Bacillus

Int J Food Microbiol, 2001 Jan 22, 63(1-2), 35 - 50
Investigation of bacterial spore structure by high resolution solid-state nuclear magnetic resonance spectroscopy and transmission electron microscopy; Leuschner RG et al.; High resolution solid-state nuclear magnetic resonance spectroscopy (NMR) in combination with transmission electron microscopy (TEM) of spores of Bacillus cereus, an outer coatless mutant B . subtilis 322, an inner coatless mutant B . subtilis 325 and of germinated spores of B . subtilis CMCC 604 were carried out . Structural differences in the coats, mainly protein of spores were reflected by NMR spectra which indicated also differences in molecular mobility of carbohydrates which was partially attributed to the cortex . Dipicolinic acid (DPA) of spores of B . cereus displayed a high degree of solid state order and may be crystalline . Heat activation was studied on spores of B . subtilis 357 lux + and revealed a structural change when analysed by TEM but this was not associated with increases in molecular mobility since no effects were measured by NMR.

Int J Food Microbiol, 2001 Jan 22, 63(1-2), 29 - 34
Modelling the influence of pH and organic acid types on thermal inactivation of Bacillus cereus spores; Leguerinel I et al.; A model is proposed to describe the influence pH on the heat resistance of Bacillus cereus spores . In addition to the conventional z value, the effect of pH on the thermal resistance of spores is characterised by a z(pH) value (z(pH) is the distance of pH from a reference pH*, which leads to a 10-fold reduction of D value) . The type of organic acid used for acidifying the heating medium, influences the z(pH) value . For nine organic acids, a linear relationship between the calculated z(pH) value and its lower acid pKa is observed . This relationship showed that the acid form (dissociated or undissociated) modifies the thermal spore resistance in addition to the H+ ion . The influence of acetic acid concentration on the D value at pH 7 shows the protective effect of the dissociated acid form on the heat resistance of spores . The acid concentration in the medium modified the heat resistance of spore and the z(pH) value.

Arch Virol, 2000, 145(12), 2643 - 57
Genetic composition and complexity of virus populations at tungro-endemic and outbreak rice sites; Azzam O et al.; We have recently demonstrated the geographic isolation of rice tungro bacilliform virus (RTBV) populations in the tungro-endemic provinces of Isabela and North Cotabato, Philippines . In this study, we examined the genetic structure of the virus populations at the tungro-outbreak sites of Lanao del Norte, a province adjacent to North Cotabato . We also analyzed the virus populations at the tungro-endemic sites of Subang, Indonesia, and Dien Khanh, Vietnam . Total DNA extracts from 274 isolates were digested with EcoRV restriction enzyme and hybridized with a full-length probe of RTBV . In the total population, 22 EcoRV-restricted genome profiles (genotypes) were identified . Although overlapping genotypes could be observed, the outbreak sites of Lanao del Norte had a genotype combination distinct from that of Subang or Dien Khanh but a genotype combination similar to that identified earlier from North Cotabato, the adjacent endemic province . Sequence analysis of the intergenic region and part of the ORF1 RTBV genome from randomly selected genotypes confirms the geographic clustering of RTBV genotypes and, combined with restriction analysis, the results suggest a fragmented spatial distribution of RTBV local populations in the three countries . Because RTBV depends on rice tungro spherical virus (RTSV) for transmission, the population dynamics of both tungro viruses were then examined at the endemic and outbreak sites within the Philippines . The RTBV genotypes and the coat protein RTSV genotypes were used as indicators for virus diversity . A shift in population structure of both viruses was observed at the outbreak sites with a reduced RTBV but increased RTSV gene diversity.

In Vivo, 2000 Nov-Dec, 14(6), 721 - 4
Oxidative stress of red blood cells during Bacillus Calmette-Guerin intravesical instillations; Mitropoulos D et al.; Although Bacillus Calmette-Guerin (BCG) intravesical instillation is widely accepted as a very effective modality in treating bladder carcinoma in situ, and in preventing superficial bladder cancer recurrence, its mechanism of action is not yet fully understood . The antitumor effects of BCG are mostly related to local immunological events but a systemic activation of the immune system cannot be excluded . The objective of the present study was to estimate the systemic production of oxidants during intravesical BCG treatment . Systemic production of oxidants was estimated by assessing the red blood cells (RBC) oxidative stress in twelve patients undergoing BCG immunotherapy for bladder carcinoma in situ . RBC oxidative stress induced by peroxynitrite was determined by luminol-enhanced chemiluminescence . During the treatment period, the RBC oxidative stress revealed a biphasic curve of changes: after an initial 5-fold increase, it dropped to pretreatment levels following the 4th instillation . Intravesical BCG administration induced systemic production of oxygen free radicals that may reflect a systemic activation of the immune system.

Z Naturforsch {C}, 2000 Nov-Dec, 55(11-12), 987 - 90
PCR-based identification of Bacillus thuringiensis isolated from soil samples in Nigeria; Ogunjimi AA et al.; Six isolates of Bacillus thuringiensis isolated from soil samples confirmed to be toxic to mosquito larvae were differentiated using a PCR-Based technique . Three of these isolates initially identified using a serological technique were further differentiated with the PCR amplification of the delta-endotoxin target sequences . Using the total DNA of isolates as template, at least four isolates yielded amplicons one or all the crystal protein genes, cryI a, b, c, or II with sizes ranging from 238-1070 bp . None of these isolates yielded an amplicon for any of Cry IV A, B and D tested . Of the four isolates identified by PCR technique one isolate remained unidentified by serology.

Lipids, 2000 Dec, 35(12), 1371 - 5
Unusual lipid composition of a Bacillus sp . isolated from Lake Pomorie in Bulgaria; Carballeira NM et al.; The lipid composition of a Bacillus sp., isolated from Lake Pomorie in Bulgaria, was unusual and consisted of 26 different fatty acids between C12 and C26, with anteiso C15-C17 saturated fatty acids predominating . The furan fatty acid, 10,13-epoxy-11-methyloctadeca-10,12-dienoic acid, was also identified, a new finding for this genus . The hydrocarbons consisted of 30 different monounsaturated hydrocarbons, between C25 and C30, with the iso-iso, iso-anteiso, anteiso-anteiso, iso-normal, and anteiso-normal methyl branching for odd-numbered chains, and the iso-iso, iso-anteiso, iso-normal, and anteiso-normal methyl branching for even-numbered chains . The double bond positions in these hydrocarbons were determined by dimethyl disulfide derivatization followed by GC-MS, and the double-bond cis configuration was confirmed by infrared spectroscopy . Some previously unknown hydrocarbons in bacteria, such as (Z)-3,21-dimethyl-9-tricosene, (Z)-3,21-dimethyl-10-tricosene, (Z)-2,24-dimethyl-11-pentacosene, and (Z)-2,25-dimethyl-13-hexacosene were identified . Sterols were detected and were based on the sitosterol nucleus.

Nagoya J Med Sci, 2000 Nov, 63(3-4), 123 - 8
Severe disseminated BCG infection in an 8-year-old girl; Yamanaka K et al.; An 8-year-old girl died of sepsis due to staphylococcal infection one year and 8 months after Bacille Calmette-Guerin (BCG) revaccination . Two months after the vaccination in accordance with the school health program, she was hospitalized with a high fever, skin rash over the face and lower limbs, and leukopenia . Her clinical and laboratory pictures were not compatible with those of any established type of immunodeficiency . The polymerase chain reaction (PCR) test for M . tuberculosis complex was positive for bone marrow, pleural fluid, and peripheral blood . The strain recovered from a mycobacterial culture of the blood was identical to the BCG strains with which the patient was vaccinated, based on restriction fragment length polymorphism (RFLP) and a pulse-field gel electrophoresis (PFGE) analyses of DNA . She developed finally a lung abscess due to staphylococcal septicemia, which was the direct cause of her death.

