Bacillus

Curr Microbiol, 2003 Oct, 47(4), 295 - 9
Cloning and expression of the insecticidal crystal protein gene Cry1Ca9 of Bacillus thuringiensis G10-01A from Taiwan granaries; Kao SS et al.; A new cry gene (cry1Ca9) was cloned and sequenced from a Bacillus thuringiensis isolate native to Taiwan (G10-01A) . The cry1C-type gene, designated cry1Ca9, consisted of an open reading frame of 3,567 bp, encoding a protein of 1,189 amino acid residues . The polypeptide has the deduced amino acid sequences predicting molecular masses of 134.7 kDa . The gene sequence was compared against the GenBank nucleotide sequence data base . It was found that the cry1Ca9 gene coded for a 134.7-kDa protoxin which had greater than 99.8% homology with the previously reported cry1Ca1 gene, as only three mismatches were found between the two amino acid sequences . When the Cry1Ca9 toxin was expressed in a crystal-negative strain of B . thuringiensis (cryB-), elliptical crystals were produced . Cell extracts from this recombinant strain appear to have high insecticidal activity against lepidopteran larvae (Plutella xylostella).

J Food Prot, 2003 Nov, 66(11), 2070 - 5
Heat resistance of Bacillus spores when adhered to stainless steel and its relationship to spore hydrophobicity; Simmonds P et al.; Twenty-one strains of Bacillus (10 B . stearothermophilus, 3 B . cereus, and 8 B . licheniformis strains) were assayed for spore surface hydrophobicity on the basis of three measures: contact angle measurement (CAM), microbial adhesion to hydrocarbons (MATH), and hydrophobic interaction chromatography (HIC) . On the basis of the spore surface characteristics obtained from these assays, along with data on the heat resistance of these spores in water, eight strains of Bacillus (three B . stearothermophilus, three B . cereus, and two B . licheniformis strains) either suspended in water or adhering to stainless steel were exposed to sublethal heat treatments at 90 to 110 degrees C to determine heat resistance (D-value) . Significant increases in heat resistance (ranging from 3 to 400%) were observed for the eight strains adhering to stainless steel . No significant correlation was found between these heat resistance increases and spore surface characteristics as determined by the three hydrophobicity assays . There was a significant positive correlation between the hydrophobicity data obtained by the MATH assay and those obtained by the HIC assay, but these data did not correlate with those obtained by the CAM assay.

Biofouling, 2003 Aug, 19(4), 257 - 67
Microfouling studies on experimental test blocks of steel-making slag and concrete exposed to seawater off Chiba, Japan; Nandakumar K et al.; Microfouling studies with the emphasis on microalgae (Bacillariophyceae) were carried out on test blocks of steel-making slag in comparison with concrete . Two types of slag test blocks, with and without fly-ash as an additional source of silica, and concrete test blocks of size 75 x 26 x 26 mm were used to study microfouling build-up for a period of 30 d, with intermittent samplings after 1, 2, 3, 7, 14 and 21 d . The species composition, cell density, biomass and surface pH of the test pieces were determined, in addition to the hydrographic parameters of the water column . Microfouling studies showed higher numbers of algal species as well as a greater cell density on the slag than on the concrete blocks . This was true with respect to biomass measured as dry weight also . Colonization was significantly delayed in the case of concrete . Navicula spp . and Nitzschia spp . were the initial colonizers on all three types of substrata and were the dominant genera throughout the study period . While the number of species increased, several disappeared after colonization, as a part of community build-up . The surface pH of the slag blocks was near neutral, whilst that of the concrete was highly alkaline during the initial period of exposure . This alkaline surface reduced the rate of species colonization on the concrete blocks initially . The study showed severe biofouling on the slag blocks compared to concrete and thus they were considered an environmentally benign construction material for land protection . The use of slag as the construction material for land protection would greatly reduce the expense compared to concrete.

Biotechnol Lett, 2003 Oct, 25(20), 1747 - 50
Biodegradation of exploded cotton stalk by Bacillus sp; Zheng L et al.; The exploded bast, branch and stem of cotton stalk were degraded by alkalophilic Bacillus NT-19, with weight losses of 24%, 20% and 14%, respectively, after 14 d . Compared with a white-rot fungus (Phanerochaete chrysosporium), Bacillus NT- 19 preferentially degraded the non-cellulose components of cotton stem . The relative degree of crystallinity of bast fibers decreased by 8% and the middle lamella was partially removed from the fiber bundle by the Bacillus.

Biotechnol Lett, 2003 Oct, 25(20), 1709 - 12
Antifreeze activities of poly(gamma-glutamic acid) produced by Bacillus licheniformis; Shih IL et al.; Various enantiomeric isomers, metals salts and molecular sizes of poly(gamma-glutamic acid), gamma-PGA, produced by Bacillus licheniformis CCRC 12826, were prepared and their antifreeze activities were studied by differential scanning calorimetry . The antifreeze activity of gamma-PGA increased as its molecular weight decreased but was indifferent to its D/L-glutamate composition . The antifreeze activity was cation dependent decreasing in the order Mg2+ >> Ca2+ approximately Na+ >> K+ which follows that of inorganic chlorides in that high ionic charge leads to high antifreeze activity . The mechanism by which the cryoprotective effects of gamma-PGA can be explained is still yet to be determined.

FEMS Immunol Med Microbiol, 2003 Nov 28, 39(2), 173 - 80
Mycobacterium bovis bacillus Calmette-Guérin infection promotes SOCS induction and inhibits IFN-gamma-stimulated JAK/STAT signaling in J774 macrophages; Imai K et al.; The resurgence in mycobacterial infection worldwide has led to renewed attention to the pathogenesis of Mycobacterium species . Although interferon-gamma (IFN-gamma) is a principal mediator of macrophage activation, macrophages infected with Mycobacterium are poor in response at the cytokine . However, the molecular mechanisms underlying mycobacterial infection remain unclear . The purpose of this study was to elucidate the mechanism of the poor response to IFN-gamma in mycobacterial infection . Our data clearly demonstrate that this is due to induction of suppressor of cytokine signal (SOCS) negative regulators of IFN-gamma signal transduction that closely correlates with the inhibition of JAK/STAT signaling and gene expression stimulated by IFN-gamma . Mycobacterium bovis bacillus Calmette-Guerin infection induces the production of SOCS-1 and SOCS-3 in murine J774 macrophages . The level of SOCS-1 mRNA increased 1 h and reached a maximum 3 h after the addition of the bacteria . SOCS-3 mRNA expression appeared as early as 1 h after the infection . We also observed that trehalose 6,6'-dimycolate/cord factor, a major component of the Mycobacterium tuberculosis cell wall, induces expression of SOCS and inhibits IFN-gamma-stimulated phosphorylation of STAT1 extensively in the cells . The results in this study suggest that a molecular mechanism of mycobacterial infection affects the unresponsiveness to IFN-gamma in the subsequent growth and spread of macrophages.

Tuberculosis (Edinb), 2003, 83(6), 351 - 9
Catalase-peroxidase activity has no influence on virulence in a murine model of tuberculosis; Cardona PJ et al.; The capacity to generate a chronic and persistent infection in the experimental murine model of tuberculosis induced aerogenically by a low-dose inoculum was determined in eight isoniazid-resistant clinical strains of Mycobacterium tuberculosis showing different catalase-peroxidase (C-P) activities . Determination of bacillary concentration in lung and spleen and the percentage of pulmonary parenchyma occupied by granulomas were monitored . Data showed no relation between the lack of C-P activity and the ability to develop a persistent infection, highlighting the potential of C-P negative strains to spread through the community.

Biotechnol Adv, 2003 Dec, 22(1-2), 45 - 69
Sustainability of insect resistance management strategies for transgenic Bt corn; Glaser JA et al.; Increasing interest in the responsible management of technology in the industrial and agricultural sectors of the economy has been met thorough the development of broadly applicable tools to assess the "sustainability" of new technologies . An arena ripe for application of such analysis is the deployment of transgenic crops . The new transgenic pesticidal or plant-incorporated protectant (PIP) crops have seen widespread application in the United States based on the features of higher yield, lower applications of insecticides, and control of mycotoxin content . However, open rejection of these new crops in Europe and in other countries has been a surprising message and has limited their worldwide acceptance . The US Environmental Protection Agency's (USEPA) Office of Pesticide Programs (OPP) has worked on the development and analysis of insect resistance management (IRM) strategies and has mandated specific IRM requirements for Bacillus thuringiensis (Bt) crops since 1995 under the Food, Fungicide, Insecticide, and Rodenticide Act . Improvement of data quality and sustainability of IRM strategies have been targeted in an ongoing partnership between the USEPA Office of Research and Development and the Office of Pesticide Programs that will further enhance the agency's ability to develop sustainable insect resistance management strategies for transgenic field corn (Bt corn) producing B . thuringiensis (Bt) insecticidal proteins.

Eur J Biochem, 2003 Nov, 270(22), 4488 - 96
Interactions of the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus; Jung HI et al.; The enzymes pyruvate decarboxylase (E1) and dihydrolipoyl dehydrogenase (E3) bind tightly but in a mutually exclusive manner to the peripheral subunit-binding domain (PSBD) of dihydrolipoyl acetyltransferase in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus . The use of directed mutagenesis, surface plasmon resonance detection and isothermal titration microcalorimetry revealed that several positively charged residues of the PSBD, most notably Arg135, play an important part in the interaction with both E1 and E3, whereas Met131 makes a significant contribution to the binding of E1 only . This indicates that the binding sites for E1 and E3 on the PSBD are overlapping but probably significantly different, and that additional hydrophobic interactions may be involved in binding E1 compared with E3 . Arg135 of the PSBD was also replaced with cysteine (R135C), which was then modified chemically by alkylation with increasingly large aliphatic groups (R135C -methyl, -ethyl, -propyl and -butyl) . The pattern of changes in the values of DeltaG degrees, DeltaH degrees and TDeltaS degrees that were found to accompany the interaction with the variant PSBDs differed between E1 and E3 despite the similarities in the free energies of their binding to the wild-type . The importance of a positive charge on the side-chain at position 135 for the interaction of the PSBD with E3 and E1 was apparent, although lysine was found to be an imperfect substitute for arginine . The results offer further evidence of entropy-enthalpy compensation ('thermodynamic homeostasis') - a feature of systems involving a multiplicity of weak interactions.

J Environ Health, 2003 Nov, 66(4), 16 - 21
Decontamination of Bacillus thuringiensis spores on selected surfaces by chlorine dioxide gas; Han Y et al.; This work examined the efficacy of chlorine dioxide (ClO2) gas for the decontamination of Bacillus thuringiensis spores on paper, wood, epoxy, and plastic surfaces . Spores representing an inoculation level of approximately 6 log colony-forming units (CFU) per surface were treated with 5, 10, 15, 20, 25, or 30 milligrams per liter (mg/L) ClO2 gas for 12 hours under 85-92 percent relative humidity and at 22 +/- 1 degrees C . Under the tested treatment conditions, the highest population of surviving spores was found on the paper surface and the lowest was found on the plastic surface (p < .05) . The 5 mg/L ClO2 gas treatment inactivated 2.5, 3.6, 4.0, and 4.9 log spores per surface on paper, wood, epoxy, and plastic surfaces, respectively . A greater than 5-log reduction of spores was achieved on each surface after the 15 mg/L ClO2 gas treatment . The minimum ClO2 gas concentration needed to completely inactivate the inoculated spores was 30 mg/L for paper and wood surfaces, 25 mg/L for epoxy surfaces, and 20 mg/L for plastic surfaces . The results of this study may provide insight into the parameters of effective decontamination procedures for Bacillus spores.

Kekkaku, 2003 Oct, 78(10), 653 - 9
{Role of oral fluoroquinolones in patients with respiratory diseases}; Ishida T; The characteristics of recently developed oral fluoroquinolones include their broad spectrum involving gram-positive/gram-negative bacteria and atypical pathogens, potent antimicrobial activity against Pneumococcus, rapid tissue/sputum transfer, prolonged half-life, and reduction of their interaction with other agents . However, it has been reported that the common use of oral fluoroquinolones increases the number of fluoroquinolone-resistant bacterial strains . We review the appropriate use of these agents in patients with respiratory infections . In most cases, upper respiratory inflammation is a viral infection . Generally, antimicrobial agents are not necessary, and should not be administered . In Japan, a large number of antimicrobial agents, especially quinolones, are frequently prescribed to treat upper respiratory infection . This tendency must not be corrected . With respect to treatment for community-acquired pneumonia, it is controversial whether oral fluoroquinolones should be prescribed under various guidelines . In elderly patients and those with an underlying disease, oral fluoroquinolones may be a first-choice treatment at the outpatient clinic, because it is difficult to differentiate atypical pneumonia from bacterial pneumonia, and because the risk of drug-resistant Pneumococcus or gram-negative bacteria is high . With respect to treatment for hospital-acquired pneumonia, oral fluoroquinolones are recommended for patients with moderate or mild conditions without risk factors under the Guidelines established by the Japanese Respiratory Society . Bacteria causing acute infectious exacerbation in patients with chronic pulmonary diseases include gram-positive/gram-negative bacteria and anaerobic bacteria . Therefore, oral fluoroquinolones may be the most appropriate treatment for such patients . New oral fluoroquinolones show potent antimicrobial activity against tubercle bacillus, and may also be effective for infection with bacteria resistant to standard antitubercular agents . It may be controversial whether these agents should be indicated for atypical acid-fast bacterial infection.

Pest Manag Sci, 2003 Nov, 59(11), 1197 - 202
Inheritance of resistance to Bt canola in a field-derived population of Plutella xylostella; Sayyed AH et al.; Crops expressing Bacillus thuringiensis (Bt) insecticidal Cry proteins are grown on millions of hectares . Recommendations to delay resistance are based on a high expression/refugia strategy that aims to kill resistant heterozygotes and enable some susceptible insects to survive . Leaf-dip bioassays on F1 crosses of Malaysian populations of diamondback moth (Plutella xylostella (L)) showed that CrylAc resistance was not fully recessive . The survival of ca 50% of heterozygotes on Bt canola (Brassica napus L) leaves expressing low concentrations of CrylAc agreed with a non-fully-recessive model for resistance . Extrapolations based on log dose-logit mortality regressions for heterozygotes using leaf-dip bioassays showed that a relatively high level of expression, of ca 2000 ng CrylAc mg(-1) total leaf protein, would be required to give 90% mortality to heterozygotes . If high enough levels of expression of Bt toxin to kill heterozygotes cannot be achieved and maintained under field conditions, the effectiveness of the high-dose/refugia strategy would be reduced.

Sichuan Da Xue Xue Bao Yi Xue Ban, 2003 Oct, 34(4), 614 - 7
{Preliminary study of the molecular regulation of BCG-mediated enhancement of hBD-1 gene expression in human pulmonary gland epithelial cells}; Zhu B et al.; OBJECTIVE: To define the regulation mechanism of the enhancement expression of human beta-defensin-1 (hBD-1) stimulated by bacille Calmette-Guerin (BCG) at transcription level . METHODS: A series of 5'-flanking deletions of hBD-1 gene were ligated into pEGFP-1 and pCAT basic vector . pEGFP hBD-1 recombinants were transfected into SPC-A-1 cells and the green fluorescence protein (GFP) expression was observed by fluorescence microscopy . SPC-A-1 cells were co-transfected with pCAT hBD-1 constructs and pSV-beta-Galactosidase control vector and were stimulated with effective BCG cell wall components . CAT and beta-Gal protein expressions were determined by ELISA . RESULTS: The promoting activity of the -69 region was lower than that of the region -575 and -314 . After being stimulated with the active fraction of BCG cell wall proteins, the relative CAT expression of pCAT hBD-1/-69 still showed the increasing tendency of -2161 construct . Computer consensus match analysis indicated that the nucleotides between -63 bp to -50 bp was the potential binding site for C/EBP beta . CONCLUSION: The nucleotides from -69 to bp is essential to the enhancement of hBD-1 gene transcription by BCG.

Bioprocess Biosyst Eng, 2003 Dec, 26(2), 123 - 32 Epub 2003 Nov 14.
Monte Carlo simulation of the alpha-amylolysis of amylopectin potato starch . 2 . alpha-amylolysis of amylopectin; Marchal LM et al.; A model is presented that describes all the saccharides that are produced during the hydrolysis of starch by an alpha-amylase . Potato amylopectin, the substrate of the hydrolysis reaction, was modeled in a computer matrix . The four different subsite maps presented in literature for alpha-amylase originating from Bacillus amyloliquefaciens were used to describe the hydrolysis reaction in a Monte Carlo simulation . The saccharide composition predicted by the model was evaluated with experimental values . Overall, the model predictions were acceptable, but no single subsite map gave the best predictions for all saccharides produced . The influence of an alpha(1-->6) linkage on the rate of hydrolysis of nearby alpha(1-->4) linkages by the alpha-amylase was evaluated using various inhibition constants . For all the subsites considered the use of inhibition constants led to an improvement in the predictions (a decrease of residual sum of squares), indicating the validity of inhibition constants as such . As without inhibition constants, no single subsite map gave the best fit for all saccharides . The possibility of generating a hypothetical subsite map by fitting was therefore investigated . With a genetic algorithm it was possible to construct hypothetical subsite maps (with inhibition constants) that gave further improvements in the average prediction for all saccharides . The advantage of this type of modeling over a regular fit is the additional information about all the saccharides produced during hydrolysis, including the ones that are difficult to measure experimentally.

J Bacteriol, 2003 Dec, 185(23), 6985 - 9
The Bacillus thuringiensis linear double-stranded DNA phage Bam35, which is highly similar to the Bacillus cereus linear plasmid pBClin15, has a prophage state; Stromsten NJ et al.; Bam35, a 15-kbp double-stranded DNA phage, infects Bacillus thuringiensis . Recently, sequencing of the related Bacillus cereus revealed a 15.1-kbp linear plasmid, pBClin15 . We show that pBClin15 closely resembles Bam35 and demonstrate conversion of Bam35 to a prophage . This state is common, as several B . thuringiensis strains release Bam35-related viruses.

Mol Microbiol, 2003 Nov, 50(3), 751 - 62
Disruption of mptpB impairs the ability of Mycobacterium tuberculosis to survive in guinea pigs; Singh R et al.; Protein tyrosine kinases and tyrosine phosphatases from several bacterial pathogens have been shown to act as virulence factors by modulating the phosphorylation and dephosphorylation of host proteins . The identification and characterization of two tyrosine phosphatases namely MptpA and MptpB from Mycobacterium tuberculosis has been reported earlier . MptpB is secreted by M . tuberculosis into extracellular mileu and exhibits a pH optimum of 5.6, similar to the pH of the lysosomal compartment of the cell . To determine the role of MptpB in the pathogenesis of M . tuberculosis, we constructed a mptpB mutant strain by homologous recombination and compared the ability of parent and the mutant strain to survive intracellularly . We show that disruption of the mptpB gene impairs the ability of the mutant strain to survive in activated macrophages and guinea pigs but not in resting macrophages suggesting the importance of its role in the host-pathogen interaction . Infection of guinea pigs with the mutant strain resulted in a 70-fold reduction in the bacillary load of spleens in infected animals as compared with the bacillary load in animals infected with the parental strain . Upon reintroduction of the mptpB gene into the mutant strain, the complemented strain was able to establish infection and survive in guinea pigs at rates comparable to the parental strain . These observations demonstrate a role of MptpB in the pathogenesis of M . tuberculosis.

Clin Exp Allergy, 2003 Nov, 33(11), 1512 - 7
Age at bacille Calmette-Guérin vaccination and risk of allergy and asthma; Bager P et al.; BACKGROUND: It has been proposed that early age at bacille Calmette-Guerin (BCG) vaccination protects against the development of allergy . OBJECTIVE: To study whether early age at BCG vaccination was associated with a decreased risk of atopy, allergic rhinitis, and asthma compared to BCG vaccination at later ages in childhood . METHODS: The occurrence of atopy, allergic rhinitis, and asthma was studied in nearly 2000 women participating in the Danish National Birth Cohort study . Detailed information on age at BCG vaccination (age 0-15 years) was available from school health records . Atopic status was assessed serologically by a specific response to 11 common inhalant allergens using serum samples obtained from the women during the period 1997-2001 . Information on allergic rhinitis and asthma was available from telephone interviews . RESULTS: Approximately 85% of the women had been BCG-vaccinated . Age at BCG vaccination was not associated with risk of atopy, allergic rhinitis, or asthma . The odds ratio of atopy, allergic rhinitis, and asthma associated with being vaccinated during the first year of life was 1.05 (95% CI 0.71-1.56), 1.42 (95% CI 0.85-2.36), and 1.71 (95% CI 0.91-3.20), respectively, compared with being vaccinated at the age of 7 years . Adjustment for birth cohort, sibship size, age of the woman's mother at birth, and social class in childhood did not affect the results . CONCLUSION: Our findings suggest that age at BCG vaccination in childhood does not influence the development of allergy or asthma.

Clin Exp Immunol, 2003 Nov, 134(2), 303 - 8
Immune response measured in human volunteers vaccinated with autoclaved Leishmania major vaccine mixed with low dose of BCG; Mahmoodi M et al.; The immune responses induced against Leishmania antigens in volunteers who were vaccinated in a double-blind, randomized field efficacy trial of a preparation of autoclaved Leishmania major (ALM) mixed with a low dose of Bacille Calmette-Guerin vaccine (BCG) who developed either a cutaneous leishmaniasis (CL) lesion due to exposure to infected sandfly bite(s) or did not develop a lesion during the course of the trial were studied and compared with those of non-vaccinated controls . Blood samples were also assayed from different groups including volunteers with history of CL and volunteers with previous positive or negative leishmanin skin test (LST) without a history of CL . The vaccinated volunteers had received a single dose of either ALM mixed with a low dose of BCG or the same dose of BCG alone . The LST and in vitro proliferative response (stimulation index, SI), interferon gamma (IFN-gamma) production and, in a few cases, interleukin (IL)-4 production of peripheral blood mononuclear cells to soluble Leishmania antigens were measured . The results indicated that volunteers who developed CL in the vaccine arm showed a slightly higher SI than cases who received BCG alone . Volunteers with history of CL and volunteers with positive LST demonstrated the strongest proliferation indices and IFN-gamma production . The data suggest that a single dose of ALM + BCG induces a weak Th1 response in vaccinated volunteers that is far lower than that in volunteers with prior subclinical infection or volunteers with history of CL, who are presumed to be immune.

Clin Exp Immunol, 2003 Nov, 134(2), 285 - 94
Interferon-gamma and skin test responses of schoolchildren in southeast England to purified protein derivatives from Mycobacterium tuberculosis and other species of mycobacteria; Weir RE et al.; The immune responses of schoolchildren in southeast England to Mycobacterium tuberculosis and other species of mycobacteria were studied prior to vaccination with bacille Calmette-Guerin (BCG) . Data are presented for tuberculin (Heaf) skin test and interferon-gamma (IFN-gamma) responses to M . tuberculosis purified protein derivative (PPD), and IFN-gamma responses to PPDs from eight other environmental mycobacteria, measured in 424 schoolchildren (13-15 years of age) . Responses to M . tuberculosis PPD were detected in 27% of schoolchildren by in vitro IFN-gamma response and in 20% by the Heaf test . IFN-gamma responses were more prevalent to PPDs from species of mycobacteria other than M . tuberculosis, predominantly those of the MAIS complex and M . marinum (45-60% responders) . Heaf test and IFN-gamma responses were associated (P<0.001) for M . tuberculosis, MAIS and M . marinum . These findings have implications for appropriate implementation of vaccination against tuberculosis.

Clin Microbiol Infect, 2003 Sep, 9(9), 973 - 5
Nosocomial bacteremia and catheter infection by Bacillus cereus in an immunocompetent patient; Hernaiz C et al.; We present a case of Bacillus cereus bacteremia and catheter infection in an immunocompetent patient subjected to abdominal surgery, who recovered following central catheter removal and treatment with piperacillin/tazobactam.

Allergy, 2003 Nov, 58(11), 1114 - 6
Effects of BCG revaccination on asthma; Choi IS et al.; In a study conducted 1 year ago, we found that Th1 immune enhancement following Bacille Calmette-Guerin (BCG) vaccination effectively suppressed human asthma . To investigate whether revaccination would further improve lung function, BCG vaccine was given again . Current lung function tended to improve in the Repeated BCG group (n = 9), but not in the Single BCG group (previously the placebo group) (n = 11), compared with that 1 year ago . The BCG vaccination improved lung function in both groups, and the Repeated BCG group showed a significant increase in the peripheral blood interferon gamma/interleukin 4 ratio . These findings suggest that repeated BCG vaccinations might be effective in asthma therapy.

J Theor Biol, 2003 Dec 21, 225(4), 493 - 6
DNA-membrane interactions can localize bacterial cell center; Rabinovitch A et al.; In actively growing bacterial cells, the DNA exerts stress on the membrane, in addition to the turgor caused by osmotic pressure . This stress is applied through coupled transcription/translation and insertion of membrane proteins (so-called "transertion" process) . In bacillary bacteria, the strength of this interaction varies along cell length with a minimum at its midpoint, and hence can locate the cell's equator for the assembly of the FtsZ-ring.

Proc R Soc Lond B Biol Sci, 2003 Nov 7, 270(1530), 2263 - 70
Rapid evolution and the cost of resistance to Bacillus thuringiensis in greenhouse populations of cabbage loopers, Trichoplusia ni; Janmaat AF et al.; The microbial insecticide Bacillus thuringiensis (Bt) has become the mainstay of non-chemical control of Lepidopteran pests, either as sprays or through the incorporation of Bt toxins into transgenic crops . Given the wide use of Bt, it is striking that currently only one pest species, Plutella xylostella, has been reported to have developed significant resistance to Bt outside the laboratory . By contrast, we report here the frequent and rapid development of resistance to B . thuringiensis kurstaki (Dipel, Abbott) in populations of cabbage loopers, Trichoplusia ni, in commercial greenhouses . Resistance to Bt appears to be costly and there is a rapid decline of resistance in populations collected from greenhouses and maintained in the laboratory without selection . Management of pests resistant to Bt in vegetable greenhouses will require sporadic use of Bt-based sprays or alternatively use of sprays containing other Bt toxins.

Curr Opin Allergy Clin Immunol, 2003 Dec, 3(6), 481 - 6
Immunotherapy with mycobacteria; Walker C et al.; PURPOSE OF REVIEW: To summarize and evaluate critically recent progress with mycobacteria as a potential novel disease modifying treatment strategy in asthma . RECENT FINDINGS: The link between exposure to pathogenic or saprophytic mycobacteria and protection from allergic diseases is still controversial, and recent epidemiological studies, which addressed only exposure to Mycobacterium tuberculosis or bacillus Calmette-Guerin, did not help to clarify this issue . Moreover, the clear efficacy of mycobacterial treatment seen in animal models has not been reproduced in human asthma, and a recent small study testing the hypothesis that heat-killed Mycobacterium vaccae attenuates asthmatic reactions after allergen challenge did not provide convincing results . However, it has been shown that treatment of mice with M . vaccae induces the generation of allergen-specific T regulatory cells capable of suppressing allergen-mediated eosinophilic lung inflammation, suggesting that a general deficiency of T regulatory cell activity might be responsible for the increased prevalence of asthma . This hypothesis is supported by findings that a lack of T regulatory cells, as found in genetic disorders of man and mouse attributable to a mutation of Foxp3, a transcription factor specifically expressed by T regulatory cells, is associated with manifestations of severe atopy and autoimmunity, precisely the spectrum of diseases linked to the hygiene hypothesis . SUMMARY: Further studies on the relationship between mycobacteria and atopic disorders are needed, but there is reason to believe that the novel findings and molecular mechanisms associated with mycobacterial infections will further strengthen the currently unproved therapeutic value of immunotherapy with mycobacteria.

FEMS Microbiol Lett, 2003 Nov 7, 228(1), 21 - 6
CbiX-homologous protein (CbiXhp), a metal-binding protein, from Streptomyces seoulensis is involved in expression of nickel-containing superoxide dismutase; Kim IK et al.; To find the accessory proteins participating in expression and maturation of nickel-containing superoxide dismutase (NiSOD), a metal-binding protein (CbiXhp) homologous to cobaltochelatase (CbiX) of Bacillus megaterium was isolated by nickel-nitrilotriacetic acid resin from Streptomyces seoulensis . The deduced amino acid sequence of cbiXhp showed 87% and 39% identity to CbiX of Streptomyces coelicolor and that of B . megaterium, respectively . Overexpression of CbiXhp increased the activity and the expression of NiSOD in the presence and absence of nickel, but to a lesser extent in its absence . This result indicates that CbiXhp is involved in the expression of NiSOD.

FEMS Microbiol Lett, 2003 Nov 7, 228(1), 1 - 9
Genes for an alkaline D-stereospecific endopeptidase and its homolog are located in tandem on Bacillus cereus genome; Komeda H et al.; Alkaline D-peptidase (Adp) from Bacillus cereus DF4-B is a D-stereospecific endopeptidase acting on oligopeptides composed of D-phenylalanine and the primary structure deduced from its gene, adp, shows a similarity with D-stereospecific hydrolases from Ochrobactrum anthropi strains . We have isolated DNA fragments covering the flanking region of adp from DF4-B genome and found an additional gene, adp2, located upstream of adp . The deduced amino acid sequence of Adp2 showed 96% and 85% identity with those of Adp from B . cereus strains AH559 and DF4-B, respectively . The recombinant Adp2 expressed in Escherichia coli was purified to homogeneity and characterized . It had hydrolyzing activity toward (D-Phe)3, (D-Phe)4, and (D-Phe)6 but did not act on (L-Phe)4, D-Phe-NH2, and L-Phe-NH2, some characteristics that are closely related to those of Adp from strain DF4-B . These results indicate that highly homologous genes encoding D-stereospecific endopeptidases are arranged in a tandem manner on the genomic DNA of B . cereus DF4-B.

Biochemistry, 2003 Nov 18, 42(45), 13304 - 18
Glycosynthase activity of Bacillus licheniformis 1,3-1,4-beta-glucanase mutants: specificity, kinetics, and mechanism; Faijes M et al.; Glycosynthases are engineered retaining glycosidases devoid of hydrolase activity that efficiently catalyze transglycosylation reactions . The mechanism of the glycosynthase reaction is probed with the E134A mutant of Bacillus licheniformis 1,3-1,4-beta-glucanase . This endo-glycosynthase is regiospecific for formation of a beta-1,4-glycosidic bond with alpha-glycosyl fluoride donors (laminaribiosyl as the minimal donor) and oligosaccharide acceptors containing glucose or xylose on the nonreducing end (aryl monosaccharides or oligosaccharides) . The pH dependence of the glycosynthase activity reflects general base catalysis with a kinetic pK(a) of 5.2 +/- 0.1 . Kinetics of enzyme inactivation by a water-soluble carbodiimide (EDC) are consistent with modification of an active site carboxylate group with a pK(a) of 5.3 +/- 0.2 . The general base is Glu138 (the residue acting as the general acid-base in the parental wild-type enzyme) as probed by preparing the double mutant E134A/E138A . It is devoid of glycosynthase activity, but use of sodium azide as an acceptor not requiring general base catalysis yielded a beta-glycosyl azide product . The pK(a) of Glu138 (kinetic pK(a) on k(cat)/K(M) and pK(a) of EDC inactivation) for the E134A glycosynthase has dropped 1.8 pH units compared to the pK(a) values of the wild type, enabling the same residue to act as a general base in the glycosynthase enzyme . Kinetic parameters of the E134A glycosynthase-catalyzed condensation between Glcbeta4Glcbeta3GlcalphaF (2) as a donor and Glcbeta4Glcbeta-pNP (15) as an acceptor are as follows: k(cat) = 1.7 s(-)(1), K(M)(acceptor) = 11 mM, and K(M)(donor) < 0.3 mM . Donor self-condensation and elongation reactions are kinetically evaluated to establish the conditions for preparative use of the glycosynthase reaction in oligosaccharide synthesis . Yields are 70-90% with aryl monosaccharide and cellobioside acceptors, but 25-55% with laminaribiosides, the lower yields (and lower initial rates) due to competitive inhibition of the beta-1,3-linked disaccharide acceptor for the donor subsites of the enzyme.

Rev Esp Salud Publica, 2003 Sep-Oct, 77(5), 553 - 65
{Risk factors for tuberculous disease in AIDS cases reported in Brazil, from 1980 to 2000}; Laguardia J et al.; BACKGROUND: Aids affects the epidemiological characteristics of tuberculosis, due to both the changes in the clinical progress of the jointly affected patients, as well as the increase in the number of tuberculosis cases among individuals having tested positive for hiv/aids . This may lead to an increase in the transmission of the bacillus among the population . this study is aimed at identifying the factors related to the occurrence of tuberculosis among aids cases reported to the brazilian ministry of health . METHODS: a cross-sectional epidemiological study was conducted, the aids cases initially having been classified into 2 categories (1) with tuberculosis and (2) without tuberculosis, a comparison was subsequently drawn among 4 groups: (1) no opportunist infection, (2) only tuberculosis (pulmonary and/or disseminated), (3) with other opportunist infections except tuberculosis and (4) with tuberculosis in conjunction with other opportunist infections . The bivariate analysis was adjusted by logistic regression . RESULTS: In the regression analysis, the highest tuberculosis prevalences were significantly linked to age under 40, little schooling, living in the southeast region of the country and diagnosis made prior to 1996 . The means of transmission most closely associated to the presence of tuberculosis was the use of intravenous drugs, followed by heterosexual relations . when 4 consecutive periods in the evolution of the reporting aids case definition, there had been a decrease in cases with tuberculosis since 1996, as well as significant increases in the proportion of patients with no opportunist infection . CONCLUSIONS: Gender, age and school attendance are predictors of the presence of tuberculosis is among opportunist infections.

Nat Biotechnol, 2003 Dec, 21(12), 1493 - 7 Epub 2003 Nov 09.
Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution; Zhao JZ et al.; Preventing insect pests from developing resistance to Bacillus thuringiensis (Bt) toxins produced by transgenic crops is a major challenge for agriculture . Theoretical models suggest that plants containing two dissimilar Bt toxin genes ('pyramided' plants) have the potential to delay resistance more effectively than single-toxin plants used sequentially or in mosaics . To test these predictions, we developed a unique model system consisting of Bt transgenic broccoli plants and the diamondback moth, Plutella xylostella . We conducted a greenhouse study using an artificial population of diamondback moths carrying genes for resistance to the Bt toxins Cry1Ac and Cry1C at frequencies of about 0.10 and 0.20, respectively . After 24 generations of selection, resistance to pyramided two-gene plants was significantly delayed as compared with resistance to single-gene plants deployed in mosaics, and to Cry1Ac toxin when it was the first used in a sequence . These results have important implications for the development and regulation of transgenic insecticidal plants.

Am J Infect Control, 2003 Oct, 31(6), 347 - 53
Comparison of a whole-blood interferon-gamma assay and tuberculin skin testing in patients with active tuberculosis and individuals at high or low risk of Mycobacterium tuberculosis infection; Fietta A et al.; BACKGROUND: QuantiFeron-TB (QIFN) is a whole-blood interferon-;gamma assay for the recognition of cell-mediated immune response to Mycobacterium tuberculosis infection . OBJECTIVES: To compare the QIFN assay with the tuberculin skin test (TST) in patients with newly diagnosed culture-proven tuberculosis (TB) and healthy volunteers with high or low risk of latent M tuberculosis infection and to identify factors associated with discordance between tests . METHOD: Two-hundred fifty-eight subjects underwent both assays . All participants completed a detailed questionnaire, and data from TB patients' medical records were collected . RESULTS: In the entire study population, agreement between tests was moderate and the correlation between the magnitude of QIFN response and the TST induration diameter was significant . In volunteers with no known risk of exposure to M tuberculosis, the specificity of the assays was comparable . However, in subjects with active TB or those vaccinated with bacille Calmette-Guerin, the QIFN assay detected more reactors than did the TST . In these individuals, agreement between assays was poor and no correlation or only a weak correlation was found between the diameter of TST induration and the magnitude of the interferon-gamma responses . CONCLUSIONS: The sensitivity of the QIFN assay is greater than that of the TST in patients with active TB before the initiation of anti-TB chemotherapy, but its specificity is influenced more by bacille Calmette-Guerin vaccination . The QIFN assay may provide an improvement over the current practice of the use of the TST to support diagnosis of active M tuberculosis infection in the clinic; however, QIFN cannot be considered an adequate replacement for the TST in the screening for latent infection.

J Biochem (Tokyo), 2003 Oct, 134(4), 543 - 50
Deciphering the molecular basis of the broad substrate specificity of alpha-glucosidase from Bacillus sp . SAM1606; Noguchi A et al.; The alpha-glucosidase of Bacillus sp . strain SAM1606 is a member of glycosyl hydrolase family 13, and shows an extraordinarily broad substrate specificity and is one of very few alpha-glucosidases that can efficiently hydrolyze the alpha-1,1-glucosidic linkage of alpha,alpha'-trehalose (trehalose) . Phylogenetic analysis of family-13 enzymes suggests that SAM1606 alpha-glucosidase may be evolutionally derived from an alpha-1,6-specific ancestor, oligo-1,6-glucosidase (O16G) . Indeed, replacement of Pro(273*) and Thr(342*) of B . cereus O16G by glycine and asparagine (the corresponding residues in the SAM1606 enzyme), respectively, was found to cause 192-fold enhancement of the relative catalytic efficiency for trehalose, suggesting that O16G may easily "evolved" into an enzyme with an extended substrate specificity by substitution of a limited number of amino acids, including that at position 273* (an asterisk indicates the amino-acid numbering of the SAM1606 sequence) . To probe the role of the amino acid at position 273* of alpha-glucosidase in determination of the substrate specificity, the amino acid at position 273 of SAM1606 alpha-glucosidase was replaced by all other naturally occurring amino acids, and the resultant mutants were kinetically characterized . The results showed that substitution of bulky residues (e.g., isoleucine and methionine) for glycine at this position resulted in large increases in the K(m) values for trehalose and maltose, whereas the affinity to isomaltose was only minimally affected by such an amino-acid substitution at this position . Three-dimensional structural models of the enzyme-substrate complexes of the wild-type and mutant SAM1606 alpha-glucosidases were built to explore the mechanism responsible for these observations . It is proposed that substitution by glycine at position 273* could eliminate steric hindrance around subsite +1 that originally occurred in parental O16G and is, at least in part, responsible for the acquired broad substrate specificity of SAM1606 alpha-glucosidase.

Biochemistry (Mosc), 2003 Sep, 68(9), 1012 - 9
Expression, isolation, purification, and biochemical properties of trehalose-6-phosphate hydrolase from thermoresistant strain Bacillus sp . GP16; Karelov DV et al.; Here we describe cloning, expression, and purification of the enzyme trehalose-6-phosphate hydrolase from thermoresistant strain Bacillus sp . GP16 . Principal biochemical properties of the enzyme at different pH and temperature values were determined . Entropy and enthalpy of activation of the enzyme for substrates trehalose-6-phosphate and p-nitrophenyl glucoside were calculated, and the dependence of the kinetic parameters from ionic strength was established.

Biochemistry (Mosc), 2003 Sep, 68(9), 984 - 7
Cloning and sequencing of the gene of site-specific nickase N.BspD6I; Perevyazova TA et al.; A fragment of chromosomal DNA from Bacillus species D6 containing the gene of nickase N.BspD6I and the regions adjacent to its 5;- and 3;-ends was cloned and sequenced . The nucleotide sequence of the nickase gene, except of one neutral change, is homologous to the nicking endonuclease N.BstNBI gene sequenced by Higgens et al . (2001) . After integration of a PCR-copy of the nickase gene into an expression vector pET28b under the control of the phage T7 promoter, specific nicking activity was detected in the lysates of transformed E . coli cells.

Biochemistry (Mosc), 2003 Sep, 68(9), 967 - 75
M.BstF5I-2 and M.BstF5I-4 DNA methyltransferases from BstF5I restriction-modification system of Bacillus stearothermophilus F5; Chernukhin VA et al.; The BstF5I restriction-modification system from Bacillus stearothermophilus F5 includes four site-specific DNA methyltransferases, thus differing from all known restriction-modification systems . Here we demonstrated for the first time that one bacterial cell can possess two pairs of methylases with identical substrate specificities (methylases BstF5I-1 and BstF5I-3 recognize GGATG, whereas methylases BstF5I-2 and BstF5I-4 recognize CATCC) that modify adenine residues on both DNA strands . Different chromatographic methods provide homogenous preparations of methylases BstF5I-2 and BstF5I-4 . We estimated the principal kinetic parameters of the reaction of transfer of methyl group from the donor S-adenosyl-L-methionine to the recognition site 5;-CATCC-3; catalyzed by BstF5I-2 and BstF5I-4 DNA {N6-adenine}-methyltransferases from the BstF5I restriction-modification system.

