration of action of sporocrystalline mass of Bacillus thuringiensis spp . Israelensis (Bti) and Bacillus sphaericus (Bsph) against three-age larvae of An . stephensi mosquitoes was studied in the laboratory setting . The duration of action depends on its initial concentration . The optimal concentration should be at least 10(5) spores/ml for An . stephensi larvae . Decreasing the concentration reduces the duration of larvicidal action . Increasing the number of the larvae placed also diminishes the duration of action shown by Bti . This has no significant influence on the duration of action by Bsph . The presence of dead larvae in the test vessel increases the duration of action by Bti only by 4 times whereas Bsph acts rather long . The follow-ups lasted 60 weeks with full death of the larvae placed again . A significant water resistance of Bti sporocrystalline mixture was found . The mixture retained its larvicidal activity 6 months following its presence in the dechlorinated water without larval placement . Bsph was less resistant and its complete inactivity was noted under the same conditions just following 2 months.

Biosci Biotechnol Biochem, 2000 Dec, 64(12), 2727 - 30
Purification and characterization of nitrite-oxidizing enzyme from heterotrophic Bacillus badius 1-73, with special concern to catalase; Sakai K et al.; Nitrite-oxidizing enzyme I (NiOx I) was purified from a heterotrophic bacterium, Bacillus badius I-73 . The enzyme was a homotetramer of a heme-containing peptide, and was similar to catalases from various sources in its N-terminal amino acid sequence . The purified enzyme also catalyzed H2O2 degradation . The nitrite oxidation reaction required ascorbic acid and oxygen . Successive H2O2 feeding could be substituted for ascorbic acid . These indicated that NiOx I is a catalase and nitrite was oxidized by a peroxidase-like reaction.

Biosci Biotechnol Biochem, 2000 Dec, 64(12), 2682 - 5
cDNA cloning of the Cry1Aa receptor variants from Bombyx mori and their expression in mammalian cells; Ikawa S et al.; We cloned cDNA of three variants of BtR175, a putative Bombyx mori receptor for Bacillus thuringiensis Cry1Aa delta-endotoxin by PCR . These variants were likely to be allelic to BtR175 . cDNA of BtR175b, the most distant variant from BtR175, was introduced into mammalian cells . BtR175b protein was expressed in the plasma membrane of the cells and showed binding activity to Cry1Aa.

Biosci Biotechnol Biochem, 2000 Dec, 64(12), 2530 - 7
Gene cloning and characterization of alpha-glucuronidase of Bacillus stearothermophilus no . 236; Choi ID et al.; The alpha-glucuronidase gene of Bacillus stearothermophilus No . 236 was cloned, sequenced, and expressed in Escherichia coli . The gene, designated aguA, encoded a 691-residue polypeptide with calculated molecular weight of 78,156 and pI of 5.34 . The alpha-glucuronidase produced by a recombinant E . coli strain containing the aguA gene was purified to apparent homogeneity and characterized . The molecular weight of the alpha-glucuronidase was 77,000 by SDS-PAGE and 161,000 by gel filtration; the functional form of the alpha-glucuronidase therefore was dimeric . The optimal pH and temperature for the enzyme activity were pH 6.5 and 40 degrees C, respectively . The enzyme's half-life at 50 degrees C was 50 min . The values for the kinetic parameters of Km and Vmax were 0.78 mM and 15.3 U/mg for aldotriouronic acid {2-O-alpha-(4-O-methyl-alpha-D-glucopyranosyluronic)-D-xylobiose} . The alpha-glucuronidase acted mainly on small substituted xylo-oligomers and did not release methylglucuronic acid from intact xylan . Nevertheless, synergism in the release of xylose from xylan was found when alpha-glucuronidase was added to a mixture of endoxylanase and beta-xylosidase.

An R Acad Nac Med (Madr), 2000, 117(2), 227 - 39; discussion 239-43
{Cutaneous tuberculosis yesterday and today}; Garcia Perez A; Cutaneous tuberculosis were very frequent in Europe until the middle of the 20th . Century in which their incidence decreased drastically as a consequence of the specific treatments . On the other hand, the M . bovis caused infections which were previously very common have also been disappearing due to control of milk and the livestock produced for eating purposes . Cutaneous tuberculosis have classically been divided into two groups: typical tuberculosis, with follicular structure and demonstrated bacillus by culture or inoculation and atypical tuberculosis (tuberculids), in which there are no follicular structure and the bacillus is not isolated . Its relationship with tuberculosis is principally based on the personal and/or familial background and on the strong positivity of the Mantoux reaction . The concept of tuberculide has always been under debate . Above all in the second half of the 20th . Century, most of the authors were skeptical in regards to its tuberculous etiology . However, since the 1990's, determinations of bacillary DNA in the lesions by the PCR technique has made it possible to demonstrate the M . tuberculosis in them . At present, due to the probably re-emergence of tuberculosis in general as consequence of the immunodeficiency (AIDS), of the M . tuberculosis strains resistant to treatment, and of the cases imported by immigration, some increase in the incidence of cutaneous tuberculosis can be predicted in the future, although it need be feared that this will reach the amounts of other periods . On the other hand, immunodeficiences have made the anergic forms, such as tuberculosis cutis miliaris diseminata or tuberculous gumma, which were previously rare, less rare al present.

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