Exp Appl Acarol, 2000 Jul, 24(7), 497 - 560
Diseases of mites; van der Geest LP et al.; An overview is given of studies on diseases of mites . Knowledge of diseases of mites is still fragmentary but in recent years more attention has been paid to acaropathogens, often because of the economic importance of many mite species . Most research on mite pathogens concerns studies on fungal pathogens of eriophyoids and spider mites especially . These fungi often play an important role in the regulation of natural mite populations and are sometimes able to decimate populations of phytophagous mites . Studies are being conducted to develop some of these fungi as commercial acaricides . Virus diseases are known in only a few mites, namely, the citrus red mite and the European red mite . In both cases, non-occluded viruses play an important role in the regulation of mite populations in citrus and peach orchards, respectively, but application of these viruses as biological control agents does not seem feasible . A putative iridovirus has been observed in association with Varroa mites in moribund honeybee colonies . The virus is probably also pathogenic for honeybees and may be transmitted to them through this parasitic mite . Few bacteria have been reported as pathogens of the Acari but in recent years research has been concentrated on intracellular organisms such as Wolbachia that may cause distorted sex ratios in offspring and incompatibility between populations . The role of these organisms in natural populations of spider mites is in particular discussed . The effect of Bacillus thuringiensis on mites is also treated in this review, although its mode of action in arthropods is mainly due to the presence of toxins and it is, therefore, not considered to be a pathogen in the true sense of the word . Microsporidia have been observed in several mite species especially in oribatid mites, although other groups of mites may also be affected . In recent years, Microsporidia infections in Phytoseiidae have received considerable attention, as they are often found in mass rearings of beneficial arthropods . They affect the efficacy of these predators as biological control agent of insect and mite pests . Microsporidia do not seem to have potential for biological control of mites.

J Mol Microbiol Biotechnol, 2001 Jan, 3(1), 73 - 82
Protein-protein interaction between Bacillus stearothermophilus tyrosyl-tRNA synthetase subdomains revealed by a bacterial two-hybrid system; Karimova G et al.; We have recently developed a bacterial two-hybrid system (BACTH), based on functional complementation between two fragments of the catalytic domain of Bordetella pertussis adenylate cyclase (AC), that allows an easy in vivo screening and selection of functional interactions between two proteins in Escherichia coli . In this work, we have further explored the potentialities of the BACTH system to study protein-protein interactions, using as a model, the interactions between various subdomains of the dimeric tyrosyl-tRNA synthetase (TyrRS) of Bacillus stearothermophilus . Using the BACTH system we confirmed the known interactions of the alpha/beta domains and those between the alpha/beta domain and the alpha domain that could be anticipated from the three-dimensional structure of TyrRS . Interestingly, the BACTH system revealed the unexpected interaction between the TyrRS alpha domains which is presumably mediated by a pseudo-leucine zipper motif . This study illustrates the interest of the bacterial two-hybrid system to delineate interacting domains of proteins and shows that it can reveal interactions that occur in vivo and that were not anticipated from the three-dimensional structure of the protein of interest.

J Leukoc Biol, 2001 Jan, 69(1), 138 - 48
Impaired IL-15 production associated with susceptibility of murine AIDS to mycobacterial infection; Umemura M et al.; LP-BM5 murine leukemia virus (MuLV) injection causes murine AIDS (MAIDS), a disease characterized by many functional abnormalities of immunocompetent cells . We show that MAIDS mice are susceptible to Mycobacterium bovis Bacille Calmette-Guerin (BCG) infection as assessed by survival rate and bacterial counts . The peritoneal exudate macrophages from MAIDS mice produced a significant level of interleukin (IL)-12 soon after inoculation with BCG, whereas IL-15 and tumor necrosis factor (TNF) production were severely impaired in BCG-infected MAIDS mice . The appearance of natural killer (NK) and CD4+ T helper type 1 (Th1) cells specific for mycobacterial antigen were depressed in MAIDS mice after BCG infection . Thus, it appeared that impaired production of IL-15, besides other inflammatory cytokines, in MAIDS mice may be involved in the poor responses of the NK and Th1 cells, resulting in an increased susceptibility to BCG.

J Basic Microbiol, 2000, 40(5-6), 385 - 8
Diversity of Bacillus thuringiensis in different habitats of northern Jordan; Obeidat M et al.; A survey of Bacillus thuringiensis was conducted for 17 locations in Northern Jordan representing 12 different habitats . Eighty isolates were identified as B . thuringiensis in the majority of the tested samples . Results showed that soils contaminated with the slaughterhouses waste materials had the highest content of spore-forming bacteria {(4.05-2.2) 10(7) CFU/g)} and B . thuringiensis {(4.05-7.9) 10(7) CFU/g)} with a (5.5%-14.9%) and (5.2%-7.7%) of the total viable bacterial count, respectively . These bacteria were more abundant in soils contaminated with such animal by-products.

J Basic Microbiol, 2000, 40(5-6), 363 - 8
New enzymatic method for determining D-arabinitol in serum; Hino M et al.; A new reagent has been developed to determine D-arabinitol . This utilizes D-arabinitol 2-oxidoreductase derived from Bacillus sp . with high stability, and water-soluble tetrazolium salt, that can detect NADH with high sensitivity . Since this enzyme does not react to D-mannitol, elimination of D-mannitol is unnecessary . Thus, this is a much simpler process than currently available with commercial kits use D-arabinitol 4-oxidoreductase . The within-run and between-run precisions (CV) were 2.4-6.9% and 3.1-8.7%, respectively, whilst the correlation (r) between the results obtained with our proposed method (y) and those obtained with the commercial "Arabinitec-auto" kit (x) was 0.964 (y = 1.02x + 0.933 mumol/l; n = 69) . However, some samples deviated remarkably from correlation in both methods . Our analyzing accuracy is satisfactory in clinical application, as it does not miss positive sample over cut-off value . We are refining this method by investigating why some specimens are apart from correlation significantly.

J Am Mosq Control Assoc, 2000 Dec, 16(4), 342 - 5
Evaluation of methylated soy oil and water-based formulations of Bacillus thuringiensis var . Israelensis and Golden Bear Oil (GB-1111) against anopheles quadrimaculatus larvae in small rice plots; Dennett JA et al.; The efficacy of formulations containing methylated soybean oil (MSO) alone and with technical-grade Bacillus thuringiensis var . israelensis (Bti) were compared to Golden Bear Oil (GB-1111) and a water-based Bti formulation against 3rd- to 4th-stage Anopheles quadrimaculatus larvae confined to sentinel cages in small rice plots . Three replicates each of MSO with 2% Pyroter added as a surfactant (MSO + PYR), MSO with 2% Pyroter and 4 g of Bti technical powder (MSO + PYR + Bti), GB-1111, a water-based formulation with 4 g of Bti technical powder (Bti + water), and untreated controls were performed . Mosquito larvae were introduced on the 1st day of treatment and at 4 days posttreatment . Mortality was recorded at 24 and 48 h posttreatment for the 1st installation and at 5 days posttreatment for the 2nd installation . The Bti + water formulation provided 71% control and the MSO + PYR + Bti formulation achieved 64% control, whereas MSO + PYR and GB-1111 produced 16 and 18% control, respectively, at 24 h posttreatment . With the exception of MSO + PYR + Bti, which decreased by 2%, the mean percent control increased slightly at 48h posttreatment across remaining treatments, with Bti + water obtaining 72% control . This was significantly higher than GB-1111, which achieved 23% control at 48 h posttreatment . The MSO + PYR and MSO + PYR + Bti formulations yielded 56 and 62% control, respectively, during the same interval and were not significantly different from one another . Formulations containing MSO + PYR exhibited delayed activity similar to GB-1111, with all formulations except MSO + PYR + Bti providing greatest control at 48 h posttreatment . Both MSO formulations (MSO + PYR + Bti and MSO + PYR) were statistically comparable to Bti + water and GB-1111, respectively, at 24 and 48 h posttreatment . None of the formulations exhibited a residual activity adequate enough to control An . quadrimaculatus larvae for up to 5 days.

J Am Mosq Control Assoc, 2000 Dec, 16(4), 321 - 3
Control of aedes aegypti breeding in desert coolers and tires by use of Bacillus thuringiensis var . Israelensis formulation; Batra CP et al.; Three different formulations of Bacillus thuringiensis var . israelensis (Bti) were evaluated for their efficacy against immature Aedes aegypti in desert coolers and tires . Three formulations, viz., VectoBac tablets, VectoBac granules, and Bacticide powder, at the application rate of 0.75, 2, and 1 g per cooler, respectively, and VectoBac tablets at 0.75 and 0.375 g per tire, were evaluated . In coolers and tires, 100% reduction in the abundance of late larval instars of Ae . aegypti was observed for a period of 2 and 3 wk, respectively . The possibility of using tablets and capsules filled with Bti granules and powder formulation by individuals or communities for control of Ae . aegypti breeding has been discussed in view of the increasing outbreaks of dengue and dengue hemorrhagic fever in India . Use of these formulations over conventional methods is better and more user-friendly.