J Clin Microbiol, 2003 Nov, 41(11), 5291 - 3
Pericardial effusion in a homeless man due to Bartonella quintana; Levy PY et al.; Bartonella quintana may cause trench fever, endocarditis, bacillary angiomatosis, and chronic bacteremia, and a reemergence among homeless populations in cities has been noted . Pericarditis from Rickettsia conorii and Coxiella burnetii infection has been described, but there have been no reports of pericarditis due to Bartonella spp . We report a case of pericardial effusion due to Bartonella quintana in a homeless man, diagnosed on the basis of PCR detection of Bartonella quintana in a pericardial biopsy sample and a fourfold rise in antibody titers . The patient recovered within 2 weeks with antibiotics active against bartonellae.

Vaccine, 2003 Dec 8, 22(1), 70 - 6
Oral vaccination with Mycobacterium bovis BCG in a lipid formulation induces resistance to pulmonary tuberculosis in brushtail possums; Aldwell FE et al.; A method was developed for formulating Mycobacterium bovis bacille Calmette-Guerin (BCG) for oral vaccination against tuberculosis . Selected lipid-based formulations of BCG were tested in the brushtail possum for their ability to elicit immune responses and protection against bovine tuberculosis . Formulation of BCG in lipid matrices maintained bacteria in a dormant but viable state . Oral delivery of 2 x 10(8) colony forming units of formulated BCG to possums induced strong lymphocyte proliferation responses to bovine purified protein derivative (PPD) in peripheral blood lymphocytes . Oral vaccination of possums also reduced the severity of disease following aerosol challenge with virulent M . bovis compared with animals vaccinated with non-formulated BCG . In a second experiment, levels of protection with lipid-formulated oral BCG were similar to those seen with subcutaneous BCG vaccination . Our data shows that formulated oral BCG is an efficient means of inducing protection against bovine tuberculosis in possums and should be a practical means of vaccinating wildlife against tuberculosis.

J Immunol Methods, 2003 Nov, 282(1-2), 45 - 52
Generation of an affinity column for antibody purification by intein-mediated protein ligation; Sun L et al.; Coupling an antigenic peptide to a solid support is a crucial step in the affinity purification of a peptide-specific antibody . Conventional methods for generating reactive agarose, cellulose or other matrices for peptide conjugation are laborious and can result in a significant amount of chemical waste . In this report, we present a novel method for the facile production of a peptide affinity column by employing intein-mediated protein ligation (IPL) in conjunction with chitin affinity chromatography . A reactive thioester was generated at the C-terminal of the chitin binding domain (CBD) from the chitinase A1 of Bacillus circulans WL-2 by thiol-induced cleavage of the peptide bond between the CBD and a modified intein . Peptide epitopes possessing an N-terminal cysteine were ligated to the chitin bound CBD tag . We demonstrate that the resulting peptide columns permit the highly specific and efficient affinity purification of antibodies from animal sera.

J Indian Med Assoc, 2003 Mar, 101(3), 198, 200 - 3
Tuberculosis and general practitioners; Tripathy SN; In spite of advancement of knowledge in diagnosis and management, tuberculosis is still the biggest health problem . There are more than 400 million people infected with tuberculous bacillus and more than 14 million cases are suffering from the disease . Prevention, control and care of tuberculosis are possible . General practitioners play a major role in combating the disease . Quick and right diagnosis and treating the cases effectively should be the motto . Sputum microscopy is the backbone of diagnosis of tuberculosis . With the availability of modern chemotherapy the outcome of a tuberculosis case has undergone dramatic changes . Short-course chemotherapy is the standard choice while tackling cases of tuberculosis . Results of directly observed treatment (DOT) strategy are very good provided execution is proper . HIV/AIDS, another dreaded disease if becomes co-infected along with tuberculosis, mortality and morbidity become very high . The general practitioners are the major strength of healthcare system in any society . They should be good enough to know all about the control programmes well implemented in the country.

Parasitol Res, 2004 Jan, 92(1), 53 - 7 Epub 2003 Nov 05.
Protection of mice infected with Plasmodium berghei by Bacillus thuringiensis crystal proteins; Xu Z et al.; Eight Bacillus thuringiensis strains were used to test their activity against Plasmodium berghei . When crystal proteins extracted from strains 007, 017, 020, 021, 030, 032, and 037 were injected into plasmodium-infected mice through the tail vein at a rate of 0.45-1.5 mg per mouse, the lengths of survival for the mice were extended up to 5 days (from 8.5 days to 13.5-15 days) . Blood-cell staining demonstrated that normal erythrocytes were lightly stained and regularly shaped while the erythrocytes from plasmodia-infected mice swelled, lost shape and even lysed . This means that the crystal proteins could protect erythrocytes from the plasmodium's attack . Proteins analysis revealed that most of the proteins are homologues of classic crystal proteins, with the exception of the 120-kDa protein of strain 020, a surface-layer protein . This study suggested a novel way to control plasmodial infections and even malaria.

Microbiology, 2003 Nov, 149(Pt 11), 3213 - 20
Koch's bacillus - a look at the first isolate of Mycobacterium tuberculosis from a modern perspective; Taylor GM et al.; Using molecular methods the authors have studied mycobacterial DNA taken from a 19th century victim of tuberculosis . This was the case from which Robert Koch first isolated and cultured the organism responsible for tuberculosis . The mycobacteria were preserved within five glass culture tubes as abundant bacterial colonies on slopes of a gelatinous culture medium of unknown composition . Originally presented by Koch to surgical laryngologist Walter Jobson Horne in London in 1901, the relic has, since 1983, been in the care of the Royal College of Surgeons of England . Light and electron microscopy established the presence of acid-fast mycobacteria but showed that morphological preservation was generally poor . Eleven different genomic loci were successfully amplified by PCR . This series of experiments confirmed that the organisms were indeed Mycobacterium tuberculosis and further showed that the original strain was in evolutionary terms similar to 'modern' isolates, having undergone the TB D1 deletion . Attempts to determine the genotypic group of the isolate were only partially successful, due in part to the degraded nature of the DNA and possibly also to a truncation in the katG gene, which formed part of the classification scheme . Spoligotyping resulted in amplification of DR spacers consistent with M . tuberculosis but with discrepancies between independent extracts, stressing the limitations of this typing method when applied to poorly preserved material.

J Clin Pathol, 2003 Nov, 56(11), 868 - 70
Inflammatory pseudotumour associated with chronic persistent Eikenella corrodens infection: a case report and brief review; Lee SH et al.; Inflammatory pseudotumour is an uncommon mass forming lesion, representing the histological expression of an infective or reactive/reparative process (pseudotumour) in most cases, and a bona fide neoplasm (for example, inflammatory myofibroblastic tumour) in a minority of cases . This report describes the case of an inflammatory pseudotumour with a pathology that unveiled proliferative CD68 positive and actin negative spindle shaped cells, with a mild mixed inflammatory infiltrate, and a culture that yielded an uncommon fastidious bacillus, Eikenella corrodens . The clinical course was indolent but protracted, with insidious progression to multifocal non-contiguous lesions, involving the lungs, liver, spleen, left kidney, and deep neck tissue, all of which responded to medical treatment with appropriate antibiotics . It is of paramount importance that clinicians search for an infective cause of an inflammatory pseudotumour, to ensure appropriate treatment.

Toxicol In Vitro, 2003 Oct-Dec, 17(5-6), 745 - 51
A new method for in vitro detection of microbially produced mitochondrial toxins; Hoornstra D et al.; Sperm motility inhibition assay, earlier shown valuable for the detection of food poisoning non-protein toxins of Bacillus species was developed into an assay useful for specific detection of mitochondria damaging toxins . This was done by assessing the dissipation of the mitochondrial inner membrane transmembrane potential, Deltapsim under conditions where the plasma membrane permeability barrier remained intact . The Deltapsim was estimated as the intensity of orange JC-1 fluorescence in the mitochondrial sheath of the exposed spermatozoa . The plasma membrane integrity of the same cells was assessed by observing the exclusion of propidium iodide from the cytoplasm . Three types of mitochondrial toxic responses to microbially made bioactive substances were recognised . Mitochondrial toxicity by gramicidin (A, B, C, D), nigericin, salinomycin, narasin, monensin, calcimycin and antimycin A was characterised by gradual fading of the JC-1 fluorescence in the mitochondria . Dissipation of the Deltapsim by cereulide, valinomycin and enniatin (A, A1, B, B1) was visible as spotwise quenching of the mitochondrial JC-1 fluorescence . In addition these substances caused hyperpolarisation of the plasma membrane . Oligomycin (A, B, C), ionomycin and staurosporine inhibited the spermatozoan motility, but Deltapsim was fully preserved . Surfactin and lichenysin A caused mitochondrial damage at concentrations where the plasma membrane was also damaged.

Toxicol In Vitro, 2003 Oct-Dec, 17(5-6), 737 - 44
In vitro assay for human toxicity of cereulide, the emetic mitochondrial toxin produced by food poisoning Bacillus cereus; Jaaskelainen EL et al.; The in vitro boar spermatozoon test was compared with the LC ion trap MS analysis for measuring the cereulide content of a pasta dish, implemented in serious emetic food poisoning caused by Bacillus cereus . Both assays showed that the poisonous food contained approximately 1.6 microg of cereulide g(-1) implying the toxic dose in human as < or =8 microg kg(-1) body weight . The threshold concentration of cereulide provoking visible mitochondrial damage in boar sperm exposed in vitro was 2 ng of cereulide ml(-1) of extended boar sperm . The same threshold value was found for cereulide extracted from the food and from the cultured bacteria . This shows that other constituents of the food did not enhance or mask the effects of cereulide . Exposure of four human cell lines (HeLa, Caco-2, Calu-3 and Paju) to cereulide showed that the threshold concentration for the loss of mitochondrial membrane potential in human cells was similar to that observed in boar sperm . Human cells and boar sperm were equally sensitive to cereulide . The results show that boar spermatozoan assay is useful for detecting cereulide concentrations toxic to humans . Spermatozoa in commercially available extended fresh boar and cryopreserved bull semen were compared, boar sperms were 100 times more sensitive to cereulide than bull sperms.

Toxicol In Vitro, 2003 Oct-Dec, 17(5-6), 623 - 8
Tamoxifen induces ultrastructural alterations in membranes of Bacillus Stearothermophilus; Luxo C et al.; Tamoxifen (TAM), a non-steroid antiestrogen, is the mostly used drug for chemotherapy and chemoprevention of breast cancer . However, the mechanisms by which TAM inhibits cell proliferation in breast cancer are not fully understood . TAM strongly incorporates in biomembranes and a variety of effects have been assigned to biophysical and biochemical interactions with membranes . Therefore, a better understanding of the physicochemical basis of interaction of TAM with biomembranes is essential to elucidate the molecular mechanisms of action . A strain of Bacillus stearothermophilus has been used as a model to clarify the interaction of TAM with the cell membrane . TAM effects on the ultrastructure of membranes of this bacterium were evaluated by electron microscopy . Important ultrastructural alterations were observed in B . stearothermophilus treated with TAM, namely change in the geometry of the membrane profile from asymmetric to symmetric, disaggregation of ribosomes, coagulation of the cytoplasmic matrix, occurrence of mesossomes, appearance of fractures in membranes and the alteration of the ultrastructure of cell wall . These ultrastructural alterations confirm that TAM is a membrane-active drug and that membrane damage may be involved in molecular mechanisms of cell death induced by this drug.

Rev Clin Esp, 2003 Nov, 203(11), 532 - 5
{High resolution computerized tomography in pulmonary tuberculosis with negative sputum bacilloscopy}; Gonzalez Constan E et al.; CONTEXT: Pulmonary tuberculosis with negative sputum bacilloscopy involves diagnostic difficulties when there are not available liquid culture media due to the slow growth of Mycobacterium tuberculosis in the traditional culture media, and also due to the need for carrying out invasive examinations . OBJECTIVE: Evaluate the high resolution computerized tomography findings (HRCT) in patients with pulmonary tuberculosis and negative bacilloscopy of sputum . METHOD: Prospectively 28 patients (12 with active tuberculosis and 16 with inactive tuberculosis) were evaluated through HRCT, assessing the following findings: centrolobular nodules, multiple branched linear structures, macronodules, cavitation, consolidation, enlargement of interlobular septums, ground-glass change, bronchiectases, emphysema, broncovascular distortion, fibrotic changes, calcified mediastinal adenopathies, parenchymous calcification, pleural enlargement, and pleural effusion . RESULTS: The findings that were associated significantly to the active disease were: consolidation (67%), macronodules (67%) and centrolobular nodules (67%) . The presence of centrolobular nodules and/or consolidation had a sensitivity of 83% and a specificity of 87% . The findings significantly associated to inactive disease were bronchiectases (87%) and broncovascular distortion (62%) . CONCLUSION: Our results support the value of HRCT in patients with pulmonary tuberculosis and negative sputum bacilloscopy, since the finding of centrolobular nodules and/or consolidation has good sensitivity and specificity for the diagnosis of active pulmonary disease.

J Med Entomol, 2003 Sep, 40(5), 672 - 7
Susceptibility profile of Culex quinquefasciatus (Diptera: Culicidae) to Bacillus sphaericus on selection with rotation and mixture of B . sphaericus and B . thuringiensis israelensis; Zahiri NS et al.; Several strains of Bacillus sphaericus (Neide) (Bsph), a spore-forming bacterium that produces potent mosquitocidal toxins, have been widely used against mosquito larvae globally . In some locations in the field and in the laboratory, moderate to high levels of resistance in Culex pipiens complex mosquito larvae to Bsph have been reported . Practical strategies for dealing with the resistance problem and more importantly to delay or possibly prevent development of resistance to Bsph in the first place are urgently needed . From published reports, we deduced that Bsph-resistant mosquito larvae are completely susceptible to Bacillus thuringiensis ssp . isreaelensis (Bti) and that there is no appearance of cross-resistance to Bti . On the basis of this information, we hypothesized that Bti offers a good potential for the management of resistance to Bsph, especially when it comes to delaying or preventing emergence of resistance . We conducted laboratory studies to see whether rotation of Bsph and Bti or mixture of both delays or prevents resistance development in Bsph-susceptible California Cx . quinquefasciatus . Development of resistance using Bsph alone increased after F15 and fluctuated slightly on further selections . Rotation of Bsph and Bti resulted in much higher level and rapid emergence of resistance to Bsph . However, selection with mixtures of Bti and Bsph for 36 generations showed no emergence of resistance . On the basis of this information, we believe mixtures have a good potential in delaying or preventing Bsph resistance.

J Dairy Sci, 2003 Oct, 86(10), 3075 - 81
Inactivation of spores of Bacillus cereus in cheese by high hydrostatic pressure with the addition of nisin or lysozyme; Lopez-Pedemonte TJ et al.; The objective of this work was to study high hydrostatic pressure (HHP) inactivation of spores of Bacillus cereus ATCC 9139 inoculated in model cheeses made of raw milk, together with the effects of the addition of nisin or lysozyme . The concentration of spores in model cheeses was approximately 6-log10 cfu/g of cheese . Cheeses were vacuum packed and stored at 8 degrees C . All samples except controls were submitted to a germination cycle of 60 MPa at 30 degrees C for 210 min, to a vegetative cells destruction cycle of 300 or 400 MPa at 30 degrees C for 15 min, or to both treatments . Bacillus cereus counts were measured 24 h and 15 d after HHP treatment . The combination of both cycles improved the efficiency of the whole treatment . When the second pressure-cycle was of 400 MPa, the highest inactivation (2.4 +/- 0.1 log10 cfu/g) was obtained with the presence of nisin (1.56 mg/L of milk), whereas lysozyme (22.4 mg/L of milk) did not increase sensitivity of the spores to HHP . For nisin (0.05 and 1.56 mg/L of milk), no significant differences were found between counts at 24 h and 15 d after treatment . Considering that mesophilic spore counts usually range from 2.6 to 3.0 log10 cfu/ml in raw milk, HHP at mild temperatures with the addition of nisin may be useful for improving safety and preservation of soft curd cheeses made from raw milk.

Prikl Biokhim Mikrobiol, 2003 Sep-Oct, 39(5), 536 - 41
{Isolation and characterisation of chitosanase from Bacillus sp . 739 strain}; Aktuganov GE et al.; The specific nature of the chitosanase activity of the strain Bacillus sp . 739 has been determined . Maximum enzyme activity was observed in a medium containing the biomass of the fruiting bodies of the fungus Macrolepiota procera . The chitosanase was purified to homogeneity using chromatography on DEAE-Sephadex A-50 and Toyopearl HW-50 . The molecular weight of the enzyme, assessed by electrophoresis (the Laemmli procedure) approximated 46 kDa . Temperature and pH optima of the purified chitosanase were in the ranges 45-55 degrees C and 6.0-6.5, respectively . Time to half-maximum inactivation of the enzyme at 50 degrees C was equal to 1 h . With colloidal chitosan as the substrate, the value of K(M) of the purified chitosanase was equal to 25 mg/ml . The enzyme also exhibited a weak ability to hydrolyze colloidal chitin.

J Infect Dis, 2003 Nov 1, 188(9), 1332 - 5 Epub 2003 Oct 15.
Nosocomial Mycobacterium bovis-bacille Calmette-Guérin infections due to contamination of chemotherapeutics: case finding and route of transmission; Vos MC et al.; We studied nosocomial infections due to Mycobacterium bovis bacille Calmette-Guerin (BCG) Onco-TICE bacteria, transmitted by contamination of medication prepared in BCG Onco-TICE-contaminated hoods in the pharmacy, in 5 immunocompromised patients at 3 hospitals . The BCG strains cultured from the patients had the same DNA profile as the BCG Onco-TICE strain used for bladder instillation . To prevent these infections, a change from open to closed preparation was made; strictly separated preparation in time of BCG Onco-TICE instillation and chemotherapy was enforced, the biological safety cabinet was disinfected between preparations, and gloves were changed between preparations.

Mycorrhiza, 2003 Oct, 13(5), 249 - 56 Epub 2003 Feb 15.
Influence of a Bacillus sp . on physiological activities of two arbuscular mycorrhizal fungi and on plant responses to PEG-induced drought stress; Vivas A et al.; The effects of bacterial inoculation (Bacillus sp.) on the development and physiology of the symbiosis between lettuce and the arbuscular mycorrhizal (AM) fungi Glomus mosseae (Nicol . and Gerd.) Gerd . and Trappe and Glomus intraradices (Schenck and Smith) were investigated . Plant growth, mineral nutrition and gas-exchange values in response to bacterial inoculation after PEG-induced drought stress were also evaluated . In AM plants, inoculation with Bacillus sp . enhanced fungal development and metabolism, measured as succinate dehydrogenase (SDH) and alkaline phosphatase (ALP) activities, more than plant growth . Under non-stressed conditions, G . intraradices colonization increased all plant physiological values to a higher extent when in dual inoculation with the bacterium . Under stress conditions, the bacterium had an important stimulatory effect on G . intraradices development . Under such conditions, the effects of the bacterium on photosynthetic rate, water use efficiency (WUE) and stomatal conductance of lettuce plants differed with the fungus species . Plant-gas exchange was enhanced in G . intraradices- and reduced in G . mosseae-colonized plants when co-inoculated with Bacillus sp . Thus, the effects of each fungus on plant physiology were modulated by the bacterium . Stress was detrimental, particularly in G . intraradices-colonized plants without the bacterium, reducing intra and extraradical mycelium growth and vitality (SDH), as well as plant-gas exchange . Nevertheless, Bacillus sp . inoculation improved all these plant and fungal parameters to the same level as in non-stressed plants . The highest amount of alive and active AM mycelium for both fungi was obtained after co-inoculation with Bacillus sp . These results suggest that selected free-living bacteria and AM fungi should be co-inoculated to optimize the formation and functioning of the AM symbiosis in both normal and adverse environments.

Appl Microbiol Biotechnol, 2004 May, 64(4), 497 - 504 Epub 2003 Oct 31.
Diglucosyl-glycerolipids from the marine sponge-associated Bacillus pumilus strain AAS3: their production, enzymatic modification and properties; Ramm W et al.; The marine strain Bacillus pumilus strain AAS3, isolated from the Mediterranean sponge Acanthella acuta, produced a diglucosyl-glycerolipid, 1,2-O-diacyl-3-{beta-glucopyranosyl-(1-6)-beta-glucopyranosyl)}glycerol, with 14-methylhexadecanoic acid and 12-methyltetradecanoic acid as the main fatty acid moieties (GGL11) . On a 30 l scale, using artificial seawater supplemented with glucose (20 g/l), yeast extract (10 g/l), and suitable nitrogen/phosphate sources, growth-associated glycoglycerolipid production reached its maximum yield of 90 mg/l after 11 h . Lipase-catalyzed modification of the native substance led to the deacylated parent compound (GG11), which could be reacylated using the same enzyme system to afford a new dipentenoyl-diglucosylglycerol (GGL12) as the major product upon addition of 4-pentenoic acid to the medium . GGL11 decreased the surface tension of water from 72 mN/m to 29 mN/m and the interfacial tension of the water/ n-hexadecane system from 44 to 5 mN/m . Anti-tumor-promoting studies on this class of diglucosyl glycerol products showed that the carbohydrate/glycerol backbone (GG11) has a more potent inhibitory activity than the acylated compounds . The diglucosyl-glycerol GG11 strongly inhibited growth of the tumor cell lines HM02 and Hep G2 (50% inhibition at approximately 1 microg/ml), while the glycerolipids GGL11 and GGL12 were less active or had no effect .

Appl Microbiol Biotechnol, 2004 Apr, 64(2), 243 - 9 Epub 2003 Oct 31.
Identification and functional characterization of a type I signal peptidase gene of Bacillus megaterium DSM319; Nahrstedt H et al.; The sipM gene of Bacillus megaterium encoding a type I signal peptidase (SPase) was isolated and structurally characterized . RNA analysis revealed a transcript size in accordance with a bicistronic operon comprising sipM and an adjacent open reading frame . Inactivation of sipM by targeted gene disruption could not be achieved, indicating its essential role for cell viability since there might be no other type I SPase of major importance present in B . megaterium . Plasmid-assisted amplification of the gene resulted in an increase in activity of the heterologous glucanase used as an extracellular reporter, suggesting a potential bottleneck for protein secretion within this species.

Appl Microbiol Biotechnol, 2004 Apr, 64(2), 187 - 91 Epub 2003 Oct 31.
Artificial carrier for oxygen supply in biological systems; Dey ES et al.; Several poly (dimethylsiloxanes) (PDMS) copolymers of dimethylsiloxane (DMS) with ethylene or propylene oxide were tested as artificial carriers for the delivery of oxygen to biological systems . Copolymers with a DMS content of 33% or lower enhanced glucose oxidation by 200% in contrast to the 25% increase produced by the same concentration of perfluorodecalin . When 0.05% of the copolymer with 18% DMS was included in the growth media of Bacillus thuriginensis, the biomass (growth rate) increased 1.5-fold . With 0.1% of this copolymer, actinorhodin production by Streptomyces coelicolor A3 (2) occurred in half the normal time and with an increased yield . In conclusion, these PDMS copolymers are a good alternative to perfluorodecalin as oxygen carriers in biotechnological processes.

FEMS Microbiol Lett, 2003 Oct 24, 227(2), 189 - 95
Mosquito larvicidal activity of transgenic Anabaena PCC 7120 expressing toxin genes from Bacillus thuringiensis subsp . israelensis; Khasdan V et al.; Genes encoding the mosquito larvicidal toxins Cry4Aa, Cry11Aa, Cyt1Aa and the regulatory P20 from Bacillus thuringiensis subsp . israelensis were introduced into the nitrogen-fixing, filamentous cyanobacterium Anabaena PCC 7120 for expression under control of two strong promoters P(psbA) and P(A1) . The clone pRVE4-ADRC displayed toxicity against fourth-instar larvae of Aedes aegypti, the highest ever achieved in cyanobacteria . It was about 2.5-fold more toxic than the respective clone without cyt1Aa {Wu et al., Appl . Environ . Microbiol . 63 (1997) 4971-4975} . Cyt1Aa synergized the combination of Crys by about five-fold . Consistently, the lethal times exerted by pRVE4-ADRC were also reduced (it killed exposed larvae more quickly) . This clone may become a useful biological control agent which reduces the probability of resistance development in the target organisms {Wirth et al., Proc . Natl . Acad . Sci . USA 94 (1997) 10536-10540}.

Biochem Biophys Res Commun, 2003 Nov 14, 311(2), 386 - 90
Real-time molecular beacon NASBA reveals hblC expression from Bacillus spp . in milk; Gore HM et al.; Nucleic acid sequence-based amplification (NASBA) was applied in combination with a fluorescein-conjugated molecular beacon specific for a sequence flanked by transcript-specific primers in order to monitor hblC enterotoxin gene expression in real-time from milk separately contaminated with Bacillus amyloliquefaciens, Bacillus cereus, and Bacillus circulans . Maximal enterotoxin expression was noted following 16, 15, and 16 h, respectively, when grown in artificially contaminated nonfat dried milk incubated aerobically at 32 degrees C, corresponding to 1.6 x 10(5), 5 x 10(7), and 9.8 x 10(4)cfu/ml, for B . amyloliquefaciens, B . cereus, and B . circulans, respectively . This RNA amplification assay allows for simultaneous detection and confirmation of target transcripts in a closed tube format and may be performed in a high DNA background . The development of a rapid, sensitive, real-time method to quantitate the expression of virulence genes in pathogenic spore-formers is useful in shelf life determination of foods and other quality assurance measures.

Biosci Biotechnol Biochem, 2003 Oct, 67(10), 2304 - 6
Increased production of antioxidative sesaminol glucosides from sesame oil cake through fermentation by Bacillus circulans strain YUS-2; Ohtsuki T et al.; Bacillus circulans strain YUS-2 was isolated as the strongest antioxidant-producer in fermentation of sesame oil cake (SOC, defatted residue yielded from sesame seed oil production) . Two major strong antioxidants from fermented SOC were purified and identified as known sesaminol triglucoside and sesaminol diglucoside, however, our results demonstrated that the fermentation process with B . circulans YUS-2 was highly effective to gain the extraction efficiency of the sesaminol glucosides.

Biosci Biotechnol Biochem, 2003 Oct, 67(10), 2132 - 8
Endophytic colonization of balloon flower by antifungal strain Bacillus sp . CY22; Cho SJ et al.; Endophytic Bacillus sp . CY22 was previously isolated from the root interior of the balloon flower (Platycodon grandiflorum) (Cho et al., Biosci . Biotechnol . Biochem., 66, 1270-1275 (2002)) . Three-month-old balloon flower seedlings were inoculated with 10(7) cfu/ml of strain CY22R3, a rifampicin-resistant strain of CY22, and external and internal root colonization was assessed 2 and 4 weeks later . After inoculation, large numbers of bacteria were observed on the root surface by scanning electron microscopy . More detailed studies using optical and transmission electron microscopy confirmed that Bacillus sp . CY22 was endophytically established within intercellular spaces, cortical cells, and aerenchymas of root . Also, Bacillus sp . CY22 showed antibiotic activities against several phytopathogens by producing the antibiotic iturin A . In the pot test, root rot of balloon flower seedlings caused by Rhizoctonia solani was suppressed when the Bacillus sp . CY22R3 was inoculated into the soil.

Vaccine, 2003 Dec 1, 21(32), 4722 - 7
Role of L-lysine HCl in immunopotentiation towards development of suitable tuberculosis vaccination; Dasgupta S et al.; L-Lysine HCl is being proposed to be a possible biocompatible adjuvant to enhance immune response by virtue of its probable non-specific bridging action and cellular proliferation properties . This proposal has been tried to be substantiated by carrying out experimentation where L-lysine HCl has been used as an adjuvant (various groups based on mode of application and frequency of booster dose were designed) in tuberculosis vaccination experiments with heat killed Mycobacterium tuberculosis (MTB) and Bacille Calmette Guerin (BCG) . Antibody titre has been followed in all the experiments as a measure of immune response . Amongst the various groups designed, group 1A (L-lysine HCl was given at a separate site as that of the antigen; lysine booster was given to this group intermittently, i.e . lysine given on 0th, 7th, 14th, 21st days of immunization) came out as the stand-alone leader . This mode and frequency of application was then compared with a group which received a standard adjuvant, viz . alhydrogel . Results were obtained which showed the following order in terms of decreasing antibody titre: alhydrogel group > lysine group > control group . Considering the biocompatible nature of lysine in comparison with the reportedly hazardous nature of alum adjuvants, we propose L-lysine HCl as a probable adjuvant in vaccination.

J Am Chem Soc, 2003 Nov 5, 125(44), 13442 - 50
Regio- and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3; Peters MW et al.; Cytochrome P450 BM-3 from Bacillus megaterium was engineered using a combination of directed evolution and site-directed mutagenesis to hydroxylate linear alkanes regio- and enantioselectively using atmospheric dioxygen as an oxidant . BM-3 variant 9-10A-A328V hydroxylates octane at the 2-position to form S-2-octanol (40% ee) . Another variant, 1-12G, also hydroxylates alkanes larger than hexane primarily at the 2-position but forms R-2-alcohols (40-55% ee) . These biocatalysts are highly active (rates up to 400 min(-1)) and support thousands of product turnovers . The regio- and enantioselectivities are retained in whole-cell biotransformations with Escherichia coli, where the engineered P450s can be expressed at high levels and the cofactor is supplied endogenously.

J Agric Food Chem, 2003 Nov 5, 51(23), 6823 - 7
Rapid digestion of Cry34Ab1 and Cry35Ab1 in simulated gastric fluid; Herman RA et al.; Two genes were identified in Bacillus thuringiensis Berliner (Bt) that code for the proteins that comprise a Cry34Ab1/Cry35Ab1 binary insecticidal crystal protein . Maize, Zea mays L., plants have been transformed to express the Cry34Ab1/Cry35Ab1 proteins, and as a result, these plants are resistant to attack by western corn rootworm, Diabrotica virgifera virgifera LeConte, a major pest in the Midwestern corn-growing area of the U.S.A . As part of the safety assessment for the proteins, digestibility studies were conducted . Digestion experiments with both proteins demonstrated rapid degradation in simulated gastric fluid, comparable to other registered plant-incorporated protectants . Quantitative and qualitative approaches for determining digestibility are illustrated.

Biochim Biophys Acta, 2003 Nov 3, 1652(1), 52 - 63
A proposed mechanism for the thermal denaturation of a recombinant Bacillus halmapalus alpha-amylase--the effect of calcium ions; Nielsen AD et al.; The thermal stability of a recombinant alpha-amylase from Bacillus halmapalus alpha-amylase (BHA) has been investigated using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC) . This alpha-amylase is homologous to other Bacillus alpha-amylases where crystallographic studies have identified the existence of three calcium binding sites in the structure . Denaturation of BHA is irreversible with a T(m) of approximately 89 degrees C and DSC thermograms can be described using a one-step irreversible model . A 5 degrees C increase in T(m) in the presence of 10-fold excess CaCl(2) was observed . However, a concomitant increase in the tendency to aggregate was also observed . The presence of 30-40-fold excess calcium chelator (ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis{beta-aminoethyl ether} N,N,N',N'-tetraacetic acid (EGTA)) results in a large destabilization of BHA, corresponding to about 40 degrees C lower T(m) as determined by both CD and DSC . Ten-fold excess EGTA reveals complex DSC thermograms corresponding to both reversible and irreversible transitions, which probably originate from different populations of BHA/calcium complexes . Combined interpretation of these observations and structural information on homologous alpha-amylases forms the basis for a suggested mechanism underlying the inactivation mechanism of BHA . The mechanism includes irreversible thermal denaturation of different BHA/calcium complexes and the calcium binding equilibria . Furthermore, the model accounts for a temperature-induced reversible structural change associated with calcium binding.

J Biotechnol, 2003 Nov 6, 105(3), 255 - 60
Magnetic alginate microparticles for purification of alpha-amylases; Safarikova M et al.; Spherical magnetic alginate microparticles (25-60 microm in diameter) were prepared using the microemulsion system, with water-saturated 1-pentanol as the organic phase . The limited solubility of 1-pentanol in water enabled simple removal of the organic solvent from the prepared beads with water solution . The prepared alginate microparticles were used as magnetic affinity adsorbents for specific purification of alpha-amylases . Enzyme activity was eluted by 1.0 M maltose . alpha-Amylases from Bacillus amyloliquefaciens and porcine pancreatic acetone powder were purified 9- and 12-fold with 88 and 96% activity recovery, respectively.

J Transl Med . 2003 Oct 10;1(1):7.
Interferon-gamma Added During Bacillus Calmette-Guerin Induced Dendritic Cell Maturation Stimulates Potent Th1 Immune Responses; Shankar G et al.; Dendritic cells (DC) are increasingly prepared in vitro for use in immunotherapy trials . Mature DC express high levels of surface molecules needed for T cell activation and are superior at antigen-presentation than immature DC . Bacillus Calmette-Guerin (BCG) is one of several products known to induce DC maturation, and interferon (IFN)-gamma has been shown to enhance the activity of DC stimulated with certain maturation factors . In this study, we investigated the use of IFN-gamma in combination with the powerful maturation agent, BCG . The treatment of immature DC with IFN-gamma plus BCG led to the upregulation of CD54, CD80, and CD86 in comparison with BCG treatment alone . In MLR or recall immune responses, the addition of IFN-gamma at the time of BCG-treatment did not increase the number of antigen-specific T cells but enhanced the development of IFN-gamma-producing Th1 cells . In primary immune responses, on the other hand, BCG and IFN-gamma co-treated DC stimulated higher proportions of specific T cells as well as IFN-gamma secretion by these T cells . Thus the use of IFN-gamma during BCG-induced DC maturation differentially affects the nature of recall versus naive antigen-specific T-cell responses . IFN-gamma co-treatment with BCG was found to induce IL-12 and, in some instances, inhibit IL-10 secretion by DC . These findings greatly enhance the potential of BCG-matured dendritic cells for use in cancer immunotherapy.

Proteins, 2003 Nov 15, 53(3), 597 - 606
Unfolding of the cold shock protein studied with biased molecular dynamics; Morra G et al.; The cold shock protein from Bacillus caldolyticus is a small beta-barrel protein that folds in a two-state mechanism . For the native protein and for several mutants, a wealth of experimental data are available on stability and folding, so that it is an optimal system to study this process . We compare data from unfolding simulations (trajectories of 5 and up to 12 ns) obtained with a bias potential at room temperature and from unbiased thermal unfolding simulations with experimental data . The unfolding patterns derived from the trajectories starting from different native-like conformations and subject to different unfolding conditions agree . The transition state found in the simulations of unfolding is close to the native structure in agreement with experiment . Moreover, a lower value of the free energy barrier of unfolding was found for the mutant R3E than for the mutant E46A and the native protein, as indicated by experimental data . The first unfolding event involves the three-stranded beta-sheet whose decomposition corresponds to the transition state . In contrast to conclusions drawn from experiments, we found that the two-stranded beta-strand forms the most stable substructure, which decomposes very late in the unfolding process . However, assuming that this structure forms very early in the folding process, our findings would not contradict the experiments but require a different interpretation of them .

Mol Pharmacol, 2003 Nov, 64(5), 1189 - 98
Ceramide is involved in r(+)-methanandamide-induced cyclooxygenase-2 expression in human neuroglioma cells; Ramer R et al.; Cannabinoids have recently been shown to induce the expression of the cyclooxygenase-2 (COX-2) isoenzyme in H4 human neuroglioma cells . Using this cell line, the present study investigates the contribution of the second messenger ceramide to this signaling pathway . Incubation of cells with the endocannabinoid analog R(+)-methanandamide (R(+)-MA) was associated with an increase of intracellular ceramide levels . Enhancement of ceramide formation by R(+)-MA was abolished by fumonisin B1, a ceramide synthase inhibitor, whereas inhibitors of neutral sphingomyelinase (spiroepoxide, glutathione) and serine palmitoyltransferase (l-cycloserine, ISP-1) were inactive in this respect . R(+)-MA caused a biphasic activation of the p38 and p42/44 mitogen-activated protein kinases (MAPKs), with phosphorylation peaks occurring after 15-min and 4- to 8-h treatments, respectively . Inhibition of ceramide synthesis with fumonisin B1 was associated with a suppression of R(+)-MA-induced delayed phosphorylations of p38 and p42/44 MAPKs and subsequent COX-2 expression . The involvement of ceramide in COX-2 expression was corroborated by findings showing that C2-ceramide and neutral sphingomyelinase from Bacillus cereus caused concentration-dependent increases of COX-2 expression that were suppressed in the presence of 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)imidazol (SB203580, a p38 MAPK inhibitor) or 2'-amino-3'-methoxyflavone (PD98059, a p42/44 MAPK activation inhibitor) . In contrast, dihydro-C2-ceramide being used as a negative control did not induce MAPK phosphorylation and COX-2 expression . Collectively, our results demonstrate that R(+)-MA induces COX-2 expression in human neuroglioma cells via synthesis of ceramide and subsequent activation of p38 and p42/44 MAPK pathways . Induction of COX-2 expression via ceramide represents a hitherto unknown mechanism by which cannabinoids mediate biological effects within the central nervous system.

Infect Immun, 2003 Nov, 71(11), 6411 - 9
Revaccination of neonatal calves with Mycobacterium bovis BCG reduces the level of protection against bovine tuberculosis induced by a single vaccination; Buddle BM et al.; Cattle may provide a suitable model for testing ways of improving tuberculosis vaccine efficacy in human infants . A vaccination and challenge study was undertaken in calves to determine the optimal time to vaccinate neonatal animals with Mycobacterium bovis bacillus Calmette-Guerin (BCG) for protection against tuberculosis and to determine whether revaccination with BCG was beneficial . Calves (10 per group) were vaccinated with BCG within 8 h of birth or at 6 weeks of age, when immune responses to antigens of environmental mycobacteria were detectable, or vaccinated at birth and revaccinated at 6 weeks . A control group was not vaccinated . BCG vaccination at birth induced strong antigen-specific gamma interferon (IFN-gamma) and interleukin-2 (IL-2) responses and antigen-specific activation in CD4(+), CD8(+), and WC1(+) gammadelta T-cell subsets from blood . The proportions of animals per group with macroscopic tuberculous lesions after challenge were 0/10 for BCG at birth, 1/9 for BCG at 6 weeks, 4/10 for the revaccinated group, and 10/10 for the nonvaccinated group . There was no significant difference in the levels of protection between groups vaccinated at birth or at 6 weeks, while animals vaccinated both at birth and at 6 weeks had significantly less protection than those vaccinated only at birth . The revaccinated calves that subsequently developed tuberculous lesions had significantly stronger IFN-gamma and IL-2 responses to bovine purified protein derivative after the BCG booster than those in the same group that did not develop lesions . The results indicated that BCG vaccination at birth induced a high level of immunity and that the sensitization of very young animals to antigens of environmental mycobacteria by 6 weeks of age did not affect the effectiveness of BCG . However, BCG revaccination of these young animals was contraindicated.

Biotechnol Lett, 2003 Sep, 25(18), 1555 - 62
Sequence analysis of cyclodextrin glycosyltransferase from the alkaliphilic Bacillus agaradhaerens; Martins RF et al.; The gene encoding an alkaline active cyclodextrin glycosyltransferase (CGTase) from the alkaliphilic B . agaradhaerens LS-3C was cloned and sequenced . It encodes a mature polypeptide of 679 amino acids with a molecular mass of 76,488 Da . The deduced amino acid sequence of the mature CGTase revealed 99 and 95% identity to the CGTase sequences from the other B . agaradhaerens strains, DSM 8721T and 9948, respectively . The next closest identity was of 59% with B . clarkii enzyme . CGTases from B . agaradhaerens, B . clarkii, and B . firmus/lentus formed a phylogenetically separated cluster from the other CGTases of Bacillus spp . origin . A number of usually conserved residues in the CGTases were found to be replaced in the sequence of B . agaradhaerens enzyme . The sequence analysis indicated the enzyme to be close to the so-called 'intermediary enzymes' in the alpha-amylase family.

Biotechnol Lett, 2003 Sep, 25(18), 1537 - 43
Integrated immobilized cell reactor-adsorption system for beta-cyclodextrin production: a model study using PVA-cryogel entrapped Bacillus agaradhaerens cells; Martins RF et al.; Production of cyclodextrins (CDs) by immobilized cells of the alkaliphilic Bacillus agaradhaerens LS-3C with integrated product recovery was studied . The microorganism was entrapped in polyvinyl alcohol-cryogel beads and used as a convenient source of immobilized cyclodextrin glycosyltransferase (CGTase) . On activation by incubation in the cultivation medium containing 1% (w/v) starch, the entrapped cells multiplied and secreted CGTase with an activity of 2-3 mg beta-cyclodextrin h(-1) g(-1) beads . The immobilized biocatalyst exhibited maximum activity at pH 9 and 50 degrees C, and formed cyclodextrins comprising 92-94% beta-CD and remaining alpha-CD . The cyclodextrin product from the immobilized cell bioreactor was continuously recovered by adsorption to Amberlite XAD-4 in a recycle batch mode . The product adsorption was facilitated at low temperature while hot water was used for elution.

