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 norma