J Egypt Soc Parasitol, 2000 Dec, 30(3), 839 - 49
Potential for Culex pipiens to develop resistance against Bacillus sphaericus toxins; Soliman BA et al.; Susceptibility of field Culex pipiens populations to Bacillus sphaericus and the possibility of resistance development in the selected laboratory colony were investigated . 180 populations collected from Giza and Qualyobia over one year show no marked variation at LC50 and LC90 values . Larvae of laboratory colony of Cx pipiens were subjected to selection pressure with a preparation of Bacillus sphaericus strain 2362 at LC80 values for 20 successive generations . The selected laboratory colony began developing resistance at F10 (RR = 1.69) when compared with its unselected counterpart . The magnitude of resistance increased gradually and reached 33 fold by F20 . The unselected colony reared without any exposure to the B . sphaericus toxin, did not show any marked change in their susceptibility to this microbial agent.

Faraday Discuss, 2000, (116), 135 - 53; discussion 171-90
Engineering artificial redox chains by molecular 'Lego'; Sadeghi SJ et al.; This work reports on a novel approach for building artificial redox chains: the molecular 'Lego' approach . This exploits the scaffold of natural redox proteins by fusing together functional protein modules with the desired properties . The molecular 'Lego' mimics the natural molecular evolution that proceeded by modular assembly of genes/DNA segments . Non-physiological electron transfer partners, flavodoxin (fld) and cytochrome c553 (c553) from Desulfovibrio vulgaris and the haem domain of P450 BM3 (BMP) from Bacillus megaterium have been used as building blocks in different combinations to build artificial redox chains . The kinetic characterization of the electron transfer (ET) between the separate building blocks has been carried out . Under pseudo-first order conditions, a limiting ET rate, klim, of 0.48 +/- 0.05 s-1 and 43.77 +/- 2.18 s-1 and an apparent binding constant, Kapp, of 21 +/- 6 microM and 1.23 +/- 0.32 microM have been found for the fld/c553 and fld/BMP redox pairs, respectively . These results show that fld can be used as a module for transferring electrons to c553 and BMP . A 3D model of the fld/c553 and fld/BMP complexes was used to guide the construction of covalently linked assemblies via engineered disulfide bridges or by fusion of the relevant genes via an engineered loop . The first approach led to the construction, expression and characterization of the S35C and S64C mutants of fld and M23C and G51C mutants of c553 . Although the redox potentials of the separate mutants were found to be the same as those of recombinant wild type proteins (-408 mV for the semiquinone/hydroquinone couple of fld and +32 mV for the c553), the c553 homo-dimers M23C-M23C and G51C-G51C were found to have redox potentials of +88 and +105 mV, respectively . These differences have been analysed in terms of exposure of the haem cofactors to the solvent, and these lead to some interesting questions on the redox potentials of the transient redox complexes in physiological systems . The fld-c553 S64C-M23C and S35C-M23C chimeras were constructed, expressed and purified but the FMN was found to be destabilised resulting in the apo-form of these proteins . The gene fusion strategy was used to produce covalently linked assemblies of both fld-c553 and fld-BMP . The former was expressed using a seven amino acid (GPGPGPG) loop linking the C-terminus of fld to the N-terminus of c553 . The fld-BMP fusion protein was successfully expressed by using the naturally occurring loop of the P450 BM3 (residues 471-479) to link the BMP domain at the N-terminus with fld domain at the C-terminus . This fusion was found to be correctly folded and functional . Efficient ET from the FMN to the haem domain (370 s-1) was also found to be in the same region of the physiological redox partners (250 s-1) . This work demonstrates the feasibility of the molecular 'Lego' approach in generating functional multi-domain proteins with designed properties, beyond the restrictions imposed by the naturally occurring protein domains.

Arch Tierernahr, 2000, 53(4), 353 - 73
Comparative studies on the in vitro properties of phytases from various microbial origins; Igbasan FA et al.; The physical and chemical properties of six crude phytase preparations were compared . Four of these enzymes (Aspergillus A, Aspergillus R, Peniophora and Aspergillus T) were produced at commercial scale for the use as feed additives while the other two (E . coli and Bacillus) were produced at laboratory scale . The encoding genes of the enzymes were from different microbial origins (4 of fungal origin and 2 of bacterial origin, i.e., E . coli and Bacillus phytases) . One of the fungal phytases (Aspergillus R) was expressed in transgenic rape . The enzymes were studied for their pH behaviour, temperature optimum and stability and resistance to protease inactivation . The phytases were found to exhibit different properties depending on source of the phytase gene and the production organism . The pH profiles of the enzymes showed that the fungal phytases had their pH optima ranging from 4.5 to 5.5 . The bacterial E . coli phytase had also its pH optimum in the acidic range at pH 4.5 while the pH optimum for the Bacillus enzyme was identified at pH 7.0 . Temperature optima were at 50 and 60 degrees C for the fungal and bacterial phytases, respectively . The Bacillus phytase was more thermostable in aqueous solutions than all other enzymes . In pelleting experiments performed at 60, 70 and 80 degrees C in the conditioner, Aspergillus A, Peniophora (measurement at pH 5.5) and E . coli phytases were more heat stable compared to other enzymes (Bacillus enzyme was not included) . At a temperature of 70 degrees C in the conditioner, these enzymes maintained a residual activity of approximately 70% after pelleting compared to approximately 30% determined for the other enzymes . Incubation of enzyme preparations with porcine proteases revealed that only E . coli phytase was insensitive against pepsin and pancreatin . Incubation of the enzymes in digesta supernatants from various segments of the digestive tract of hens revealed that digesta from stomach inactivated the enzymes most efficiently except E . coli phytase which had a residual activity of 93% after 60 min incubation at 40 degrees C . It can be concluded that phytases of various microbial origins behave differently with respect to their in vitro properties which could be of importance for future developments of phytase preparations . Especially bacterial phytases contain properties like high temperature stability (Bacillus phytase) and high proteolytic stability (E . coli phytase) which make them favourable for future applications as feed additives.

Mikrobiologiia, 2000 Nov-Dec, 69(6), 796 - 800
{Characterization of a new strain of Bacillus thuringiensis, a producer of an endotoxin against coleoptera}; Dobrzhanskaia EO et al.; A new strain of Bacillus thuringiensis 2-7 was found to belong to the serotype H8 . Cells of this strain contained irregular and flat crystalline inclusions and two large plasmids . The gene responsible for crystal formation is most likely located on the large plasmid greater than 105 MDa in size . Comparison of the cry gene of B . thuringiensis 2-7 and the cryIIIA gene of B . thuringiensis subsp . tenebrionis showed that their nucleotide sequences are identical.

Acta Paediatr, 2000 Dec, 89(12), 1495 - 7
Sternal tuberculosis in a 9-month-old infant after BCG vaccination; Kato Y et al.; Tuberculosis osteitis is mainly observed as a late complication of the pulmonary infection . We describe a 9-mo-old Japanese infant who became infected with Mycobacterium tuberculosis even after receiving Bacillus Calmette-Guerin (BCG) vaccination at 3 mo of age . This is the first report that the sternum is the localization for tuberculous osteitis in infants . He developed a localized tumor in the sternum without any respiratory symptom . Mycobacterium tuberculosis complex was detected by means of nucleic acid amplification test . Gastric aspirates also yielded Mycobacterium tuberculosis . The source of the infection was unclear . CONCLUSION: Tuberculous osteitis should be excluded in infants with undiagnosed bone lesions, even if they have been vaccinated with BCG.

Sci Total Environ, 2000 Dec 18, 263(1-3), 155 - 60
The measurement of volatile constituents in Foray 48B, an insecticide prepared from Bacillus thuringiensis var . kurstaki; van Netten C et al.; Foray 48B, an insecticide prepared from Bacillus thuringiensis var . kurstaki (Btk), has been used for many years to combat infestations of Gypsy moths . Foray 48B also contains a large number of 'inert ingredients' which are not disclosed by the manufacturer . Gypsy moths usually enter the country through marine- and airports in close proximity to urban areas, which consequently need to be sprayed . The population affected often demands more detailed information than what is available including the potential presence of volatile organic agents which could be released during spraying, posing a potential health hazard . Four different methods were investigated using GC/mass spectrometry regarding their ability to capture volatile agents associated with Foray 48B . It was found that solid phase micro-extraction was most efficient in capturing volatile agents from the head-space of Foray 48B . Separate trials using 95:5% ethanol/isopropanol mixture and toluene in an impinger configuration were much less efficient . Standard techniques using activated charcoal tubes in the laboratory setting as well as in a field trial did not capture any compounds . It was concluded that the volatile agents associated with Foray 48B did not appear to constitute a significant health hazard and no one agent was a likely candidate to serve as a tracer for Foray 48B exposure.