Arch Mal Coeur Vaiss, 2003 Sep, 96(9), 923 - 6
{Cardiobacterium hominis endocarditis . A case report}; Balcou-Leroy E et al.; We report the case of a Cardiobacterium hominis endocarditis causing an acute mitral insufficiency complicated of left heart failure . The patient has been treated after a few days by surgical valvuloplasty . Cardiobacterium hominis is a bacteria of the HACCEK group, bacille gram-negative, sometimes anaerobic, difficult to isolate . Recently, Polymerase Chain Reaction analysis appears to be effective for the the diagnosis in the identification of fastidious micro-organisms like Cardiobacterium hominis . We have reviewed in the literature 71 cases of Cardiobacterium hominis endocarditis; clinical presentation is often sub-acute, the bacteriological diagnosis is based on hemocultures for which the culture is slow and require enriched environments . Hemodynamic and thrombo-embolic complications are frequent because of the high pathogenicity of the bacteria which provides big and friable vegetations . Despite a high sensibility to antibiotherapy, surgical intervention is often required.

AIDS, 2003 Nov 7, 17(16), 2351 - 5
Immunogenicity of an inactivated mycobacterial vaccine for the prevention of HIV-associated tuberculosis: a randomized, controlled trial; Vuola JM et al.; OBJECTIVE: Prior to the widespread use of Mycobacterium bovis, Bacille Calmette-Guerin (BCG), inactivated whole cell mycobacterial vaccines had been shown effective in the prevention of tuberculosis . The present study was conducted to determine the safety and immunogenicity of an inactivated whole cell mycobacterial vaccine in persons with HIV infection.DESIGN Randomized, controlled trial . METHODS: A total of 39 HIV-positive patients with prior BCG immunization and CD4 cell counts >/= 200 x 10(6) cells/l were randomized to five doses of inactivated Mycobacterium vaccae (MV) vaccine or control vaccine (CV) . Lymphocyte proliferation (LPA) and interferon gamma (IFN-gamma) responses to mycobacterial antigens were assayed at baseline, after three and five doses of vaccine and > 1 year later . Parallel studies were conducted in 10 HIV-negative subjects with prior BCG immunization . RESULTS: Among HIV-positive patients, 19 MV recipients had higher LPA and IFN-gamma responses to MV sonicate than 20 CV recipients after three and five doses of vaccine and > 1 year later . LPA responses to Mycobacterium tuberculosis whole cell lysate increased over time in both groups consistent with prior BCG immunization and current antiretroviral therapy; after three doses, responses were boosted to higher levels in MV subjects than CV subjects . LPA responses to WCL were also boosted in HIV-negative MV recipients . Immunization was safe and had no adverse effects on HIV viral load or CD4 cell count . CONCLUSIONS: In BCG-primed, HIV-positive and HIV-negative subjects, MV induces durable cellular immune responses to a new mycobacterial antigen and boosts pre-existing responses to WCL . MV is a candidate for clinical trials for the prevention of HIV-associated tuberculosis.

Curr Microbiol, 2003 Sep, 47(3), 226 - 30
Toxicity of Bacillus sphaericus LP1-G against susceptible and resistant Culex quinquefasciatus and the cloning of the mosquitocidal toxin gene; Shi YX et al.; Bacillus sphaericus LP1-G, belonging to flagellar serotype H3, has been found to have moderate toxicity against two resistant Culex quinquefasciatus colonies (RLCq1 and RLCq2) and the susceptible contrast (SLCq) . With an aim of screening mosquitocidal acting factor, a partial genome library was prepared from a partial HindIII digest of the total DNA from Bacillus sphaericus LP1-G . Two thousand twenty Escherichia coli clones were screened for toxicity against susceptible SLCq, and a toxic clone, designated E-UL68, was chosen for further study . The recombinant E-UL68 performed toxicity against both susceptible and two resistant colonies, having the same level of toxicity as that of wide-type strain LP1-G . Sequence analysis revealed that the inserted fragment was composed of 3876 nucleotides and contained a complete gene, whose sequence was identical to that of the mtx gene from B . sphaericus SSII-1 . Because the binary toxin produced during sporulation of strain LP1-G has no activity against the target mosquitoes, this indicates that the Mtx toxin or other active factors might perhaps be responsible for the toxicity of LP1-G against different colonies of mosquito larvae.

Am J Clin Pathol, 2002 Dec, 118 Suppl, S3 - 17
Tuberculosis . Advances in laboratory diagnosis and drug susceptibility testing; Eichbaum Q et al.; Laboratory diagnosis of mycobacterial infection is complicated by the fastidious growth requirements of the bacillus organism . Delays in diagnosis can impede effective treatment and surveillance of the disease . Control of Mycobacterium tuberculosis has also been aggravated by the emergence of multidrug-resistant tuberculosis, and enhanced methods of antibiotic susceptibility testing are needed . Molecular laboratory techniques and advances in rapid culture methods have led to the development of diagnostic and drug susceptibility tests that are rapid and reliable . This article describes recent innovations in the molecular diagnosis and antibiotic susceptibility testing of M tuberculosis.

J Immunol, 2003 Nov 1, 171(9), 4689 - 99
CD4+ T cells mediate IFN-gamma-independent control of Mycobacterium tuberculosis infection both in vitro and in vivo; Cowley SC et al.; Although IFN-gamma is necessary for survival of Mycobacterium tuberculosis infection in people and animal models, it may not be sufficient to clear the infection, and IFN-gamma is not a reliable correlate of protection . To determine whether IFN-gamma-independent mechanisms of immunity exist, we developed a murine ex vivo culture system that directly evaluates the ability of splenic or lung lymphocytes to control the growth of M . tuberculosis within infected macrophages, and that models in vivo immunity to tuberculosis . Surprisingly, CD4(+) T cells controlled >90% of intracellular M . tuberculosis growth in the complete absence of IFN-gamma stimulation of macrophages, via a NO-dependent mechanism . Furthermore, bacillus Calmette-Guerin-vaccinated IFN-gamma-deficient mice exhibited significant protection against M . tuberculosis challenge that was lost upon depletion of CD4(+) T cells . These findings demonstrate that CD4(+) T cells possess IFN-gamma-independent mechanisms that can limit the growth of an intracellular pathogen and are dominant in secondary responses to M . tuberculosis.

J Mol Biol, 2003 Oct 31, 333(4), 721 - 45
Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy; Rosenthal PB et al.; A computational procedure is described for assigning the absolute hand of the structure of a protein or assembly determined by single-particle electron microscopy . The procedure requires a pair of micrographs of the same particle field recorded at two tilt angles of a single tilt-axis specimen holder together with the three-dimensional map whose hand is being determined . For orientations determined from particles on one micrograph using the map, the agreement (average phase residual) between particle images on the second micrograph and map projections is determined for all possible choices of tilt angle and axis . Whether the agreement is better at the known tilt angle and axis of the microscope or its inverse indicates whether the map is of correct or incorrect hand . An increased discrimination of correct from incorrect hand (free hand difference), as well as accurate identification of the known values for the tilt angle and axis, can be used as targets for rapidly optimizing the search or refinement procedures used to determine particle orientations . Optimized refinement reduces the tendency for the model to match noise in a single image, thus improving the accuracy of the orientation determination and therefore the quality of the resulting map . The hand determination and refinement optimization procedure is applied to image pairs of the dihydrolipoyl acetyltransferase (E2) catalytic core of the pyruvate dehydrogenase complex from Bacillus stearothermophilus taken by low-dose electron cryomicroscopy . Structure factor amplitudes of a three-dimensional map of the E2 catalytic core obtained by averaging untilted images of 3667 icosahedral particles are compared to a scattering reference using a Guinier plot . A noise-dependent structure factor weight is derived and used in conjunction with a temperature factor (B=-1000A(2)) to restore high-resolution contrast without amplifying noise and to visualize molecular features to 8.7A resolution, according to a new objective criterion for resolution assessment proposed here.

J Mol Biol, 2003 Oct 31, 333(4), 711 - 20
Solution structural study of BlaI: implications for the repression of genes involved in beta-lactam antibiotic resistance; Melckebeke HV et al.; beta-Lactamase and penicillin-binding protein PBP2' mediate staphylococcal resistance to beta-lactam antibiotics, which are otherwise highly clinically effective . Two repressors (BlaI and MecI) regulate expression of these inducible proteins . Here, we present the first solution structure of the 82 amino acid residue DNA-binding domain of Bacillus licheniformis BlaI which is very similar in primary sequence to the medically significant Staphyloccocal BlaI and MecI proteins . This structure is composed of a compact core of three alpha-helices and a three-stranded beta-sheet typical of the winged helix protein (WHP) family . The protein/DNA complex was studied by NMR chemical shift comparison between the free and complexed forms of BlaI . Residues involved in DNA interaction were identified and a WHP canonical model of interaction with the operators is proposed . In this model, specific contacts occur between the base-pairs of the TACA motif and conserved amino acid residues of the repressor helix H3 . These results help toward understanding the repression and induction mechanism of the genes coding for beta-lactamase and PBP2'.

Med Parazitol (Mosk), 2003 Jul-Sep, (3), 18 - 20
{Indication of Borrelia burgdorferi sensu lato in the taiga tick larvae obtained from spontaneously infected females}; Nefedova VV; Hungry larvae from 17 clutches by spontaneously infected Ixodes persulcatus females were examined by inoculation of the BSK II medium, by microscopy of fixed smears, and by polymerase chain reaction (PCR) with the primers specific toward the conserved sequences of Borrelia burgdorferi 16S ribosomal RNA gene . A study of 781 larvae individually or as pools could not reveal Borrelia DNA by PCR . Inoculations of the BSK II medium with 600 larvae yielded no positive results either; immobile spiral forms of Bacillus spp . were detected in 16 (26.7 +/- 5.7%) cultures of the larvae obtained from 7 females . Microscopy of 1416 fixed smears showed typical Borrelia in 7 (0.5 +/- 0.4%) specimens of larvae resulting from the same hatch; spirochaete-like cells were present in 13 (0.9 +/- 0.5%) cases . The single female from those of the 17 hatches transmitted Borrelia through eggs to filial-generation larvae . The infection rate of the larvae emerged from the eggs of this hatch was 7% and their individual infection rates were 0.4-0.8 Borrelia per 100 fields of vision . The findings suggest that there may a transovarian transmission of Borrelia in I . persulcatus ticks, but its likelihood is very little.

J Ind Microbiol Biotechnol, 2003 Oct, 30(10), 606 - 12 Epub 2003 Oct 16.
Solubilization of cobalt from ocean nodules at neutral pH-a novel bioprocess; Mukherjee A et al.; A marine organism ( Bacillus M1) isolated from Indian Ocean manganese nodules was characterized . The organism grew well in artificial seawater medium, at near neutral pH, 30 degrees C and 0.25 M NaCl, and showed MnO(2)-reducing activity . Growing cultures of Bacillus M1 as well as cell-free spent liquor from fully-grown cultures were employed to extract metals from the nodules . The spent liquor of cultures of the organism could dissolve around 45% cobalt (Co) at a pH of 8.2 in 2 h . Co recovery by this treatment was comparable to that in acidic leaching with 2.5 M hydrochloric acid solutions, and was independent of pulp density (w/v ratio) . The amount of Co dissolved was beyond the thermodynamic solubility limit in aqueous solution at a pH of 8.2 . It is inferred that the metabolites present in the spent liquor played a pivotal role in complexing the Fe (III) phase, solubilizing Co in the process . Partial characterization of spent liquor by spot tests, UV visible spectroscopy and FTIR spectroscopy, showed the presence of siderophore-like phenolic compound(s) with an attached carboxyl group that might form soluble organic complexes with Fe (III).

Mol Immunol, 2003 Dec, 40(8), 555 - 62
Major linear IgE epitopes of mountain cedar pollen allergen Jun a 1 map to the pectate lyase catalytic site; Midoro-Horiuti T et al.; Resolution of the 3D structures and IgE epitopes of allergens may identify common or conserved features of allergens . Jun a 1, the predominant allergen in mountain cedar pollen, was chosen as a model for identifying common structural and functional features among a group of plant allergens . In this study, synthetic, overlapping peptides of Jun a 1 and sera from patients allergic to mountain cedar pollen were used to identify linear epitopes . A 3D model of Jun a 1 was produced using the Bacillus subtiles pectate lyase (PL) as a template and validated with biophysical measurements . This allowed mappings of four IgE binding sites on Jun a 1 . Two of the epitopes mapped to turns or loops on the surface of the model structure . The other two epitopes mapped to the beta-sheet region, homologous to the catalytic site of PL . This region of Jun a 1 is highly conserved in the group 1 allergens from other cedar trees as well as microbial PLs . The finding that two out of three major IgE epitopes map to highly conserved catalytic regions of group 1 cedar allergens may help to explain the high degree of cross-reactivity between cedar pollen allergens and might represent a pattern of reactivity common to other allergens with catalytic activity.

J Antibiot (Tokyo), 2003 Aug, 56(8), 716 - 20
SNA-60-367 components, new peptide enzyme inhibitors of aromatase: structure of the fatty acid side chain and amino acid sequence by mass spectrometry; Esumi Y et al.; SNA-60-367 components, new peptide enzyme inhibitors of aromatase, were isolated from the culture broth of soil bacterium, Bacillus sp . SNA-60-367 . These inhibitors are a family of acylated decapeptides that differ from each other in terms of amino acid composition and the nature of the fatty acid side chain . The structures of the fatty acid moieties were shown to be (3-hydroxy)heptadecanoic acid and (3-hydroxy)hexadecanoic acid that possess normal-, iso- or anteiso-type alkyl groups . The amino acid sequence of the open form of the lactone ring of the acylpeptides is RCO-L-Glu-D-Orn-L(or D)-Tyr3-D-allo-Thr-L-Glu-D-X1 (Ala, Aba or Val)-L-Pro-L-Gln-D(or L)-Tyr-L-X2(10)(Ile or Val)-OH . The lactone ring of SNA-60-367 components is formed between Tyr3 and X2(10).

Hum Pathol, 2003 Sep, 34(9), 929 - 38
Pseudomonas pneumonia in infants: an autopsy study; Bonifacio SL et al.; Pseudomonas pneumonia is an uncommon but serious infection in infants, occurring mainly in infants of low birth weight . In this retrospective clinicopathologic correlation study, we reviewed the clinical records and analyzed postmortem lung pathology in 8 infants with pneumonia due to P . aeruginosa . From the histopathology, 2 different pneumonic patterns emerged: a distinctive paucicellular coagulative confluent bronchopneumonia with perivascular bacillary infiltration (7 cases) and a more usual cellular pneumonia without evidence of perivascular organisms (1 case) . Clinically, infants with the first type could be considered immunocompromised and had a precipitous course characterized by signs of sepsis, whereas the infant with the second type (who likely had a more normal immune system) had a relatively protracted course with respiratory failure . We conclude that (1) the pattern of pneumonic inflammation correlates with the immune state of infants, similar to what has been reported in adults; (2) among immunocompromised infants, histopathologic signs of bacteremia are prevalent; and (3) the clinical signs do not correlate with the severity of the pathology at autopsy and may reflect sepsis rather than pneumonia . We speculate that the histopathology in this population reflects the virulence of the organism, as well as the immune status of the host.

Protein Pept Lett, 2003 Oct, 10(5), 521 - 4
Purification, crystallization and preliminary X-ray studies of a p-nitrophenylphosphatase from Bacillus stearothermophilus; Ji CN et al.; Thermostable p-nitrophenylphosphatase from Bacillus stearothermophilus has been expressed in Escherichia coli, purified and crystallized . The crystals belong to space group C(2), with unit-cell parameters a = 67.17 A, b = 57.84 A, c = 62.49 A and alpha = 90.0 degrees, beta = 95.4 degrees, gamma = 90.0 degrees . Diffraction data were collected to 1.40 A resolution with a completeness of 94.7% (96.6% for the last shell), an R(fac) value of 0.074 (0.341) and an I/sigma (I) value of 30.1 (2.67).

J Theor Biol, 2003 Nov 7, 225(1), 91 - 7
Growth dynamics of Bacillus circulans colony; Komoto A et al.; We have investigated the growth dynamics of Bacillus circulans colony exhibiting the knotted-branching pattern by swarming on a hard agar medium . The knotted-branching pattern consists of many circular clusters, so-called subcolonies, and their trajectories . We analysed the processes of a subcolony because they are presumably the key elements for the formation of knotted-branching pattern . It was found that a subcolony has three processes, i.e . "generation", "growth", and "migration" by microscopic and time-resolved observations . An embryonic small subcolony (child subcolony) formed around an existing subcolony (parent subcolony) grows larger and migrates away from the parent subcolony . We proposed a simple model to explain the migration and the growth processes . It is assumed that the internal part of the subcolony is unfavorable for the bacteria and that the motion of the child subcolony on the agar medium can be modeled using a frictional force . The experimental data were quantitatively analysed in order to compare with models . Our models are consistent with the experimental results on following three points: (1) the radius of a subcolony increases linearly with the incubation time, (2) a subcolony stops just after formation and then starts to migrate suddenly, and (3) the trajectory of a subcolony predicted by the model agrees with the experimental one.

Clin Infect Dis, 2003 Nov 1, 37(9), 1226 - 33 Epub 2003 Oct 06.
Danish bacille Calmette-Guérin vaccine-induced disease in human immunodeficiency virus-infected children; Hesseling AC et al.; An analysis of isolates of Mycobacterium tuberculosis complex was performed to determine the prevalence of bacille Calmette-Guerin (BCG) disease among human immunodeficiency virus (HIV)-infected children . Speciation was done with polymerase chain reaction; 183 isolates from mycobacterial cultures for 49 HIV-infected patients were analyzed . The Danish Mycobacterium bovis BCG strain was isolated from 5 patients . No cases of Tokyo M . bovis BCG strain disease were detected . All patients were asymptomatic at birth, <12 months of age, and severely immunodeficient at presentation . Four patients had regional axillary adenitis ipsilateral to the vaccination site, and 2 had pulmonary BCG disease . Two patients with regional BCG disease had simultaneous pulmonary M . tuberculosis infection . Although chest radiographic features were similar to those seen in patients with tuberculosis, BCG disease should be considered in HIV-infected infants with right axillary adenitis ipsilateral to the vaccination site . Young, symptomatic, HIV-infected infants are at risk for BCG-related complications . Controlled, population-based studies are needed to assess the risk of BCG in HIV-infected children.

Proc Natl Acad Sci U S A, 2003 Oct 14, 100(21), 12420 - 5 Epub 2003 Oct 13.
The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue; Hsu T et al.; Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine . Bacillus Calmette-Guerin (BCG), the tuberculosis vaccine, is an attenuated mutant of Mycobacterium bovis that was isolated after serial subcultures, yet the functional basis for this attenuation has never been elucidated . A single region (RD1), which is absent in all BCG substrains, was deleted from virulent M . bovis and Mycobacterium tuberculosis strains, and the resulting DeltaRD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice . The M . tuberculosis DeltaRD1 mutants were also shown to protect mice against aerosol challenge, in a similar manner to BCG . Interestingly, the DeltaRD1 mutants failed to cause cytolysis of pneumocytes, a phenotype that had been previously used to distinguish virulent M . tuberculosis from BCG . A specific transposon mutation, which disrupts the Rv3874 Rv3875 (cfp-10 esat-6) operon of RD1, also caused loss of the cytolytic phenotype in both pneumocytes and macrophages . This mutation resulted in the attenuation of virulence in mice, as the result of reduced tissue invasiveness . Moreover, specific deletion of each transcriptional unit of RD1 revealed that three independent transcriptional units are required for virulence, two of which are involved in the secretion of ESAT-6 (6-kDa early secretory antigenic target) . We conclude that the primary attenuating mechanism of bacillus Calmette-Guerin is the loss of cytolytic activity mediated by secreted ESAT-6, which results in reduced tissue invasiveness.

Trends Microbiol, 2003 Oct, 11(10), 452 - 4
Bacterial spore germination and protein mobility; Moir A; Fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) has been used to report on protein mobility in single spores . Proteins found in dormant Bacillus spores are not mobile; however, mobility is restored when germination occurs and the core rehydrates . Spores of a cwlD mutant, in which the cortex is resistant to hydrolysis, are able to complete the earliest stages of germination in response to a specific germinant stimulus; in these circumstances, the protein in the spore remains immobile . Therefore, the earliest stages of spore germination, including loss of resistance to extreme heat and the complete release of the spore component dipicolinic acid, are achieved without the restoration of protein mobility.

Cancer Sci, 2003 Oct, 94(10), 914 - 8
Helicobacter pylori risk associated with childhood home environment; Ueda M et al.; Helicobacter pylori (H . pylori) is considered to be a cause of gastric ulcer, gastric cancer and other diseases . The relationship between infection and the hygiene or housing circumstances of such patients in their childhood was explored . The study subjects were those who applied for a H . pylori antibody test, and were asked to fill out a questionnaire enclosed with a test kit, inquiring as to their hygiene and housing conditions when they were 10 years old . Of 5971 applicants, 5854 agreed to participate in the study . Associations between the six factors in the questionnaire and infection were calculated, and adjusted for sex, age and district . Drinking water, type of toilet, residential area, number of people in the house, and birth order showed significant correlations with H . pylori infection . The odds ratios (95% confidence intervals) were 0.73 (0.55-0.96) for tap water, 0.72 (0.63-0.84) for flush toilets, 0.74 (0.66-0.83) for urban location, 1.34 (1.09-1.64) for 7 or more people in the household, 1.19 (1.00-1.41) for 4th or 5th in birth order, and 1.47 (1.17-1.85) for 6th or more in birth order . No significant association with breast feeding was observed . These results suggest that infection with H . pylori may be associated with water-related sanitary factors in childhood, and that the bacillus may be transmitted within a family.

Cell Death Differ, 1997 Oct, 4(7), 560 - 9
A Bacillus thuringiensis delta-endotoxin induces programmed cell death in mosquito larvae; Smouse D et al.; We present evidence that a delta-endotoxin isolated from Bacillus thuringiensis subsp.israelensis induces programmed cell death in polytene midgut cells of Culex pipiens larvae . After exposure to toxin, polytene nuclei in the anterior region of the larval midgut undergo many of the morphological and physiological changes which are characteristic of apoptosis, including the ability to stain with the vital dye, acridine orange, and fragmentation of nuclear DNA as demonstrated by agarose gel electrophoresis and in situ TUNEL labeling . The temporal sequence of toxin ingestion, acridine orange staining and larval death suggests a cause and effect relationship between programmed cell death and larval death . Amino sugars that interfere with toxicity also interfere with the time course of acridine orange staining of larval polytene nuclei . The toxin first causes programmed cell death of anterior midgut and gastric caeca cells and, subsequently, posterior midgut cells . This pattern is similar to the temporal sequence of larval polytene cell death that occurs during metamorphosis . From the size and distribution of the nuclei that are stained with acridine orange, it appears that only polytene midgut cells are affected by toxin and that the diploid regenerative cell are not affected.

Chest, 2003 Oct, 124(4), 1476 - 81
Comparative roles of levofloxacin and ofloxacin in the treatment of multidrug-resistant tuberculosis: preliminary results of a retrospective study from Hong Kong; Yew WW et al.; OBJECTIVE: To compare levofloxacin and ofloxacin in the treatment of multidrug-resistant tuberculosis (MDR-TB) . PATIENTS AND METHODS: A retrospective analysis of 106 patients with MDR-TB (February 1990 through December 2000) receiving directly observed therapy with fluoroquinolone and accompanying drugs, which mainly included aminoglycosides, cycloserine, ethionamide/prothionamide, and pyrazinamide, was performed . Clinical data from 99 suitable patients were subjected to univariate analysis, stratification, and multiple logistic regression to compare the roles of levofloxacin and ofloxacin in multidrug regimens . RESULTS: Forty patients received 612.5 +/- 79.0 mg qd levofloxacin (mean +/- SD), and 59 patients received 628.8 +/- 101.8 mg qd ofloxacin together with similar active second-line drugs for similar durations . The times to sputum smear (both 1.8 months) and culture conversion (both 2.1 months) were equivalent . Adverse reactions occurred at similar rates (10.0% vs 11.9%) . The combined treatment success rate was 83.8%, being higher among ofloxacin-susceptible than ofloxacin-resistant cases (90.5% vs 64.0%, p < 0.01) . The success rates for the levofloxacin group were 90.0% (overall), 96.2% (ofloxacin-susceptible cases), and 78.6% (ofloxacin-resistant cases) in comparison with 79.7%, 87.5%, and 45.5%, respectively, for the ofloxacin group (Mantel-Haenszel common odds ratio estimate, 4.0; p < 0.05) . Bacillary susceptibility to ofloxacin, good adherence, radiographic extent of one lung or less, and use of levofloxacin were independent predictors of favorable outcome (odds ratios, 7.6 to 21.3) . One patient each from both groups relapsed . CONCLUSION: Levofloxacin was found to be more efficacious than ofloxacin when incorporated into multidrug regimens used for treatment of MDR-TB.

Carbohydr Res, 2003 Oct 10, 338(21), 2213 - 20
A synergistic reaction mechanism of a cycloalternan-forming enzyme and a D-glucosyltransferase for the production of cycloalternan in Bacillus sp . NRRL B-21195; Kim YK et al.; Cycloalternan-forming enzyme (CAFE) was first described as the enzyme that produced cycloalternan from alternan . In this study, we found that a partially purified preparation of CAFE containing two proteins catalyzed the synthesis of cycloalternan from maltooligosaccharides, whereas the purified CAFE alone was unable to do so . In addition to the 117 kDa CAFE itself, the mixture also contained a 140 kDa protein . The latter was found to be a disproportionating enzyme (DE) that catalyzes transfer of a D-glucopyranosyl residue from the non-reducing end of one maltooligosaccharide to the non-reducing end of another, forming an isomaltosyl residue at the non-reducing end . CAFE then transfers the isomaltosyl residue to the non-reducing end of another isomaltosyl maltooligosaccharide, to form an alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-(1-->4)-maltooligosaccharide, and subsequently catalyzes a cyclization to produce cycloalternan . Thus, DE and CAFE act synergistically to produce cycloalternan directly from maltodextrin or starch.

J Anim Sci, 2003 Oct, 81(10), 2546 - 51
Detection of corn intrinsic and recombinant DNA fragments and Cry1Ab protein in the gastrointestinal contents of pigs fed genetically modified corn Bt11; Chowdhury EH et al.; Genetically modified corn has been approved as an animal feed in several countries, but information about the fate of genetically modified DNA and protein in vivo is insufficient . Genetically modified corn Bt11 is developed by inserting a recombinant DNA sequence encoding insecticidal Cry1Ab protein from Bacillus thuringiensis subsp . kurstaki . We examined the presence of corn intrinsic and recombinant cry1Ab gene by PCR, and the Cry1Ab protein by immunological tests in the gastrointestinal contents of five genetically modified corn Bt11-fed and five nongenetically modified corn-fed pigs . Fragments of corn zein (242 bp), invertase (226 bp) and of ribulose-1,5-bisphosphate carboxylase/ oxygenase genes (1,028 bp) were detected in the gastrointestinal contents of both Bt11 and nongenetically modified corn-fed pigs . Fragments of recombinant cry1Ab gene (110 bp and 437 bp) were detected in the gastrointestinal contents of the Bt11-fed pigs but not in the control pigs . Neither corn intrinsic nor cry1Ab gene fragments were detected in the peripheral blood by PCR . The gastrointestinal contents were positive for Cry1Ab protein by ELISA, immunochromatography, and immunoblot; however, these methods did not work for blood and precluded conclusions about any potential absorption of the protein . These results suggest that ingested corn DNA and Cry1Ab protein were not totally degraded in the gastrointestinal tract, as shown by their presence in a form detectable by PCR or immunological tests.

Arch Virol, 2003 Oct, 148(10), 1957 - 68
Sequence diversity of South Pacific isolates of Taro bacilliform virus and the development of a PCR-based diagnostic test; Yang IC et al.; We have analysed the sequence variability in the putative reverse transcriptase (RT)/ribonuclease H (RNaseH) and the C-terminal coat protein (CP)-coding regions from Taro bacilliform virus (TaBV) isolates collected throughout the Pacific Islands . When the RT/RNaseH-coding region of 22 TaBV isolates from Fiji, French Polynesia, New Caledonia, Papua New Guinea (PNG), Samoa, Solomon Islands and Vanuatu was examined, maximum variability at the nucleotide and amino acid level was 22.9% and 13.6%, respectively . Within the CP-coding region of 13 TaBV isolates from Fiji, New Caledonia, PNG, Samoa and the Solomon Islands, maximum variability at the nucleotide and amino acid level was 30.7% and 19.5%, respectively . Phylogenetic analysis showed that TaBV isolates from the Solomon Islands showed greatest variability while those from New Caledonia and PNG showed least variability . Based on the sequences of the TaBV RT/RNaseH-coding region, we have developed a PCR-based diagnostic test that specifically detects all known TaBV isolates . Preliminary indexing has revealed that TaBV is widespread throughout Pacific Island countries . A sequence showing approximately 50% nucleotide identity to TaBV in the RT/RNaseH-coding region was also detected in all taro samples tested . The possibility that this may represent either an integrated sequence or the genome of an additional badnavirus infecting taro is discussed.

Biotechnol Adv, 1988, 6(4), 697 - 724
Bacillus thuringiensis insecticidal crystal toxins: gene structure and mode of action; Brousseau R et al.; Thanks to the techniques of recombinant DNA, there is now abundant sequence information on several endotoxin genes of Bacillus thuringiensis . The task of correlating this sequence information with the economically important aspects of the toxins such as insect specificity, LD(50) and speed of kill is now under worldwide investigation . Progress has also been made on understanding the mechanism of action of the toxins and on identifying the parts of the protoxin which are important in toxicity . Taken together, the mechanistic data and the sequence information allow the first attempts at rational design of mutant endotoxin genes and greatly facilitate the transfer of those genes to other organisms such as plants . More information is still needed, however, as to the nature of the binding site of the toxin and on the three-dimensional structure of the activated toxins.

Biotechnol Adv, 2000 Apr, 18(2), 121 - 40
Genetic immobilization of proteins on the yeast cell surface; Ueda M et al.; A genetic system has been exploited to immobilize proteins in their active and functional forms on the cell surface of yeast, Saccharomyces cerevisiae . DNAs encoding proteins with a secretion signal peptide were fused with the genes encoding yeast agglutinins, a- and alpha-type proteins involved in mating . The fusion gene was introduced into S . cerevisiae and expressed under the control of several promoters . Appearance of the fused proteins expressed on the cell surface was demonstrated biochemically and by immunofluorescence and immunoelectron microscopy techniques . Alpha-galactosidase from Cyamopsis tetragonoloba seeds, peptide libraries including scFv and variable regions of the T cell receptor from mammalian cells have been successfully immobilized on the yeast cell wall in the active form . Recently, surface-engineered yeasts have been constructed by immobilizing the enzymes and a functional protein, for example, green fluorescent protein (GFP) from Aequorea victoria . The yeasts were termed 'arming yeasts' with biocatalysts or functional proteins . Such arming cells displaying glucoamylase from Rhizopus oryzae and alpha-amylase from Bacillus stearothermophilus, or carboxymethylcellulase and beta-glucosidase from Aspergillus acleatus, could assimilate starch or cellooligosaccharides as the sole carbon source, although S . cerevisiae cannot intrinsically assimilate these substrates . GFP-arming cells can emit green fluorescence from the cell surface in response to the environmental conditions . The approach described in this review will enable us to endow living cells, including yeast cells, with novel additional abilities and to open new dimensions in the field of biotechnology.

J Biochem Mol Biol, 2003 Sep 30, 36(5), 508 - 13
Identification of two isoforms of aminopeptidase N in Aedes aegypti larval midgut; Pootanakit K et al.; The bacterium Bacillus thuringiensis produces toxin inclusions that are deleterious to target insect larvae . These toxins are believed to interact with a specific receptor protein(s) that is present on the gut epithelial cells of the larvae . In various insect species (in particular those belonging to the lepidopteran class), aminopeptidase N (APN) is one of the two receptor proteins that are considered to be involved in toxin-receptor interactions . However, in mosquitoes, the nature and identity of the receptor protein is unknown . Here, using RT-PCR, we identified two isoforms of the APN transcripts in the Aedes aegypti mosquito larval midgut . These results are congruent with a previous report of multiple isoforms of the APN gene expression in lepidopteran larvae . Which of the two isoforms (or other yet unidentified receptor proteins) is involved in the killing of mosquito larvae remains to be elucidated.

J Urol, 2003 Nov, 170(5), 2009 - 13
Androgen dependent regulation of bacillus Calmette-Guerin induced interleukin-6 expression in human transitional carcinoma cell lines; Chen F et al.; PURPOSE: Autocrine expression of interleukin (IL)-6 by transitional cell carcinoma (TCC) in response to bacillus Calmette-Guerin (BCG) may have an important role in promoting BCG adherence to TCC and consequently in BCG treatment efficacy . IL-6 expression in response to BCG requires nuclear factor (NF)-kappaB mediated signal transduction . We evaluated the influence of androgens on BCG induced, NF-kappaB mediated IL-6 expression . MATERIALS AND METHODS: Reverse transcriptase-polymerase chain reaction was used to confirm androgen receptor expression in the human TCC lines 253J and T24 . A reporter construct containing an androgen response element was used to establish the integrity of androgen mediated signal transduction . Subsequently the dose dependent effect of dihydrotestosterone (DHT) on BCG induced IL-6 expression and NF-kappaB signaling was evaluated . Two pharmacological androgen receptor blockers were used to determine if receptor blockade inhibited the effect of DHT on activation of the androgen response element, NF-kappaB signaling and BCG induced IL-6 expression . RESULTS: The 2 human TCC lines expressed androgen receptor and demonstrated intact androgen stimulated signaling pathways . DHT suppressed BCG induced, NF-kappaB mediated signaling and IL-6 expression in a dose dependent manner . DHT decreased mRNA levels of IL-6, expression of the full-length IL-6 promoter construct and expression of an NF-kappaB specific reporter construct in response to BCG relative to controls . Competitive pharmacological blockade of androgen receptor inhibited the effect of DHT on BCG induced signaling in dose dependent fashion . CONCLUSIONS: DHT down-regulates NF-kappaB mediated IL-6 expression by human TCC lines in response to BCG . This effect depends on a functional androgen receptor signaling pathway and it can be blocked by the inhibition of androgen/androgen receptor binding.

J Clin Microbiol, 2003 Oct, 41(10), 4783 - 6
Rapid and specific detection of Mycobacterium tuberculosis by using the Smart Cycler instrument and a specific fluorogenic probe; Cleary TJ et al.; A procedure using the Smart Cycler instrument and a fluorescence quencher (FQ) probe for the specific identification of Mycobacterium tuberculosis complex (MTB) was used to detect organisms in 366 acid-fast bacillus smear-positive respiratory specimens . It was compared to culture and the AMPLICOR M . tuberculosis PCR test . MTB was isolated from 198 of these samples . The FQ PCR assay was sensitive (197 of 198, 99.5%) and specific (165 of 168, 98.2%); no significant difference was observed between the two PCR protocols . After DNA extraction, a final result was available within 1.5 h with the real-time PCR protocol.

Appl Environ Microbiol, 2003 Oct, 69(10), 6208 - 15
Use of bromodeoxyuridine immunocapture to identify active bacteria associated with arbuscular mycorrhizal hyphae; Artursson V et al.; Arbuscular mycorrhizae are beneficial for crops grown under low-till management systems . Increasingly, it is becoming apparent that bacteria associated with mycorrhizae can enhance the beneficial relationship between mycorrhizae and plants . However, it has been difficult to study these relationships by conventional techniques . In this study actively growing bacteria were identified in soil from an undisturbed fallow field known to contain arbuscular mycorrhizae by using molecular tools to eliminate the need for cultivation . A thymidine analog, bromodeoxyuridine (BrdU), was added to the soil and incubated for 2 days . DNA was extracted, and the newly synthesized DNA was isolated by immunocapture of the BrdU-containing DNA . The active bacteria in the community were identified by 16S rRNA gene PCR amplification and DNA sequence analysis . Based on 16S rRNA gene sequence information, a selective medium was chosen to isolate the corresponding active bacteria . Bacillus cereus strain VA1, one of the bacteria identified by the BrdU method, was isolated from the soil and tagged with green fluorescent protein . By using confocal microscopy, this bacterium was shown to clearly attach to arbuscular mycorrhizal hyphae . This study was the first to use this combination of molecular and traditional approaches to isolate, identify, and visualize a specific bacterium that is active in fallow soil and associates with arbuscular mycorrhizal hyphae.

Appl Environ Microbiol, 2003 Oct, 69(10), 5898 - 906
Dual resistance to Bacillus thuringiensis Cry1Ac and Cry2Aa toxins in Heliothis virescens suggests multiple mechanisms of resistance; Jurat-Fuentes JL et al.; One strategy for delaying evolution of resistance to Bacillus thuringiensis crystal (Cry) endotoxins is the production of multiple Cry toxins in each transgenic plant (gene stacking) . This strategy relies upon the assumption that simultaneous evolution of resistance to toxins that have different modes of action will be difficult for insect pests . In B . thuringiensis-transgenic (Bt) cotton, production of both Cry1Ac and Cry2Ab has been proposed to delay resistance of Heliothis virescens (tobacco budworm) . After previous laboratory selection with Cry1Ac, H . virescens strains CXC and KCBhyb developed high levels of cross-resistance not only to toxins similar to Cry1Ac but also to Cry2Aa . We studied the role of toxin binding alteration in resistance and cross-resistance with the CXC and KCBhyb strains . In toxin binding experiments, Cry1A and Cry2Aa toxins bound to brush border membrane vesicles from CXC, but binding of Cry1Aa was reduced for the KCBhyb strain compared to susceptible insects . Since Cry1Aa and Cry2Aa do not share binding proteins in H . virescens, our results suggest occurrence of at least two mechanisms of resistance in KCBhyb insects, one of them related to reduction of Cry1Aa toxin binding . Cry1Ac bound irreversibly to brush border membrane vesicles (BBMV) from YDK, CXC, and KCBhyb larvae, suggesting that Cry1Ac insertion was unaffected . These results highlight the genetic potential of H . virescens to become resistant to distinct Cry toxins simultaneously and may question the effectiveness of gene stacking in delaying evolution of resistance.

Protein Pept Lett, 2003 Aug, 10(4), 361 - 8
Mutation of the hydrophobic residue on helix alpha5 of the Bacillus thuringiensis Cry4B affects structural stability; Krittanai C et al.; Cry4B toxin is a mosquito-larvicidal protein from the Bacillus thuringiensis subsp . israelensis . We have investigated the role of two conserved hydrophobic residues of Cry4B in structural stabilization . Substitutions of the leucine-175 and isoleucine-189 on helix alpha5 with valine and leucine did not affect the expression level, solubility and proteolytic processing . Steady state analysis of an unfolding experiment as monitored by circular dichroism and fluorescence spectroscopy demonstrated a typical two-state transition . The determined unfolding free energy for the L175V mutant revealed a structural destabilization of 10.49 kcal/mol relative to the wild type . However unfolding kinetic analysis gave identical activation energy for wild type and both mutants . Our findings suggested that a perturbation on the close packing of the hydrophobic side chains in protein interior could lead to a significant destabilization of the native conformation.

Comb Chem High Throughput Screen, 2003 Sep, 6(6), 541 - 56
Diversity among microbial cyclic lipopeptides: iturins and surfactins . Activity-structure relationships to design new bioactive agents; Bonmatin JM et al.; A prominent group of bioactive lipopeptides produced by Bacillus species is constituted by iturins, surfactins and lichenysins . Interest in such substances results in their exceptional surfactant power, and their valuable antifungal, antibacterial, antitumoral and anti-Mycoplasma properties . As is typical for peptidic secondary-metabolites synthesized by the polyenzymic pathway, they are produced as mixtures of components varying in the peptidic and/or in the lipidic structure . In the context of structure-activity relationships, it is possible to take advantage of the adaptability of the biosynthesis system by systematically adding selected amino acids in the culture medium of the producing bacterium . When an amino acid is used as the sole nitrogen source, it is inserted directly into selected positions of the peptide sequence, thus amplifying the original structural microheterogeneity via a production of variants . This method revealed very efficient for increasing the amounts of preexisting variants and for building new variants of surfactins and lichenysins but totally inefficient with iturins . In this group, the peptidic diversity strictly depends on the selected strain . So far the screening remained the only method to discover new iturins . Another interesting peculiarity is the common occurrence in a single strain of two lipopeptides with different core structures such as surfactins and iturins . Taken together, these features led to an extensive metabolite pattern . Besides, engineered variants and chemical derivatives enlarged the array of available molecules . Despite the high degree of chemical similarity, the separation of variants and/or homologues was successfully achieved by reversed-phase HPLC leading to well-separated compounds ideally suited to investigation of structure-activity relationships . Improved physical techniques such as 2D-NMR and mass spectrometry allowed to describe efficiently and rapidly the composition of cyclic lipopeptides even in mixtures containing several variants . From NMR, the 3D structure and dynamics gave crucial data for fine structure-activity relationships as well as for understanding of the properties at the membrane and/or at the air/water interface . Here the role of residues was identified in the context of hydrophobic and electrostatic interactions that play a leader role . Such a comprehensive approach, based on both structural and biosynthesis knowledge, opened the way to rational design for enhanced properties and its validity was confirmed with 10 fold higher surfactant efficacy.