AIDS, 2001 Jan 5, 15(1), 55 - 60
Bacteremia due to Mycobacterium tuberculosis or M . bovis, Bacille Calmette-Guérin (BCG) among HIV- positive children and adults in Zambia; Waddell RD et al.; BACKGROUND: Among adults with advanced HIV infection in developing countries, bacteremia due to Mycobacterium tuberculosis (MTB) is common and bacteremia due to M . bovis (bacille Calmette-Guerin; BCG) is rare . Comparable data are not available for children with HIV . OBJECTIVE: To compare the prevalence of bacteremia due to M . tuberculosis or M . bovis BCG in hospitalized children and adults with HIV infection in a developing country with a high prevalence of tuberculosis and HIV and > 95% BCG immunization coverage . DESIGN: Descriptive cross-sectional study . METHODS: Prospectively hospitalized patients in Lusaka, Zambia who were suspected to have HIV infection underwent phlebotomy for HIV ELISA, HIV viral load, and lysis-centrifugation blood culture for mycobacteria . Histories were obtained and patients were examined for BCG scars . Mycobacterial isolates were identified using DNA probes for MTB complex (MTBC), multiplex PCR and IS6110 typing . RESULTS: The median age of 387 HIV-positive children was 15 months; 98% were BCG immunized . The median age of 344 HIV-positive adults was 32 years; 44% were BCG immunized . Blood cultures were positive for mycobacteria in six children (2%) and 38 adults(11%) (P < 0.001) . The six pediatric isolates included five MTBC (40% clustered) and one BCG . The 38 adult isolates included 36 MTBC (16% clustered) and two M . avium complex . CONCLUSION: Bacteremia due to MTB is less common among children than adults with advanced HIV infection in Zambia . Bacteremia due to M . bovis BCG is rare even among children with recent BCG immunization and symptomatic HIV infection.

J Med Microbiol, 2001 Jan, 50(1), 23 - 8
Binding of alpha2-laminins by pathogenic and non-pathogenic mycobacteria and adherence to Schwann cells; Marques MA et al.; The ability of Mycobacterium leprae to specifically bind alpha2-laminins of Schwann cells has been described recently as being an important property of the leprosy bacillus, which could explain the neural tropism of M . leprae . Therefore, the extent of the expression of alpha2-laminin-binding properties among mycobacteria was investigated . In an ELISA-based assay, all three species of Mycobacterium tested (M . tuberculosis, M . chelonae and M . smegmatis) expressed laminin-binding capacity, suggesting that the ability to bind alpha2-laminins is conserved within the genus Mycobacterium . This report also demonstrated that not only M . leprae but all the mycobacterial species tested readily interacted with the ST88-14 cells, a human schwannoma cell line, and that the addition of soluble alpha2-laminins significantly increased their adherence to these cells . These results failed to demonstrate the presence in M . leprae of a unique system based on alpha2-laminins for adherence to Schwann cells.

Yi Chuan Xue Bao, 2000, 27(10), 932 - 8
{Cloning of plasmid pBMB2062 in Bacillus thuringiensis strain YBT-1520 and construction of plasmid vector with genetic stability}; Sun M et al.; A small plasmid pBMB2062 in Bacillus thuringiensis strain YBT-1520 was cloned and sequenced . Its 2,062 bp sequence contains two potential open reading frames (orfs) . The orf1 and orf2 encode a tentative replication initial protein consisting of 289 amino acid residues and a tentative replication protein consisting of 80 amino acid residues, respectively . Two homological plasmids were found by Blast searching . There are 23 nucleotides difference occurring among three of the plasmids . The difference occurred in the orf1 causes different encoding capability . Comparing with the orf1 in pBMB2062, the orf1 in the homological plasmids are truncated, one at the N-terminal and another at the C-terminal . cDNA synthesis and PCR detection showed that the mRNA corresponding to orf1 in pBMB2062 really occurs . Shuttle vectors were constructed based on pBMB2062 and showed the ability to express insecticidal crystal gene . Under nonselective condition, recombinant plasmids based on pBMB2062 were genetically stable.

Sheng Wu Gong Cheng Xue Bao, 2000 Sep, 16(5), 587 - 90
{Transfer of cry1C gene into Bacillus thuringiensis by electroporation to construct strain with broader insecticidal activity}; Lu SQ et al.; Three Transformants were selected by transferring cry1C into Bacillus thuringiensis strain YBT1535 . Plasmid profiles, PCR and Southern blot result all proved that cry1C had been transferred into strain YBT1535 . Bioassay results showed that the transformants of strain YBT1535 displayed significantly higher toxicity against Spodoptera exigua than strain YBT1535, but the toxicities against Heliothis armigera and Plutella xylostella did not rise except transformant YBT1535-2.

Med Clin North Am, 2001 Jan, 85(1), 79 - 114
Nosocomial pneumonia . Diagnostic and therapeutic considerations; Cunha BA; Many patients with presumed nosocomial pneumonia probably have infiltrates on the chest radiograph, fever, and leukocytosis resulting from noninfectious causes . Because of the high mortality and morbidity associated with nosocomial pneumonias, however, most clinicians treat such patients with a 2-week empiric trial of antibiotics . Before therapy is initiated, the clinician should rule out other causes of pulmonary infiltrates, fever, and leukocytosis that mimic a nosocomial pneumonia (e.g., pre-existing interstitial lung disease, primary or metastatic lung carcinomas, pulmonary emboli, pulmonary drug reactions, pulmonary hemorrhage, collagen vascular disease affecting the lungs, or congestive heart failure) . If these disorders can be eliminated from diagnostic consideration, a 2-week trial of empiric monotherapy is indicated . The clinician should treat cases of presumed nosocomial pneumonia as if P . aeruginosa were the pathogen . Although P . aeruginosa is not the most common cause of nosocomial pneumonia, it is the most virulent pulmonary pathogen associated with nosocomial pneumonia . Coverage directed against P . aeruginosa is effective against all other aerobic gram-negative bacillary pathogens causing hospital-acquired pneumonia . The clinician should select an antibiotic for empiric monotherapy that is highly effective against P . aeruginosa, has a good side-effect profile, has a low resistance potential, and is relatively inexpensive in terms of its cost to the institution . The preferred agents for empiric monotherapy for nosocomial pneumonia are cefepime, meropenem, and piperacillin . Single organisms are responsible for nosocomial pneumonia, not multiple pathogens . S . aureus rarely, if ever, causes nosocomial pneumonia but is mentioned frequently in studies based on cultures of respiratory tract secretions . S . aureus, unless accompanied by a necrotizing pneumonia with rapid cavitation within 72 hours, in the sputum indicates colonization rather than infection and should not be addressed therapeutically . Antibiotics associated with a high resistance potential should not be used as monotherapy or included in combination therapy regimens (i.e., ceftazidime, ciprofloxacin, imipenem, or gentamicin) . Combination therapy is more expensive than monotherapy and is indicated only when P . aeruginosa is extremely likely, based on its characteristic clinical presentation, or is proved by tissue biopsy . Therapy should not be based on respiratory secretion cultures regardless of technique . Optimal combination regimens include cefepime or meropenem plus levofloxacin or piperacillin or aztreonam or amikacin . Nosocomial pneumonias usually are treated for 14 days . Lack of radiographic or clinical response to appropriate empiric nosocomial pneumonia monotherapy after 14 days suggests an alternate diagnosis . In these patients, a tissue biopsy specimen should be obtained to determine the cause of the persistence of pulmonary infiltrates unresponsive to appropriate antimicrobial therapy.

Rev Sci Tech, 2000 Apr, 19(1), 136 - 50
Cat-scratch disease; Chomel BB; Cat-scratch disease (CSD) was first described by Debre in 1950, yet the causative bacterial agent of CSD remained obscure until 1992, when Bartonella (formerly Rochalimaea) henselae was implicated in CSD by serological and microbiologic studies . Bartonella henselae had initially been linked to bacillary angiomatosis (BA), a vascular proliferative disease most commonly associated with long-standing human immunodeficiency virus infection or other significant immunosuppression . Bartonella henselae has also been associated with bacillary peliosis, relapsing bacteraemia and endocarditis in humans . Cats are healthy carriers of B . henselae, and can be bacteraemic for months or years . Cat-to-cat transmission of the organism by the cat flea, with no direct contact transmission, has been demonstrated . Two new Bartonella species have been identified in the cat reservoir, namely: B . clarridgeiae and B . koehlerae . The role of these species in the aetiology of CSD still needs to be confirmed by isolation or DNA identification from lesions in humans . The author discusses the present state of knowledge on the aetiology, clinical features and epidemiological characteristics of CSD/BA, in addition to diagnosis, treatment and prevention.

Wei Sheng Wu Xue Bao, 1997 Dec, 37(6), 480 - 2
{The preparing of Bacillus penetrans preparation and their controlling to root-knot nematodes}; Pan C et al.; In order to make use of B . penetrans the preparation of these bacteria was prepared successfully . Each gram of the specimen contains more than 3.1 x 10(8) spores according to determination by bloodcount plate . The method of its production are: First, infect the larvae of root-knot nematodes with the spores of B . penetrans . Second, inoculate infectable plants with the infected larvae . Third, beat the root-systems of inoculated plants after adding some water . The flowed liquid from a bronze sieve was mixed with the clear water, filtered, precipitated, air-dried under 5-10 degrees C, and grinded into fine powder, When these powder was added into the water which contained the larvae of root-knot nematodes, the spores of B . penetrans adhered to the cuticles of these larvae in 24 h . The pot experiments of tomato, chilli and cockscomb showed that the galls, egg-sacs, females, males and all-stages of larvae produced by the plant inoculated with the larvae infected with B . penetrans are about 75%-80% less than that without these bacteria . Thus it can be seen that the spores of B . penetrans can depress the populations of root-knot nematodes obviously . Therefore, it is a promising preparation for biological control.

Wei Sheng Wu Xue Bao, 1997 Dec, 37(6), 473 - 6
{Fermentation conditions of engineering strain utilizing starch for production of alkaline proteinase}; Feng Y et al.; The highest activity of alkaline proteinase produced by Bacillus pumilus c172(pBX 96) transformant was 9,000 U/ml in shaking flask where corn meal and bean cake meal were carbon and nitrogen sources, respectively . The enzyme activity was raised under the conditions of initial pH 7.0, MgCl2 instead of MgSO4, and glucose (0.1%) in the substrate . The fermentation of c172(pBx 96) transformant was carried out with parameters of pH, reducing sugar, total sugar and enzyme activity.

Wei Sheng Wu Xue Bao, 1997 Oct, 37(5), 397 - 400
{The properties of protease from Bacillus sphaericus C3-41}; Sun F et al.; The production and properties of protease from Bacillus sphaericus strain C3-41 were reported in this paper . It was found out that the secrete of extracellular protease began at the exponential phase, reached a maximum at the early phase of sporangium and then decrease rapidly . The production conditions of the protease have been studied . The protease preparation was purified by salting out with ammonium sulfate and by chromatography fractionating on Sephadex G-100 . The purified enzyme have a specific activity of 6741.5 U/mg protein and a molecular weight of 42,000 . The optimal activities of the protease were around pH11.0 and at 4 degrees C respectively . The enzyme was stable at pH5.0-12.0 . The proteolytic activity was inhibited by phenylmethylsulphony fluoride (PMSF) and EDTA, but not by IAA, SA and DTT . The activity was inhibited in the presence of Al3+, Hg2+, Fe3+, Cu2+ and Fe2+ . The enzyme was sensitive to higher temperature, but was quite stable in the presence of Ca2+.

Med Sci Monit, 2000 Jan-Feb, 6(1), 8 - 12
Factors influencing the cellular location of proteolytic enzymes of Bacillus intermedius; Sharipova MR et al.; Thiol-dependent serine proteinase and glutamylendopeptidase of Bacillus intermedius 3-19 being prevailing enzymes in the total pool of extracellular proteinases (95%) of this microorganism in catalytic active form were detected on the membrane of the cells . Production of these enzymes was maximum on the medium containing inorganic phosphate and gelatin and decreased 2-4-fold on the medium with glucose and lactate . The level of the activity of extracellular enzymes correlated with that of corresponding membrane-bound proteins . The addition of CoCl2 (2 mM) into the medium caused essential increase in extracellular glutamylendopeptidase activity and promoted the release of membrane-bound enzyme into cultural fluid . Proteolytic activity was detected in cytoplasm also . Proteinases localized in cytoplasm were shown to differ in properties from those secreted.

Environ Microbiol, 1999 Dec, 1(6), 503 - 15
Climatic influence on mesophilic Bacillus cereus and psychrotolerant Bacillus weihenstephanensis populations in tropical, temperate and alpine soil; von Stetten F et al.; Bacillus weihenstephanensis strains are psychrotolerant and grow from below 7 degrees C to 38 degrees C . Closely related mesophilic Bacillus cereus strains can grow from above 7 degrees C to 46 degrees C . We classified 1060 B . cereus group isolates from different soil samples with respect to their psychrotolerant and mesophilic genotypes by polymerase chain reaction (PCR) targeting of specific 16S rDNA and cold shock protein A gene signatures . In parallel, growth tests at 7 degrees C were carried out to determine the thermal phenotype . The geographic distribution of psychrotolerant and mesophilic isolates was found to depend significantly on the prevalent annual average temperature . In one tropical, one temperate and two alpine habitats, the proportion of psychrotolerant cspA genotypes was found to be 0%, 45% and 86% and 98%, respectively, with the corresponding annual average temperatures being 28 degrees C, 7 degrees C, 4 degrees C and 1 degrees C . In the tropical habitat, only the mesophilic B . cereus was found, characterized by correspondence of thermal genotype and phenotype . In the alpine habitat, almost only the psychrotolerant B . weihenstephanensis was isolated . In the temperate habitat, mesophilic B . cereus and psychrotolerant B . weihenstephanensis as well as 'intermediate thermal types' occurred, the latter having opposite thermal genotypes and phenotypes or opposing sets of thermal DNA signatures, characterized by the coexistence of mesophilic and psychrotolerant 16S rDNA operon copies within a single isolate . Both sugar utilization and DNA fingerprinting patterns revealed a high, probably non-clonal microsite diversity within the population of the temperate habitat . We interpret our observations in terms of a temperature-dependent selection regime, acting on recombining B . cereus/ B . weihenstephanensis populations in soil.

Clin Exp Immunol, 2001 Feb, 123(2), 264 - 70
Recombinant bacille Calmette-Guérin (BCG) expressing human interferon-alpha 2B demonstrates enhanced immunogenicity; Luo Y et al.; To increase its immunostimulatory properties, BCG was genetically engineered to secrete recombinant human interferon-alpha 2B (rhIFN-alpha) under control of the mycobacterial heat shock protein (hsp)60 promoter and the alpha antigen signal sequence . Expression of rhIFN-alpha was readily detectable by ELISA and on Western blotting . When compared with control BCG, rhIFN-alpha BCG was substantially more active in inducing the production of IFN-gamma and IFN-inducible protein 10 (IP-10) from human peripheral blood mononuclear cells, while IL-10 production was correspondingly decreased . These effects were reversible upon antibody neutralization of rhIFN-alpha . Among 10 patients tested, rhIFN-alpha BCG enhanced IFN-gamma production in all patients ranging from 1.4- to 23.7-fold with a general trend toward greatest enhancement among those with weakest baseline responses to control BCG . Correspondingly, rhIFN-alpha BCG decreased IL-10 production in all patients by 1.2-4.8-fold . The onset of IFN-gamma production induced by rhIFN-alpha BCG was also more rapid, occurring within 4 h after stimulation versus > 24 h with wild-type BCG . The observation that the maximum IFN-gamma induction depends on the simultaneous presence of both IFN-alpha and BCG highlights the advantages of rhIFN-alpha BCG . Taken together, these immunostimulatory properties of rhIFN-alpha BCG suggest that it may be a superior agent for immunotherapeutic protocols involving live BCG in humans.

Clin Exp Immunol, 2001 Feb, 123(2), 219 - 25
Effect of deworming on human T cell responses to mycobacterial antigens in helminth-exposed individuals before and after bacille Calmette-Guérin (BCG) vaccination; Elias D et al.; The protective efficacy of BCG vaccination against pulmonary tuberculosis (TB) is highly variable in different populations . The reason remains to be elucidated . This study aims to investigate the possible effect of intestinal helminths on the immune response to PPD in naturally immunized or BCG-vaccinated humans . The study population was assessed for helminthic infection and those found to be positive were randomly assigned to either an albendazole treatment group or a control group who received a placebo . The immune response to PPD was compared between the two groups . In addition, subjects who were tuberculin skin test-negative in both groups were BCG vaccinated and later on tested for PPD-specific responses . Albendazole induced elimination/or reduction in intestinal worms resulting in a significant improvement in T cell proliferation and in interferon-gamma production by peripheral blood mononuclear cells (PBMC) stimulated with PPD . Moreover, BCG vaccination significantly improved PPD-specific immune responses in the treated group but not in the placebo group . The differences in the in vivo skin test responses were not significant . The data show that cellular immune responses to PPD are reduced in persons with concurrent helminthic infections, perhaps reflecting a lowered resistance to mycobacterial infections . This could explain, at least in part, the reduced efficacy of BCG against TB in helminth-endemic areas of the world.