Comb Chem High Throughput Screen, 2003 Sep, 6(6), 527 - 40
Combinatorial biosynthesis of non-ribosomal peptides; Keller U et al.; Non-ribosomal peptide synthetases (NRPS) are modular assembly lines catalysing the synthesis of many small peptides in microbes . Genetic replacements of domains or modules in NRPS encoded by gene clusters in Bacillus sp . with corresponding domains or modules from foreign NRPS have led in several cases to the in vivo synthesis of peptides with predicted amino acid substitutions . Fusion points were in variable regions between C- and A- or between T- and C-domains . Successful insertions of whole modules using fusion points in conserved regions internal to functional domains have also been reported . For studying the role of C-, A-, T- and TE (thioesterase)-domains in NRPS, several bi- and trimodular model-NRPS derived from natural NRPS systems were constructed and obtained after expression in E . coli with coexpression of a 4'- phosphopantetheine transferase or in suitable hosts such as the Streptomyces . Such enzymes were shown to catalyse in vitro synthesis of di- and tripeptides, respectively, with and without turnover depending on the presence of Te-domains . The enzymatic analysis revealed the mechanisms of the domains and proved their functional autonomy suggesting the possibility to use any NRPS interdomain region for fusions . Nevertheless, recombinant synthesis of longer and more complex peptides will still be restricted to alteration of existing structures by manipulations of NRPS gene clusters located on chromosomes or artificial chromosomes . Besides targeted replacements of domains and modules, reprogramming of NRPS by altering the substrate specificities of A-domains is a promising tool for the future to get novel peptides.

Annu Rev Microbiol, 2003, 57, 517 - 49
Molecular genetics of Mycobacterium tuberculosis pathogenesis; Clark-Curtiss JE et al.; Tuberculosis (TB) has afflicted humankind throughout history . Approximately one third of the world's population is currently infected with Mycobacterium tuberculosis and nearly two million people die of TB annually . Although much has been learned about the structure of the tubercle bacillus, the epidemiology of TB, the physiological and immunological responses of the host to infection, and the physiology of M . tuberculosis in laboratory broth cultures, much of the basic biology of M . tuberculosis in its natural setting (the infected human) remains to be elucidated . Within the past decade, there have been remarkable advances in the development of genetic and molecular biological tools with which to study M . tuberculosis . This review discusses the approaches that have been employed and the progress that has been made in discovering how M . tuberculosis has achieved its prowess as a successful pathogen.

Biotechnol Prog, 2003 Sep-Oct, 19(5), 1439 - 43
Specific oxygen uptake rate variations during batch fermentation of Bacillus thuringiensis subspecies kurstaki HD-1; Rowe GE et al.; The specific oxygen uptake rate (q(O)2, respiration rate) of Bacillus thuringiensis subsp . kurstaki HD-1 was very high at inoculation and was found to decrease essentially monotonically throughout both vegetative growth phase and transition phase under different batch culture conditions . Average q(O)2 values decreased from 8-10 mmol/g h at 1 h after inoculation to less than 2 mmol/g h by the time growth ended . The results are shown to be consistent with the few previous reports on q(O)2 in B . thuringiensis in the literature but also novel in that this pattern of monotonic decline has not been described previously . Both pH control and EDTA in low concentration shortened the vegetative growth phase and reduced the 10 h biomass concentration . Using plots of q(O)2 versus specific growth rate, mu, biomass yield based on the oxygen used for growth, was calculated for transition phase to be 0.041-0.047 g/mmol, consistent with literature values . The same plot also showed that the presence of EDTA resulted in an atypical q(O)2-mu trajectory and apparently much higher biomass yield from the oxygen consumed.

J Am Mosq Control Assoc, 2003 Sep, 19(3), 251 - 8
Efficacy of Bacillus thuringiensis israelensis, Bacillus sphaericus and temephos for managing Anopheles larvae in Eritrea; Shililu JI et al.; We evaluated the larvicidal activity of the granular formulation of Bacillus thuringiensis israelensis (Bti) serotype H-14 (Vectobac G, 200 ITU/mg) and Bacillus sphaericus (Bsph) serotype H5a5b (Vectolex CG, 670 Bs ITU/mg) against Anopheles arabiensis and other mosquitoes in breeding habitats in 3 sites, Gash-Barka, Anseba, and Debub zones, in Eritrea . The primary objective was to determine the optimal application rate and duration of effect for Bti and Bsph in representative larval habitats as compared with the organophosphate temephos . The biolarvicides were tested at 100% (high) and 50% (low) of the maximum recommended application rate . Temephos was applied at a rate of 100 ml/ha . At least 4 replicate experiments with Vectobac G (5.6 and 11.2 kg/ha), Vectolex CG (11.2 and 22.4 kg/ha) were conducted in each study site . All 3 larvicides caused significant mortality of the main malaria vector species, An . arabiensis, and other mosquito species (Anopheles cinereus, Anopheles pretoriensis, Culex quinquefasciatus) . The larvicidal activity for Bti and Bsph was variable depending upon breeding habitat, mosquito species, and general ecology of the area . Both biopesticides had a similar duration of activity (2-3 wk) and were generally as effective as temephos for these time periods . In some cases, the high and low application rates for Bti and Bsph produced equivalent control over 2-3 wk . The 2 Bacillus biopesticides were less effective in habitats with high algal content and in fast flowing streams primarily because of the inability to penetrate algal mats and dilution effect, respectively . The results show that application of the 2 biolarvicides bimonthly to streambed pools, rain pools, and similar habitats would maintain control of the anopheline mosquito population.

Bull Hist Med, 2003 Fall, 77(3), 636 - 61
"Unfit for human consumption": tuberculosis and the problem of infected meat in late Victorian Britain; Waddington K; By the 1890s, questions about tuberculous meat in Britain served to transform the issue of infected meat from an ill-defined to a concrete threat . Veterinarians, building on European inoculation (or transmission) experiments, played a prominent part in constructing the debate, with medical officers of health following . With the emergence of bacteriology in the 1880s, a consensus emerged about the dangers of tuberculous meat: Robert Koch's identification of the tubercle bacillus in 1882, and the connection he saw between bovine tuberculosis and the disease in man, provided confirmation of the disease's danger to man . It was from this point that veterinary and public health interests diverged . Whereas a general agreement had been reached, the extent of the problem remained open to doubt . Confusion revolved around two issues: the localization of infection, and the question of cooking . The latter was thought to make tuberculous meat "safe," as attention shifted to the problem of milk; whereas the former frustrated efforts to combat the sale of meat showing signs of infection.

Pediatrics . 2003 Oct;112(4):e298.
A prospective study of bacillus Calmette-Guérin scar formation and tuberculin skin test reactivity in infants in Lima, Peru; Santiago EM et al.; OBJECTIVES: To determine the sensitivity of the bacillus Calmette-Guerin (BCG) scar as an indicator of previous vaccination and to ascertain the tuberculin skin test (TST) response in infancy after vaccination in a community from an area hyperendemic for tuberculosis (TB) . METHODS: In a birth cohort of healthy term infants from Lima, Peru, a single dose of BCG vaccine was administered within the first month of life . Scar formation was assessed biweekly during the first 6 months and again at 3 years after vaccination . TST response was evaluated 6 months after vaccination . RESULTS: Six months after vaccination, 99% (68) of the newborns exhibited a BCG scar (>2 mm) . Scar size did not differ by sex, birth weight, age at vaccination, or nutritional status in the first 2 months . Eighty percent of the participants were found 3 years after vaccination, and all of them had a BCG scar . Mean TST reaction size 6 months after vaccination was 2.9 +/- 0.3 mm . No association was found between sex or age at BCG vaccination and TST size . Only 3 children had a TST >10 mm, and the 3 had a TB contact at home . CONCLUSIONS: The BCG scar was a sensitive indicator of vaccination status up to 3 years after the administration of the vaccine in the first month of life . Although nearly a quarter of the children had a TST response >5 mm 6 months after vaccination, TST reactions >10 mm did not occur in the absence of exposure to a person with tuberculosis . A cutoff of 10 mm should be used for disease control purposes in people who are born in countries where TB is endemic.

Zhonghua Jie He He Hu Xi Za Zhi, 2003 Sep, 26(9), 526 - 30
{Relationships between the diameters of Bacille Calmette-Guerin scars and asthma, atopy in urban and rural Beijing children}; Ma Y et al.; OBJECTIVE: To compare the diameters of Bacille Calmette-Guerin (BCG) scars between asthmatic (allergic) students and normal students, and to compare the diameters of BCG scars in children in rural Beijing area with those in urban Beijing area, and to compare the prevalence of asthmatic (allergic) symptoms and atopy between rural and urban Beijing students . METHODS: Sampled by cluster-random Method, 819 urban school children and 1 044 rural school children aged 13 - 14 years were recruited for the Beijing arm of the phase III international study of asthma and allergies in childhood . The children answered the core questionnaires, and underwent skin prick test for 13 allergens, and were checked for BCG scars on their arms . RESULTS: (1) In both rural and urban Beijing students, the mean diameters of BCG scars in those with asthma (allergy) (asthma ever, wheeze ever, current wheeze within 12 months, hay fever ever, repeated rash ever, eczema ever, allergic rhinitis, atopy, high-sensitivity or were diagnosed asthma) were (7.0 +/- 2.1) mm-(8.8 +/- 2.8) mm, which were not significantly different from those of (7.2 +/- 2.7) mm-(8.2 +/- 2.2) mm in normal students (P > 0.05) . (2) Compared to the urban students, the mean diameter of BCG scars in the rural students was (8.2 +/- 2.3) mm, significantly larger than that of (7.3 +/- 2.9) mm in the urban students (P < 0.01) . The prevalence of asthma (allergy) (asthma ever, wheeze ever, current wheeze within 12 months, hay fever ever, repeated rash ever, eczema ever, allergic rhinitis, atopy, high-sensitivity or diagnosed asthma) were 0.6% - 11.4% in the rural Beijing students, significantly lower than that of 3.1% - 35.7% in urban Beijing schoolchildren (P < 0.01) . CONCLUSIONS: The diameters of BCG scars were not significantly different between asthmatic (allergic) students and normal students . The diameters of BCG scars in the rural students were significantly larger than those in the urban students . The prevalence of asthma and allergy in the rural children were significantly lower than that in the urban students . The results suggest that there is difference in immunity between rural and urban children from early life, which may be caused by the different living environments.

Biosci Biotechnol Biochem, 2003 Sep, 67(9), 1976 - 82
Occurrence of a specific protein in basidiomycete-lytic enzyme preparation produced by Bacillus circulans KA-304 inductively with a cell-wall preparation of Schizophyllum commune; Yano S et al.; KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation (CWP) of Schizophyllum commune, has been reported to have an activity to form protoplasts from S . commune mycelia . The SDS-polyacrylamide gel electrophoreses described here demonstrated that a specific proteinous component (molecular weight: 150,000) occurred in KA-prep . The protein (P150T) was also formed in culture filtrates with CWP of several basidiomycetes, which could release the protoplasts, suggesting that the component was an indispensable factor for protoplast formation . P150T, isolated from an ammonium sulfate fraction of KA-prep (0-30% saturation), did not have any protoplast-forming activity . Results were obtained indicating that P150T participates in protoplast formation together with chitinase(s) and beta-glucanase(s) in KA-prep . The N-terminal amino acid sequence indicated an analogy of P150T to mutanase (alpha-1,3-glucanase) from Bacillus sp . RM1, and actually P150T hydrolyzed mutan as well as S-(alpha-1,3) glucan from S . commune.

Biosci Biotechnol Biochem, 2003 Sep, 67(9), 1875 - 82
Characterization and kinetics of 45 kDa chitosanase from Bacillus sp . P16; Jo YY et al.; An extracellular 45 kDa endochitosanase was purified and characterized from the culture supernatant of Bacillus sp . P16 . The purified enzyme showed an optimum pH of 5.5 and optimum temperature of 60 degrees C, and was stable between pH 4.5-10.0 and under 50 degrees C . The Km and Vmax were measured with a chitosan of a D.A . of 20.2% as 0.52 mg/ml and 7.71 x 10(-6) mol/sec/mg protein, respectively . The enzyme did not degrade chitin, cellulose, or starch . The chitosanase digested partially N-acetylated chitosans, with maximum activity for 15-30% and lesser activity for 0-15% acetylated chitosan . The chitosanase rapidly reduced the viscosity of chitosan solutions at a very early stage of reaction, suggesting the endotype of cleavage in polymeric chitosan chains . The chitosanase hydrolyzed (GlcN)7 in an endo-splitting manner producing a mixture of (GlcN)(2-5) . Time course studies showed a decrease in the rate of substrate degradation from (GlcN)7 to (GlcN)6 to (GlcN)5, as indicated by the apparent first order rate constants, k1 values, of 4.98 x 10(-4), 2.3 x 10(-4), and 9.3 x 10(-6) sec(-1), respectively . The enzyme hardly catalyzed degradation of chitooligomers smaller than the pentamer.

Biopolymers, 2003 Oct, 70(2), 186 - 200
Molecular dynamics studies of alanine racemase: a structural model for drug design; Mustata GI et al.; Alanine racemase (AlaR) is a bacterial enzyme that catalyzes the interconversion of L- and D-alanine, which is an essential constituent of the peptidoglycan layer of the bacterial cell wall and requires pyridoxal 5'-phosphate (PLP) as a cofactor . The enzyme is universal to bacteria, including mycobacteria, making it an attractive target for drug design . To investigate the effects of flexibility on the binding modes of the substrate and an inhibitor and to analyze how the active site is affected by the presence of the substrate versus inhibitor, a molecular dynamics simulation on the full AlaR dimer from Bacillus stearothermophilus (pdb code: 1SFT) with a D-alanine molecule in one active site and the noncovalent inhibitor, propionate, in the second site has been carried out . Within the time scale of the simulation, we show that the active site becomes more stabilized in the presence of substrate versus inhibitor . The results of this simulation are in agreement with the proposed mechanism of alanine racemase reaction in which the substrate carboxyl group directly participates in the catalysis by acting cooperatively with Tyr 265' and Lys 39 . A structural water molecule in contact with both substrate and inhibitor (i.e., in both active sites) and bridging residues in both active sites was identified . It shows a remarkably low mobility and does not exchange with bulk water . This water molecule can be taken into account for the design of specific AlaR inhibitors by either utilizing it as a bridging group or displacing it with an inhibitor atom . The results presented here provide insights into the dynamics of the alanine racemase in the presence of substrate/inhibitor, which will be used for the rational design of novel inhibitors .

J Exp Med, 2003 Oct 6, 198(7), 987 - 97 Epub 2003 Sep 29.
MyD88 primes macrophages for full-scale activation by interferon-gamma yet mediates few responses to Mycobacterium tuberculosis; Shi S et al.; Macrophages are activated from a resting state by a combination of cytokines and microbial products . Microbes are often sensed through Toll-like receptors signaling through MyD88 . We used large-scale microarrays in multiple replicate experiments followed by stringent statistical analysis to compare gene expression in wild-type (WT) and MyD88-/- macrophages . We confirmed key results by quantitative reverse transcription polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay . Surprisingly, many genes, such as inducible nitric oxide synthase, IRG-1, IP-10, MIG, RANTES, and interleukin 6 were induced by interferon (IFN)-gamma from 5- to 100-fold less extensively in MyD88-/- macrophages than in WT macrophages . Thus, widespread, full-scale activation of macrophages by IFN-gamma requires MyD88 . Analysis of the mechanism revealed that MyD88 mediates a process of self-priming by which resting macrophages produce a low level of tumor necrosis factor . This and other factors lead to basal activation of nuclear factor kappaB, which synergizes with IFN-gamma for gene induction . In contrast, infection by live, virulent Mycobacterium tuberculosis (Mtb) activated macrophages largely through MyD88-independent pathways, and macrophages did not need MyD88 to kill Mtb in vitro . Thus, MyD88 plays a dynamic role in resting macrophages that supports IFN-gamma-dependent activation, whereas macrophages can respond to a complex microbial stimulus, the tubercle bacillus, chiefly by other routes.

Chang Gung Med J, 2003 Jul, 26(7), 520 - 4
Aeromonas hydrophila bacteremia presenting as non-traumatic acute osteomyelitis in a cirrhotic patient; Lee CH et al.; Aeromonas hydrophila is a low-virulence gram-negative bacillus . It has never been reported as a pathogen of non-traumatic acute osteomyelitis in a cirrhotic patient . Herein, we reported on a case of decompensated liver cirrhosis with Aeromonas hydrophila infection presenting as acute gastroenteritis and non-traumatic acute osteomyelitis . It has been shown that Aeromonas bacteremia usually affects immunocompromised subjects, such as those with liver cirrhosis . Non-traumatic acute osteomyelitis should be highly suggested when a cirrhotic patient with Aeromonas bacteremia presents with severe low back pain and no associated trauma.

Biotechnol Lett, 2003 Aug, 25(15), 1219 - 23
Characterization of a novel cry2Aa-type gene from Bacillus thuringiensis subsp . kurstaki; Tounsi S et al.; A 4 kb BamHI-HindIlI fragment, corresponding to the cry2A operon of Bacillus thuringiensis subsp . kurstaki strain BNS3, was cloned . The sequencing of the corresponding cry2Aa-type gene, termed crybns3-4, revealed an open reading frame of 1902 bp, encoding a protein of 633 amino-acid residues . Both nucleotide and amino-acid sequences similarity analysis revealed that crybns3-4 is a new cry2Aa-type gene which has several differences from the reported cry2Aa-type genes . The transfer of the cloned operon to an acrystalliferous mutant of BNS3, revealed an expression of the new cry2Aa-type gene and a production of parasporal crystal inclusions in the transformants.

Biotechnol Lett, 2003 Sep, 25(17), 1445 - 9
Overproduction and secretion of Bacillus circulans endo-beta-1,3-1,4-glucanase gene (bglBC1) in B . subtilis and B . megaterium; Kim JY; A gene coding for endo-beta-1,3-1,4-glucanase (lichenase) containing a recombinant plasmid, pLL200K, was transferred from Bacillus circulans into a new shuttle plasmid, pLLS920, by ligating linearized DNAs of pLL200K and pUB110 . B . subtilis RM125 and B . megaterium ATCC14945 transformed with pLLS920 produced the endo-beta-1,3-1,4-glucanase . The enzyme was produced during active growth with maximum activity . The B . subtilis (pLLS920) enzyme was 83 times (8522 mU ml(-1)) more active than that of the gene donor cells (103 mU ml(-1)) . The B . megaterium (pLLS920) enzyme was 7 times (735 mU ml(-1)) more active than that of the gene donor cells . While E . coli secreted only about 10% of the produced enzyme, B . subtilis excreted the enzyme completely into the medium and B . megaterium by about 98% . The plasmid pLLS920 was stable in B . megaterium (98%), and in B . subtilis (51%) but not in E . coli (29%).

Rev Alerg Mex, 2003 Jul-Aug, 50(4), 161 - 5
{The difficulty of obtaining immunologic responses in mucosae . Use of coadjuvants}; Rodriguez Orozco AR; The search of immunogenicity represents a real problem to be solved with the new techniques to vaccines design . Despite of numerous initiatives to increase the immune response at mucosal sites, there are few substances available to exert an adequate immunogenicity in mucosal compartments . On this hand the research on mucosal adjuvants has arise interest . Mechanism of both: immunogenicity and adjuvanticity, were reviewed putting emphasis mainly in two mucosal adjuvants, cholera toxin and the protoxin Cry1Ac from Bacillus thuringiensis.

Biotechniques, 2003 Sep, 35(3), 488 - 92
Epitope tagging: a monoclonal antibody specific for recombinant fusion proteins in plants; Lawrence SD et al.; The easy identification of a recombinant protein in plant material becomes increasingly relevant as more transgenic plants are used for research and commercial applications . Tagging recombinant proteins with a small peptide (epitope) can perform such a task . However, available epitope antibodies will also cross-react with endogenous plant proteins at a level that may be unacceptable . Here we describe the new epitope antibody AcV5 . Whether it is attached to the carbooxyl terminal end of enhanced green fluorescent protein (EGFP) or the Bacillus thuringiensis endotoxin Cry3A, these proteins remain functional . In addition, using less than 250 pg AcV5-tagged EGFP produces a strong signal on Western blots with no cross-reactivity of proteins from a broad range of plants of agronomic importance.

Biochem Biophys Res Commun, 2003 Oct 10, 310(1), 158 - 62
Relocating expression of vegetative insecticidal protein into mother cell of Bacillus thuringiensis; Arora N et al.; Vegetative insecticidal protein (VIP) is a class of insecticidal proteins produced by some strains of Bacillus thuringiensis during the vegetative stage of their growth . Unlike delta-endotoxins which are produced as parasporal inclusion bodies within the cell during sporulation, VIP is secreted into the culture medium . Here we report the relocation of the expression of VIP into the mother cell compartment in a manner similar to well-characterized Cry proteins . Relocation of VIP is directed to mother cell by placing its synthesis under sporulation-dependent promoters, BtI and BtII . The insertion of cry preferred transcription termination sequence at the 3(') region and a STAB-SD sequence at the 5(') region of the gene provided stability to the vip transcript and enhanced its yield . The demonstrated expression of VIP within the cells in the form of inclusion bodies would facilitate development of a suitable formulation for the application of this class of insecticidal proteins in the field.

Environ Microbiol, 2003 Oct, 5(10), 997 - 1001
Enduring toxicity of transgenic Anabaena PCC 7120 expressing mosquito larvicidal genes from Bacillus thuringiensis ssp . israelensis; Manasherob R et al.; Persistence of biological control agents against mosquito larvae was tested under simulated field conditions . Mosquito larvicidal activity of transgenic Anabaena PCC 7120 expressing cry4Aa, cry11Aa and p20 from Bacillus thuringiensis ssp . israelensis was greater than B . thuringiensis ssp . israelensis primary powder (fun 89C06D) or wettable powder (WP) (Bactimos products) when either mixed with silt or exposed to sunlight outdoors . Reduction of Bactimos primary powder toxicity was at least 10-fold higher than Anabaena's after mixing with silt . In outdoors experiments, Bactimos WP remained toxic (over 30% mortality of 3rd instar Aedes aegypti larvae) for 2-4 days only, while transgenic Anabaena's toxicity endured 8-21 days.

Nucleic Acids Res Suppl, 2003, (3), 315 - 6
Directed evolution of xylanase J from alkaliphilic Bacillus sp . strain 41M-1: restore of alkaliphily of a mutant with an acidic pH optimum; Inami M et al.; Alkaliphilic Bacillus sp . strain 41M-1 produces an alkaliphilic xylanase (xylanase J) . The newly constructed mutant E177Q deltaJC had an acidic pH optimum and showed almost no activity at pH 8.0 . The alkaliphily of the enzyme was restored by directed evolution . The evolved mutants, Y176S/E177Q deltaJC and G32V/Y176D/E177Q deltaJC, retained about 30% and 43% activity of their maximal activities at pH 6.0, respectively.

Braz J Biol, 2003 May, 63(2), 301 - 6 Epub 2003 Aug 15.
Pathogenicity of Bacillus thuringiensis isolated from two species of Acromyrmex (Hymenoptera, Formicidae); Pinto LM et al.; The control of Acromyrmex leaf-cutting ants is necessary due to the severe damage they cause to diverse crops . A possibility was to control them using the bacterium Bacillus thuringiensis (Bt) that characteristically produces insecticidal crystal proteins (ICPs) . The ICPs have been effective in controlling lepidopterans, dipterans, and coleopterans, but their action against hymenopterans is unknown . This paper describes an attempt to isolate Bt from ants of two Acromyrmex species, to evaluate its pathogenicity towards these ants, and to test isolates by PCR . Bacterial isolates of Bt obtained from A . crassispinus and A . lundi have been assayed against A . lundi in the laboratory . The bioassays were carried out in BOD at 25 degrees C, with a 12-hour photoperiod, until the seventh day after treatment . The Bt isolates obtained were submitted to total DNA extraction and tested by PCR with primers specific to cry genes . The results showed Bt presence in 40% of the assessed samples . The data from the in vivo assays showed a mortality rate higher than 50% in the target population, with the Bt HA48 isolate causing 100% of corrected mortality . The PCR results of Bt isolates showed a magnification of DNA fragments relative to cry1 genes in 22% of the isolates, and cry9 in 67% . Cry2, cry3, cry7, and cry8 genes were not detected in the tested samples, and 22% had no magnified DNA fragments corresponding to the assessed cry genes . The results are promising not only regarding allele identification in new isolates, but also fort the assays aimed at determining the Bt HA48 LC50's, which can eventually be applied in controlling of Acromyrmex leaf-cutting ants.

Am J Pathol, 2003 Oct, 163(4), 1321 - 7
Infectious angiogenesis: Bartonella bacilliformis infection results in endothelial production of angiopoetin-2 and epidermal production of vascular endothelial growth factor; Cerimele F et al.; Pathological angiogenesis, the development of a microvasculature by neoplastic processes, is a critical component of the development of tumors . The role of oncogenes in the induction of angiogenesis has been extensively studied in benign and malignant tumors . However, the role of infection in inducing angiogenesis is not well understood . Verruga peruana is a clinical syndrome caused by the bacterium Bartonella bacilliformis, and is characterized by the development of hemangioma-like lesions, in which bacteria colonize endothelial cells . To gain insight into how this bacteria induces angiogenesis in vivo, we performed in situ hybridization of clinical specimens of verruga peruana for the angiogenesis factors vascular endothelial growth factor (VEGF), its receptors VEGFR1 and VEGFR2, and angiopoietin-2 . High-level expression of angiopoietin-2 and VEGF receptors was observed in the endothelium of verruga peruana . Surprisingly, the major source of VEGF production in verruga peruana is the overlying epidermis . Infection of cultured endothelium with B . bacilliformis also resulted in induction of angiopoetin-2 in vitro . These findings imply a collaboration between infected endothelium and overlying epidermis to induce angiogenesis.

Scand J Immunol, 2003 Oct, 58(4), 449 - 61
Modulation of host immune responses by overexpression of immunodominant antigens of Mycobacterium tuberculosis in bacille Calmette-Guérin; Rao V et al.; Based on their immunodominant nature and ability to induce appropriate immune responses in the host, several antigens of Mycobacterium tuberculosis have shown promise of protection . However, most of the candidate vaccines developed by employing various strategies have afforded protection that is at best comparable with bacillus Calmette-Guerin (BCG) in animal models . Due to the inherent ability of BCG to prime cellular responses in the host, it has become an attractive vehicle for development of a vaccine against intracellular infections . In this study, we have cloned the genes of three immunodominant antigens of M . tuberculosis viz . the ESAT6 (Rv3875), the 19 kDa lipoprotein (Rv3763) and the 38 kDa antigen (Pst homolog) (Rv0934) in pSD5 under the transcriptional control of Trrn, a strong mycobacterial promoter, and expressed them in BCG . The19 kDa antigen and the 38 kDa antigen were expressed at levels that were approximately five and eightfolds higher in the cytosols of recombinant BCG strains rBCG19T and rBCG38T, respectively, as compared with their corresponding levels in M . bovis BCG . Both these antigens were also secreted into the extracellular medium at enhanced levels (19 kDa antigen fourfold and 38 kDa antigen twofold) by rBCG strains in comparison with the wild type BCG . ESAT6 antigen, which is absent in M . bovis BCG, was also expressed at a very high level in the cytosol of the rBCG strain (rBCGE6T) . Evaluation of immune responses induced by these three rBCG strains in mice shows a markedly different pattern . The rBCG strain overexpressing the 38 kDa antigen exhibited a predominant T helper 1 (Th1) response with high levels of interferon-gamma (IFN-gamma) production, whereas overexpression of the 19 kDa antigen resulted in completely polarized Th2 responses against the BCG sonicate . The rBCG-expressing ESAT6 antigen induced a mixed Th1/Th2 response . Our observations suggest that the 38 kDa antigen may hold excellent promise in the rBCG approach for the development of a vaccine against tuberculosis.

Curr Microbiol, 2003 Aug, 47(2), 144 - 5
Identification of a novel DNA methyltransferase activity from Bacillus thuringiensis; Jenkinson E et al.; A DNA methyltransferase activity was identified in a strain of Bacillus thuringiensis that was found to protect DNA from cleavage by the restriction endonuclease HaeIII at overlapping sites . Site-directed mutagenesis was used to confirm therecognition sequence of the methyltransferase as ACGGC.

Curr Microbiol, 2003 Aug, 47(2), 102 - 8
An antifungal chitinase produced by Bacillus cereus with shrimp and crab shell powder as a carbon source; Chang WT et al.; The production of inexpensive chitinolytic enzymes is an element in the utilization of shellfish processing wastes . In this study, shrimp and crab shell powder prepared by treating shrimp and crab processing wastes with boiling and crushing was used as a substrate for the isolation of an antifungal chitinase-producing microorganism . Bacillus cereus YQ 308, a strain isolated from the soil samples, excreted one chitinase when cultured in a medium containing 2% (wt/vol) shrimp and crab shell powder as major carbon source . The chitinase, purified by sequential chromatography, had an Mr of 48 kDa and pI of 5.2 . The purified chitinase (2 mg/ml) inhibited the hyphal extension of the fungi Fusarium oxysporum and Pythium ultimum.

J Exp Biol, 2003 Nov, 206(Pt 21), 3877 - 85
Recombinant bacteria for mosquito control; Federici BA et al.; Bacterial insecticides have been used for the control of nuisance and vector mosquitoes for more than two decades . Nevertheless, due primarily to their high cost and often only moderate efficacy, these insecticides remain of limited use in tropical countries where mosquito-borne diseases are prevalent . Recently, however, recombinant DNA techniques have been used to improve bacterial insecticide efficacy by markedly increasing the synthesis of mosquitocidal proteins and by enabling new endotoxin combinations from different bacteria to be produced within single strains . These new strains combine mosquitocidal Cry and Cyt proteins of Bacillus thuringiensis with the binary toxin of Bacillus sphaericus, improving efficacy against Culex species by 10-fold and greatly reducing the potential for resistance through the presence of Cyt1A . Moreover, although intensive use of B . sphaericus against Culex populations in the field can result in high levels of resistance, most of this can be suppressed by combining this bacterial species with Cyt1A; the latter enables the binary toxin of this species to enter midgut epithelial cells via the microvillar membrane in the absence of a midgut receptor . The availability of these novel strains and newly discovered mosquitocidal proteins, such as the Mtx toxins of B . sphaericus, offers the potential for constructing a range of recombinant bacterial insecticides for more effective control of the mosquito vectors of filariasis, Dengue fever and malaria.

Vaccine, 2003 Oct 1, 21(27-30), 4270 - 4
The in vitro evolution of BCG vaccines; Mostowy S et al.; The bacillus Calmette-Geurin (BCG) family of vaccines currently implemented to prevent tuberculosis (TB) consist of clonal bacterial strains independently shaped by nearly a half-century of evolution . Derived from virulent Mycobacterium bovis, daughter strains of BCG were additionally passaged under the same laboratory conditions that resulted in its original attenuation . Genomic loss of the RD1 region has been demonstrated to coincide with attenuation from virulence, while deletions occurring after the loss of RD1 are speculated to be responsible for BCG's over-attenuation . To provide a more complete description of their total genomic variation, the genomic content of BCG strains are investigated by Affymetrix GeneChip . Because clinical isolates of M . tuberculosis have previously been characterized via GeneChip interrogation, analysis permits the comparison of in vivo versus in vitro evolution of M . tuberculosis complex subspecies . The contrast between the two modes of evolution are discussed in its relevance towards TB pathogenicity.

Insect Biochem Mol Biol, 2003 Oct, 33(10), 999 - 1010
Identification of novel Bacillus thuringiensis Cry1Ac binding proteins in Manduca sexta midgut through proteomic analysis; McNall RJ et al.; The crystal proteins of Bacillus thuringiensis are widely used in transgenic crops and commercially available insecticides . Manduca sexta, the tobacco hornworm, is the model insect for B . thuringiensis studies . Although brush border vesicles prepared from larval M . sexta midgut have been used in numerous mode-of-action studies of B . thuringiensis toxins, their protein components are mostly unknown . Vesicles prepared from the brush border of M . sexta midgut were analyzed using one- and two-dimensional gel electrophoresis to establish a midgut brush border proteome . Sub-proteomes were also established for B . thuringiensis Cry1Ac binding proteins and glycosylphosphatidyl inositol (GPI) anchored proteins . Peptide mass fingerprints were generated for several spots identified as Cry1Ac binding proteins and GPI-anchored proteins and these fingerprints were used for database searches . Results generally did not produce matches to M . sexta proteins, but did match proteins of other Lepidoptera . Actin and alkaline phosphatase were identified as novel proteins that bind Cry1Ac in addition to the previously reported aminopeptidase N . Aminopeptidase N was the only GPI-anchored protein identified . Actin, aminopeptidase N, and membrane alkaline phosphatase were confirmed as accurate protein identifications through western blots.

Bioorg Med Chem Lett, 2003 Oct 20, 13(20), 3507 - 12
Complexation of cyclic dodecadepsipeptide, cereulide with ammonium salts; Pitchayawasin S et al.; Cereulide is a principal toxin causing emetic syndrome which is produced by Bacillus cereus and has been known as potassium selective ionophore . This paper deals with its complexation with inorganic and organic ammonium ions to assign the higher structures similar to the complex with potassium ion by means of NMR and ESI-MS spectroscopy . Of particular interest, the detectable ions are not only at m/z 1191.8 as K(+) complex but also (or sometimes exclusively) at m/z 1170.8 as NH(4)(+) complex in its LC-MS analyses depending upon the conditions . This difference is due to the sample preparation and measurement condition.

J Hosp Infect, 2003 Sep, 55(1), 47 - 52
Evaluation of a low-temperature steam and formaldehyde sterilizer; Kanemitsu K et al.; We evaluated a low-temperature steam and formaldehyde (LTSF) sterilizer based on the draft European Standard prEN 14180 . Microbiological tests were conducted on small and full loads using process challenge devices in five programs (P1-P5) . With small loads all tests showed no growth of Bacillus stearothermophilus (ATCC7953) spores . However, positive cultures were observed with full-load tests using P5 (sterilization temperature, 50 degrees C) . Our data indicated that the load influenced the efficacy of the LTSF sterilizer . Desorption tests were conducted to determine residual formaldehyde in indicator strips . The mean concentrations of formaldehyde in P1-P5 were 31.9, 56.3, 54.9, 82.2 and 180.6 microg, respectively, which are below the limits allowed by the draft Standard . Our results indicate that the LTSF sterilizer is useful for sterilization because of its excellent efficacy, short handling time, and safety.

J Hosp Infect, 2003 Sep, 55(1), 33 - 8
Pseudo-outbreak of toxigenic Bacillus cereus isolated from stools of three patients with diarrhoea after oral administration of a probiotic medication; Kniehl E et al.; From December 2000 to January 2001 toxigenic Bacillus cereus was isolated from stools of three patients with diarrhoea at two tertiary hospitals in southwest Germany . Two cases with nosocomial diarrhoea were apparently epidemiologically related (same time and ward), a third case was unrelated with respect to time and location . In order to investigate the epidemiology of these three cases, clinical isolates and isolates from an unexpected, possible common source (probiotic medication) were compared by toxin assay, biotyping and randomly amplified polymorphic DNA (RAPD) analysis . The three clinical isolates, as well as the two isolates from different lots of the probiotic medication (Bactisubtil containing 'Bacillus IP 5832'; Cassella-med, Cologne, Germany), were indistinguishable by toxin assay, biotyping and RAPD, when compared with other distinguishable clinical B . cereus strains . As the diarrhoeal disease had begun before the probiotic medication had been administered to overcome it, the isolated B . cereus probably was at least initially, not the cause of the observed diarrhoeal disease . Isolation of toxigenic B . cereus from stools appeared to be a diagnostically misleading epiphenomenon after oral medication with the probiotic . We conclude, that probiotic medication with Bactisubtil (Bacillus IP 5832) may result in diagnostically misleading results when culturing stool specimens from patients with diarrhoea . The clonal identity of isolates may be misinterpreted as an outbreak . Stool specimens should be taken before start of probiotic treatment and clinicians should state probiotic medication when ordering stool examinations to allow correct interpretation of results . Nevertheless, it is noteworthy that a probiotic medication contains potentially toxigenic material.

Bioprocess Biosyst Eng, 2003 Jan, 25(4), 229 - 33 Epub 2002 Sep 14.
Neural networks applied to the prediction of fed-batch fermentation kinetics of Bacillus thuringiensis; Valdez-Castro L et al.; This paper proposes using a new recurrent neural network model (RNNM) to predict and control fed batch fermentations of Bacillus thuringiensis . The control variables are the limiting substrate and the feeding conditions . The multi-input multi-output RNNM proposed has twelve inputs, seven outputs, nineteen neurons in the hidden layer, and global and local feedbacks . The weight update learning algorithm designed is a version of the well known backpropagation through time algorithm directed to the RNNM learning . The error approximation for the last epoch of learning is 2% and the total learning time is 51 epochs, where the size of an epoch is 162 iterations . The RNNM generalization was carried out reproducing a B . thuringiensis fermentation not included in the learning process . It attains an error approximation of 1.8%.

Bioprocess Biosyst Eng, 2003 Jan, 25(4), 213 - 6 Epub 2002 Oct 16.
Recovery of Bacillus sphaericus 2362 spores from growth medium by flocculation/sedimentation; Luna CL et al.; Bacillus sphaericus 2362 spore sedimentation was evaluated through batch settling experiments, using tannin as the flocculating agent . The flocculation was demonstrated to be efficient up to the tannin concentration of 150 ppm . The floc diameter averaged 208 micro m, and its density was found to be 1.09 g/mL . The sedimentation process using tannin as a flocculating agent is promising, and a spore concentrate was obtained without loss of biological activity, presenting a LC(50) equal to 0.22 ppm . The result indicates that the larvicide activity is appropriate for controlling populations of Culex quinquefasciatus.

J Econ Entomol, 2003 Aug, 96(4), 1322 - 8
Efficacy of transgenic cotton containing a cry1Ac gene from Bacillus thuringiensis against Helicoverpa armigera (Lepidoptera: Noctuidae) in northern China; Wu K et al.; NuCOTN 33B, a Bt transgenic variety of upland cotton (Gossypium hirsutum L.) expressing the insecticidal protein Cry1Ac from Bacillus thuringiensis Berliner sp . kurstaki, was evaluated for resistance to Helicoverpa armigera (Hubner) during 1998-2000 in northern China . The results indicated that there was no significant difference in egg densities between NuCOTN 33B and three nontransgenic varieties (DP5415, Zhongmian12, and Shiyuan321) during the season, although the survival of larvae on NuCOTN 33B seemed significantly reduced . High larval densities observed on non-Bt cotton appeared in great contrast to the low larval populations observed on NuCOTN 33B plants during the seasons . In an environment without insecticide sprays, the annual ginned cotton yields in NuCOTN 33B plots, ranging from 1391.17 to 1511.35 kg/ha, were significantly higher than those in non-Bt cotton (340.34-359.58 kg/ha) . These high levels of field efficacy for NuCOTN 33B against H . armigera in northern China may pave the way for reduced pesticide applications and an expansion of alternative pest-control strategies.