Cell Microbiol, 2000 Oct, 2(5), 431 - 41
Interaction of Bartonella henselae with endothelial cells results in rapid bacterial rRNA synthesis and replication; Kempf VA et al.; Bartonella henselae is a slow-growing microorganism and the causative pathogen of bacillary angiomatosis in man . Here, we analysed how interaction of B . henselae with endothelial cells might affect bacterial growth . For this purpose, bacterial rRNA production and ribosome content was determined by fluorescence in situ hybridization (FISH) using rRNA-targeted fluorescence-labelled oligonucleotide probes . B . henselae grown on agar plates showed no detectable rRNA content by means of FISH, whereas B . henselae co-cultured with endothelial cells showed a rapid increase of rRNA production within the first 18 h after inoculation . The increased rRNA synthesis was paralleled by a approximately 1000-fold intracellular bacterial replication, whereas bacteria grown on agar base showed only a approximately 10-fold replication within the first 48 h of culture . Pretreatment of host cells with paraformaldehyde prevented adhesion, invasion, intracellular replication and bacterial rRNA synthesis of B . henselae . In contrast, inhibition of host cell protein synthesis by cycloheximide did not affect bacterial adhesion and invasion, but prevented intracellular replication although bacterial rRNA content was increased . Inhibition of actin polymerization by cytochalasin D did not affect adhesion, invasion, increased rRNA content or intracellular replication of B . henselae . These results demonstrate that rRNA synthesis and replication of B . henselae is promoted by viable host cells with intact de novo protein synthesis.

Oncol Rep, 2001 Mar-Apr, 8(2), 257 - 61
Changes in mucosal immune cells of bladder tumor patient after BCG intravesical immunotherapy; Chang SG et al.; The Bacillus Calmette-Guerin (BCG) is considered to be at least as effective, and perhaps superior to chemotherapy in the prophylaxis of recurrent superficial tumors . However, the mechanism of the antitumor effect of BCG is still not exactly known . We have conducted investigations to examine changes in bladder mucosal immune cells in patients with superficial bladder carcinoma treated with a first cycle of BCG . The study group included 15 BCG and 5 doxorubicin instillation patients, most in the intermediate or high risk group for recurrent tumor . Grossly normal bladder mucosal cold cup biopsies were performed at initial TUR and one week after six consecutive weekly instillations of BCG or doxorubicin . All specimens underwent immunohistochemical staining, both pre-treatment and post-treatment, including CD20, CD45RO, CD8, CD4 and CD57 . Immunoreactive cell counts were evaluated from three different microscopic fields (x400) under the grid . The mean duration of follow-up was 52.8 months . The post-treatment bladder mucosal B-cells (CD20) and T-cells (CD45RO, CD4, CD8) were significantly increased compared to pre-treatment in patients treated with BCG instillation, but NK-cells (CD57) were not changed . However, there was no change in B-cells or T-cells in patient treated with doxorubicin . The CD20 cells in pre-treatment specimens did not correlate with any other cells . However, it was a statistically significant correlation with CD45RO in post-treatment specimens . The CD4 correlated with CD45RO and CD8 in pre-treatment, but it was correlated with CD45RO and CD57 in post-treatment specimens . There was no tumor recurrence in cases with significantly increased B-cells after BCG instillation . The results of these studies suggest that intravesical BCG immunotherapy for superficial bladder tumor induces a significant increase in T-cells as well as B-cells and that B-cells have a preventive effect on tumor recurrence . Further studies with a larger number of patients are needed to confirm the value of the B-cell increment after BCG instillation as a clinically independent prognostic factor.

Biotechnol Bioeng, 2001 Feb 20, 72(4), 434 - 40
Orthophosphate anion enhances the stability and activity of endoxylanase from Bacillus sp; Park MT et al.; Endoxylanase, for which the optimum temperature is 60 degrees C (optimum pH 7), is labile to heat . Because the isoelectric point (pI) value of this xylanase is 10.6, the net charge of this enzyme is positive at pH 7 . Thus, ions are likely to influence its enzyme structure and the thermal stability of endoxylanase may improve . Among the various ions tested, orthophosphate anion (HPO(4)(2-)) was found to significantly improve not only the stability but the activity of xylanase . When K(2)HPO(4) concentration was increased from 50 mM to 1.2 M, the T(m )value of xylanase was increased from 60.0 degrees C to 74.5 degrees C . The affinity of xylanase on xylan also increased along with K(2)HPO(4) concentration . Thus, the xylanase activity at 0.6 M K(2)HPO(4) was 2.3-fold higher than that at 50 mM K(2)HPO(4), and 120.2-fold higher than that in 40 mM MOPS buffer . This enhanced activity in the presence of K(2)HPO(4 )probably takes place because the orthophosphate anion affects the binding and catalytic residues of endoxylanase .

Eur J Biochem, 2001 Feb, 268(4), 895 - 902
Properties of the prophenoloxidase activating enzyme of the freshwater crayfish, Pacifastacus leniusculus; Wang R et al.; The prophenoloxidase activating enzyme (ppA), a serine proteinase catalyzing the conversion of prophenoloxidase to an active phenoloxidase, has a molecular mass of about 36 kDa in its active form . This protein was cloned from a blood cell cDNA library and its corresponding cDNA of 1736 base pairs encodes a zymogenic protein (proppA) of 468 amino acids . An antibody raised against a synthetic peptide derived from a region of the cDNA sequence could efficiently inhibit the beta-1,3-glucan triggered activation of prophenoloxidase in vitro . The C-terminal half of the proppA is composed of a typical serine proteinase domain, with a sequence similar to other invertebrate and vertebrate serine proteinases . The N-terminal half contains a cationic glycine-rich domain, a cationic proline-rich domain and a clip-domain, in which the disulfide-bonding pattern is likely to be identical to those of the horseshoe crab big defensin and mammalian beta-defensins . Antibodies made against both the C- and the N-terminal halves recognize two proppAs under reducing conditions . However, under nonreducing conditions only the anti-C antibody recognized the two proppAs, which suggests that a conformational change takes place upon reduction that allows the anti-N to react with the N-terminal half of proppA . The recombinant clip-domain in crayfish proppA was overexpressed in Escherichia coli and the resulting peptide exhibited antibacterial activity against Gram-positive bacterial strains such as Micrococcus luteus Ml11 and Bacillus megaterium Bm11 with 50% growth inhibitory concentrations of 1.43 microM and 17.9 microM, respectively . These results suggest that the clip-domains in proppAs may function as antibacterial peptides.

Infect Immun, 2001 Mar, 69(3), 1847 - 55
Effects of tumor necrosis factor alpha on host immune response in chronic persistent tuberculosis: possible role for limiting pathology; Mohan VP et al.; Reactivation of latent tuberculosis contributes significantly to the incidence of disease caused by Mycobacterium tuberculosis . The mechanisms involved in the containment of latent tuberculosis are poorly understood . Using the low-dose model of persistent murine tuberculosis in conjunction with MP6-XT22, a monoclonal antibody that functionally neutralizes tumor necrosis factor alpha (TNF-alpha), we examined the effects of TNF-alpha on the immunological response of the host in both persistent and reactivated tuberculous infections . The results confirm an essential role for TNF-alpha in the containment of persistent tuberculosis . TNF-alpha neutralization resulted in fatal reactivation of persistent tuberculosis characterized by a moderately increased tissue bacillary burden and severe pulmonic histopathological deterioration that was associated with changes indicative of squamous metaplasia and fluid accumulation in the alveolar space . Analysis of pulmonic gene and protein expression of mice in the low-dose model revealed that nitric oxide synthase was attenuated during MP6-XT22-induced reactivation, but was not totally suppressed . Interleukin-12p40 and gamma interferon gene expression in TNF-alpha-neutralized mice was similar to that in control mice . In contrast, interleukin-10 expression was augmented in the TNF-alpha-neutralized mice . In summary, results of this study suggest that TNF-alpha plays an essential role in preventing reactivation of persistent tuberculosis, modulates the pulmonic expression of specific immunologic factors, and limits the pathological response of the host.

Infect Immun, 2001 Mar, 69(3), 1704 - 7
Immune response to infection with Mycobacterium ulcerans; Gooding TM et al.; Mycobacterium ulcerans is a slow-growing, acid-fast bacillus that causes chronic necrotizing skin ulcers known as Buruli ulcers . Previously reported information on immunity to this mycobacterium is limited . We examined immune responses to M . ulcerans and M . bovis BCG in patients with M . ulcerans disease and in 20 healthy control subjects (10 tuberculin test positive and 10 tuberculin test negative) . Cell-mediated immunity was assessed by stimulating peripheral blood mononuclear cells (PBMC) with whole mycobacteria and then measuring PBMC proliferation and the production of gamma interferon (IFN-gamma) . Humoral immunity was assessed by immunoblotting . PBMC from all subjects showed significantly greater proliferation and IFN-gamma production in response to stimulation with living mycobacteria compared with killed cells . However, PBMC from subjects with past or current M . ulcerans disease showed significantly reduced proliferation and production of IFN-gamma in response to stimulation with live M . ulcerans or M . bovis than PBMC from healthy, tuberculin test-positive subjects (P < 0.001) and showed results in these assays comparable to those of tuberculin test-negative subjects (P > 0.2) . Serum from 9 of 11 patients with M . ulcerans disease, but no control subject, contained antibodies to M . ulcerans . The results indicate that patients with M . ulcerans infection mount an immune response to M . ulcerans as evidenced by antibody production, but they demonstrate profound systemic T-cell anergy to mycobacterial antigens . These findings may explain some of the distinct clinical and pathological features of M . ulcerans-induced disease.

Biochem Biophys Res Commun, 2001 Feb 16, 281(1), 151 - 8
Molecular cloning of an alpha-glucosidase-like gene from Penicillium minioluteum and structure prediction of its gene product; Garcia B et al.; The dexC cDNA, which is expressed in dextran-containing medium by the filamentous fungus Penicillium minioluteum, was cloned and sequence characterized . The cDNA sequence comprises 1859 bp plus a poly (A) tail, coding for a predicted protein of 597 amino acids . The genomic counterpart was isolated by PCR, finding three introns in its sequence . The dexC gene was located by Southern blot in the same 9-kb fragment that the previously isolated dextranase-encoding gene (dexA) . Sequence analysis revealed that the deduced DexC protein belongs to glycosyl hydrolase family 13, showing a high sequence identity (58%) with Aspergillus parasiticus alpha-1,6-glucosidase . In addition, the high sequence identity (51%) between DexC protein and oligo-1,6-glucosidase of Bacillus cereus, with three-dimensional (3D) structure determined, leads us to proposed a 3D model for the structural core of DexC protein.

Curr Microbiol, 2001 Apr, 42(4), 237 - 40
Cloning and characterization of a bacterial cell-bound type B carboxylesterase from Bacillus sp . BP-7; Prim N et al.; A clone producing halos on tributyrin plates was isolated from a genomic library of Bacillus sp . BP-7 . The insert contained an open reading frame that coded for a protein of 487 amino acids with homology to carboxylesterases . The cloned enzyme showed clear preference for esters of short-chain fatty acids, being classified as an esterase . Maximum activity was found at 45 degrees C and pH 7.5 . The enzyme displayed stability in the pH range from 6 to 9.5, and at temperatures from 4 degrees to 45 degrees C . Zymogram analysis of the protein revealed a molecular mass of 53 kDa and a pI of 5.1 . The enzyme showed homology to members of the bacterial subclass of type B carboxylesterases, a set of proteins potentially useful for biotechnological applications.

J Urol, 2001 Mar, 165(3), 834 - 6
Primary treatment of condylomata acuminata with viable bacillus Calmette-Guerin; Bohle A et al.; PURPOSE: Condylomata acuminata or genital warts are caused by human papillomavirus . Prevalence data show that infection rates are increasing . To our knowledge we report the first successful primary treatment of genital warts with topical bacillus Calmette-Guerin (BCG) and provide long-term followup in a group of adjuvant treated patients with recurrent condylomata acuminata . MATERIALS AND METHODS: In 10 consecutive men viable BCG was directly applied to the condylomata acuminata lesions once weekly for 6 weeks . In nonresponding patients another course of 9 applications was administered for 3 weeks . RESULTS: A complete response was achieved in 6 of the 10 men after 1 or 2 treatment cycles . All responding patients are disease-free at a median followup of 9.2 months (range 4 to 12) . One patient achieved partial regression of the lesions and in 3 the condylomata did not disappear . Side effects were rare and mild . Long-term followup in 6 adjuvant treated patients with rapidly recurrent condylomata acuminata showed no further recurrence after topical BCG in 5 at a median of 30.8 months (range 29 to 50) . CONCLUSIONS: Topical application of viable BCG has therapeutic activity as adjuvant and primary treatment for penile condylomata acuminata with negligible side effects . Long-term followup implies the prevention of recurrent disease.

J Urol, 2001 Mar, 165(3), 745 - 56
The limits of bacillus Calmette-Guerin for carcinoma in situ of the bladder; Kim JC et al.; PURPOSE: Historically carcinoma in situ of the bladder has been treated with radical cystectomy based on the aggressive and potentially invasive nature of this disease . The introduction in the late 1970s of intravesical bacillus Calmette-Guerin (BCG) has made this therapy the gold standard in the management of carcinoma in situ . Cases that are refractory or resistant to BCG therapy are a management dilemma with various available treatment options . MATERIALS AND METHODS: A comprehensive literature review of the current management of carcinoma in situ of the bladder was performed using MEDLINE, a review of current urology journals and abstracts from recent urology meetings . Data focused on BCG resistant carcinoma in situ of the bladder and current approaches in use for refractory disease . RESULTS: Complete and durable response rates have been reported in more than 70% of patients with carcinoma in situ who are treated with intravesical BCG . To our knowledge the optimal therapeutic regimen has not been established, although extended periods of treatment beyond the originally described 6-week course have not been shown to improve complete response rates . Prolonged administration of BCG is associated with adverse side effects . Various prognostic indicators of recurrence and progression exist that may identify a subset of cases unlikely to respond favorably to a conservative approach, including carcinoma in situ with associated stage T1 bladder lesions, diffuse and multifocal carcinoma in situ, multiple recurrences with intravesical therapy and extravesical involvement . Current molecular markers may also predict the response of carcinoma in situ to therapy . Treatment options available for BCG refractory carcinoma in situ of the bladder include intravesical chemotherapy, combined immuno-chemotherapy and radical cystectomy . Intravesical valrubicin and oral bropirimine have been shown to induce a complete response rate of 21% to 50%, although data on long-term followup are forthcoming . Radical cystectomy remains effective therapy for aggressive carcinoma in situ of the bladder . CONCLUSIONS: The current management of carcinoma in situ of the bladder is ill defined due to the variable natural history and unpredictable response of this disease to therapy . Controversy exists as to the optimal treatment of carcinoma in situ of the bladder since different forms of carcinoma in situ may exist that complicate therapeutic decisions for appropriate therapy . Some tumor characteristics are associated with more aggressive behavior and may be predictive of treatment outcome.

Curr Opin Pediatr, 2001 Feb, 13(1), 56 - 9
Treatment of cat-scratch disease; Conrad DA; Cat-scratch disease is an infection caused by Bartonella henselae, a fastidious gram-negative bacillus acquired from exposure to an infected kitten or cat . The most common manifestation of human disease is lymphadenitis . Atypical forms of infection include Parinaud oculoglandular syndrome, stellate neuroretinitis, persistent fever without localizing signs, hepatosplenic infection, encephalopathy, osteomyelitis, and endocarditis . Immunocompromised individuals with B . hensalae infection may develop bacillary angiomatosis, bacillary peliosis, and relapsing bacteremia with fever syndrome . The bacillus is susceptible to several antibacterial agents in vitro, including penicillins, cephalosporins, aminoglycosides, tetracyclines, macrolides, quinolones, trimethoprim and sulfamethoxazole, and rifampin . Greatest clinical efficacy has been observed following treatment with rifampin, ciprofloxacin, gentamicin, trimethoprim and sulfamethoxazole, clarithromycin, and azithromycin . In one placebo-controlled study, azithromycin therapy was associated with more rapid diminution in size of infected lymph nodes . The majority of cases of cat-scratch disease occurring in normal hosts do not require anti-infective therapy for resolution of infection.

J Am Acad Dermatol, 2001 Feb, 44(2), 261 - 4
Utility of anti-bacillus Calmette-Guérin antibodies as a screen for organisms in sporotrichoid infections; Byrd J et al.; BACKGROUND: Sporotrichoid infections often present a diagnostic challenge for the dermatopathologist . Therefore an affordable, reliable method of easier recognition of organisms is desired . Recently, immunohistochemical staining with antibodies against Mycobacterium bovis (bacillus Calmette-Guerin, BCG) was found to be useful in identifying various fungi and mycobacteria with minimal background staining . OBJECTIVE: The purpose of this study was to demonstrate the usefulness of anti-BCG antibodies as a screen for organisms in sporotrichoid infections . METHODS: Thirteen specimens of suspected sporotrichosis were selected for staining with anti-BCG antibody . Sporotrichoid infection was confirmed by histochemical staining, biopsy, and follow-up results . RESULTS: Twelve of the 13 specimens stained positively using anti-BCG antibody . Of the 5 cultures done, 2 were positive for M . marinum, and 1 grew Sporothrix schenckii . CONCLUSION: Immunohistochemical staining with anti-BCG antibody offers another screening method to identify organisms in sporotrichoid infections because of its ease, cost-effectiveness, and simplicity.