J Econ Entomol, 2003 Aug, 96(4), 1290 - 9
Resistance to the Cry1Ac delta-endotoxin of Bacillus thuringiensis in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae); Akhurst RJ et al.; Three laboratory strains of Helicoverpa armigera (Hubner) were established by mating of field-collected insects with an existing insecticide-susceptible laboratory strain . These strains were cultured on artificial diet containing the Cry1Ac protoxin of Bacillus thuringiensis using three different protocols . When no response to selection was detected after 7-11 generations of selection, the three strains were combined by controlled mating to preserve genetic diversity . The composite strain (BX) was selected on the basis of growth rate on artificial diet containing Cry1Ac crystals . Resistance to Cry1Ac was first detected after 16 generations of continuous selection . The resistance ratio (RR) peaked approximately 300-fold at generation 21, after which it declined to oscillate between 57- and 111-fold . First-instar H . armigera from generation 25 (RR = 63) were able to complete their larval development on transgenic cotton expressing Cry1Ac and produce fertile adults . There appeared to be a fitness cost associated with resistance on cotton and on artificial diet . The BX strain was not resistant to the commercial Bt spray formulations DiPel and XenTari, which contain multiple insecticidal crystal proteins, but was resistant to the MVP formulation, which only contains Cry1Ac . The strain was also resistant to Cry1Ab but not to Cry2Aa or Cry2Ab . Toxin binding assays showed that the resistant insects lacked the high affinity binding site that was detected in early generations of the strain . Genetic analysis confirmed that resistance in the BX strain of H . armigera is incompletely recessive.

J Econ Entomol, 2003 Aug, 96(4), 1039 - 44
Novel fermentation media for production of Bacillus thuringiensis subsp . israelensis; Poopathi S et al.; The production of Bacillus thuringiensis subsp . israelensis (deBarjac) (Bti) as a biopesticide is not cost-effective using existing fermentation technology . In this study, we explored the use of several less expensive alternative culture media (potato, common sugar, and Bengal gram) for the growth and production of Bti . Growth was obtained in all tested media and was comparable to that obtained in conventional medium (Luria-Bertani) . Toxicity assays showed that the toxin produced from the novel growth media were effective in killing larvae of Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti and toxicity was comparable to that produced from Luria-Bertani medium . These observations suggest that potato can be used as a cheap source of culture medium for the production of Bti toxin in mosquito control programs.

J Econ Entomol, 2003 Aug, 96(4), 1031 - 8
Insect resistance to transgenic Bt crops: lessons from the laboratory and field; Tabashnik BE et al.; Transgenic crops that produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) grew on >62 million ha worldwide from 1996 to 2002 . Despite expectations that pests would rapidly evolve resistance to such Bt crops, increases in the frequency of resistance caused by exposure to Bt crops in the field have not yet been documented . In laboratory and greenhouse tests, however, at least seven resistant laboratory strains of three pests (Plutella xylostella {L.}, Pectinophora gossypiella {Saunders}, and Helicoverpa armigera {Hubner}) have completed development on Bt crops . In contrast, several other laboratory strains with 70- to 10,100-fold resistance to Bt toxins in diet did not survive on Bt crops . Monitoring of field populations in regions with high adoption of Bt crops has not yet detected increases in resistance frequency . Resistance monitoring examples include Ostrinia nubilalis (Hubner) in the United States (6 yr), P . gossypiella in Arizona (5 yr), H . armigera in northern China (3 yr), and Helicoverpa zea (Boddie) in North Carolina (2 yr) . Key factors delaying resistance to Bt crops are probably refuges of non-Bt host plants that enable survival of susceptible pests, low initial resistance allele frequencies, recessive inheritance of resistance to Bt crops, costs associated with resistance that reduce fitness of resistant individuals relative to susceptible individuals on non-Bt hosts ("fitness costs"), and disadvantages suffered by resistant strains on Bt hosts relative to their performance on non-Bt hosts ("incomplete resistance") . The relative importance of these factors varies among pest-Bt crop systems, and violations of key assumptions of the refuge strategy (low resistance allele frequency and recessive inheritance) may occur in some cases . The success of Bt crops exceeds expectations of many, but does not preclude resistance problems in the future.

Microb Ecol, 2004 Feb, 47(2), 159 - 63 Epub 2003 Sep 17.
Extreme spore UV resistance of Bacillus pumilus isolates obtained from an ultraclean Spacecraft Assembly Facility; Link L et al.; Recent environmental microbial sampling of the ultraclean Spacecraft Assembly Facility at NASA Jet Propulsion Laboratory (JPL-SAF) identified spores of Bacillus pumilus as major culturable bacterial contaminants found on and around spacecraft . As part of an effort to assess the efficacy of various spacecraft sterilants, purified spores of 10 JPL-SAF B . pumilus isolates were subjected to 254-nm UV and their UV resistance was compared to spores of standard B . subtilis biodosimetry strains . Spores of six of the 10 JPL-SAF isolates were significantly more resistant to UV than the B . subtilis biodosimetry strain, and one of the JPL-SAF isolates, B . pumilus SAFR-032, exhibited the highest degree of spore UV resistance observed by any Bacillus spp . encountered to date .

Acta Crystallogr D Biol Crystallogr, 2003 Oct, 59(Pt 10), 1869 - 70 Epub 2003 Sep 19.
Purification, crystallization and preliminary X-ray analysis of the novel DEAD protein BstDEAD from Bacillus stearothermophilus; Carmel AB et al.; DEAD proteins are members of a large and diverse family of RNA helicases that use energy from ATP hydrolysis to unwind short regions of duplex RNA . BstDEAD from Bacillus stearothermophilus is a 436-amino-acid protein and a representative member of the DEAD protein family . In addition to the mechanistic core common to DEAD proteins, BstDEAD has a unique approximately 60-amino-acid C-terminal extension that may denote a specific biological role . BstDEAD has been crystallized in space group P4(1/3)2(1)2, with unit-cell parameters a = b = 100.3, c = 110.6 A and one molecule per asymmetric unit . It is the first DEAD protein to be crystallized containing a unique extension outside of the core.

Protein Sci, 2003 Oct, 12(10), 2141 - 9
Directed evolution of a bacterial alpha-amylase: toward enhanced pH-performance and higher specific activity; Bessler C et al.; alpha-Amylases, in particular, microbial alpha-amylases, are widely used in industrial processes such as starch liquefaction and pulp processes, and more recently in detergency . Due to the need for alpha-amylases with high specific activity and activity at alkaline pH, which are critical parameters, for example, for the use in detergents, we have enhanced the alpha-amylase from Bacillus amyloliquefaciens (BAA) . The genes coding for the wild-type BAA and the mutants BAA S201N and BAA N297D were subjected to error-prone PCR and gene shuffling . For the screening of mutants we developed a novel, reliable assay suitable for high throughput screening based on the Phadebas assay . One mutant (BAA 42) has an optimal activity at pH 7, corresponding to a shift of one pH unit compared to the wild type . BAA 42 is active over a broader pH range than the wild type, resulting in a 5-fold higher activity at pH 10 . In addition, the activity in periplasmic extracts and the specific activity increased 4- and 1.5-fold, respectively . Another mutant (BAA 29) possesses a wild-type-like pH profile but possesses a 40-fold higher activity in periplasmic extracts and a 9-fold higher specific activity . The comparison of the amino acid sequences of these two mutants with other homologous microbial alpha-amylases revealed the mutation of the highly conserved residues W194R, S197P, and A230V . In addition, three further mutations were found K406R, N414S, and E356D, the latter being present in other bacterial alpha-amylases.

Infect Immun, 2003 Oct, 71(10), 6045 - 8
Interleukin-15 as an immune adjuvant to increase the efficacy of Mycobacterium bovis bacillus Calmette-Guérin vaccination; Umemura M et al.; Interleukin-15 (IL-15) transgenic mice which had been inoculated with Mycobacterium bovis bacillus Calmette-Guerin (BCG) 24 weeks previously showed resistance against airborne infection with Mycobacterium tuberculosis H37Rv accompanied by an increased CD8(+)-Tc1-cell response . IL-15 may be used as an immune adjuvant given with BCG vaccination to enhance its biologic efficacy.

J Microbiol Methods, 2003 Oct, 55(1), 1 - 10
Application of sonication to release DNA from Bacillus cereus for quantitative detection by real-time PCR; Fykse EM et al.; A rapid sonication method for lysis of Gram-positive bacteria was evaluated for use in combination with quantitative real-time polymerase chain reaction (PCR) analyses for detection . Other criteria used for evaluation of lysis were microscopic cell count, colony forming units (cfu), optical density at 600 nm and total yield of DNA measured by PicoGreen fluorescence . The aim of this study was complete disruption of cellular structures and release of DNA without the need for lysing reagents and time-consuming sample preparation . The Gram-positive bacterium Bacillus cereus was used as a model organism for Gram-positive bacteria . It was demonstrated by real-time PCR that maximum yield of DNA was obtained after 3 to 5 min of sonication . The yield of DNA was affected by culture age and the cells from a 4-h-old culture in the exponential phase of growth gave a higher yield of DNA after 5 min of sonication than a 24-h-old culture in the stationary phase of growth . The 4-h-old culture was also more sensitive for lysis caused by heating . The maximum yield of DNA, evaluated by real-time PCR, from a culture of the Gram-negative bacterium Escherichia coli, was obtained after 20 s of sonication . However, the yield of target DNA from E . coli rapidly decreased after 50 s of sonication due to degradation of DNA . Plate counting (cfu), microscopic counting and absorbance at 600 nm showed that the number of viable and structurally intact B . cereus cells decreased rapidly with sonication time, whereas the yield of DNA increased as shown by PicoGreen fluorescence and real-time PCR . The present results indicate that 3-5 min of sonication is sufficient for lysis and release of DNA from samples of Gram-positive bacteria.

Carbohydr Res, 2003 Sep 10, 338(19), 1969 - 80
Study of the inhibition of four alpha amylases by acarbose and its 4IV-alpha-maltohexaosyl and 4IV-alpha-maltododecaosyl analogues; Yoon SH et al.; Acarbose analogues, 4IV-maltohexaosyl acarbose (G6-Aca) and 4IV-maltododecaosyl acarbose (G12-Aca), were prepared by the reaction of cyclomaltodextrin glucanyltransferase with cyclomaltohexaose and acarbose . The inhibition kinetics of acarbose and the two acarbose analogues were studied for four different alpha-amylases: Aspergillus oryzae, Bacillus amyloliquefaciens, human salivary, and porcine pancreatic alpha-amylases . The three inhibitors showed mixed, noncompetitive inhibition, for all four alpha-amylases . The acarbose inhibition constants, Ki, for the four alpha-amylases were 270, 13, 1.27, and 0.80 microM, respectively; the Ki values for G6-Aca were 33, 37, 14, and 7 nM, respectively; and the G12-Aca Ki constants were 59, 81, 18, and 11 nM, respectively . The G6-Aca and G12-Aca analogues are the most potent alpha-amylase inhibitors observed, with Ki values one to three orders of magnitude more potent than acarbose, which itself was one to three orders of magnitude more potent than other known alpha-amylase inhibitors.

Appl Microbiol Biotechnol, 2004 Apr, 64(2), 263 - 9 Epub 2003 Sep 16.
Isolation and evaluation of antagonistic bacteria towards the cucurbit powdery mildew fungus Podosphaera fusca; Romero D et al.; Powdery mildew is one of the most important limiting factors for cucurbits production in Spain, its management being strongly dependent on chemicals . The aim of this work was to evaluate the possibility of exploiting antagonistic bacteria in the biological control of the cucurbit powdery mildew fungus Podosphaera fusca (syn . Sphaerotheca fusca) . Among a collection of bacterial strains isolated from distinct cucurbit powdery mildew diseased plants and rhizospheric soils, four isolates were selected, by means of a screening method based on antibiotic production, and identified as Bacillus spp . These isolates proved to be efficacious in the control of cucurbit powdery mildew in in vitro detached leaves and seedling biocontrol assays, where reductions of disease severity of up to 80% were obtained . Furthermore, bacterial populations on melon leaves remained at similar levels (10(5) cfu cm(-2)) over the 16-day period studied and, as observed by scanning electron microscopy analysis, they were able to establish microcolonies associated with an extracellular matrix, which reveals that these isolates efficiently colonize melon phylloplane . These results indicate that the bacterial isolates selected are promising candidates for biological control agents of cucurbit powdery mildew in southern Spain.

Theor Appl Genet, 2003 Nov, 107(8), 1533 - 40 Epub 2003 Sep 06.
Additive transgene expression and genetic introgression in multiple green-fluorescent protein transgenic crop x weed hybrid generations; Halfhill MD et al.; The level of transgene expression in crop x weed hybrids and the degree to which crop-specific genes are integrated into hybrid populations are important factors in assessing the potential ecological and agricultural risks of gene flow associated with genetic engineering . The average transgene zygosity and genetic structure of transgenic hybrid populations change with the progression of generations, and the green fluorescent protein (GFP) transgene is an ideal marker to quantify transgene expression in advancing populations . The homozygous T(1) single-locus insert GFP/ Bacillus thuringiensis (Bt) transgenic canola ( Brassica napus, cv Westar) with two copies of the transgene fluoresced twice as much as hemizygous individuals with only one copy of the transgene . These data indicate that the expression of the GFP gene was additive, and fluorescence could be used to determine zygosity status . Several hybrid generations (BC(1)F(1), BC(2)F(1)) were produced by backcrossing various GFP/Bt transgenic canola ( B . napus, cv Westar) and birdseed rape ( Brassica rapa) hybrid generations onto B . rapa . Intercrossed generations (BC(2)F(2) Bulk) were generated by crossing BC(2)F(1) individuals in the presence of a pollinating insect ( Musca domestica L.) . The ploidy of plants in the BC(2)F(2) Bulk hybrid generation was identical to the weedy parental species, B . rapa . AFLP analysis was used to quantify the degree of B . napus introgression into multiple backcross hybrid generations with B . rapa . The F(1) hybrid generations contained 95-97% of the B . napus-specific AFLP markers, and each successive backcross generation demonstrated a reduction of markers resulting in the 15-29% presence in the BC(2)F(2) Bulk population . Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC(2)F(2) Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid generations (F(1), BC(1)F(1) and BC(2)F(1)) . These data demonstrate that the formation of homozygous individuals within hybrid populations increases the average level of transgene expression as generations progress . This phenomenon must be considered in the development of risk-management strategies.

J Invertebr Pathol, 2003 Sep, 84(1), 30 - 7
An improved baculovirus insecticide producing occlusion bodies that contain Bacillus thuringiensis insect toxin; Chang JH et al.; Baculovirus occlusion bodies, large proteinaceous structures which contain virions, have recently been engineered to incorporate foreign proteins . The major constituent protein of occlusion bodies from the baculovirus Autographa californica nucleopolyhedrovirus is polyhedrin, and assembly of recombinant occlusion bodies which incorporate a foreign protein depends on an interaction between native polyhedrin and a polyhedrin-foreign protein fusion . This technology has now been applied to the generation of a recombinant baculovirus (ColorBtrus) that produces occlusion bodies incorporating the Bacillus thuringiensis (Bt) insecticidal Cry1Ac toxin protein . ColorBtrus coexpresses native polyhedrin and a fusion protein in which polyhedrin is fused to the Bt toxin, which is in turn fused to green fluorescent protein (GFP) . Analysis of ColorBtrus occlusion bodies confirmed that they include both Bt toxin and GFP, yet still incorporate virions . Bioassay of ColorBtrus demonstrated that its speed of action and pathogenicity are strikingly enhanced compared to wild-type virus . ColorBtrus represents a novel, powerful biological insecticide that combines positive attributes of both Bt toxin and baculovirus based systems.

Biochem J, 2003 Dec 15, 376(Pt 3), 773 - 9
Arginyl-tRNA synthetase with signature sequence KMSK from Bacillus stearothermophilus; Li J et al.; ArgRS (arginyl-tRNA synthetase) belongs to the class I aaRSs (aminoacyl-tRNA synthetases), though the majority of ArgRS species lack the canonical KMSK sequence characteristic of class I aaRSs . A DNA fragment of the ArgRS gene from Bacillus stearothermophilus was amplified using primers designed according to the conserved regions of known ArgRSs . Through analysis of the amplified DNA sequence and known tRNA(Arg)s with a published genomic sequence of B . stearothermophilus, the gene encoding ArgRS ( argS ') was amplified by PCR and the gene encoding tRNA(Arg) (ACG) was synthesized . ArgRS contained 557 amino acid residues including the canonical KMKS sequence . Recombinant ArgRS and tRNA(Arg) (ACG) were expressed in Escherichia coli . ArgRS purified by nickel-affinity chromatography had no ATPase activity . The kinetics of ArgRS and cross-recognition between ArgRSs and tRNA(Arg)s from B . stearothermophilus and E . coli were studied . The activities of B . stearothermophilus ArgRS mutated at Lys(382) and Lys(385) of the KMSK sequence and at Gly(136) upstream of the HIGH loop were determined . From the mutation results, we concluded that there was mutual compensation of Lys(385) and Gly(136) for the amino acid-activation activity of B . stearothermophilus ArgRS.

Nahrung, 2003 Aug, 47(4), 226 - 7
Preliminary study of L-lysine production by Bacillus species using various agricultural by-products; Ekwealor IA et al.; The production of lysine by Bacillus megaterium SP-14 and Bacillus circulans Tx-22 using agricultural by-products as carbon and nitrogen sources was assessed . Among the carbon substrates used were potato, sorghum, plantain, millet, yam, cassava, and corn starches, while the nitrogen sources include cowpea, bambara-nut, cotton seed, groundnut, soybean, and blood meals . The effect of natural nitrogen sources (1.0% w/v) and synthetic nitrogen source (4.0% w/v (NH4)2SO4) on lysine production by the Bacillus strains showed that natural nitrogen sources gave better lysine yields.

Am J Trop Med Hyg, 2003 Aug, 69(2), 206 - 12
Enhancement of the efficacy of a combination of Mesocyclops aspericornis and Bacillus thuringiensis var . israelensis by community-based products in controlling Aedes aegypti larvae in Thailand; Kosiyachinda P et al.; Prolonged efficacy of a combination of bacteria (Bacillus thuringiensis var . israelensis {Bti} and copepods (Mesocyclops aspericornis) in controlling immature forms of Aedes aegypti in peridomestic water containers was achieved by adding various products from local villages as supplementary food for copepods . In all experiments, 100 first-instar larvae were added into the breeding containers every day for eight weeks . Combinations of biological control agents and each local supplementary food were applied once at the beginning of the experiment . At the end of the experiment, the average number of mosquito larvae in containers with a combination of copepods and Bti with one gram of rice grain had decreased to only 0.5% of that with no control agent . In comparison, the average numbers of mosquito larvae in containers with Bti only, or copepods only, were approximately 10% and 33% of those in containers with no control agents, respectively . In addition, the number of copepods in containers with mosquito larvae and supplementary food was at least three times higher than those with mosquito larvae alone.

Indian J Environ Health, 2002 Oct, 44(4), 258 - 65
Distribution and occurrence of diatom community along three estuaries of Dakshina Kannada, west coast of India; Shashikumar KC et al.; The present paper deals with the collection and identification of Diatoms (Bacillariophyceae) of three estuaries at Dakshina Kannada district, (Karnataka) . The studies were carried out for a period of one year extending between November 1994 to December 1995 . A total of 33 taxa belonging to 24 genera were recorded from three different estuaries . The highest number of diatom taxa were recorded from H1 & G1 estuaries and the most common diatom genera observed at all the stations were Coscinodiscus sp., and Cheatoceros sp., . The various ecological parameters analysed are discussed.

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1537 - 44
Alicyclobacillus pomorum sp . nov., a novel thermo-acidophilic, endospore-forming bacterium that does not possess omega-alicyclic fatty acids, and emended description of the genus Alicyclobacillus; Goto K et al.; A thermo-acidophilic endospore-forming bacterium was isolated from a mixed fruit juice . The organism, strain 3A(T), was rod-shaped, grew aerobically at 30-60 degrees C (optimum 45-50 degrees C), pH 3.0-6.0 (optimum pH 4.0-4.5) and produced acid from various sugars . It contained menaquinone-7 as the major isoprenoid quinone . The G+C content of the DNA was 53.1 mol% . The predominant cellular fatty acids of the strain were iso-C(15 : 0), anteiso-C(15 : 0), iso-C(16 : 0), iso-C(17 : 0) and anteiso-C(17 : 0), but omega-alicyclic fatty acids, which are characteristic of the genus Alicyclobacillus, were not found in the strain . Phylogenetic analyses based on both 16S rRNA and gyrB (DNA gyrase B subunit gene) gene sequences showed that strain 3A(T) falls into the Alicyclobacillus cluster, validated by significant bootstrap values . However, strain 3A(T) did not show a close relationship to the other species of the cluster . The level of 16S rDNA similarity between strain 3A(T) and other strains of the cluster was between 92.5 and 95.5 % . The level of gyrB sequence similarity between strain 3A(T) and other strains of the cluster was between 68.5 and 74.4 % . DNA-DNA hybridization values between strain 3A(T) and phylogenetically related strains of the genera Alicyclobacillus, Bacillus and Sulfobacillus were under 13 %, indicating that strain 3A(T) represents a distinct species . On the basis of these results, strain 3A(T) should be classified as a novel Alicyclobacillus species . The name Alicyclobacillus pomorum is proposed for this organism . The type strain of Alicyclobacillus pomorum is strain 3A(T) (=DSM 14955(T)=IAM 14988(T)).

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1531 - 6
Bacillus krulwichiae sp . nov., a halotolerant obligate alkaliphile that utilizes benzoate and m-hydroxybenzoate; Yumoto I et al.; Obligate alkaliphilic strains, AM31D(T) and AM11D, that utilize benzoate and m-hydroxybenzoate were isolated from soil obtained from Tsukuba, Ibaraki, Japan . The isolates grew at pH 8-10, but not at neutral pH . They were Gram-positive, facultatively anaerobic, straight rods with peritrichous flagella and produced ellipsoidal spores . The isolates reduced nitrate to nitrite and grew in 0-14 % NaCl, but not in higher concentrations . The major isoprenoid quinones were menaquinone-5, -6 and -7, and the cellular fatty acid profile consisted of significant amounts of 15-C branched-chain acids, isoC(15 : 0) and anteisoC(15 : 0) . Phylogenetic analysis based on 16S rRNA gene sequencing indicated that strain AM31D(T) was a member of group 6 (alkaliphiles) in the genus BACILLUS: DNA-DNA hybridization revealed a low relatedness of the isolates with several phylogenetically close neighbours, including Bacillus alcalophilus and Bacillus pseudalcaliphilus (less than 19.3 %) . Based on phenotypic characteristics, phylogenetic data and DNA-DNA relatedness data, it was concluded that these isolates merited classification as a new species, for which the name Bacillus krulwichiae is proposed . The type strain of this species is AM31D(T) (=NCIMB 13904(T)=JCM 11691(T)=IAM 15000(T)).

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1305 - 14
Tuberculosis in seals caused by a novel member of the Mycobacterium tuberculosis complex: Mycobacterium pinnipedii sp . nov; Cousins DV et al.; A comparison of Mycobacterium tuberculosis complex isolates from seals (pinnipeds) in Australia, Argentina, Uruguay, Great Britain and New Zealand was undertaken to determine their relationships to each other and their taxonomic position within the complex . Isolates from 30 cases of tuberculosis in six species of pinniped and seven related isolates were compared to representative and standard strains of the M . tuberculosis complex . The seal isolates could be distinguished from other members of the M . tuberculosis complex, including the recently defined 'Mycobacterium canettii' and 'Mycobacterium caprae', on the basis of host preference and phenotypic and genetic tests . Pinnipeds appear to be the natural host for this 'seal bacillus', although the organism is also pathogenic in guinea pigs, rabbits, humans, Brazilian tapir (Tapirus terrestris) and, possibly, cattle . Infection caused by the seal bacillus is predominantly associated with granulomatous lesions in the peripheral lymph nodes, lungs, pleura, spleen and peritoneum . Cases of disseminated disease have been found . As with other members of the M . tuberculosis complex, aerosols are the most likely route of transmission . The name Mycobacterium pinnipedii sp . nov . is proposed for this novel member of the M . tuberculosis complex (the type strain is 6482(T)=ATCC BAA-688(T)=NCTC 13288(T)).

J Bacteriol, 2003 Oct, 185(19), 5722 - 34
Listeria monocytogenes sigma B regulates stress response and virulence functions; Kazmierczak MJ et al.; While the stress-responsive alternative sigma factor sigma(B) has been identified in different species of Bacillus, Listeria, and Staphylococcus, the sigma(B) regulon has been extensively characterized only in B . subtilis . We combined biocomputing and microarray-based strategies to identify sigma(B)-dependent genes in the facultative intracellular pathogen Listeria monocytogenes . Hidden Markov model (HMM)-based searches identified 170 candidate sigma(B)-dependent promoter sequences in the strain EGD-e genome sequence . These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted sigma(B)-dependent promoters as well as selected virulence and stress response genes . RNA for the microarray experiments was isolated from both wild-type and Delta sigB null mutant L . monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl) . Microarray analyses identified a total of 55 genes with statistically significant sigma(B)-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type) . Of the 55 genes exhibiting sigma(B)-dependent expression, 54 were preceded by a sequence resembling the sigma(B) promoter consensus sequence . Rapid amplification of cDNA ends-PCR was used to confirm the sigma(B)-dependent nature of a subset of eight selected promoter regions . Notably, the sigma(B)-dependent L . monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh) . Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, sigma(B) also contributes to regulation of virulence gene expression in L . monocytogenes . These findings strongly suggest that sigma(B) contributes to L . monocytogenes gene expression during infection.

FEMS Microbiol Lett, 2003 Sep 12, 226(1), 107 - 12
The respiratory arsenate reductase from Bacillus selenitireducens strain MLS10; Afkar E et al.; The respiratory arsenate reductase from the Gram-positive, haloalkaliphile, Bacillus selenitireducens strain MLS10 was purified and characterized . It is a membrane bound heterodimer (150 kDa) composed of two subunits ArrA (110 kDa) and ArrB (34 kDa), with an apparent K(m) for arsenate of 34 microM and V(max) of 2.5 micromol min(-1) mg(-1) . Optimal activity occurred at pH 9.5 and 150 g l(-1) of NaCl . Metal analysis (inductively coupled plasma mass spectrometry) of the holoenzyme and sequence analysis of the catalytic subunit (ArrA; the gene for which was cloned and sequenced) indicate it is a member of the DMSO reductase family of molybdoproteins.

FEMS Microbiol Lett, 2003 Sep 12, 226(1), 73 - 7
Bacillus thuringiensis serovar israelensis is highly toxic to the coffee berry borer, Hypothenemus hampei Ferr . (Coleoptera: Scolytidae); Mendez-Lopez I et al.; A native collection of Bacillus thuringiensis strains was screened, once a reliable bioassay technique to assess the toxicity against the coffee berry borer (CBB) first-instar larvae was developed . A first round of bioassays with 170 strains indicated that the great majority of them showed no or very little insecticidal activity and that very few showed significant levels of toxicity . Interestingly, only those strains that had previously been associated with mosquitocidal activity were also toxic to CBB . Qualitative bioassays (using one high dose) were carried out only with those native mosquitocidal strains, corroborating their significant toxicity towards the CBB first-instar larvae . Most of these strains belong to serovar israelensis . In a second approach, strains from the Institut Pasteur type collection, whose mosquitocidal activity had been previously demonstrated, were also subjected to bioassays . Only those strains that showed a comparable protein content in their parasporal crystals to the israelensis type strain also showed high levels of toxicity towards CBB . Finally, an accurate LC(50) was estimated, using purified parasporal crystals from B . thuringiensis serovar israelensis type strain, at 219.5 ng cm(-2) of diet . All the statistical requirements for a reliable estimator were fulfilled . This is the first report of B . thuringiensis serovar israelensis being active against a coleopteran species.

Indian J Exp Biol, 2002 Dec, 40(12), 1378 - 81
Antimicrobial activity of crude extracts from plant parts and corresponding calli of Bixa orellana L; Castello MC et al.; Ethanol extracts from the different parts of B . orellana showed differential antimicrobial activity . It was found that the extracts of in vitro leaves showed maximum activity against Bacillus pumilus followed by the extracts from the roots and hypocotyls . The callus derived from different explants too showed antimicrobial activity . The leaf callus showed maximum activity . The zone of inhibition for the diluted extracts of in vitro hypocotyls and roots and their corresponding calli showed minimum zone of inhibition at concentration 24 mg/ml, whereas the diluted extract of in vitro leaves and leaf derived callus showed minimum zone of inhibition at 16 mg/ml.

Mem Inst Oswaldo Cruz, 2003 Jul, 98(5), 697 - 702 Epub 2003 Sep 08.
Evaluation of the impact of Bacillus thuringiensis serovar israelensis and Temephos, used for the control of Simulium (Chirostilbia) pertinax Kollar, 1832 (Diptera, Simuliidae) on the associated entomofauna, Paraty, state of Rio de Janeiro, Brazil; de Araujo-Coutinho CJ et al.; The study was set up to evaluate the impact of two commercial larvicide formulations, Bacillus thuringiensis serovar israelensis base (Bti) at 15 ppm/1 min and temephos at 0.03 ppm of active ingredient, used to control Simulium pertinax populations, on associated non-target entomofauna occupying the same breeding sites . The experiments were carried out on the Pedra Branca and Muricana rivers, on the slopes of Serra do Mar massif, municipality of Paraty, state of Rio de Janeiro, Brazil . Bti was applied to the river Pedra Branca and temephosto theriver Muricana . On both rivers, treatment and control sections were labeled as such, each one with two observation posts: slow moving water and fast water regions respectively . Artificial substrata was used to evaluate the abundance of associated entomofauna . Attached immature stages of arthropods were removed from both of its surfaces fortnightly . Were collected, from the two rivers, 28 477 specimens of the entomofauna associated with S . pertinax . The families Hydropsychidae, Chironomidae, Bactidae, Simuliidae, Blephariceridae and Megapodagrionidae were represented . These was an impact of temephos on the entomofauna associated with S . pertinax only in Simuliidae and Chironomidae, and to Bti only in Simuliidae . However, the reduction in their numbers was not statistically significant.

J Struct Biol, 2003 Aug, 143(2), 135 - 44
Automated image acquisition and processing using a new generation of 4K x 4K CCD cameras for cryo electron microscopic studies of macromolecular assemblies; Zhang P et al.; We have previously reported the development of AutoEM, a software package for semi-automated acquisition of data from a transmission electron microscope . In continuing efforts to improve the speed of structure determination of macromolecular assemblies by electron microscopy, we report here on the performance of a new generation of 4 K CCD cameras for use in cryo electron microscopic applications . We demonstrate that at 120 kV, and at a nominal magnification of 67000 x, power spectra and signal-to-noise ratios for the new 4 K CCD camera are comparable to values obtained for film images scanned using a Zeiss scanner to resolutions as high as approximately 1/6.5A(-1) . The specimen area imaged for each exposure on the 4 K CCD is about one-third of the area that can be recorded with a similar exposure on film . The CCD camera also serves the purpose of recording images at low magnification from the center of the hole to measure the thickness of vitrified ice in the hole . The performance of the camera is satisfactory under the low-dose conditions used in cryo electron microscopy, as demonstrated here by the determination of a three-dimensional map at 15 A for the catalytic core of the 1.8 MDa Bacillus stearothermophilus icosahedral pyruvate dehydrogenase complex, and its comparison with the previously reported atomic model for this complex obtained by X-ray crystallography.

Tuberculosis (Edinb), 2003, 83(5), 287 - 97
Mycobacterium bovis bacille Calmette-Guerin vaccination of cattle: activation of bovine CD4+ and gamma delta TCR+ cells and modulation by 1,25-dihydroxyvitamin D3; Waters WR et al.; SETTING: 1,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) is a potent modulator of immune responses and may be beneficial in the treatment of tuberculosis . Recent evidence suggest that 1,25(OH)(2)D(3) may affect T-dependent responses in cattle; however, mechanisms by which this vitamin modulates activation of bovine T cells are unclear . OBJECTIVE: Determine the effects of 1,25(OH)(2)D(3) on the expression of CD25, CD44, and CD62L by bovine T cell subsets proliferating in response to antigen stimulation . DESIGN: Antigen-specific recall responses of Mycobacterium bovis bacille Calmette-Guerin (BCG) vaccinated cattle were used as a model system to evaluate effects of 1,25(OH)(2)D(3) on the proliferation and activation of bovine T cell subsets . RESULTS: CD4(+) and gamma delta TCR(+) cells were the predominant T cell subsets responding to soluble crude M . bovis-derived antigens (i.e., purified protein derivative and a BCG whole cell sonicate) by proliferation and activation-induced alterations in phenotype . These subsets exhibited increased CD25 and CD44 mean fluorescence intensity (mfi) and decreased CD62L mfi upon antigen stimulation . Addition of 1,25(OH)(2)D(3) inhibited proliferation of CD4(+) cells and decreased the expression of CD44 on responding (i.e., proliferating) CD4(+) and gamma delta TCR(+) cells . CONCLUSION: These findings suggest that the production of 1,25(OH)(2)D(3) by macrophages within tuberculous lesions would inhibit proliferation and CD44 expression by co-localized CD4(+) and gamma delta TCR(+) cells.

Lett Appl Microbiol, 2003, 37(4), 354 - 9
Isolation and characterization of Bacillus sp . BP-6 LipA, a ubiquitous lipase among mesophilic Bacillus species; Ruiz C et al.; AIMS: The aim of this study was to perform the isolation, cloning and characterization of a lipase from Bacillus sp . BP-6 bearing the features of a biotechnologically important group of enzymes . METHODS AND RESULTS: Strain Bacillus sp . BP-6, showing activity on tributyrin plates, was used for isolation of lipase-coding gene lipA by means of inverse and direct PCR . The complete 633 nucleotide ORF isolated was cloned in Escherichia coli for further characterization . The amino acid sequence of the cloned protein was 98% identical to B . subtilis and B . megaterium lipases, the enzyme also showing similar molecular and biochemical features . CONCLUSIONS: The gene coding for Bacillus sp . BP-6 LipA was found in all mesophilic Bacillus species assayed, indicating its ubiquity in the genus . The cloned enzyme displayed the same properties as those of homologous lipases . SIGNIFICANCE AND IMPACT OF THE STUDY: The overall profile of Bacillus sp . BP-6 LipA was found to be that of a ubiquitous and highly conserved subfamily I.4 bacterial lipase . Previously described lipases within this family have shown to be well suited for biotechnological applications, suggesting that the cloned enzyme could be used accordingly.

J Org Chem, 2003 Sep 19, 68(19), 7298 - 307
Design, synthesis, and evaluation of water-soluble phospholipid analogues as inhibitors of phospholipase C from Bacillus cereus; Franklin CL et al.; The rate of hydrolysis of natural phospholipids by the phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLC(Bc)) follows the order phosphatidylcholine > phosphatidylethanolamine >> phosphatidyl-l-serine . To probe the structural basis for this substrate specificity, a series of water-soluble, nonhydrolyzable substrate analogues were needed so their complexes with the enzyme could be studied via X-ray crystallography and isothermal titration calorimetry (ITC) . Accordingly the water-soluble dithiophospholipids 2-10 having choline, ethanolamine, and l-serine headgroups were synthesized, and the inhibitory activity of each was determined in an assay using 1,2-dihexanoyl-sn-glycero-3-phosphocholine (C6PC) as the monomeric substrate . The 1,2-dibutanoyl dithiophosphocholine 2 was a weak inhibitor, whereas the related 1,2-dipentanoyl dithiophosphocholine 3 and the ethylene glycol dithiophosphocholines 4 and 5 were moderate inhibitors . The 1,2-omega-hydroxydiacyl dithiophosphocholines 6 and 7 were potent inhibitors, while the related compound 8, which had shorter acyl side chains, was a weak inhibitor . The dithiophosphoethanolamine 9 was a modest inhibitor, whereas the dithiophospho-l-serine 10 was a somewhat weaker inhibitor . Overall, the phospholipid analogues had increasing K(i) values according to the order 2 << 10 < 3 < 4 approximately 5 approximately 8 < 9 << 6 << 7 and increasing solubility according to the sequence 5 approximately 7 < 4 approximately 6 approximately 9 < 3 < 10 < 8 < 2.

J Biol Chem, 2003 Nov 28, 278(48), 47545 - 53 Epub 2003 Sep 10.
Phenol hydroxylase from Bacillus thermoglucosidasius A7, a two-protein component monooxygenase with a dual role for FAD; Kirchner U et al.; A novel phenol hydroxylase (PheA) that catalyzes the first step in the degradation of phenol in Bacillus thermoglucosidasius A7 is described . The two-protein system, encoded by the pheA1 and pheA2 genes, consists of an oxygenase (PheA1) and a flavin reductase (PheA2) and is optimally active at 55 degrees C . PheA1 and PheA2 were separately expressed in recombinant Escherichia coli BL21(DE3) pLysS cells and purified to apparent homogeneity . The pheA1 gene codes for a protein of 504 amino acids with a predicted mass of 57.2 kDa . PheA1 exists as a homodimer in solution and has no enzyme activity on its own . PheA1 catalyzes the efficient ortho-hydroxylation of phenol to catechol when supplemented with PheA2 and FAD/NADH . The hydroxylase activity is strictly FAD-dependent, and neither FMN nor riboflavin can replace FAD in this reaction . The pheA2 gene codes for a protein of 161 amino acids with a predicted mass of 17.7 kDa . PheA2 is also a homodimer, with each subunit containing a highly fluorescent FAD prosthetic group . PheA2 catalyzes the NADH-dependent reduction of free flavins according to a Ping Pong Bi Bi mechanism . PheA2 is structurally related to ferric reductase, an NAD(P)H-dependent reductase from the hyperthermophilic Archaea Archaeoglobus fulgidus that catalyzes the flavin-mediated reduction of iron complexes . However, PheA2 displays no ferric reductase activity and is the first member of a newly recognized family of short-chain flavin reductases that use FAD both as a substrate and as a prosthetic group.

Biotechnol Lett, 2003 Jul, 25(14), 1143 - 6
Kinetics of beta-mannanase fermentation by Bacillus licheniformis; Feng YY et al.; Bacterial growth, konjac powder utilization and beta-mannanase production by Bacillus licheniformis NK-27 in batch fermentation were used to develop a model of the process . The optimal set of parameters was estimated by fitting the model to experimental data . The results predicted by the model were in good agreement with the experimental data . NOMENCLATURE: fs, fraction parameter; Kp, constant in Equation (6) (g l(-1)); Ks, Monod constant for bacteria growth (g l(-1)); m, maintenance coefficient (g g(-1) h(-1)); P, beta-mannanase concentration (g 1(-1)); qm, constant in Equation (6) (h(-1)); S, substrate concentration (g l(-1)); S0, initial substrate concentration (g l(-1)); Si, insoluble substrate concentration (g l(-1)); Ss, soluble substrate concentration (g l(-1)); t, fermentation time (h); tL, lag time (h); mu, specific growth rate (h(-1)); mu(max), maximum specific growth rate (h(-1)); X, biomass concentration (g l(-1)); YP/S, beta-mannanase yield on carbon substrate (g g(-1)); YX/S, biomass yield on carbon substrate (g g(-1)).

Comp Biochem Physiol C Toxicol Pharmacol, 2003 Aug, 135(4), 405 - 14
Effects of Bacillus thuringiensis toxin on hepatic lipid peroxidation and free-radical scavengers in rats given alpha-tocopherol or acetylsalicylate; Shaban NZ et al.; The effect of Dipel (D), a Bacillus thuringiensis-based bioinsecticide, on hepatic antioxidant enzyme activities and lipid peroxidation in rat liver was investigated . Administration of D in a dose of 1 mg/100 g body mass for 4 successive days increased the activities of glutathione peroxidase (GPx), glutathione reductase (GR) and the level of malondialdehyde (MDA) in rat hepatocytes . The activity of superoxide dismutase (SOD) and glutathione (GSH) level were decreased . Administration of D in rats pretreated with alpha-tocopherol (alphaT) or acetylsalicylic acid (ASA) decreased the activities of GPx, GR and MDA levels, while the GSH level was increased compared with rats treated with D alone . The SOD activity was increased in rats pretreated with alphaT before D, but decreased on pretreatment with ASA, compared with rats treated with D alone . The results indicated that D induced oxidative stress in rat liver that has been protected by prior administration of alphaT or ASA.

Br Poult Sci, 2003 Jul, 44(3), 438 - 49
Supplements of transgenic malt or grain containing (1,3-1,4)-beta-glucanase increase the nutritive value of barley-based broiler diets to that of maize; Von Wettstein D et al.; 1 . A diet with addition to normal barley of malt from transgenic barley expressing a protein engineered, thermotolerant Bacillus (1,3-1,4)-beta-glucanase during germination has previously been demonstrated to provide a broiler chicken weight gain comparable to maize diets . It also reduced dramatically the number of birds with adhering sticky droppings, but did not entirely eliminate sticky droppings . One of the objectives of the broiler chicken trials reported here was to determine if higher concentrations of transgenic malt could alleviate the sticky droppings . 2 . Another aim was to investigate the feasibility of using mature transgenic grain containing the thermotolerant (1,3-1,4)-beta-glucanase as feed addition and to compare diets containing transgenic grain to a diet with the recommended amount of a commercial beta-glucanase-based product . 3 . Inclusion of 75 or 151 g/kg transgenic malt containing 4.7 or 98 mg/kg thermotolerant (1,3-1,4)-beta-glucanase with 545 or 469 g/kg non-transgenic barley instead of maize yielded a weight gain in Cornish Cross broiler chickens indistinguishable from presently used maize diets . The gene encoding the enzyme is expressed in the aleurone with a barley alpha-amylase gene promoter and the enzyme is synthesised with a signal peptide for secretion into the endosperm of the malting grain . 4 . Equal weight gain was achieved, when the feed included 39 g/kg transgenic barley grain {containing 66 mg/kg thermotolerant (1,3-1,4)-beta-glucanase} and 581 g/kg non-transgenic barley instead of maize . In this case, the gene encoding the enzyme has been expressed with the D-hordein gene (Hor3-1) promoter during grain maturation . The enzyme is synthesised as a precursor with a signal peptide for transport through the endoplasmic reticulum and targeted into the storage vacuoles . Deposition of the enzyme in the prolamin storage protein bodies of the endosperm protects it from degradation during the programmed cell death of the endosperm in the final stages of grain maturation and provides extraordinary heat stability . The large amount of highly active (1,3-1,4)-beta-glucanase in the mature grain allowed the reduction of the transgenic grain ingredient to 0.2 g/kg diet, thus making the ingredient comparable to that of the trace minerals added to standard diets . 5 . A direct comparison using transgenic grain supplement at the level of 1 g/kg of feed with the standard recommended addition of the commercial enzyme preparation Avizyme 1100 at 1 g/kg yielded equal weight gain, feed consumption and feed efficiency in birds fed a barley-based diet . 6 . The production of sticky droppings characteristic of broilers fed on barley diets was avoided with all 9 experimental diets and reduced to the level observed with a standard maize diet by supplementation with transgenic barley . 7 . The excellent growth and normal survival of the 400 broilers tested on barley diets supplemented with transgenic grain or malt showed the grain and malt not to be toxic . 8 . The barley feed with added transgenic grain or malt containing thermotolerant (1,3-1,4)-beta-glucanase provides an environmentally friendly alternative to enzyme additives, as it uses photosynthetic energy for production of the enzyme in the grain and thus avoids use of non-renewable energy for fermentation . The deposition of the enzyme in the protein bodies of the grain in the field makes coating procedures for stabilisation of enzyme activity superfluous . 9 . Barley feed with the small amount of transgenic grain as additive to normal barley provides an alternative for broiler feed in areas where grain maize cannot be grown for climatic reasons or because of unsuitable soil and thus has to be imported.

J Biol Chem, 2003 Nov 21, 278(47), 46961 - 7 Epub 2003 Sep 08.
Induction of p38 mitogen-activated protein kinase reduces early endosome autoantigen 1 (EEA1) recruitment to phagosomal membranes; Fratti RA et al.; Mycobacterium tuberculosis survives in the infected host by parasitizing macrophages in which the bacillus resides in a specialized phagosome sequestered from the phagolysosomal degradative pathway . Here we report a role of the stress-induced p38 mitogen-activated protein kinase (p38 MAPK) in the component of M . tuberculosis phagosome maturation arrest that has been linked previously to the reduced recruitment of the endosomal and phagosomal membrane-tethering molecule called early endosome autoantigen 1 (EEA1; Fratti, R . A., Backer, J . M., Gruenberg, J., Corvera, S., and Deretic, V . (2001) J . Cell Biol . 154, 631-644) . A pharmacological inhibition of M . tuberculosis var . bovis Bacillus Calmette-Guerin-induced p38 MAPK activity caused a marked increase in EEA1 colocalization with mycobacterial phagosomes . Consistent with the increase in EEA1 association and its role in phagosomal maturation, the pharmacological block of p38 activity caused phagosomal acidification and enrichment of the late endocytic markers lysobisphosphatidic acid and CD63 (lysosomal integral membrane protein 1) on mycobacterial phagosomes . A negative regulatory role of p38 MAPK activation in phagosome maturation was further demonstrated by converse experiments with latex bead phagosomes . Artificial activation of p38 MAPK caused a decrease in EEA1 colocalization with model latex bead phagosomes, which normally acquire EEA1 and subsequently mature into the phagolysosome . These findings show that p38 MAPK activity contributes to the arrest of M . tuberculosis phagosome maturation and demonstrate a negative regulatory role of p38 in phagolysosome biogenesis.

Biochemistry, 2003 Sep 16, 42(36), 10545 - 53
Do enzymes change the nature of transition states? Mapping the transition state for general acid-base catalysis of a serine protease; Bott RR et al.; The properties of the transition state for serine protease-catalyzed hydrolysis of an amide bond were determined for a series of subtilisin variants from Bacillus lentus . There is no significant change in the structure of the enzyme upon introduction of charged mutations S156E/S166D, suggesting that changes in catalytic activity reflect global properties of the enzyme . The effect of charged mutations on the pK(a) of the active site histidine-64 N(epsilon)(2)-H was correlated with changes in the second-order rate constant k(cat)/K(m) for hydrolysis of tetrapeptide anilides at low ionic strength with a Bronsted slope alpha = 1.1 . The solvent isotope effect (D)2(O)(k(cat)/K(m))(1) = 1.4 +/- 0.2 . These results are consistent with a rate-limiting breakdown of the tetrahedral intermediate in the acylation step with hydrogen bond stabilization of the departing amine leaving group . There is an increase in the ratio of hydrolysis of succinyl-Ala-Ala-Pro-Phe-anilides for p-nitroaniline versus aniline leaving groups with variants with more basic active site histidines that can be described by the interaction coefficient p(xy) = delta beta(lg)/delta pK(a) (H64) = 0.15 . This is attributed to increased hydrogen bonding of the active site imidazolium N-H to the more basic amine leaving group as well as electrostatic destabilization of the transition state . A qualitative characterization of the transition state is presented in terms of a reaction coordinate diagram that is defined by the structure-reactivity parameters.

J Leukoc Biol, 2003 Oct, 74(4), 523 - 30 Epub 2003 Jul 01.
Surfactant protein A enhances Mycobacterium avium ingestion but not killing by rat macrophages; Lopez JP et al.; Mycobacterium avium complex (MAC) is a significant cause of opportunistic infection in patients with acquired immunodeficiency syndrome . Although the major route of entry of MAC is via the gastrointestinal tract, MAC can infect humans through the respiratory tract and eventually encounter alveolar macrophages within the lung . Once in the lung, MAC can potentially interact with surfactant protein A (SP-A), an important component of the pulmonary innate-immune response . Previous work on other pulmonary pathogens including Mycobacterium bovis Bacillus Calmette-Guerin (BCG) suggests that SP-A participates in promoting efficient clearance of these organisms by alveolar macrophages . In the present study, we investigated the role of SP-A in clearance of MAC by cultured rat macrophages . SP-A bound to MAC organisms and enhanced the ingestion of the mycobacteria by macrophages . Infection of macrophages with SP-A-MAC complexes induced the production of nitric oxide (NO) and tumor necrosis factor-alpha . However, intracellular survival of MAC was not altered by preopsonization with SP-A . In addition, inhibitors of inducible NO synthase did not alter MAC clearance . These results suggest that SP-A can bind to and enhance the uptake of MAC by alveolar macrophages, similar to previous findings with BCG and Mycobacterium tuberculosis.However, unlike BCG and other pulmonary pathogens that are cleared effectively in the presence of SP-A via a NO-dependent pathway, macrophage-mediated clearance of MAC is not enhanced by SP-A.

Appl Environ Microbiol, 2003 Sep, 69(9), 5343 - 53
Introduction of Culex toxicity into Bacillus thuringiensis Cry4Ba by protein engineering; Abdullah MA et al.; Bacillus thuringiensis mosquitocidal toxin Cry4Ba has no significant natural activity against Culex quinquefasciatus or Culex pipiens (50% lethal concentrations {LC(50)}, >80,000 and >20,000 ng/ml, respectively) . We introduced amino acid substitutions in three putative loops of domain II of Cry4Ba . The mutant proteins were tested on four different species of mosquitoes, Aedes aegypti, Anopheles quadrimaculatus, C . quinquefasciatus, and C . pipiens . Putative loop 1 and 2 exchanges eliminated activity towards A . aegypti and A . quadrimaculatus . Mutations in a putative loop 3 resulted in a final increase in toxicity of >700-fold and >285-fold against C . quinquefasciatus (LC(50) congruent with 114 ng/ml) and C . pipiens (LC(50) 37 ng/ml), respectively . The enhanced protein (mutein) has very little negative effect on the activity against Anopheles or AEDES: These results suggest that the introduction of short variable sequences of the loop regions from one toxin into another might provide a general rational design approach to enhancing B . thuringiensis Cry toxins.

Appl Environ Microbiol, 2003 Sep, 69(9), 5269 - 74
Diversity of Bacillus thuringiensis strains from Latin America with insecticidal activity against different mosquito species; Ibarra JE et al.; The characterization of selected Bacillus thuringiensis strains isolated from different Latin America countries is presented . Characterization was based on their insecticidal activity against Aedes aegypti, Culex quinquefasciatus, and Anopheles albimanus larvae, scanning electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and plasmid profiles as well as PCR analysis using novel general and specific primers for cry and cyt genes encoding proteins active against mosquitoes (cyt1, cyt2, cry2, cry4A, cry4B, cry10, cry11, cry17, cry19, cry24, cry25, cry27, cry29, cry30, cry32, cry39, and cry40) . Strains LBIT315, LBIT348, and IB604 showed threefold higher mosquitocidal activity against A . aegypti and C . quinquefasciatus larvae than B . thuringiensis subsp . israelensis and displayed high similarities with the B . thuringiensis subsp . israelensis used in this study with regard to protein and plasmid profiles and the presence of cry genes . Strain 147-8906 has activity against A . aegypti similar to that of B . thuringiensis subsp . israelensis but has different protein and plasmid profiles . This strain, harboring cry11, cry30, cyt1, and cyt2 genes, could be relevant for future resistance management interventions . Finally, the PCR screening strategy presented here led us to identify a putative novel cry11B gene.

Appl Environ Microbiol, 2003 Sep, 69(9), 5207 - 11
Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene; Song F et al.; A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1I-type gene fragments . Amplification products were digested with the Bsp119I and BanI enzymes, and four kinds of known cry1I-type genes were successfully identified . The results showed that cry1I-type genes appeared in 95 of 115 B . thuringiensis isolates and 7 of 13 standard strains . A novel cry1I-type gene was found in one standard strain and six isolates . The novel cry1I gene was cloned from B . thuringiensis isolate Btc007 and subcloned into vector pET-21b . Then it was overexpressed in Escherichia coli BL21(DE3) . The expressed product was shown to be toxic to the diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia furnacalis), and soybean pod borer (Leguminivora glycinivorella) . However, it was not toxic to the cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), or elm leaf beetle (Pyrrhalta aenescens) in bioassays . Subsequently, the Cry protein encoded by this novel cry gene was designated Cry1Ie1 by the B . thuringiensis delta-endotoxin nomenclature committee.

Gene, 2003 Aug 14, 313, 103 - 9
Rapid and efficient transposon mutagenesis of Bartonella henselae by transposome technology; Riess T et al.; Molecular genetics are difficult to perform in Bartonella henselae, the causative agent of cat scratch disease and the vasculoproliferative disorders bacillary angiomatosis and bacillary peliosis . To elucidate the underlying bacterial pathogenic mechanisms, genetic manipulation of B . henselae is the method of choice . We describe how to perform transposon mutagenesis in B . henselae using transposome technology . B . henselae mutants revealed by this technique showed random transpositional insertion into the chromosome . In contrast to transposon mutagenesis by conjugational transfer, transposome technology allows transposon mutagenesis of early passaged Bartonella spp . with approximately 100-fold higher efficiency . The results show that transposome technique is a rapid, efficient and simple method to generate transposon mutants of B . henselae.

Acta Pharmacol Sin, 2003 Sep, 24(9), 907 - 12
Mycobacterium bovis bacille Calmette-Guérin (BCG) enhances human beta-defensin-1 gene transcription in human pulmonary gland epithelial cells; Zhu BD et al.; AIM: To examine the stimulatory effect of bacille Calmette-Gu rin (BCG) cell wall components on human beta-defensin-1 (hBD-1) gene expression and analyze the response element in the 5'-flanking region of the gene . METHODS: BCG cell wall proteins were fractionated by Sephadex G-150 chromatography . Using reverse-transcription polymerase chain reaction (RT-PCR) and Northern hybridization analysis, hBD-1 mRNA expression was detected in a human pulmonary gland epithelial cell line SPC-A-1 cells . Progressive deletions of 5'-flanking region of hBD-1 gene were produced by PCR and ligated into promoterless chloramphenicol acetyltransferase (CAT) expression plasmid to construct pCAT reporter plasmids . Reporter gene expression was determined by ELISA . RESULTS: There was an obvious enhancement of hBD-1 mRNA expression after stimulation with heat-inactivated BCG whole cells (50 mg/L), or the cell wall components with a molecular weight of 18-30 kDa (3 mg/L) for 8 h . The upstream sequence between -314 bp and +54 bp had the inducible activity by BCG, which contained CCAAT/enhancer binding protein-beta (C/EBP beta), activator protein-1 (AP-1), and CP2 cis element . CONCLUSION: BCG cell wall components (18-30 kDa) can stimulate hBD-1 mRNA expression in pulmonary gland epithelial cells . The sequence (-314/+54) containing C/EBP beta, AP-1, and CP2 binding sites in the upstream of hBD-1 is involved in this induction.

Lancet Infect Dis, 2003 Sep, 3(9), 578 - 90
Latent tuberculosis: mechanisms of host and bacillus that contribute to persistent infection; Tufariello JM et al.; Most people infected with Mycobacterium tuberculosis contain the initial infection and develop latent tuberculosis . This state is characterised by evidence of an immune response against the bacterium (a positive tuberculin skin test) but no signs of active infection . It can be maintained for the lifetime of the infected person . However, reactivation of latent infection occurs in about 10% of infected individuals, leading to active and contagious tuberculosis . An estimated 2 billion people worldwide are infected with M tuberculosis--an enormous reservoir of potential tuberculosis cases . The establishment and reactivation of latent infection depend on several factors, related to both host and bacterium . Elucidation of the host immune mechanisms that control the initial infection and prevent reactivation has begun . The bacillus is well adapted to the human host and has a range of evasion mechanisms that contribute to its ability to avoid elimination by the immune system and establish a persistent infection . We discuss here current understanding of both host and bacterial factors that contribute to latent and reactivation tuberculosis.

Drugs R D, 2003, 4(5), 293 - 9
Pharmacokinetic interaction of tetracycline with centchroman in healthy female volunteers; Khurana M et al.; OBJECTIVES: We aimed to investigate the effect of tetracycline coadministration, with and without lactic acid bacillus spores supplementation, on the pharmacokinetics of centchroman, a nonsteroidal oral contraceptive, in healthy female volunteers . PARTICIPANTS AND METHODS: The study was a single-centre, single-blinded, randomised, parallel treatment study in healthy female subjects of reproductive age randomised to two groups (11 subjects in each group) . On day 1, subjects were given either a single oral dose of centchroman 30 mg with tetracycline 250 mg (group A) or a single dose of centchroman 30 mg, tetracycline 250 mg and one tablet containing 60 million lactic acid bacillus spores (group B) . Tetracycline (250 mg three times daily) and lactic acid bacillus spores (one tablet three times daily) were continued for 3 days . Serial blood samples were collected and analysed by high performance liquid chromatography . The pharmacokinetic parameters were compared with the control data reported previously from this laboratory . RESULTS: Coadministration of tetracycline yielded significantly higher maximum plasma concentrations (C(max)) {35%} and a shorter time to reach C(max) (t(max)) values for centchroman (42%) than those obtained in the control group of females (p < 0.05) . Inclusion of lactic acid bacillus spores in the regimen resulted in similar effects with increased C(max) (47%) and area under the concentration-time curve from time zero to infinity (34%) of centchroman (p < 0.05) with a significant decrease in t(max) . Other parameters such as half-life, apparent clearance, apparent volume of distribution and mean residence time of centchroman were not affected by either of the treatments . CONCLUSIONS: The apparent effects of either of the regimens on centchroman pharmacokinetics seem to be of little clinical relevance in terms of increased rate or extent of availability . It can be concluded that this tetracycline-containing regimen is unlikely to alter the contraceptive efficacy of centchroman in humans.

Int J Vitam Nutr Res, 2003 Jul, 73(4), 235 - 44
In vitro effects of 1,25-dihydroxyvitamin D3 on interferon-gamma and tumor necrosis factor-alpha secretion by blood leukocytes from young and adult cattle vaccinated with Mycobacterium bovis BCG; Nonnecke BJ et al.; Interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) are critical in the development of an effective immune response . Vitamin D, essential in short-term calcium homeostasis and recently shown to modulate proliferation and function of blood mononuclear cells from adult dairy cattle, may be an effective modulator of the calf's immune system . Effects of antigen sensitization and 1,25-dihydroxyvitamin D3{1,25-(OH)2D3} on cytokine secretion by cells from calves vaccinated with Bacille Calmette-Guerin (BCG) were examined . One-week-old dairy calves (n = 6) and yearling heifers (n = 4) were vaccinated concurrently with BCG and boosted six weeks later . Ten weeks after primary vaccination, cells from vaccinated calves and adults, and nonvaccinated, age-matched calves (n = 4) were evaluated in vitro for their capacity to produce IFN-gamma and TNF-alpha . Cells were stimulated with pokeweed mitogen (PWM) or recall antigen {Mycobacterium bovis-derived purified protein derivative (PPD)} in the presence of 0, 0.1, 1.0, and 10 nM of 1,25-(OH)2D3 for 20, 44, and 68 hours, respectively . IFN-gamma and TNF-alpha concentrations in culture supernatants harvested at these times were quantified by enzyme-linked immunosorbent assay (ELISA) . PPD-induced IFN-gamma and TNF-alpha responses of cells from vaccinated calves and adults were greater than responses of autologous unstimulated cells . In contrast, PPD-specific responses of calf and adult cells collected immediately before primary vaccination were substantially lower and comparable to responses in resting (i.e., unstimulated) cultures . At ten weeks, the PPD-specific response of vaccinates exceeded the response of nonvaccinated calves; however, responses of vaccinated calves were more vigorous than corresponding responses of vaccinated adults . Incubation period also influenced the magnitude of both IFN-gamma and TNF-alpha, responses in PPD- and PWM-stimulated cultures . Effects of 1,25-(OH)2D3 on antigen-induced secretion of IFN-gamma and TNF-alpha were marginal . Only IFN-gamma responses of vaccinated adults were affected by 1,25-(OH)2D3 . Vitamin D caused a concentration-dependent decrease in IFN-gamma response and an increase in TNF-alpha response in PWM-stimulated cultures . These results indicate that animal maturity (i.e., age) and antigenic experience affect IFN-gamma and TNF-alpha secretion by bovine leukocytes and suggest that 1,25-(OH)2D3 can alter secretion of both cytokines under specific conditions of culture.

Biosci Biotechnol Biochem, 2003 Aug, 67(8), 1719 - 24
Inhibition of beta-fructofuranosidases and alpha-glucosidases by synthetic thio-fructofuranoside; Kiso T et al.; A synthetic beta-thio-fructofuranoside of mercaptoethanol inhibited not only beta-fructofuranosidases but also alpha-glucosidases . The compound was hardly hydrolyzed by the glycosidases . The thio-fructoside competitively inhibited beta-fructofuranosidases from Aspergillus niger, Candida sp., and Saccharomyces cerevisiae, but not Arthrobacter beta-fructofuranosidase at all . Sucrase activity of rat intestinal sucrase/isomaltase complex was also suppressed in the presence of the thio-fructoside . The thio-fructoside showed noncompetitive inhibition toward maltase activity of the rat intestinal enzyme complex and Saccharomyces sp . alpha-glucosidase . Inhibition against the Bacillus stearothermophilus alpha-glucosidase, Rhizopus glucoamylase, and porcine kidney trehalase were more slight than that against these two alpha-glucosidases.

FEMS Microbiol Lett, 2003 Aug 29, 225(2), 249 - 56
Phylogenetic analysis and confirmation of the endospore-forming nature of Pasteuria penetrans based on the spo0A gene; Trotter JR et al.; Pasteuria penetrans is an obligate parasite of plant parasitic nematodes and has yet to be grown in vitro . We have cloned the pivotal sporulation gene, spo0A, which is the first whole gene yet to come from this organism . Partial spo0A sequences were also obtained from the related bacteria, Pasteuria ramosa and Alicyclobacillus acidocaldarius . Phylogenetic analyses using the spo0A sequence data from this and previous studies confirmed the closeness of the genera Pasteuria and members of the supergenus Bacillus . A segment of the spo0A gene was also used to show that genetic heterogeneity exists within and between populations of P . penetrans . This may explain, partly at least, the variability of P . penetrans as a biological control agent of nematodes.

J Virol Methods, 2003 Sep, 112(1-2), 153 - 6
Sodium sulphite yields improved DNA of higher stability for PCR detection of Citrus yellow mosaic virus from citrus leaves; Baranwal VK et al.; Citrus yellow mosaic virus (CYMV), a non-enveloped bacilliform DNA virus causes a severe mosaic disease in sweet oranges in India . CYMV is weakly immunogenic, thus serodiagnosis is not a preferred method for its detection . As an alternative a rapid and reliable detection protocol by polymerase chain reaction (PCR) was developed . However, high levels of polyphenolics and tannins in citrus leaves generally interfered with obtaining good quality DNA, and thus affected the reliable detection of virus by PCR . Consequently, we evaluated the addition of sodium sulphite to a DNA extraction protocol used previously and compared the two methods with a commercially available plant DNeasy Kit (Qiagen) . The addition of sodium sulphite improved the yield, quality and stability of DNA . The CYMV DNA was not only amplified at lower template DNA concentration, but also provided better DNA yields . In addition, the sodium sulphite extracted DNA survived at various temperatures much longer than those extracted without addition of sodium sulphite or with the commercial kit . The amplified product of CYMV DNA was cloned, sequenced and found to have 89% sequence identity with the only other sequenced Indian isolate of CYMV.

Biochemistry, 2003 Sep 9, 42(35), 10482 - 9
Molecular basis for Bacillus thuringiensis Cry1Ab toxin specificity: two structural determinants in the Manduca sexta Bt-R1 receptor interact with loops alpha-8 and 2 in domain II of Cy1Ab toxin; Gomez I et al.; The identification of epitopes involved in Cry toxin-receptor interaction could provide insights into the molecular basis of insect specificity and for designing new toxins to overcome the potential problem of insect resistance . In previous works, we determined that the Manduca sexta Cry1A cadherin-like receptor (Bt-R(1)) interacts with Cry1A toxins through epitope (865)NITIHITDTNN(875) and by loop 2 of domain II in the toxin (Gomez, I., Miranda-Rios, J., Rudino-Pinera, E., Oltean, D . I., Gill, S . S., Bravo, A., and Soberon, M . (2002) J . Biol . Chem . 277, 30137-30143.) . In this work, we narrowed to 12 amino acids a previously identified Bt-R(1) 66 amino acids epitope (Dorsch, J . A., Candas, M., Griko, N . B., Maaty, W . S . A., Midbo, E . G., Vadlamudi, R . K., and Bulla, L . A., Jr . (2002) Insect Biochem . Mol . Biol . 32, 1025-1036) and identified loop alpha-8 of Cry1Ab domain II as its cognate binding epitope . Two amino acid Bt-R(1) toxin binding regions of 70 residues, one comprised of residues 831-900 containing the (865)NITIHITDTNN(875) epitope (TBR1) and the other comprised of residues 1291-1360 (TBR2) were cloned by RT-PCR and produced in Escherichia coli . Cry1A toxins bind with the two TBR regions in contrast with the nontoxic Cry3A toxin . The loop 2 synthetic peptide competed with the binding of Cry1Ab toxin to both TBR regions in contrast to the alpha-8 synthetic peptide that only competed with Cry1Ab binding to TBR2 . Western blots and competition ELISA analysis showed that the Cry1Ab loop 2 RR368-9EE mutant did not show observable binding to TBR1 but still bound the TBR2 peptide . This result suggests that loop alpha-8 interacts with the TBR2 region . Competition ELISA analysis of Cry1Ab binding to the two TBR peptides revealed that the toxin binds the TBR1 region with 6-fold higher affinity than the TBR2 region . The amino acid sequence of TBR2 involved on Cry1Ab interaction was narrowed to 12 amino acids, (1331)IPLPASILTVTV(1342), by using synthetic peptides as competitors for Cry1Ab binding to Bt-R(1) . Our results show that the specificity of Cry1A involves at least two structural determinants on both molecules.

Water Res, 2003 Oct, 37(17), 4231 - 5
Removal of metal ions from aqueous solution by polysaccharide produced from Bacillus firmus; Salehizadeh H et al.; This paper deals with adsorption of Pb, Cu and Zn on the polysaccharide produced by Bacillus firmus . The adsorption of metal ions was significantly affected by the initial pH of solution, initial metal ion and polysaccharide concentrations, and presence of other ions in solution . At optimum pH, the uptakes of Pb, Cu and Zn were 98.3%, 74.9% and 61.8%, respectively . The metal ions removal was lower at neutral and generally the initial adsorption rate was rapid and reached equilibrium after 10 min . The process of uptake obeys both Langmuir and Freundlich isotherms.

Mikrobiol Z, 2003 May-Jun, 65(3), 14 - 20
{Effect of mineral nutrition sources on the growth and toxin formation of the entomopathogenic bacteria Bacillus thuringiensis}; Drehval' OA et al.; Mineral composition of culture medium has been studied for its effect on the processes of growth, spore-and-toxin formation of entomopathogenic bacteria Bacillus thuringiensis . It has been shown that salts Na2SO4, Na2SiO3.9H2O, MgSO4.7H2O increase the yield of biomass and endotoxin; FeSO4.7H2O, CaCl2, MnSO4.4H2O increase only the yield of biomass; CdSO4.7H2O and CoCl2.6H2O--inhibit the processes of spore-and-toxin formation, but increase the cell titre . A complex of mineral salts has been found which takes positive effect on the biomass accumulation, delta-endotoxin synthesis and insecticide activity of the studied bacteria . It is shown that the drug "Gumat" besides other elements of the culture medium completely provides bacteria with necessary mineral compounds.

J Biol Chem, 2003 Nov 14, 278(46), 45594 - 602 Epub 2003 Aug 27.
Resistance to a bacterial toxin is mediated by removal of a conserved glycosylation pathway required for toxin-host interactions; Griffitts JS et al.; Crystal (Cry) proteins made by the bacterium Bacillus thuringiensis are pore-forming toxins that specifically target insects and nematodes and are used around the world to kill insect pests . To better understand how pore-forming toxins interact with their host, we have screened for Caenorhabditis elegans mutants that resist Cry protein intoxication . We find that Cry toxin resistance involves the loss of two glycosyltransferase genes, bre-2 and bre-4 . These glycosyltransferases function in the intestine to confer susceptibility to toxin . Furthermore, they are required for the interaction of active toxin with intestinal cells, suggesting they make an oligosaccharide receptor for toxin . Similarly, the bre-3 resistance gene is also required for toxin interaction with intestinal cells . Cloning of the bre-3 gene indicates it is the C . elegans homologue of the Drosophila egghead (egh) gene . This identification is striking given that the previously identified bre-5 has homology to Drosophila brainiac (brn) and that egh-brn likely function as consecutive glycosyltransferases in Drosophila epithelial cells . We find that, like in Drosophila, bre-3 and bre-5 act in a single pathway in C . elegans . bre-2 and bre-4 are also part of this pathway, thereby extending it . Consistent with its homology to brn, we demonstrate that C . elegans bre-5 rescues the Drosophila brn mutant and that BRE-5 encodes the dominant UDP-GlcNAc:Man GlcNAc transferase activity in C . elegans . Resistance to Cry toxins has uncovered a four component glycosylation pathway that is functionally conserved between nematodes and insects and that provides the basis of the dominant mechanism of resistance in C . elegans.

Clin Infect Dis, 2003 Sep 1, 37(5), 628 - 33 Epub 2003 Aug 07.
American cutaneous leishmaniasis, lepromatous leprosy, and pulmonary tuberculosis coinfection with downregulation of the T-helper 1 cell response; Delobel P et al.; Cutaneous leishmaniasis, leprosy, and tuberculosis are caused by intracellular pathogens whose development depends on impaired cell-mediated immunity . We report an exceptional triple association of American cutaneous leishmaniasis, lepromatous leprosy, and pulmonary tuberculosis in a man with no recognized immunodeficiency . Normal immunological assessment of the interferon-gamma pathway does not support the hypothesis of a genetic defect in any of the genes involved in the T helper (Th)-1 cytokine cascade in this patient . Unresponsiveness to interleukin (IL)-12 of his T cells after stimulation with Leishmania guyanensis, Mycobacterium bovis bacille Calmette-Guerin, and Mycobacterium leprae antigens suggested the inability to mount an appropriate Th cell response to upregulate the IL-12 receptor expression.

J Prosthet Dent, 2003 Sep, 90(3), 282 - 8
Calcium hypochlorite as a disinfecting additive for dental stone; Twomey JO et al.; STATEMENT OF PROBLEM: Dental casts come into direct contact with impression materials and other items that are contaminated by saliva and blood from a patient's mouth, leaving the casts susceptible to cross-contamination . Topical methods of disinfecting casts are difficult to control, while immersion methods are potentially destructive . Thus, an additional method to control cross-contamination between patients and laboratory personnel is needed . PURPOSE: This study was undertaken in an attempt to develop a dental stone with disinfecting properties and adequate compressive and tensile strengths . MATERIAL AND METHODS: Calcium hypochlorite {Ca(OCl)(2)} in aqueous solution in concentrations from 0 to 1.5% was tested as a disinfecting additive to type V dental stone . The compressive and tensile strength properties of the modified stone were measured (MPa) using a universal testing machine at a consistency similar to unmodified stone . Strength data were analyzed by 1-way ANOVA and post hoc Tukey-Kramer procedure (alpha < or =.05) . To measure the disinfecting ability, the effect on Bacillis subtilis bacteriophage phi29 was tested in triplicate to find the minimum concentration at which no phage was detected . Additionally, 3 impressions were disinfected with CaviCide, and 3 impressions rinsed in water served as controls . RESULTS: In general, the effect of adding the disinfectant to the stone was a decrease in strength . Exceptions were the dry compressive strength, for which there was a significant increase in strength (P=.048) at 0.5%, and the wet compressive and wet tensile strength, which showed no significant difference between the 1.5% and the control . When Ca(OCl)(2) was added at the concentration 0.5% (2765 ppm available chlorine), the gypsum had acceptable mechanical properties; dry compressive strength was 78.86 +/- 4.12 MPa, and dry tensile strength was 10.64 +/- 1.27 MPa, compared to control values of 67.85 +/- 6.28 and 13.41 +/- 1.24 MPa, respectively . At concentrations of 0.3% and higher (36 1650 ppm of available chlorine), calcium hypochlorite was able to completely inactivate phi29 . CONCLUSION: It is possible to prepare a type V dental stone that contains a disinfectant, has adequate mechanical properties, and will reduce numbers of residual microorganisms . For example, stone mixed with water containing 0.5% Ca(OCl)(2) meets these criteria.

Immunol Lett, 2003 Sep 8, 88(3), 175 - 84
Skewing of the Th1/Th2 responses in mice due to variation in the level of expression of an antigen in a recombinant BCG system; Dhar N et al.; In spite of rapid developments in the study of mycobacteria during the last two decades, tuberculosis (TB) has maintained its status as the leading killer among all infectious diseases . Extensive evidence exists to support a central role for a T-helper type 1 (Th1) immune response for protection against TB in mice and humans . Bacille Calmette-Guerin (BCG), the only vaccine against TB, although not perfect in its ability to protect against the adult form of TB, is a strong inducer of Th1 responses and is being increasingly used as a delivery vehicle for the presentation of foreign antigens to the immune system . It has been proposed that expression of immunodominant antigens or cytokine genes in BCG can enhance the ability of BCG to induce a Th1 immune response . Since dose of the antigen is considered as one of the parameters that influence the Th cell responses, the level of expression of the candidate antigen should influence the final Th response against the recombinant BCG (rBCG) . In the present study, the effect of over-expression of a candidate antigen Antigen 85B (Ag 85B) in a rBCG system, on the Th-priming ability of BCG has been investigated in the murine model . BALB/c mice were immunized with three different rBCG constructs expressing Ag 85B to various levels . Induction of Th1/Th2 responses was analyzed by measuring levels of interferon-gamma (Th1) and interleukin-10 (Th2) in antigen-stimulated splenocyte cultures and by quantifying the antigen-specific IgG2a (Th1) and IgG1 (Th2) antibody responses . By varying the level of expression of Ag 85B, specific immune responses against Ag 85B were observed to range from mixed Th1/Th2 to Th1 . However, the BCG-specific immune responses in case of all rBCG-immunized animals remained predominantly Th1.

DNA Cell Biol, 2003 May, 22(5), 347 - 53
Molecular cloning and analysis of a region of the Bartonella bacilliformis genome encoding NlpD, L-isoaspartyl methyltransferase and YajC homologs; Padmalayam I et al.; The NlpD/LppB homolog of the human pathogen, Bartonella bacilliformis, is an immunogenic 43-kDa protein that is encoded by a 1206-bp open reading frame (ORF-401) . The regions flanking the nlpD/lppB gene of B . bacilliformis were sequenced to determine if it is located within the rpoS operon, as it is in most bacteria . We report that the B . bacilliformis nlpD/lppB gene is located immediately downstream of pcm, a gene encoding a 25-kDa protein, L-isoaspartyl protein carboxyl methyltransferase, that is a component of the rpoS operon in other bacteria . However, the genomic organization downstream of the B . bacilliformis nlpD/lppB gene appears to be distinct . In other bacteria, the third gene in the operon is rpoS, a gene that codes for an alternative sigma factor of RNA polymerase . In B . bacilliformis, an open reading frame encoding a protein homologous to the immunodominant YajC protein is located directly downstream of the nlpD/lppB gene . We show that Bartonella henselae, a close relative of B . bacilliformis, also shares this unusual organizational feature . Thus, the genomic organization of the nlpD/lppB genes of B . bacilliformis, and B . henselae appears to be unique among all bacteria for which the sequence of this region has been reported.

Med Vet Entomol, 2003 Sep, 17(3), 251 - 6
Cross-resistance between strains of Bacillus sphaericus but not B . thuringiensis israelensis in colonies of the mosquito Culex quinquefasciatus; Yuan ZM et al.; Two colonies of Culex quinquefasciatus Say (Diptera: Culicidae) were selected with Bacillus sphaericus strains C3-41 and IAB59 in the laboratory for 13 and 18 generations; they attained 145,000- and 48.3-fold resistance, respectively, in comparison with a susceptible laboratory colony (SLCq) and showed very high levels of cross-resistance (8500- to 145,000-fold) to B . sphaericus strains C3-41, 1593, 2297 and 2362 . They were relatively susceptible to B . sphaericus strains LP1-G and 47-6B (only 0.8- to 2.8-fold tolerance), with 24.8- to 48.3-fold cross-resistance to strain IAB59 . B . sphaericus-resistant mosquito colonies remained highly susceptible to B . thuringiensis israelensis, suggesting that B.t.i . would be of value in the management of B . sphaericus-resistant Cx . quinquefasciatus colonies . The demonstration of low or no cross-resistance of two selected resistant Cx . quinquefasciatus colonies to IAB59, LP1-G and 47-6B strains of B . sphaericus and the finding of a major 49 kDa protein in these strains suggest that there is likely to be another mosquitocidal factor in the three strains.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2003 Sep, 38(9), 1837 - 48
The biodegradation of 1,3-dichlorobenzene by an adapted strain Bacillus cereus PF-11 derived from town-gas industrial effluent; Wang L et al.; In the present study, an adapted bacterium PF-11 with high 1,3-dichlorobenzene degradation capability was isolated from town-gas industrial effluent through continuous introducing of N-methyl-N'-nitro-N-nitrosoquanidine (NTG) . In suitable condition, a degradation rate of 32 mg L(-1) d(-1) of 1,3-dichlorobenzene was obtained by strain PF-11 with effective chlorion release . Strain PF-11 was tentatively identified as gram-positive Bacillus cereus . The substrate specificity of the strain PF-11 was relatively low, and the degradation rate for different chlorobenzenes was in the order of monochlorobenzene > 1,3-dichlorobenzene > 1,2-dichlorobenzene . Initial oxidation step was molecular oxygen attacking chlorobenzene ring catalyzed by dioxygenase.

J Clin Lab Anal, 2003, 17(5), 155 - 63
Diagnosis of tuberculosis: available technologies, limitations, and possibilities; Garg SK et al.; Rapid diagnosis and treatment are important for preventing transmission of Mycobacterium tuberculosis . However, the diagnosis of tuberculosis continues to pose serious problems, mainly because of difficulties in differentiating between patients with active tuberculosis and those with healed lesions, normal mycobacterium boris BCG (Bacillus Calmette Guerin) vaccinated individuals, and unvaccinated Manteux positives . Physicians still rely on conventional methods such as Ziehl-Neelsen (ZN) staining, fluorochrome staining, sputum culture, gastric lavage, and other non-traditional methods . Although the tuberculin test has aided in the diagnosis of tuberculosis for more than 85 years, its interpretation is difficult because sensitization with nontuberculous mycobacteria leads to false-positive tests . There have been numerous unsuccessful attempts to develop clinically useful serodiagnostic kits for tuberculosis . A number of proteinaceous and nonprotein antigens (such as acyltrehaloses and phenolglycolipids) have been explored from time to time for the development of such assays but they have not proved to be clinically useful . It has been difficult to develop an ELISA utilizing a suitable antigen because M . tuberculosis shares a large number of antigenic proteins with other microorganisms that may or may not be pathogenic . With the advent of molecular biology techniques, there have been significant advances in nucleic acid-based amplification and hybridization, which are helping to rectify existing flaws in the diagnosis of tuberculosis . The detection of mycobacterial DNA in clinical samples by polymerase chain reaction (PCR) is a promising approach for the rapid diagnosis of tuberculous infection . However, the PCR results must be corrected for the presence of inhibitors as well as for DNA contamination . In the modern era of genetics, marked by proteomics and genomics, the day is not far off when DNA chip-based hybridization assays will instantly reveal mycobacterial infections .

Braz J Med Biol Res, 2003 Sep, 36(9), 1221 - 6 Epub 2003 Aug 19.
A method for multiple sequential analyses of macrophage functions using a small single cell sample; Nascimento FR et al.; Microbial pathogens such as bacillus Calmette-Guerin (BCG) induce the activation of macrophages . Activated macrophages can be characterized by the increased production of reactive oxygen and nitrogen metabolites, generated via NADPH oxidase and inducible nitric oxide synthase, respectively, and by the increased expression of major histocompatibility complex class II molecules (MHC II) . Multiple microassays have been developed to measure these parameters . Usually each assay requires 2-5 x 10(5) cells per well . In some experimental conditions the number of cells is the limiting factor for the phenotypic characterization of macrophages . Here we describe a method whereby this limitation can be circumvented . Using a single 96-well microassay and a very small number of peritoneal cells obtained from C3H/HePas mice, containing as little as <=2 x 10(5) macrophages per well, we determined sequentially the oxidative burst (H2O2), nitric oxide production and MHC II (IAk) expression of BCG-activated macrophages . More specifically, with 100 micro l of cell suspension it was possible to quantify H2O2 release and nitric oxide production after 1 and 48 h, respectively, and IAk expression after 48 h of cell culture . In addition, this microassay is easy to perform, highly reproducible and more economical.

J Endotoxin Res, 2003, 9(4), 215 - 24
Beta-glucosidase enzymatic activity of crystal polypeptide of the Bacillus thuringiensis strain 1.1; Papalazaridou A et al.; The crystals of Bacillus thuringiensis strain 1.1 consist of the 140 kDa delta-endotoxin, which exhibits beta-glucosidase enzymatic activity, based on the following data . (i) Purified crystals exhibit beta-glucosidase enzymatic activity . When the crystals are reacted with specific antibodies directed either against the commercial (almond purified) beta-glucosidase or against the 140 kDa polypeptide, then considerable reduction of enzymatic activity is observed almost at the same level with both antibodies . (ii) Commercial beta-glucosidase and the 140 kDa crystal polypeptide share antigenic similarities; in Western immunoblots, the 140 kDa crystal polypeptide is recognized by anti-beta-glucosidase antibodies, and commercial beta-glucosidase is recognized by anti-140-kDa antibodies . (iii) The enzymatic properties of commercial beta-glucosidase and that resident in the crystals of B . thuringiensis strain 1.1 are very similar . Thus, both enzymes hydrolyze a wide range of substrates (aryl-beta-glucosides, disaccharides with alpha- or beta-linkage polysaccharides) and have an optimum activity at 40 degrees C and pH 5 . Both enzymes are relatively thermostable and are resistant to end-product inhibition by glucose . Additionally, they show the same pattern of inhibition or activation by several chemical compounds . (iv) The crystals and commercial beta-glucosidase show almost equivalent levels of insecticidal activity against Drosophila melanogaster larvae and, furthermore, cause reduction in adult flies that emerge from larvae surviving treatment.

Adv Biochem Eng Biotechnol, 2003, 84, 211 - 73
Recent advances in tuberculosis research in India; Tyagi AK et al.; Tuberculosis (TB) continues to be the leading killer of mankind among all infectious diseases, especially in the developing countries . Since the discovery of tubercle bacillus more than 100 years ago, TB has been the subject of research in an attempt to develop tools and strategies to combat this disease . Research in Indian laboratories has contributed significantly towards developing the DOTS strategy employed worldwide in tuberculosis control programmes and elucidating the biological properties of its etiologic agent, M . tuberculosis . In recent times, the development of tools for manipulation of mycobacteria has given a boost to researchers working in this field . New strategies are being employed towards understanding the mechanisms of protection and pathogenesis of this disease . Molecular methods are being applied to develop new tools and reagents for prevention, diagnosis and treatment of tuberculosis . With the sequencing of the genome of M . tuberculosis, molecules are being identified for the development of new drugs and vaccines . In this chapter, the advances made in these areas by Indian researchers mainly during the last five years are reviewed.

Infect Immun, 2003 Sep, 71(9), 4901 - 7
A DNA prime-Mycobacterium bovis BCG boost vaccination strategy for cattle induces protection against bovine tuberculosis; Skinner MA et al.; The variable efficacy of bacillus Calmette-Guerin (Mycobacterium bovis BCG) in protecting humans and cattle against tuberculosis has prompted a search for a more effective vaccination regimen . A prime-boost strategy was investigated in cattle naturally sensitized to environmental mycobacteria by using a combination of three DNA vaccines coding for Hsp 65, Hsp 70, and Apa for priming, followed by a boost with BCG prior to experimental challenge with virulent M . bovis . Controls were vaccinated with DNA or BCG alone or were not vaccinated . The immune responses were monitored throughout the study, and protection was assessed based on reductions in the numbers of lesions and viable mycobacteria in lymph node samples . Vaccination with BCG alone or with a DNA prime-BCG boost regimen induced high levels of antigen-specific gamma interferon (IFN-gamma) in whole-blood cultures . In the prime-boost group there were fewer animals with severe lung lesions, fewer lymph nodes with lesions per animal, a smaller proportion of animals with lesions, lower mean lung and lymph node lesion scores, and less M . bovis isolated from retropharyngeal and thoracic lymph nodes compared to the results obtained for the nonvaccinated animals . The prime-boost regimen induced significant enhancement of protection in six parameters, compared with significant enhancement of protection in only two parameters for BCG alone . In addition, following challenge, in vitro IFN-gamma responses against ESAT-6 and CFP-10, as well as bovine tuberculin-induced skin test and in vitro IFN-gamma responses, were identified as immunological markers that predicted protection . The use of the prime-boost strategy suggested that a combination of vaccines may be better than a single vaccine for protection against tuberculosis.

J Environ Qual, 2003 Jul-Aug, 32(4), 1228 - 33
Chromate reduction by chromium-resistant bacteria isolated from soils contaminated with dichromate; Camargo FA et al.; Extensive use of hexavalent chromium {Cr(VI)} in various industrial applications has caused substantial environmental contamination . Chromium-resistant bacteria isolated from soils can be used to remove toxic Cr(VI) from contaminated environments . This study was conducted to isolate chromium-resistant bacteria from soils contaminated with dichromate and describes the effects of some environmental factors such as pH, temperature, and time on Cr(VI) reduction and resistance . We found that chromium-resistant bacteria can tolerate 2500 mg L(-1) Cr(VI), but most of the isolates tolerated and reduced Cr(VI) at concentrations lower than 1500 mg L(-1) . Chromate reduction activity of whole cells was detected in five isolates . Most of these isolates belong to the genus Bacillus as identified by the 16S rRNA gene sequencing . Maximal Cr(VI) reduction was observed at the optimum pH (7.0-9.0) and temperature (30 degrees C) of growth . One bacterial isolate (Bacillus sp . ES 29) was able to aerobically reduce 90% of Cr(VI) in six hours . The Cr(VI) reduction activity of the whole cells of five isolates had a K(M) of 0.271 (2.61 mM) to 1.51 mg L(-1) (14.50 mM) and a V(max) of 88.4 (14.17 nmol min(-1)) to 489 mg L9-1) h(-1) (78.36 nmol min(-1)) . Our consortia and monocultures of these isolates can be useful for Cr(VI) detoxification at low and high concentrations in Cr(VI)-contaminated environments and under a wide range of environmental conditions.

BJU Int, 2003 Sep, 92(4), 393 - 9
Superficial bladder tumours: analysis of prognostic factors and construction of a predictive index; Ali-El-Dein B et al.; OBJECTIVES: To assess the prognostic factors that could be used to predict tumour recurrence and progression, and to construct and validate a predictive index . PATIENTS AND METHODS: Between June 1991 and December 2000, 533 patients (418 men and 115 women; mean age 55.4 years) underwent complete transurethral resection of histologically confirmed pTa and pT1 transitional cell carcinoma of the bladder, after which 377 (test series) were randomized into two subsequent studies, of six groups, to receive adjuvant intravesical sequential bacillus Calmette-Guerin (BCG) and epirubicin, BCG alone, epirubicin (50 or 80 mg), adriamycin 50 mg or no adjuvant therapy . Factors potentially affecting tumour recurrence or progression were assessed using univariate and multivariate analysis, i.e . tumour stage, histological grade, DNA ploidy, history of recurrence, multiplicity, size, tumour configuration, associated carcinoma in situ, recurrence at the first 3-month check cystoscopy and the use of adjuvant therapy . The regression coefficients determined by Cox regression analysis were used to construct a predictive index (PI) . The algebraic sum of the regression coefficients of the factors with independent and significant association with disease-free survival for each case represented a proportional hazard score (PHS) . The PI was validated in another series of 156 patients (validation series) in whom the same regression coefficients for the same significant factors as the test series were used to categorize it into three risk groups . Kaplan-Meier survival curves were plotted to compare the different risk categories in both test and validation series . RESULTS: The mean (sd, range) follow-up in the test and validation series were 58 (19, 5-96) and 28.3 (14.9, 2-94) months, respectively . In the test series, tumour stage, DNA ploidy, multiplicity, history of recurrence, tumour configuration, cystoscopy result and the type of adjuvant therapy had independent significance for recurrence on multivariate analysis . For progression, the cystoscopy result, DNA ploidy and grade were the only independent and significant predictors . The ranges of PHS for the factors affecting recurrence-free and progression-free survival were 0.0-7.14 and 0.0-5.84, respectively, which were divided equally into three risk categories with significant differences on Kaplan-Meier curves and a log-rank test (P < 0.001) . The three categories in the validation series were significantly different from each other and each was comparable with that in the test series . CONCLUSIONS: Tumour stage, DNA ploidy, multiplicity, history of recurrence, tumour configuration and type of adjuvant therapy affected independently the rate of recurrence after resecting superficial bladder tumour . Recurrence at the 3-month cystoscopy, histological grade and DNA ploidy were the only predictors of progression to muscle-invasion . The PI dividing the patients into three risk groups with different treatment and follow-up strategies for recurrence and progression was reproducible in a validation series.

Biochem J, 2003 Nov 15, 376(Pt 1), 237 - 44
Aromatic residues within the substrate-binding cleft of Bacillus circulans chitinase A1 are essential for hydrolysis of crystalline chitin; Watanabe T et al.; Bacillus circulans chitinase A1 (ChiA1) has a deep substrate-binding cleft on top of its (beta/alpha)8-barrel catalytic domain and an interaction between the aromatic residues in this cleft and bound oligosaccharide has been suggested . To study the roles of these aromatic residues, especially in crystalline-chitin hydrolysis, site-directed mutagenesis of these residues was carried out . Y56A and W53A mutations at subsites -5 and -3, respectively, selectively decreased the hydrolysing activity against highly crystalline beta-chitin . W164A and W285A mutations at subsites +1 and +2, respectively, decreased the hydrolysing activity against crystalline beta-chitin and colloidal chitin, but enhanced the activities against soluble substrates . These mutations increased the K(m)-value when reduced (GlcNAc)5 (where GlcNAc is N -acetylglucosamine) was used as the substrate, but decreased substrate inhibition observed with wild-type ChiA1 at higher concentrations of this substrate . In contrast with the selective effect of the other mutations, mutations of W433 and Y279 at subsite -1 decreased the hydrolysing activity drastically against all substrates and reduced the kcat-value, measured with 4-methylumbelliferyl chitotrioside to 0.022% and 0.59% respectively . From these observations, it was concluded that residues Y56 and W53 are only essential for crystalline-chitin hydrolysis . W164 and W285 are very important for crystalline-chitin hydrolysis and also participate in hydrolysis of other substrates . W433 and Y279 are both essential for catalytic reaction as predicted from the structure.

Genet Sel Evol, 2003, 35 Suppl 1, S67 - 81
Evaluation of immune responses of cattle as a means to identify high or low responders and use of a human microarray to differentiate gene expression; Hernandez A et al.; An immune response (IR) index to identify cows with high (H) and low (L) antibody-mediated immune responses (AMIR) had been previously devised . High AMIR associated with decreased mastitis and improved response to vaccination . Measurement of cell-mediated immune response (CMIR) was not included in the index; therefore various antigen/adjuvant combinations were evaluated as inducers of DTH to be added to the IR-index . The Bacillus Calmette Guerin (BCG)-induced/purified protein derivative (PPD)-elicited tuberculin skin test is a reliable measure of DTH; however, its use to identify livestock with high CMIR may be confounded due to previous exposure to Mycobacteria tuberculosis . DTH to BCG/PPD was therefore compared with that induced by Mycobacteria phlei (saprophyte) and its derivative phlein as the test antigen . Antibody to OVA was also evaluated . The results indicated that BCG/PPD and M . phlei/phlein induced similar DTH, but cross reaction to PPD was evident following induction of DTH using M . phlei making it a less than ideal alternative for testing livestock . Nonetheless, cows could be ranked for both AMIR and CMIR . RNA from two cows with the highest and lowest IR ranks was then used to probe a human 1.7 kD microarray to determine the ability of a human array to provide information on bovine genes associated with H and L.

Cell Microbiol, 2003 Sep, 5(9), 637 - 48
The effects of reactive nitrogen intermediates on gene expression in Mycobacterium tuberculosis; Ohno H et al.; Nitric oxide (NO) and related reactive nitrogen intermediates (RNI) are effective antimycobacterial agents and signal-transducing molecules . The present study uses microarray analysis to examine the effects of RNI on Mycobacterium tuberculosis gene expression . A common set of 53 genes was regulated by two chemically distinct nitric oxide donors . For a subset of the RNI-inducible genes, evidence exists suggesting that they may play a role in promoting survival of the tubercle bacillus in the host . Results obtained from studies based on a murine experimental tuberculosis model involving nos2-deficient mice suggest that RNI could regulate M . tuberculosis gene expression in vivo . Finally, there is a remarkable overlap between the RNI-inducible regulon and that previously reported to be regulated by hypoxia; and both reactive nitrogen species and anaerobicity upregulate the expression of one and the same putative two-component regulatory response system . Together, the results of this study provide evidence suggesting that (i) RNI play a role in regulating M . tuberculosis gene expression in vivo; (ii) the reactive nitrogen species upregulate genes that may be conducive to the survival of the tubercle bacillus in the infected host; and (iii) RNI and hypoxia may regulate mycobacterial gene expression via overlapping signal transduction pathways.

Cas Lek Cesk, 2003, 142(6), 356 - 64
{Multiresistant tuberculosis in the Czech Republic (1999-2001)}; Havelkova M et al.; BACKGROUND: Multidrug-resistant (MDR) tuberculosis, defined as a disease caused by Mycobacterium tuberculosis strains, which are resistant to more antituberculous drugs (at least to isoniazid and rifampicin), is a problem frequently discussed in the Czech Republic . Cases of specific disease refractive to causal antituberculous therapy are associated with the risk of the spread of the causative agent among the population . METHODS AND RESULTS: The National Reference Laboratory for Mycobacteria collected 2813 Mycobacterium tuberculosis strains isolated by Czech mycobacteriological laboratories in 1999 to 2001 . All strains were tested for susceptibility to basic antituberculous drugs and then the MDR strains were further tested for susceptibility/resistance to other antituberculous and antibacterial drugs . The MDR strains were studied by DNA analysis (DNA fingerprinting restriction analysis, RFLP-Restriction Fragment Length Polymorphism) as well . Thirty-nine patients who had MDR tuberculosis were excretors of 56 Mycobacterium tuberculosis strains . In average, MDR tuberculosis accounted for 1.96% (1.7-2.4) of all cases of bacillary tuberculosis . The most frequent type of the multidrug resistance was that resistant to four basic antituberculous drugs (isoniazid, rifampicin, ethambutol and streptomycin) . It was confirmed in 48.2% multidrug resistant strains . CONCLUSIONS: Isepamicin, clofazimin, capreomycin and amikacin are considered to be the most promising antituberculosis drugs . Based on RFLP profiles, 61.5% of strains were placed into 8 clusters while the other strains remained unclustered . No significant differences in geographical distribution and population structure were found between the excretors of clustered strains and those of unclustered strains . Preliminary comparison with restriction profiles of the MDR Mycobacterium tuberculosis strains in the international database suggests the uniqueness of Czech strains showing the profiles not found elsewhere to date.

J Bacteriol, 2003 Sep, 185(17), 5133 - 47
Mutational loss of a K+ and NH4+ transporter affects the growth and endospore formation of alkaliphilic Bacillus pseudofirmus OF4; Wei Y et al.; A putative transport protein (Orf9) of alkaliphilic Bacillus pseudofirmus OF4 belongs to a transporter family (CPA-2) of diverse K+ efflux proteins and cation antiporters . Orf9 greatly increased the concentration of K+ required for growth of a K+ uptake mutant of Escherichia coli . The cytoplasmic K+ content of the cells was reduced, consistent with an efflux mechanism . Orf9-dependent translocation of K+ in E . coli is apparently bidirectional, since ammonium-sensitive uptake of K+ could be shown in K+ -depleted cells . The upstream gene product Orf8 has sequence similarity to a subdomain of KTN proteins that are associated with potassium-translocating channels and transporters; Orf8 modulated the transport capacities of Orf9 . No Orf9-dependent K+(Na+)/H+ antiport activity was found in membrane vesicles . Nonpolar deletion mutants in the orf9 locus of the alkaliphile chromosome exhibited no K+ -related phenotype but showed profound phenotypes in medium containing high levels of amine-nitrogen . Their patterns of growth and ammonium content suggested a physiological role for the orf9 locus in bidirectional ammonium transport . Orf9-dependent ammonium uptake was observed in right-side-out membrane vesicles of the alkaliphile wild type and the mutant with an orf8 deletion . Uptake was proton motive force dependent and was inhibited by K+ . Orf9 is proposed to be designated AmhT (ammonium homeostasis) . Ammonium homeostasis is important in high-amine-nitrogen settings and is particularly crucial at high pH since cytosolic ammonium accumulation interferes with cytoplasmic pH regulation . Endospore formation in amino-acid-rich medium was significantly defective and germination was modestly defective in the orf9 and orf7-orf10 deletion mutants.

Arch Biochem Biophys, 2003 Sep 1, 417(1), 81 - 6
Using X-ray crystallography of the Asp55Asn mutant of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus to support the mechanistic role of Asp55 as the general base; Antikainen NM et al.; Because mutations of the ionizable Asp at position 55 of the phosphatidylcholine preferring phospholipase C from Bacillus cereus (PLC(Bc)) to a non-ionizable Asn generate a mutant enzyme (D55N) with 10(4)-fold lower catalytic activity than the wild-type enzyme, we tentatively identified Asp55 as the general base for the enzymatic reaction . To eliminate the alternate possibility that Asp55 is a structurally important amino acid, the X-ray structures of unbound D55N and complexes of D55N with two non-hydrolyzable substrate analogues have been solved and refined to 2.0, 2.0, and 2.3A, respectively . The structures of unbound wild-type PLC(Bc) and a wild-type PLC(Bc)-complex with a non-hydrolyzable substrate analogue do not change significantly as a result of replacing Asp55 with Asn . These observations demonstrate that Asp55 is not critical for the structural integrity of the enzyme and support the hypothesis that Asp55 is the general base in the PLC(Bc)-catalyzed hydrolysis of phospholipids.

Cochrane Database Syst Rev . 2003;(3):CD003231.
Intravesical bacillus Calmette-Guerin versus mitomycin C for Ta and T1 bladder cancer; Shelley MD et al.; BACKGROUND: Tumour recurrence following transurethral resection (TUR) for Ta and T1 bladder cancer is a major clinical problem . Intravesical administration of mitomycin C (MMC) or bacillus Calmette-Guerin (BCG) has proven prophylactic activity but both are associated with local and systemic side-effects . A systematic review was carried out to compare the efficacy of these two agents . OBJECTIVES: To undertake a systematic review and meta-analysis comparing intravesical mitomycin C and Bacillus Calmette-Guerin in terms of tumour recurrence, disease progression and overall survival in Ta and T1 bladder cancer . Treatment-related toxicities would also be evaluated . SEARCH STRATEGY: A comprehensive search of MEDLINE, EMBASE, Healthstar, Cochrane Controlled Trials Register, Cancerlit, and DARE was performed, and hand searching of relevant journals undertaken . SELECTION CRITERIA: Trials in any language were included in the meta-analysis if they were properly randomised, included medium to high risk patients with Ta or T1 bladder cancer and compared intravesical MMC versus BCG . DATA COLLECTION AND ANALYSIS: Trial eligibility, methodological quality and data extraction were assessed independently by two reviewers . Time to event analysis was evaluated using log hazard ratios, with a sensitivity analysis for subgroups according to patient's risk of recurrence . MAIN RESULTS: Twenty-five articles were identified but only seven were considered eligible . This represented 1901 evaluable patients in total, 820 randomised to MMC and 1081 to BCG . Six trials had sufficient data for meta-analysis and included 1527 patients, 693 in the mitomycin arm and 834 in the BCG arm . The weighted mean log hazard ratio (variance) for tumour recurrence for the six trials was - 0.022 (0.005) . This indicated no significant difference between MMC and BCG (p = 0.76) . However, the meta-analysis indicated evidence of significant heterogeneity between trials (p = 0.001) . A subgroup analysis of three trials that included only high risk Ta and T1 patients indicated no heterogeneity (p = 0.25) and a log hazard ratio (variance) for recurrence of -0.371 ( 0.012) . With MMC used as the control in the meta-analysis, a negative ratio is in favour of BCG and, in this case, is highly significant (p = 0.0008) . The seventh trial, in abstract form only, used BCG in low doses for two arms of the trial (27 mg and 13.5mg) compared to a standard dose of mitomycin C (30mg), and reported a significantly reduced recurrent rate with BCG (27mg) compared to mitomycin C (p = 0.001) . Only two trials included sufficient data to analyse disease progression and survival, representing a total of 681 patients; 338 randomised to BCG and 343 to MMC . There was no significant difference between MMC and BCG for disease progression (log hazard ratio + variance: 0.044 + 0.04, p = 0.16) or survival (-0.112 + 0.03, p = 0.50) . Local toxicities (dysuria, cystitis, frequency, and haematuria) were associated with both MMC (30%) and BCG (44%) . Systemic toxicities, such as chills, fever and malaise, were observed with both MMC and BCG (12% and 19%, respectively) although skin rash was more common with MMC . REVIEWER'S CONCLUSIONS: The data from the present meta-analysis indicate that tumour recurrence was significantly reduced with intravesical BCG compared to MMC only in the subgroup of patients at high risk of tumour recurrence . However, there was no difference in terms of disease progression or survival, and the decision to use either agent might be based on adverse events and cost.

Curr Opin Urol, 2003 Sep, 13(5), 357 - 62
The current status of intravesical therapy for superficial bladder cancer; Patard JJ et al.; PURPOSE OF REVIEW: To analyse recent advances in intravesical instillation therapy for superficial bladder cancer . RECENT FINDINGS: Although intravesical bacillus Calmette-Guerin has been used for many years in the treatment of superficial bladder cancer, its mechanism of action remains unclear, its poor tolerance remains a problem, the prediction of its efficacy has still to be validated, and its long-term effects on progression and survival are controversial . The exact timing and place of intravesical chemotherapy needs to be better defined, as well as the place of some new molecules . Finally, new approaches need to be explored for overcoming the limitations of the usual intravesical agents . SUMMARY: No dramatic advances have been made in understanding the mechanisms of action of bacillus Calmette-Guerin during the past year . However, a careful dissection of this complex immunological pathway continues and immunological criteria are promising for predicting the response to bacillus Calmette-Guerin . Evidence has been accumulating to suggest that a dose reduction during the initial treatment remains effective and reduces side-effects . In addition, bacillus Calmette-Guerin maintenance therapy is useful for high-risk patients . However, long-term tolerance remains an important issue, and the optimal protocol has not yet been defined . On the other hand, it has been proved that intravesical chemotherapy, when administered early after transurethral resection, is effective in preventing frequent recurrences, whereas maintenance chemotherapy is ineffective . Finally, new approaches, including instillations of activated immune cells or targeted gene therapy, are being explored.

J Biol Chem, 2003 Oct 24, 278(43), 41900 - 7 Epub 2003 Aug 12.
Characterization of the cobaltochelatase CbiXL: evidence for a 4Fe-4S center housed within an MXCXXC motif; Leech HK et al.; CbiX is a cobaltochelatase required for the biosynthesis of vitamin B12 and is found in Archaea as a short form (CbiXS containing 120-145 amino acids) and in some bacteria as a longer version (CbiXL containing 300-350 amino acids) . Purification of either recombinant Bacillus megaterium or Synechocystis CbiXL in Escherichia coli, which is facilitated by the presence of a naturally occurring histidine-rich region of the protein, results in the isolation of a dark brown protein solution . The UV/visible spectrum of the protein is consistent with the presence of a redox group, and the lack of definition within the spectrum is suggestive of a 4Fe-4S center . The presence of an iron-sulfur center was confirmed by EPR analysis of the proteins, which produces a pseudoaxial spectrum with g values at 2.04, 1.94, and 1.90 . The EPR spectrum was absent at 70 K, an observation that is diagnostic of a 4Fe-4S center . Redox potentiometry coupled with optical spectroscopy allowed the midpoint potential of the redox center to be determined for the CbiXL from both B . megaterium and Synechocystis . Sequence analysis of CbiXL proteins reveals only two conserved cysteine residues within the CbiXL proteins, which are part of an MXCXXC motif . Mutagenesis of the two cysteines leads to loss of both the EPR spectrum and UV/visible spectral features of the Fe-S center in the protein, clearly indicating that these residues are involved in ligating the cofactor to the apoprotein possibly in a butterfly arrangement . The potential physiological role of the iron-sulfur center is discussed.

Protein Eng, 2003 Jul, 16(7), 505 - 14
Alpha-amylase from Bacillus licheniformis mutants near to the catalytic site: effects on hydrolytic and transglycosylation activity; Rivera MH et al.; The alpha-amylase from Bacillus licheniformis is the most widely used enzyme in the starch industry owing to its hyperthermostability, converting starch to medium-sized oligosaccharides . Based on sequence alignment of homologous amylases, we found a semi-conserved sequence pattern near the active site between transglycosidic and hydrolytic amylases, which suggested that hydrophobicity may play a role in modifying the transglycosylation/hydrolysis ratio . Based on this analysis, we replaced residue Val286 by Phe and Tyr in Bacillus licheniformis alpha-amylase . Surprisingly, the two resultant mutant enzymes, Val286Phe and Val286Tyr, showed two different behaviors . Val286Tyr mutant was 5-fold more active for hydrolysis of starch than the wild-type enzyme . In contrast, the Val286Phe mutant, differing only by one hydroxyl group, was 3-fold less hydrolytic than the wild-type enzyme and apparently had a higher transglycosylation/hydrolysis ratio . These results are discussed in terms of affinity of subsites, hydrophobicity and electrostatic environment in the active site . The engineered enzyme reported here may represent an attractive alternative for the starch transformation industries as it affords direct and substantial material savings and requires no process modifications.

Am J Epidemiol, 2003 Aug 15, 158(4), 316 - 27
Population-based study of non-Hodgkin lymphoma, histology, and medical history among human immunodeficiency virus-negative participants in San Francisco; Holly EA et al.; A population-based case-control study of non-Hodgkin lymphoma was conducted in the San Francisco Bay Area of California from 1988 to 1995 . The study used Working Formulation histologic subtypes of non-Hodgkin lymphoma . Diffuse and immunoblastic large-cell lymphomas and all follicular lymphomas were combined to reflect two Revised European-American Lymphoma (REAL) classifications . Results were obtained from age- and sex-adjusted polytomous multivariable logistic models for 1,304 cases and 2,402 controls who were negative for human immunodeficiency virus . Statistically significant odds ratios for REAL large-cell lymphoma were decreased for receipt of five or more different vaccines (odds ratio (OR) = 0.65), use of calcium channel blockers (OR = 0.35), mononucleosis (OR = 0.55), plant allergies (OR = 0.72), and animal allergies (OR = 0.40) . Odds ratios for REAL follicular lymphoma were decreased for use of nonsteroidal antiinflammatory drugs (OR = 0.67), hepatitis (OR = 0.36), and having had three or more bee or wasp stings (OR = 0.79) and were increased for heart disease (OR = 1.5) . Similar elevated odds ratios for heart disease and Bacillus Calmette-Guerin vaccine and decreased odds ratios for nonsteroidal antiinflammatory drugs, plant allergies, and three or more bee or wasp stings were found for small lymphocytic lymphoma . These data provide direction for further investigation of immune function and genetic characteristics of subtypes of non-Hodgkin lymphoma in lymphomagenesis and warrant analyses in large pooled data sets.

Insect Biochem Mol Biol, 2003 Sep, 33(9), 929 - 35
Binding of Bacillus thuringiensis toxins in resistant and susceptible strains of pink bollworm (Pectinophora gossypiella); Gonzalez-Cabrera J et al.; Evolution of resistance by pests could cut short the success of transgenic plants producing toxins from Bacillus thuringiensis, such as Bt cotton . The most common mechanism of insect resistance to B . thuringiensis is reduced binding of toxins to target sites in the brush border membrane of the larval midgut . We compared toxin binding in resistant and susceptible strains of Pectinophora gossypiella, a major pest of cotton worldwide . Using Cry1Ab and Cry1Ac labeled with (125)I and brush border membrane vesicles (BBMV), competition experiments were performed with unlabeled Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ca, Cry1Ja, Cry2Aa, and Cry9Ca . In the susceptible strain, Cry1Aa, Cry1Ab, Cry1Ac, and Cry1Ja bound to a common binding site that was not shared by the other toxins tested . Reciprocal competition experiments with Cry1Ab, Cry1Ac, and Cry1Ja showed that these toxins do not bind to any additional binding sites . In the resistant strain, binding of (125)I-Cry1Ac was not significantly affected; however, (125)I-Cry1Ab did not bind to the BBMV . This result, along with previous data from this strain, shows that the resistance fits the "mode 1" pattern of resistance described previously in Plutella xylostella, Plodia interpunctella, and Heliothis virescens.

Braz J Biol, 2003 Feb, 63(1), 113 - 9 Epub 2003 Aug 14.
Valve morphology of the benthic diatom Fallacia marnieri (Manguin) Witkowski (Sellaphoraceae--Bacillariophyta); Procopiak LK et al.; The genus Fallacia Stickle & Mann includes naviculoid birraphid diatoms with H-like plastids, valves with uniseriate striae and covered by porous conopea, rounded areolae occluded by hymens, and depressed hyaline lyriform area characterizing the valvar surface . The species Fallacia marnieri was found in samples collected in King George Island, Antarctic Peninsula, on which an electron microscopy study and literature revision were carried out . From this investigation, we found that since Fallacia marnieri has the diagnostic structures of the genus, its recent inclusion in Fallacia is justified . The species was found on rocks of an intertidal region, and seems to be endemic in cold regions, as indicated in the literature.

Semin Pediatr Infect Dis, 2003 Jul, 14(3), 213 - 9
Immunization Monitoring Program, Active: a model of active surveillance of vaccine safety; Scheifele DW et al.; In Canada since 1993 the nation's passive system for reporting postimmunization adverse events has been supplemented by the active surveillance of inpatients at 10 to 12 pediatric referral centers, a system referred to as the Immunization Monitoring Program, Active . Participating centers are located from coast-to-coast and receive referrals from every province and territory . Approximately 20 percent of the population aged 0 to 12 years lives in the immediate vicinity of these centers . Nurse monitors at each center search for numerous target conditions, including postimmunization adverse events and vaccine-preventable infections . Vaccine safety observations have included (1) a substantial decrease in the risk of the development of febrile seizures and hypotonic-hyporesponsive episodes since the country switched from whole-cell to acellular pertussis-containing vaccines, (2) no evidence for encephalopathy resulting from the latter vaccines, (3) a generally benign outcome with postimmunization thrombocytopenia cases, and (4) an unexpectedly high rate of disseminated bacille Calmette-Guerin infections among aboriginal infants . Concomitant disease surveillance has been important for sustaining the surveillance system because few postimmunization adverse events require hospital admission.

J Urol, 2003 Sep, 170(3), 964 - 9
Immune mechanisms in bacillus Calmette-Guerin immunotherapy for superficial bladder cancer; Bohle A et al.; PURPOSE: Of all medical disciplines it is exclusively in urology in which immunotherapy for cancer has an established position today with intravesical bacillus Calmette-Guerin (BCG) against superficial bladder carcinoma recurrences . BCG is regarded as the most successful immunotherapy to date . However, the mode of action has not yet been fully elucidated . We provide a thorough overview of this complex field of research . MATERIALS AND METHODS: Rather than simply reporting all experimental data available for better understanding the involved immune mechanisms, we chose to provide comprehensively only information supported by several independent pathways of evidence . RESULTS: Major findings made during the last few years include systematic analyses of patient material, detailed in vitro studies and investigations in animal models, which have led to a substantially greater understanding of the mechanisms involved . CONCLUSIONS: The efficacy of BCG is based on a complex and long lasting local immune activation . The bladder as a confined compartment, in which high local concentrations of the immunotherapy agent and effective recruitment of immune cells can be achieved, serves as an ideal target organ for this type of immunotherapy approach.

J Urol, 2003 Sep, 170(3), 777 - 82
Intravesical electromotive mitomycin C versus passive transport mitomycin C for high risk superficial bladder cancer: a prospective randomized study; Di Stasi SM et al.; PURPOSE: In laboratory studies electromotive mitomycin C (MMC) demonstrated markedly increased transport rates compared with passive transport . We performed a prospective study in patients with high risk superficial bladder cancer to assess the efficacy of intravesical electromotive vs passive MMC using bacillus Calmette-Guerin (BCG) as a comparative treatment . MATERIALS AND METHODS: Following transurethral resection and multiple biopsies 108 patients with multifocal Tis, including 98 with T1 tumors, were randomized into 3 equal groups of 36 each who underwent 40 mg electromotive MMC instillation with 20 mA electric current for 30 minutes, 40 mg passive MMC with a dwell time of 60 minutes or 81 mg BCG with a dwell time of 120 minutes . Patients were scheduled for an initial 6 weekly treatments, a further 6 weekly treatments for nonresponders and a followup 10 monthly treatments for responders . Primary end points were the complete response rate at 3 and 6 months . MMC pharmacokinetics were assessed . RESULTS: The complete response for electromotive vs passive MMC at 3 and 6 months was 53% versus 28% (p = 0.036) and 58% versus 31% (p = 0.012) . For BCG the responses were 56% and 64% . Median time to recurrence was 35 vs 19.5 months (p = 0.013) and for BCG it was 26 months . Peak plasma MMC was significantly higher following electromotive MMC than after MMC (43 vs 8 ng/ml), consistent with bladder content absorption . CONCLUSIONS: Intravesical electromotive administration increases bladder uptake of MMC, resulting in an improved response rate in cases of high risk superficial bladder cancer.

Biol Pharm Bull, 2003 Aug, 26(8), 1089 - 94
Alleviating effects of si-ni-san, a traditional Chinese prescription, on experimental liver injury and its mechanisms; Jiang J et al.; The present study aims at examining the effects of Si-Ni-San, a prescription usually used for treating hepatitis in Traditional Chinese Medicine (TCM), on various experimental liver injury models and its mechanisms . The prescription showed significant hepatoprotection against CCl(4)-induced hepatic damage, both in vivo and in vitro . To the liver injury induced by Bacillus Calmette-Guerin (BCG) with lipopolysaccharide (LPS), Si-Ni-San also provided significant alleviation through enhancing nitric oxide (NO) release by macrophages . Against the liver injury induced by a delayed-type hypersensitivity reaction to picryl chloride (PCl-DTH), Si-Ni-San alleviated it remarkably when administered during either the induction or effector phase . A significant reduction of in-vitro hepatotoxicity, as measured by the inhibition of serum transaminase evaluation, was observed in nonparenchymal cells from liver-injured mice treated with Si-Ni-San . Si-Ni-San facilitated apoptosis in nonparenchymal cells from liver-injured mice, as well as in spleen cells activated by PCl in vivo or by Con A in vitro . These results suggest that Si-Ni-San provides alleviating effects against liver injury through multiple mechanisms, including protection of the hepatocyte membrane, enhancement of NO release, and dysfunction of liver-infiltrating cells mainly through causing their apoptosis.

Respirology, 2003 Sep, 8(3), 376 - 8
Control of an outbreak of BCG complications in Gaza; Daoud W; OBJECTIVE: We aim to describe Bacille Calmette Guerin (BCG) complications in Gaza using two studies: one during an outbreak and the other after control of the possible contributing factors to this outbreak . METHODOLOGY: The first study was conducted on 6145 newborn infants vaccinated in 21 primary care centres in Gaza with BCG vaccine, Pasteur Paris, batch number 5122 from July to October 2001 . The study was repeated after changing the BCG vaccine and training 63 nurses from November to December 2001 . The training program included theoretical lectures on BCG and tuberculosis and practical training in strict intradermal injection . The second study included 6877 newborn infants vaccinated with BCG vaccine, batch number 101023, Denmark, from January to April 2002 . RESULTS: During the outbreak, BCG complications occurred in 225 infants with a complication rate of 36.61 per 1000 vaccinations . The mean age at presentation was 4 months . The commonest complications were regional lymphadenitis in 138 (61.33%) infants, local abscess in 48 (21.33%) infants, local ulcer in 26 (11.56%) infants, keloid scar in 12 (5.33%) infants and one (0.44%) infant who died from disseminated disease had severe combined immunodeficiency . In the follow-up study, BCG complications occurred in 43 infants with a complication rate of 6.25 per 1000 vaccinations, which is significantly lower (P < 0.001) than that during the initial outbreak . CONCLUSION: The study supports the use of a less virulent vaccine and proper vaccination techniques to minimize the incidence of BCG complications . The training of nurses in strict intradermal injection should be maintained and the proper selection of those receiving the vaccines should be considered to avoid the vaccination of any infant with immunodeficiency.

Nippon Hinyokika Gakkai Zasshi, 2003 Jul, 94(5), 574 - 7
{A case in which severely irritable bladder following intravesical instillation of Bacillus Calmette Guerin was successfully treated by steroid therapy}; Kimura K et al.; We report a patient whose irritable bladder symptoms following Bacillus Calmette Guerin (BCG) instillation were satisfactorily treated by steroid administration . A 59-year-old male had undergone transurethral resection for the bladder carcinoma recurred three times . The histopathological examination revealed the tumor as transitional cell carcinoma, G1 to G2, and pTa . Subsequently an instillation of 80 mg BCG into the bladder was planned 8 times every 7 days . After the 5th instillation he presented with gross hematuria, painful micturition, pollakisuria, urgency and reduced bladder capacity of 15 ml . The dose was reduced to 40 mg and another 3 instillations were accomplished . Since conventional treatments of anti-cholinergics, analgesics and epidural anesthesia were of little help for the subjective symptoms, he was put on the steroid pulse therapy 2 weeks after completion of the BCG regimen . The treatment gradually improved the subjective symptoms and increased the bladder capacity up to 160 ml . In conclusion, we believe that the steroid pulse therapy deserves considering in the early stage of irritable bladder symptoms following BCG instillation.

Fortschr Neurol Psychiatr, 2003 Aug, 71(8), 406 - 14
{Neurological complications of inflammatory intestinal diseases}; Dietrich W et al.; A number of neurological symptoms have been described as complications of inflammatory bowel disorders such as coeliac disease, Whipple's disease, Crohn's disease and ulcerative colitis . The neurology of coeliac disease includes disorders of the central nervous system encompassing cerebellar ataxia, epilepsy, myoclonus, dementia and multifocal leukoencephalopathy . Peripheral neuropathies, of axonal and demyelinating types, and myopathies have also been reported . The pathomechanisms are more related to immunological-inflammatory processes than to a malresorptive vitamin deficiencies . While CNS symptoms of coeliac disease show a poor response to gluten restriction, peripheral symptoms may respond to elimination of gluten from the diet . There are few reports of a successful corticosteroid treatment in patients showing inflammatory findings in the CSF or bioptic material . Whipple's disease is caused by the gram-positive bacillus Tropheryma whipplei . Patients with a central nervous system manifestation of Whipple's disease may develop a variety of symptoms including dementia, supranuclear gaze palsy, movement disorders, hypothalamic dysfunction and myorhythmia . The CNS-infection is diagnosed by PCR of the CSF . Long-term antibiotic treatment is required, and relapses may occur after withdrawal of antimicrobial therapy . Crohn's disease and ulcerative colitis are complicated by various forms of polyneuropathies, and arterial and venous cerebrovascular diseases . In most cases with neurological complications of inflammatory gastrointestinal diseases an immune mediated inflammatory process is suspected to be the underlying pathomechanism.

Plant Cell Rep, 2003 Aug, 21(12), 1199 - 206 Epub 2003 May 15.
A promoter derived from taro bacilliform badnavirus drives strong expression in transgenic banana and tobacco plants; Yang IC et al.; Taro bacilliform virus (TaBV) is a pararetrovirus of the genus Badnavirus which infects the monocotyledonous plant, taro ( Colocasia esculenta) . A region of the TaBV genome spanning nucleotides 6,281 to 12 (T1200), including the 3' end of open reading frame 3 (ORF 3) and the intergenic region to the end of the tRNA(met)-binding site, was tested for promoter activity along with four different 5' deletion fragments (T600, T500, T250 and T100) . In transient assays, only the T1200, T600, T500 fragments were shown to have promoter activity in taro leaf, banana suspension cells and tobacco callus . When these three promoters were evaluated in stably transformed, in vitro-grown transgenic banana and tobacco plants, all were found to drive near-constitutive expression of either the green fluorescent protein or beta-glucuronidase (GUS) reporter gene in the stem (or pseudostem), leaves and roots, with strongest expression observed in the vascular tissue . In transgenic banana leaves, the T600 promoter directed four-fold greater GUS activity than that of the T1200, T500 and the maize polyubiquitin-1 promoters . In transgenic tobacco leaves, the levels of GUS expression directed by the three promoters was between four- and ten-fold lower than that of the double Cauliflower mosaic virus 35S promoter . These results indicate that the TaBV-derived promoters may be useful for the high-level constitutive expression of transgenes in either monocotyledonous or dicotyledonous species.

Gene, 2003 Jul 17, 312, 289 - 95
Polymerase chain reaction amplification of the Bag320 satellite family reveals the ancestral library and past gene conversion events in Bacillus rossius (Insecta Phasmatodea); Cesari M et al.; Polymerase chain reaction amplifications of genomic DNA in 17 individuals of bisexual and parthenogenetic populations of three subspecies of Bacillus rossius (Insecta Phasmatodea) revealed that the species still harbours the whole variability of the ancestral Bag320 satellite family, since monomers of all non-hybrid Bacillus taxa plus private sequences occur in it . Bag320 monomers had not been rescued as a major satellite component in B . rossius, but possibly represent the remnant of a set of diverging sequences present in the Bacillus ancestor . Following the library hypothesis, these monomer variants have been differently amplified along the evolutionary pathways leading to present taxa in agreement with the mitochondrial phylogeny of the genus . The putative converted tracts observed are explained as the results of past gene conversion events.

Water Res, 2003 Sep, 37(16), 4004 - 10
Adsorption of halogenated aromatic pollutants by a protein released from Bacillus pumilus; Choi SD et al.; Previous studies of the biosorption of halogenated aromatic pollutants (HAPs) have focused on the sorption of these compounds by cell bodies . However, in this study we investigated the adsorption of HAPs by biocompounds released from a bacterium, Bacillus pumilus . When B . pumilus was exposed to high temperature, it released a protein and carbohydrates, exclusively . After determining experimental conditions using 1,2,3,4-tetrachlorinated dibenzofuran (1,2,3,4-TCDF), the adsorption characteristics of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), polychlorinated biphenyls (PCBs), chlorinated benzenes, and chlorinated naphthalenes were investigated . These HAPs were adsorbed considerably not only by cells but also by the released protein . In general, highly chlorinated congeners were adsorbed to a greater extent on the protein than lowly chlorinated ones, and the amount adsorbed differed between isomers . The present results are consistent with adsorption occurring via a passive physico-chemical mechanism . Finally, the importance of biocompounds released or excreted from microorganisms for the removal of HAPs is discussed.

Rev Soc Bras Med Trop, 2003 May-Jun, 36(3), 373 - 82 Epub 2003 Jul 31.
{Leprosy in Brazil}; Araujo MG; Leprosy or Hansen's disease is a chronic infectious disease caused by the Mycobacterium leprae . The skin and nervous manifestations of the disease present a singular clinical picture that is easily recognized . After India, Brazil still is the second country with the greatest number of cases in the world . Around 94% of the known cases and 94% of the new cases reported in America, come from Brazil . The disease presents itself in two well-defined stable and opposite poles (lepromatous and tuberculoid) and two unstable groups (indeterminate and dimorphic) . The spectrum of presentation of the disease may also be classified as: tuberculoid tuberculoid (TT), borderline tuberculoid (BT), borderline borderline (BB), borderline lepromatous (BL) and lepromatous lepromatous (LL) . The finding of acid fast bacillus in tissue is the most useful method of diagnosis . The effective treatment of leprosy includes the use of specific therapy, suppression of lepra reactions, prevention of physical incapacity, and physical and psychosocial rehabilitation . Chemotherapy with rifampin, dapsone and clofazimine have produced very good results and the control of the disease in Brazil in the foreseeable future is likely.

DNA Cell Biol, 2003 Jun, 22(6), 431 - 40
The role of the host immune response in pathogenesis of Bartonella henselae; Resto-Ruiz S et al.; Bartonella henselae can infect humans resulting in a wide range of disease syndromes including cat-scratch disease, fever with bacteremia, endocarditis, bacillary angiomatosis, and bacillary peliosis hepatis, among others . The nature and severity of the clinical presentation correlates well with the status of the hosts' immune system . Individuals with impaired immune function, including HIV infection, progress to systemic infections more often . Patients with intact immune function who become infected with B . henselae usually get cat-scratch disease, a disease that usually involves lymphadenopathy resulting from a strong cellular immune response to the bacterium . However, immunocompromised patients often progress to bacillary angiomatosis or bacillary peliosis hepatis . The reduced ability of the hosts immune response to control bacterial infection apparently results in a bacteremia of longer duration, and in some patients the presence of angiogenic lesions that are unique among bacterial infections to Bartonella . Recently, the role of immune effector cells that produce angiogenic cytokines upon stimulation with B . henselae has been proposed . Here, the current status of the role of the immune response in both controlling infection and in B . henselae-triggered immunopathogenesis is presented.

Chin Med Sci J, 2002 Jun, 17(2), 81 - 4
The construction and expression of recombinant shuttle plasmid with OmpL1 gene from leptospira interrogans serovar Lai strain 017 in Bacille Calmette-Guerin; Bao L et al.; OBJECTIVE: To construct recombinant BCG against leptospirosis . METHODS: We amplified the entire open reading frame of the OmpL1 gene from the genome of the leptospire serovar Lai strain 017 . Two recombinant plasmids pBQ1 and pBQ2 were constructed by oriented ligation based on the E . coli-BCG shuttle plasmids pMV261 and pMV361 respectively . The recombinant plasmids were transformed into BCG by electroporation . The rBCGs bearing pBQ1 and pBQ2 were induced by high temperature of 45 degrees C . RESULTS: The expressed product, a 35kD protein was detected by SDS-PAGE . The result indicates that pBQ1 and pBQ2 can express OmpL1 in rBCG . CONCLUSION: The technical methods in this study may help detect the immunogenicity and immunoprotection of OmpL1 and develop more safe, highly effective rBCG bearing leptospiral antigen with long-lasting protection.

Clin Infect Dis, 2003 Aug 15, 37(4), 559 - 66 Epub 2003 Jul 31.
Prevalence of Bartonella infection among human immunodeficiency virus-infected patients with fever; Koehler JE et al.; Bartonella infection can be difficult to diagnose, especially when it manifests as bacteremia, which is usually accompanied by nonspecific symptoms, such as fever . Therefore, we hypothesized that Bartonella infection represents an underrecognized cause of febrile illness . To determine the prevalence of Bartonella infection among patients presenting with fever, we evaluated 382 patients in San Francisco . Overall, 68 patients (18%) had evidence of Bartonella infection detected by culture, indirect fluorescent antibody testing, or polymerase chain reaction (PCR) . Twelve patients (3%) had either Bartonella henselae or Bartonella quintana isolated from specimens of blood, tissue, or both or had DNA detected in tissue; all 12 had concomitant human immunodeficiency virus (HIV) infection . Bartonella antibodies were detected in 17% of febrile patients, including 75% of culture-positive or PCR-positive patients . In a nested, matched case-control study aimed at identifying clinical features of febrile illness associated with Bartonella infection, only bacillary angiomatosis and elevated alkaline phosphatase levels were associated with Bartonella infection (P< or =.03 for both) . The prevalence of Bartonella infection among patients with late-stage HIV infection and unexplained fever is much greater than has previously been documented.

Med Microbiol Immunol (Berl), 2004 Feb, 193(1), 19 - 25 Epub 2003 Aug 02.
Immunogenicity of recombinant BCG vaccine strains overexpressing components of the antigen 85 complex of Mycobacterium tuberculosis; Dhar N et al.; The components of antigen 85 complex of Mycobacterium tuberculosis (Ag 85A, Ag 85B and Ag 85C), due to their immunodominant and secretory nature, represent promising protective antigen candidates and have been used in numerous vaccine preparations . We have used recombinant Bacille Calmette Guerin (BCG) strains overexpressing Ag 85A and Ag 85C to immunize BALB/c mice to investigate the immunogenicity of these strains . Mice immunized with recombinant BCG strains exhibited an increased humoral immune response when compared to mice immunized with wild-type BCG . The recombinant BCG strain overexpressing Ag 85A also induced an increased Th1-like response, characterized by elevated levels of IFN-gamma in antigen stimulated splenocyte cultures and a strong IgG2a antibody response, when compared to wild-type BCG . Immunization with recombinant BCG strain overexpressing Ag 85C, on the other hand did not elicit increased IFN-gamma secretion on restimulation of splenocytes in vitro.

Microbiology, 2003 Aug, 149(Pt 8), 2083 - 92
pGIL01, a linear tectiviral plasmid prophage originating from Bacillus thuringiensis serovar israelensis; Verheust C et al.; Bacillus thuringiensis serovar israelensis harbours, in addition to several circular plasmids, a small linear molecule of about 15 kb . Sequence analysis of this molecule, named pGIL01, showed the presence of at least 30 ORFs, five of which displayed similarity with proteins involved in phage systems: a B-type family DNA polymerase, a LexA-like repressor, two potential muramidases and a DNA-packaging protein (distantly related to the P9 protein of the tectiviral phage PRD1) . Experimental evidence confirmed that pGIL01 indeed corresponds to the linear prophage of a temperate phage . This bacteriophage, named GIL01, produces small turbid plaques and is sensitive to organic solvents, which suggests the presence of lipid components in its capsid . Experiments using proteases and exonucleases also revealed that proteins are linked to the genomes of both pGIL01 prophage and GIL01 phage at their 5' extremities . Altogether, these features are reminiscent of those of phages found in the Tectiviridae family, and more specifically of those of PRD1, a broad-host-range phage of Gram-negative bacteria . Dot-blot hybridization, PFGE, PCR and RFLP analyses also showed the presence of pGIL01 variants in the Bacillus cereus group.

Lett Appl Microbiol, 2003, 37(3), 196 - 200
Improved production of teicoplanin using adsorbent resin in fermentations; Lee JC et al.; AIMS: To use adsorbent resins in fermentations to eliminate toxic effects on growth, reduce feedback repression of production and assist in recovery of teicoplanin . METHODS AND RESULTS: An adsorbent resin was added to the culture broth for the adsorption of teicoplanin . Amberlite XAD-16, Diaion HP-20, charcoal and silica gel were investigated as adsorbent resins . The adsorbed teicoplanin was extracted from the resin by 80% methanol after fermentation . Antibiotic activity was quantified by the disc-agar diffusion assay against Bacillus substilis, and qualitative evaluations were based on HPLC using YMC-Pack ODS-A column . Diaion HP-20 was the most effective adsorbent resin when added at a concentration of 5% (w/v) in the inoculation stage . CONCLUSIONS: Addition of Diaion HP-20 in fermentations eliminated toxic effects on growth and reduced feedback repression of teicoplanin by adsorption . There was a 4.2-fold increase in the quantities of teicoplanin . Addition of adsorbent assisted in the recovery of teicoplanin by reducing the recovery steps . SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide useful information for the production of teicoplanin, a glycopeptide antibiotic produced by Actinoplanes teicomyceticus . Addition of adsorbent in fermentation increased productivity of teicoplanin by more than five times.

Nucleic Acids Symp Ser, 2000, (44), 165 - 6
Phage display of xylan-binding module of xylanase J from alkaliphilic Bacillus sp . strain 41M-1; Miyakubo H et al.; Xylanase J from alkaliphilic Bacillus sp . strain 41M-1 has a family 11/G catalytic domain and a xylan-binding module (XBM) . The XBM of xylanase J was displayed on the surface of filamentous bacteriophage . The XBM expressed on the phage surface retained binding activity to xylan . Random mutations were introduced in the XBM gene by error-prone PCR, and the repertoire was cloned for display on phage . Sequence analysis of the xylan-binding activity-deficient mutants revealed that Phe 284 and Trp317 of the XBM would contribute to the xylan-binding activity.

Nucleic Acids Res Suppl, 2002, (2), 227 - 8
A novel chitosanase from Bacillus sp . strain K17: gene cloning and expression in Escherichia coli; Yatsunami R et al.; The gene encoding a novel chitosanase from Bacillus sp . strain K17 was cloned and sequenced . The nucleotide sequence of the gene contained an open reading frame corresponded to a protein of 453 amino acids . The deduced amino acid sequence of the K17 chitosanase exhibited the highest homology to those of family 8 glycanases, suggesting that the enzyme belonged to family 8.

Am J Clin Oncol, 2003 Aug, 26(4), 402 - 7
Impact of intravesical chemotherapy versus BCG immunotherapy on recurrence of superficial transitional cell carcinoma of the bladder: metaanalytic reevaluation; Huncharek M et al.; Bacille Calmette-Guerin (BCG) immunotherapy is currently considered the most effective agent in the management of superficial bladder cancer . Prior work suggests that the efficacy of intravesical chemotherapy in preventing tumor recurrence may be greater than previously suggested . This latter finding, therefore, brings into question the currently perceived superiority of BCG treatment for this disease . A metaanalysis was performed to rigorously examine existing data relevant to this relationship and to quantify the relative efficacy of both treatment modalities on tumor recurrence . A prospective protocol outlining the above-noted metaanalysis was initially developed followed by a thorough search of the existing published literature using strict eligibility criteria . Nine randomized trials were found that met protocol specifications . These reports contained data on 2,261 patients that were statistically combined using a fixed effects model (Peto) . The outcome of interest was the proportion of patients with recurrence at 1, 2, and 3 years following intravesical therapy (i.e., a summary odds ratio, ORp) . Combining all nine trials using 1-year recurrence as the endpoint demonstrated significant statistical heterogeneity, although the ORp favored BCG over intravesical chemotherapy (0.89 {0.74-1.07}) . This precluded statistical pooling of the data and sensitivity analyses were performed to determine the source of heterogeneity . These tests showed that the prior chemotherapy treatment in a large number of the randomized trials biased study results in favor of the BCG arms . Once the data were stratified on presence or absence of prior drug treatment, intravesical chemotherapy reduced 1-, 2-, and 3-year recurrence by 21% to 82% versus BCG, depending on the endpoint of interest . The available data suggest that clinical trials directly comparing intravesical BCG to intravesical chemotherapy must stratify on the presence or absence of prior chemotherapy . Recurrences following prior intravesical chemotherapy appear less responsive to drug therapy than those in chemotherapy-naive patients . The currently perceived superiority of BCG therapy may therefore be an artifact of this phenomenon, since most randomized trials include chemotherapy failures in their chemotherapy treatment arms.

J Immunol, 2003 Aug 15, 171(4), 1961 - 8
An anti-inflammatory role for V alpha 14 NK T cells in Mycobacterium bovis bacillus Calmette-Guérin-infected mice; Dieli F et al.; The possible contribution of NKT cells to resistance to Mycobacterium tuberculosis infection remains unclear . In this paper we characterized the Valpha14 NKT cell population following infection with Mycobacterium bovis bacillus Calmette-Guerin (BCG) . BCG infection determined an early expansion of Valpha14 NKT cells in liver, lungs, and spleen, which peaked on day 8 and was sustained until day 30 . However, an NK1.1(+) Valpha14 NKT population preferentially producing IFN-gamma predominated at an early stage (day 8), which was substituted by an NK1.1(-) population preferentially producing IL-4 at later stages (day 30) . Despite the fact that Valpha14 NKT cell-deficient mice eliminated BCG as did control mice, they had significantly higher numbers of granulomas in liver and lungs . Additionally, while control mice developed organized small granulomas, those in Valpha14 NKT-deficient mice had signs of caseation, large cellular infiltrates, and some multinucleated macrophages, suggesting that Valpha14 NKT cells may actually work as anti-inflammatory cells by limiting excessive lymphocyte influx and tissue pathology . In agreement, we found an increased spontaneous production and mRNA expression of TNF-alpha in liver and lungs of Valpha14 NKT-deficient mice, whose neutralization in vivo by anti-TNF-alpha mAbs consistently reduced the number of granulomas in liver and lungs . Together, our results support a regulatory role for Valpha14 NKT cells in the course of BCG infection through their ability to limit the extent of inflammatory response and point to an important role for this cell subset as a regulator of the balance between protective responses and immunopathology.

Appl Environ Microbiol, 2003 Aug, 69(8), 4901 - 9
Strain-specific ureolytic microbial calcium carbonate precipitation; Hammes F et al.; During a study of ureolytic microbial calcium carbonate (CaCO(3)) precipitation by bacterial isolates collected from different environmental samples, morphological differences were observed in the large CaCO(3) crystal aggregates precipitated within bacterial colonies grown on agar . Based on these differences, 12 isolates were selected for further study . We hypothesized that the striking differences in crystal morphology were the result of different microbial species or, alternatively, differences in the functional attributes of the isolates selected . Sequencing of 16S rRNA genes showed that all of the isolates were phylogenetically closely related to the Bacillus sphaericus group . Urease gene diversity among the isolates was examined by using a novel application of PCR-denaturing gradient gel electrophoresis (DGGE) . This approach revealed significant differences between the isolates . Moreover, for several isolates, multiple bands appeared on the DGGE gels, suggesting the apparent presence of different urease genes in these isolates . The substrate affinities (K(m)) and maximum hydrolysis rates (V(max)) of crude enzyme extracts differed considerably for the different strains . For certain isolates, the urease activity increased up to 10-fold in the presence of 30 mM calcium, and apparently this contributed to the characteristic crystal formation by these isolates . We show that strain-specific calcification occurred during ureolytic microbial carbonate precipitation . The specificity was mainly due to differences in urease expression and the response to calcium.

Appl Environ Microbiol, 2003 Aug, 69(8), 4794 - 805
CynD, the cyanide dihydratase from Bacillus pumilus: gene cloning and structural studies; Jandhyala D et al.; The cyanide dihydratase in Bacillus pumilus was shown to be an 18-subunit spiral structure by three-dimensional reconstruction of electron micrographs of negatively stained material at its optimum pH, 8.0 . At pH 5.4, the subunits rearrange to form an extended left-handed helix . Gel electrophoresis of glutaraldehyde cross-linked enzyme suggests that the fundamental component of the spiral is a dimer of the 37-kDa subunit . The gene was cloned, and the recombinant enzyme was readily expressed at high levels in Escherichia coli . Purification of the recombinant enzyme was facilitated by the addition of a C-terminal six-histidine affinity purification tag . The tagged recombinant enzyme has K(m) and V(max) values similar to those published for the native enzyme . This is the first cyanide dihydratase from a gram-positive bacterium to be sequenced, and it is the first description of the structure of any member of this enzyme class . The putative amino acid sequence shares over 80% identity to the only other sequenced cyanide dihydratase, that of the gram-negative Pseudomonas stutzeri strain AK61, and is similar to a number of other bacterial and fungal nitrilases . This sequence similarity suggests that the novel short spiral structure may be typical of these enzymes . In addition, an active cyanide dihydratase from a non-cyanide-degrading isolate of B . pumilus (strain 8A3) was cloned and expressed . This suggests that cynD, the gene coding for the cyanide dihydratase, is not unique to the C1 strain of B . pumilus and is not a reflection of its origin at a mining waste site.

Appl Environ Microbiol, 2003 Aug, 69(8), 4648 - 57
The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab delta-endotoxin; Lee MK et al.; The Vip3A protein, secreted by Bacillus spp . during the vegetative stage of growth, represents a new family of insecticidal proteins . In our investigation of the mode of action of Vip3A, the 88-kDa Vip3A full-length toxin (Vip3A-F) was proteolytically activated to an approximately 62-kDa core toxin either by trypsin (Vip3A-T) or lepidopteran gut juice extracts (Vip3A-G) . Biotinylated Vip3A-G demonstrated competitive binding to lepidopteran midgut brush border membrane vesicles (BBMV) . Furthermore, in ligand blotting experiments with BBMV from the tobacco hornworm, Manduca sexta (Linnaeus), activated Cry1Ab bound to 120-kDa aminopeptidase N (APN)-like and 250-kDa cadherin-like molecules, whereas Vip3A-G bound to 80-kDa and 100-kDa molecules which are distinct from the known Cry1Ab receptors . In addition, separate blotting experiments with Vip3A-G did not show binding to isolated Cry1A receptors, such as M . sexta APN protein, or a cadherin Cry1Ab ecto-binding domain . In voltage clamping assays with dissected midgut from the susceptible insect, M . sexta, Vip3A-G clearly formed pores, whereas Vip3A-F was incapable of pore formation . In the same assay, Vip3A-G was incapable of forming pores with larvae of the nonsusceptible insect, monarch butterfly, Danaus plexippus (Linnaeus) . In planar lipid bilayers, both Vip3A-G and Vip3A-T formed stable ion channels in the absence of any receptors, supporting pore formation as an inherent property of Vip3A . Both Cry1Ab and Vip3A channels were voltage independent and highly cation selective; however, they differed considerably in their principal conductance state and cation specificity . The mode of action of Vip3A supports its use as a novel insecticidal agent.

Appl Environ Microbiol, 2003 Aug, 69(8), 4502 - 10
PCR assay of the groEL gene for detection and differentiation of Bacillus cereus group cells; Chang YH et al.; Strains of species in the Bacillus cereus group are potentially enterotoxic . Thus, the detection of all B . cereus group strains is important . As 16S ribosomal DNA sequence analysis cannot adequately differentiate species of the B . cereus group, we explored the potential of the groEL gene as a phylogenetic marker . A phylogenetic analysis of the groEL sequences of 78 B . cereus group strains revealed that the B . cereus group strains were split into two major clusters, one including six B . mycoides and one B . pseudomycoides (cluster II) and the other including two B . mycoides and the rest of the B . cereus group strains (cluster I) . Cluster I was further differentiated into two subclusters, Ia and Ib . The sodA gene sequences of representative strains from different clusters were also compared . The phylogenetic tree constructed from the sodA sequences showed substantial similarity to the tree constructed from the groEL sequences . Based on the groEL sequences, a PCR assay for detection and identification of B . cereus group strains was developed . Subsequent restriction fragment length polymorphism (RFLP) analysis verified the PCR amplicons and the differentiation of the B . cereus group strains . RFLP with MboI was identical for all the B . cereus group strains analyzed, while RFLP with MfeI or PstI classified all B . cereus and B . thuringiensis strains into two groups . All cluster II B . mycoides and B . pseudomycoides strains could be discriminated from other B . cereus group bacteria by restriction analysis with TspRI.

Environ Geochem Health, 2003 Mar, 25(1), 139 - 45
A preliminary study of the phycological degradation of natural stone masonry; Welton RG et al.; For many years it has been realised that the weathering of stone is not merely determined by physical and chemical factors but also by biological agents . When the stone in question is a historic building or monument, the damage done constitutes an irretrievable loss of our heritage and history . Laboratory studies have commenced in Paisley to study the effect of photoautotrophs on the major sedimentary rock forming minerals, with a view to expanding this work to study the overall effect of these micro-organisms on heritage masonry . Tests were carried out on Albite, Calcite, Dolomite, Orthoclase, Siderite and Quartz, using axenic cultures of the following: Chlorella vulgaris, Chlorococcum tetrasporum, Scenedesmus obliquus, Oocystis marsonii, Stichococcus bacillaris . The rock chips were immersed in either water or bolds basal media and exposed to a mix of the micro-organisms listed above and then tested weekly for their pH, fortnightly for the waters chemical composition using inductively coupled plasma-atomic emission spectrometry (ICP-AES) and visually utilising the university's SEM facilities . Work so far has revealed biologically mediated etching of minerals, a well-defined pH profile over a period of 90 days, as well as a variety of elemental release patterns for the different minerals.

Mikrobiologiia, 2003 May-Jun, 72(3), 356 - 60
{Chitinolytic activity of Bacillus Cohn.--phytopathogenic fungus antagonist}; Aktuganov GE et al.; Among the 70 tested Bacillus spp . strains antagonistic to phytopathogenic fungi, 19 were found to possess chitinolytic activity when grown on solid media with 0.5% colloidal chitin . The chitinolytic activity of almost all of these 19 strains grown in liquid cultures ranged from 0.1 to 0.3 U/ml . One of the 19 strains exhibited exochitinase activity . In addition to chitinase, two strains also produced chitosanase and one strain, beta-1,3-glucanase . No correlation was found between the antifungal activity of the bacillar strains studied and their ability to synthesize extracellular chitinase . Among the 19 chitinolytic strains, the correlation between these parameters was also low (r x,y = 0.45), although the enzymatic preparations of most of these strains inhibited the growth of the phytopathogenic fungus Helminthosporium sativum.

Mikrobiologiia, 2003 May-Jun, 72(3), 338 - 42
{Synthesis and secretion of Bacillus intermedius proteinases in the late stages of sporulation}; Balaban NP et al.; In the late stages of sporulation, cells of Bacillus intermedius 3-19 secreted into the medium two proteinases, glutamyl endopeptidase and subtilisin, whose maximum activities were recorded in the 40th and 44th hours of growth, respectively . By estimating beta-galactosidase activity as a marker of cytoplasmic membrane integrity, it was revealed that the accumulation of these proteinases in the medium was a result of their secretion and not of lysis of the cell envelope . Concentrations of peptone and inorganic phosphate ensuring the maximum production of the enzymes were established . Ammonium ions were shown to inhibit the production of proteinases by the mechanism of repression by nitrogen metabolites.

Crit Rev Oncol Hematol, 2003 Aug, 47(2), 109 - 26
Intravesical therapy of superficial bladder cancer; Malmstrom PU; The aim of treatment of superficial bladder cancer with intravesical therapy is threefold: (1) Eradicate existing disease . (2) Prevention of recurrence . (3) Prevention of tumor progression . The prognostic factors allow differentiation in different risk groups and this is useful in planning treatment . Studies on pharmacokinetics have proved the efficacy of optimized drug delivery . Comparing resection with and without intravesical chemotherapy a short term approximately 15% decrease in tumor recurrence with chemotherapy can be obtained but no effect on progression was proven . No agent has proved more effective than the other . Single, early instillation of chemotherapy has proven effective but the role of maintenance therapy has been controversial . Immunotherapy in the form of Bacillus Calmette-Guerin generally have proven more efficacious than chemotherapy . The results in comparison to mitomycin C have not been as conclusive . Several new approaches are explored to improve the efficacy of this therapy.

Chin Med Sci J, 1999 Mar, 14(1), 52 - 6
Kinetic changes of antibody to M . leprae and its relation with clinical disease; Jianping S et al.; A sero-epidemiological survey on 1,833 healthy residents was carried out in 6 villages of a leprosy high-endemic area in Wenshan and Guangnan counties, Yunnan Province . The part of the residents with initially antibody-positive as well as the part of residents with initially antibody-negative have been followed up for 3 consecutive years by serology and clinical examination for studying kinetic changes of antibody to M . leprae and its relation with clinical disease . The results showed that the rates of subclinical infection of leprosy in a high-endemic area are different from village to village, and the risk of developing clinical disease does not associate with subclinical infection rate . It correlates with the number of cured accumulative leprosy cases and active cases within the village . The authors consider that in leprosy high-endemic villages, especially those cropped up new multi-bacillary leprosy cases frequently in recent years, it may be helpful to use serology to detect early leprosy cases.

Int Urol Nephrol, 2002, 34(3), 329 - 34
Evaluation of multiple recurrence events in superficial bladder cancer patients treated with intravesical bacillus Calmette-Guérin therapy using the Andersen-Gill's model; Takashi M et al.; To evaluate factors affecting recurrence after intravesical bacillus Calmette-Guerin (BCG) therapy (Tokyo 172 strain), we reviewed data for 101 patients with superficial bladder cancer (pTa {n = 80} and pT1 {n = 21}) treated between 1985 and 1999 . The median follow-up period was 58.9 months . Factors affecting the first tumour recurrence were evaluated using Cox's proportional hazards model and those affecting multiple recurrence with Andersen-Gill's model . The 5-year recurrence-free rate was 63% for all 101 patients . The recurrence frequency, defined as times per 100 patient-months of follow-up, greatly decreased from 7.3 +/- 9.6 (SD) before the instillation to 2.6 +/- 5.6 after the therapy (p < 0.0001) . Patients with pT1 tumours tended to have earlier recurrence than those with pTa tumours (p = 0.06) . Multivariate analysis using Cox's proportional hazards model revealed that a history of bladder cancer and pathological stage were independent factors affecting the first tumour recurrence after the BCG therapy . When multiple endpoints of recurrence were evaluated using the Andersen-Gill's model, number of tumours as well as a history of bladder cancer and pathological stage demonstrated significant links to tumour recurrence after the BCG therapy . The 5-year progression-free and 5-year survival rates were 89.3% and 85.3% for all the 101 patients, respectively . Because intravesical recurrence may involve multiple events during the clinical course of patients with bladder cancer, the Andersen-Gill's model appears useful for evaluation of risk factors.

Arch Virol, 2003 Aug, 148(8), 1465 - 83
Changes in level of virus accumulation and incidence of infection are critical in the characterization of Rice tungro bacilliform virus (RTBV) resistance in rice; Sta Cruz FC et al.; Analysis by enzyme-linked immunosorbent assay showed that Rice tungro bacilliform virus (RTBV) accumulated in a cyclic pattern from early to late stages of infection in tungro-susceptible variety, Taichung Native 1 (TN1), and resistant variety, Balimau Putih, singly infected with RTBV or co-infected with RTBV + Rice tungro spherical virus (RTSV) . These changes in virus accumulation resulted in differences in RTBV levels and incidence of infection . The virus levels were expressed relative to those of the susceptible variety and the incidence of infection was assessed at different weeks after inoculation . At a particular time point, RTBV levels in TN1 or Balimau Putih singly infected with RTBV were not significantly different from the virus level in plants co-infected with RTBV + RTSV . The relative RTBV levels in Balimau Putih either singly infected with RTBV or co-infected with RTBV + RTSV were significantly lower than those in TN1 . The incidence of RTBV infection varied at different times in Balimau Putih but not in TN1, and to determine the actual infection, the number of plants that became infected at least once anytime during the 4 wk observation period was considered . Considering the changes in RTBV accumulation, new parameters for analyzing RTBV resistance were established . Based on these parameters, Balimau Putih was characterized having resistance to virus accumulation although the actual incidence of infection was >75%.

Int J Surg Pathol, 2003 Jul, 11(3), 241 - 4
Upper gastrointestinal bacillary angiomatosis causing hematemesis: a case report; Chetty R et al.; A 38-year-old HIV-positive woman presented with massive hematemesis on initial admission to hospital . Endoscopy revealed ulcerated nodular lesions in the esophagus, stomach, and duodenum . The clinical impression was of Kaposi's sarcoma . The stomach was biopsied when the patient re-presented, and another endoscopy was performed . The biopsy showed mucosal ulceration with a proliferation of vascular channels associated with neutrophils and clumps of purplish, granular bacterial colonies, which were highlighted by a Warthin-Starry stain . The histopathological features were typical of bacillary angiomatosis . This case highlights bacillary angiomatosis involving the gastrointestinal tract at multiple sites, the cause of massive upper gastrointestinal hemorrhage that was the initial presentation of an HIV-positive patient, and the occurrence of visceral bacillary angiomatosis in the absence of cutaneous lesions.

Biochem Biophys Res Commun, 2003 Aug 1, 307(3), 620 - 5
A constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1; Arora N et al.; A 36 kDa chitinase was purified by ion exchange and gel filtration chromatography from the culture supernatant of Bacillus thuringiensis HD-1 . The chitinase production was independent of the presence of chitin in the growth medium and was produced even in the presence of glucose . The purified chitinase was active at acidic pH, had an optimal activity at pH 6.5, and showed maximum activity at 65 degrees C . Of the various substrates, the enzyme catalyzed the hydrolysis of the disaccharide 4-MU(GlnAc)(2) most efficiently and was therefore classified as an exochitinase . The sequence of the tryptic peptides showed extensive homology with Bacillus cereus 36 kDa exochitinase . The 1083 bp open reading frame encoding 36 kDa chitinase was amplified with primers based on the gene sequence of B . cereus 36 kDa exochitinase . The deduced amino-acid sequence showed that the protein contained an N-terminal signal peptide and consisted of a single catalytic domain . The two conserved signature sequences characteristic of family 18 chitinases were mapped at positions 105-109 and 138-145 of Chi36 . The recombinant chitinase was expressed in a catalytically active form in Escherichia coli in the vector pQE-32 . The expressed 36 kDa chitinase potentiated the insecticidal effect of the vegetative insecticidal protein (Vip) when used against neonate larvae of Spodoptera litura.

Res Microbiol, 2003 Jul-Aug, 154(6), 409 - 15
Metal tolerance and biosorption capacity of Bacillus circulans strain EB1; Yilmaz EI; A heavy-metal-resistant bacterium Bacillus sp., strain EB1 was isolated from heavy-metal-contaminated soil in the southeast region of Turkey . Based on 16S ribosomal DNA sequencing, the microorganism was closely related to Bacillus circulans . Minimal inhibitory concentrations of metals (MICs) for the bacterium were determined . Bacillus EB1 exhibited high MIC values for metals and a large spectrum of antibiotic resistance . The order of toxicity of the metals to the bacterium was Cd=Co>Cu>Ni>Zn>Mn in solid media . The effects of increasing metal concentrations to the growth rate were determined in order to obtain precise patterns of resistance in liquid cultures . From the results of heavy metal toxicity, inhibitory concentrations in solid media were higher than those in liquid media . Metal biosorption was determined during the course of growth . B . circulans strain EB1 was capable of removing 90% of Mn, 68% of Zn, 65% of Cu, 45% of Ni and 40% of Co during the active growth cycle with a specific biosorption capacity of 25, 22, 20, 13 and 12 mg/l, respectively . Since Bacillus cells could grow in the presence of significant concentrations of metals and due to high metal biosorption capacity in aerobic conditions, this bacterium may be potentially applicable in in situ bioremediation of heavy-metal-contaminated aqueous systems.

Phytochem Anal, 2003 Jul-Aug, 14(4), 245 - 50
Seasonal variability of phytoplankton at Varna Bay (Black Sea); Moncheva S et al.; Intrinsic fluorescence and SDS-PAGE analysis were employed to study the seasonal qualitative and quantitative changes of phytoplankton composition at Varna Bay (Black Sea) . Variation in the maximum emission wavelength (lambda(max)) of the phytoplankton proteins (398 nm in the summer and 340 nm in the spring) was observed . In addition, a decrease in fluorescence intensity, and a shift in lambda(max) as a result of changes in phytoplankton protein stability, according to the season, was noted . Similarly, SDS-PAGE analysis showed different protein patterns for each season, for example in summertime the major protein constituents were of 14, 37, 48 and 70 kDa, while in the springtime the sizes ranged between 38 and 48 kDa . In general, higher carbohydrate and protein contents correlated with larger phytoplankton biomass found during the summer . The dominant species, the Bacillariophyceae and Dinophyceae, were found to be present in the water body in an alternate pattern . All of these changes could be accounted for by the adaptation of the organism to seasonal variations that modify the sea environment at Varna Bay.

J Biotechnol, 2003 Aug 15, 103(3), 203 - 12
Engineering cyclodextrin glycosyltransferase into a starch hydrolase with a high exo-specificity; Leemhuis H et al.; Cyclodextrin glycosyltransferase (CGTase) enzymes from various bacteria catalyze the formation of cyclodextrins from starch . The Bacillus stearothermophilus maltogenic alpha-amylase (G2-amylase is structurally very similar to CGTases, but converts starch into maltose . Comparison of the three-dimensional structures revealed two large differences in the substrate binding clefts . (i) The loop forming acceptor subsite +3 had a different conformation, providing the G2-amylase with more space at acceptor subsite +3, and (ii) the G2-amylase contained a five-residue amino acid insertion that hampers substrate binding at the donor subsites -3/-4 (Biochemistry, 38 (1999) 8385) . In an attempt to change CGTase into an enzyme with the reaction and product specificity of the G2-amylase, which is used in the bakery industry, these differences were introduced into Thermoanerobacterium thermosulfurigenes CGTase . The loop forming acceptor subsite +3 was exchanged, which strongly reduced the cyclization activity, however, the product specificity was hardly altered . The five-residue insertion at the donor subsites drastically decreased the cyclization activity of CGTase to the extent that hydrolysis had become the main activity of enzyme . Moreover, this mutant produces linear products of variable sizes with a preference for maltose and had a strongly increased exo-specificity . Thus, CGTase can be changed into a starch hydrolase with a high exo-specificity by hampering substrate binding at the remote donor substrate binding subsites.

Am J Trop Med Hyg, 2003 Jun, 68(6), 721 - 7
Low prevalence and increased household clustering of Mycobacterium tuberculosis infection in high altitude villages in Peru; Olender S et al.; Anecdotal historical evidence suggests that tuberculosis was uncommon at high altitude, but whether transmission is affected by high altitude is not known . To test whether high altitude lowers infection by Mycobacterium tuberculosis, the prevalence of tuberculin skin test (TST) positivity was compared between two high altitude villages (3,340 meters {10,960 feet} and 3,500 meters {11,480 feet}) and three sea-level sites in Peru . High altitude villages had lower TST-positive prevalence rates (5.7% and 6.8%) than sea level areas (25-33%), and the difference remained significant (odds ratio = 4.5-6.0) after adjusting for age, education, bacille Calmette-Guerin vaccination, and contact with tuberculosis patients . The TST-positive individuals clustered within highland families more than within sea level families . These data suggest that prevention and control efforts targeted to families may be more effective at high altitude . The mechanism by which TST-positivity prevalence is decreased at high altitude is unknown, but may reflect relative hypoxia, low humidity, or an increased ultraviolet effect.

Mycol Res, 2003 May, 107(Pt 5), 516 - 22
Neophaeosphaeria and Phaeosphaeriopsis, segregates of Paraphaeosphaeria; Camara MP et al.; Two new genera, Neophaeosphaeria and Phaeosphaeriopsis, are described to accommodate species of Paraphaeosphaeria that are not congeneric based on morphological characters and results of 18S rDNA sequence analyses . Paraphaeosphaeria s . str . is restricted to species with two-septate ascospores and anamorphs that produce non-septate, smooth, pale brown conidia enteroblastically from phialides which have some periclinal thickening . Species in Neophaeosphaeria have 3-4-septate ascospores and anamorphs that produce ovoid to ellipsoid, non-septate, brown, verrucose or punctate conidia from percurrently proliferating conidiogenous cells . Paraphaeosphaeria barrii, P . conglomerata, P . filamentosa and P . quadriseptata are transferred to Neophaeosphaeria . At present all species in Neophaeosphaeria occur on Yucca (Agavaceae) . Phaeosphaeriopsis is described for species that produce 4-5-septate ascospores . Known anamorphs produce cylindrical, 0-3-septate, brown, punctate conidia from percurrently proliferating conidiogenous cells or bacillar conidia from simple phialides . P . agavensis, P . glauco-punctata, P . nolinae and P . obtusispora are transferred to Phaeosphaeriopsis . P . amblyspora is described as a new species.

Appl Microbiol Biotechnol, 2003 Aug, 62(2-3), 279 - 83 Epub 2003 Mar 06.
Degradation and detoxification of endosulfan isomers by a defined co-culture of two Bacillus strains; Awasthi N et al.; The degradation of alpha and beta isomers of endosulfan by a two-member bacterial co-culture was studied . Results were similar whether the two isomers were present individually or together, as in technical endosulfan . The degradation of both isomers was accompanied by the formation of endosulfan diol and endosulfan lactone . Accumulation of the metabolite, endosulfan sulfate was, however, not observed during the reaction with either of the isomers . The microbial degradation of endosulfan isomers was also accompanied by a decrease in its toxicity to the test organism Tubifex tubifex Muller.

Appl Microbiol Biotechnol, 2003 Aug, 62(2-3), 233 - 8 Epub 2003 Mar 12.
Antagonistic properties of two recombinant strains of Streptomyces melanosporofaciens obtained by intraspecific protoplast fusion; Agbessi S et al.; Intraspecific protoplast fusion was used to produce stable prototrophic recombinants of Streptomyces melanosporofaciens EF-76, a biocontrol agent of plant disease producing geldanamycin . Two recombinant strains (FP-54 and FP-60) that differed with regard to their antagonistic properties against Bacillus cereus ATCC 14579, Streptomyces scabies EF-35 and Phytophthora fragariae var . rubi 390 were characterized . FP-60 lost the ability to inhibit the in vitro growth of these microbial strains while FP-54 exhibited higher antagonistic activities against them . FP-60 was deficient in geldanamycin biosynthesis whereas FP-54 was shown to produce, in addition to geldanamycin, at least two other antimicrobial compounds that were absent in the culture supernatants of strain EF-76 . Like the wild-type strain EF-76, strain FP-54 reduced common scab symptoms on potato tuber but no significant difference was observed between the disease index attributed to tubers treated with strain EF-76 or with strain FP-54 . Strain FP-60 showed no protective effect against common scab . The disease index of tubers treated with this recombinant was worse than the index associated with potato tubers from control treatments.

Int Immunol, 2003 Aug, 15(8), 915 - 25
Immunization with a mycobacterial lipid vaccine improves pulmonary pathology in the guinea pig model of tuberculosis; Dascher CC et al.; Lipids and glycolipid molecules derived from Mycobacterium tuberculosis can be presented to T cells by CD1 cell-surface molecules in humans . These lipid-specific T cells are cytolytic, secrete pro-inflammatory cytokines and have bactericidal activity . Here, we describe studies in which lipids from M . tuberculosis were incorporated into liposomes with adjuvant and tested as vaccines in a guinea pig aerosol tuberculosis challenge model . Animals vaccinated with mycobacterial lipids showed reduced bacterial burdens in the lung and spleen at 4 weeks after infection . In addition, the lungs of lipid-vaccinated animals also had significantly less pathology, with granulomatous lesions being smaller and more lymphocytic . In contrast, animals receiving only vehicle control immunizations had granulomatous lesions that were larger and often contained caseous necrotic centers . Quantification of histopathology by morphometric analysis revealed that the overall percentage of lung occupied by diseased tissue was significantly smaller in lipid-vaccinated animals as compared to vehicle control animals . In addition, the mean area of individual granulomatous lesions was found to be significantly smaller in both lipid- and bacillus Calmette-Guerin-vaccinated guinea pigs . These data support an important role for lipid antigens in the immune response to M . tuberculosis infection, potentially through the generation of CD1-restricted T cells . Immunogenic lipids thus represent a novel class of antigens that might be included to enhance the protective effects of subunit vaccine formulations.

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