J Biochem (Tokyo), 2001 Feb, 129(2), 303 - 12
Crystal structure of glucose dehydrogenase from Bacillus megaterium IWG3 at 1.7 A resolution; Yamamoto K et al.; The crystal structure of glucose dehydrogenase (GlcDH) from Bacillus megaterium IWG3 has been determined to an R-factor of 17.9% at 1.7 A resolution . The enzyme consists of four identical subunits, which are similar to those of other short-chain reductases/dehydrogenases (SDRs) in their overall folding and subunit architecture, although cofactor binding sites and subunit interactions differ . Whereas a pair of basic residues is well conserved among NADP(+)-preferring SDRs, only Arg39 was found around the adenine ribose moiety of GlcDH . This suggests that one basic amino acid is enough to determine the coenzyme specificity . The four subunits are interrelated by three mutually perpendicular diad axes (P, Q, and R) . While subunit interactions through the P-axis for GlcDH are not so different from those of the other SDRs, those through the Q-axis differ significantly . GlcDH was found to have weaker hydrophobic interactions in the Q-interface . Moreover, GlcDH lacks the salt bridge that stabilizes the subunit interaction in the Q-interface in the other SDRs . Hydrogen bonds between Q-axis related subunits are also less common than in the other SDRs . The GlcDH tetramer dissociates into inactive monomers at pH 9.0, which can be attributed mainly to the weakness of the Q-axis interface.

Acta Crystallogr D Biol Crystallogr, 2001 Feb, 57(Pt 2), 292 - 5
Crystallization and preliminary X-ray analysis of the sporulation factor SpoIIAA in its native and phosphorylated forms; Seavers PR et al.; Sporulation in Bacillus begins with an asymmetric cell division producing two progeny with identical chromosomes but different developmental fates . As such, it is a simple example of cellular differentiation . The establishment of cell type is controlled by a series of alternate RNA polymerase sigma subunits . The first compartment-specific sigma factor is sigma(F), whose activity is controlled by SpoIIAB, an anti-sigma factor, and SpoIIAA, an anti-sigma factor antagonist which is phosphorylated by the kinase activity of SpoIIAB . Here, the preliminary crystallographic analysis of SpoIIAA and phosphorylated SpoIIAA from B . sphaericus in forms suitable for high-resolution structure determination are reported.

FEBS Lett, 2001 Feb 9, 490(1-2), 70 - 4
The role of a proline-induced broken-helix motif in alpha-helix 2 of Bacillus thuringiensis delta-endotoxins; Arnold S et al.; Bacillus thuringiensis delta-endotoxins (Cry proteins), are widely used for insect control and plant protection . They are water-soluble proteins that insert into membranes forming ion channels . In most Cry toxins alpha-helix 2 is broken by a highly conserved proline residue (Pro70 in Cry1Ab), generating a broken-helix motif . The flexibility of the motif was altered through site-directed mutagenesis . It was found that increasing the flexibility of the motif decreased the stability, the ion transport ability and the toxicity of the protein . By removing the broken-helix motif, the biological properties were restored to a wild type level.

Environ Health Perspect, 2001 Jan, 109(1), 47 - 54
Spatial and temporal distribution of airborne Bacillus thuringiensis var . kurstaki during an aerial spray program for gypsy moth eradication; Teschke K et al.; We measured airborne exposures to the biological insecticide Bacillus thuringiensis var . kurstaki (Btk) during an aerial spray program to eradicate gypsy moths on the west coast of Canada . We aimed to determine whether staying indoors during spraying reduced exposures, to determine the rate of temporal decay of airborne concentrations, and to determine whether drift occurred outside the spray zone . During spraying, the average culturable airborne Btk concentration measured outdoors within the spray zone was 739 colony-forming units (CFU)/m3 of air . Outdoor air concentrations decreased over time, quickly in an initial phase with a half time of 3.3 hr, and then more slowly over the following 9 days, with an overall half-time of about 2.4 days . Inside residences during spraying, average concentrations were initially 2-5 times lower than outdoors, but at 5-6 hr after spraying began, indoor concentrations exceeded those outdoors, with an average of 244 CFU/m3 vs . 77 CFU/m3 outdoors, suggesting that the initial benefits of remaining indoors during spraying may not persist as outside air moves indoors with normal daily activities . There was drift of culturable Btk throughout a 125- to 1,000-meter band outside the spray zone where measurements were made, a consequence of the fine aerosol sizes that remained airborne (count median diameters of 4.3 to 7.2 microm) . Btk concentrations outside the spray zone were related to wind speed and direction, but not to distance from the spray zone.

J Pharm Sci, 2001 Mar, 90(3), 275 - 87
Thermal sterilization of heat-sensitive products using high-temperature short-time sterilization; Mann A et al.; High-temperature short-time (HTST) sterilization with a continuous-flow sterilizer, developed for this study, was evaluated . The evaluation was performed with respect to (a) the chemical degradation of two heat-sensitive drugs in HTST range (140-160 degrees C) and (b) the microbiological effect of HTST sterilization . Degradation kinetics of two heat-sensitive drugs showed that a high peak temperature sterilization process resulted in less chemical degradation for the same microbiological effect than a low peak temperature process . Both drugs investigated could be sterilized with acceptable degradation at HTST conditions . For the evaluation of the microbiological effect, Bacillus stearothermophilus ATCC 7953 spores were used as indicator bacteria . Indicator spore kinetics (D(T), z value, k, and E(a)), were determined in the HTST range . A comparison between the Bigelow model (z value concept) and the Arrhenius model, used to describe the temperature coefficient of the microbial inactivation, demonstrated that the Bigelow model is more accurate in prediction of D(T) values in the HTST range . The temperature coefficient decreased with increasing temperature . The influence of Ca(2+) ions and pH value on the heat resistance of the indicator spores, which is known under typical sterilization conditions, did not change under HTST conditions.

Biopolymers, 2001 Mar, 58(3), 260 - 7
Conformational characterization of designed minibarnase; Takahashi K et al.; We have designed a minibarnase by removing one module from barnase, a bacterial RNase from Bacillus amyloliquefaciens . Barnase, consisting of 110 amino acid residues, is decomposed into six modules, M1-M6 . Module is defined as a peptide segment consisting of contiguous amino acid residues that makes a small compact conformation within a globular domain . To understand the role of module in protein architecture, we analyzed NMR and CD spectra of a minibarnase, which lacked 26 amino acid residues corresponding to module M2 . We demonstrated the formation of hydrophobic cores in the minibarnase similar to those of barnase . Although its conformational stability against acids and heat was reduced in comparison with barnase, the minibarnase retained cooperative folding character (two-state folding) . Therefore, the folding of the minibarnase consisting of modules M1 and M3-M6 is independent to some extent of module M2 . This finding may be useful for future module-based protein design .

Mol Microbiol, 2001 Feb, 39(3), 813 - 21
An increase in expression of a Mycobacterium tuberculosis mycolyl transferase gene (fbpB) occurs early after infection of human monocytes; Wilkinson RJ et al.; Changes in the mRNA levels of two Mycobacterium tuberculosis genes (fbpB known as antigen 85B, and hspX known as Acr) were studied in infected human monocytes . Antigen 85B is an enzyme involved in cell wall biosynthesis and is also a major target of the immune response . Acr is a stress protein believed to be involved in the bacillary response to adverse conditions and in non-replicating persistence . During the first 24 h of intracellular infection, the intramonocyte 85B mRNA level increased 54-fold (P = 0.00001) and 14.6 times in comparison with the 16S ribosomal rRNA . In contrast, the Acr mRNA fell 14.3 times . Although monocyte cytokine production was very variable, the 24 h secretion of tumour necrosis factor (TNF)-alpha correlated with the 85B-16S RNA ratio at 24 h (r = 0.77, Pcorr < 0.01) . Furthermore, the addition of exogenous TNF-alpha to cultures was associated with a twofold increase in the 85B-16S ratio and, conversely, neutralization of endogenous TNF-alpha reduced the ratio . As antigen 85B also induces TNF-alpha, the positive feedback implied by our findings suggests a previously unsuspected role for this protein in the immunopathogenesis of tuberculosis.

Environ, Toxicol . Pharmacol. . 2001 Jan 1, 9(3), 79 - 85
Domoic acid: a fascinating marine toxin; Mos L; There are indications that toxic algal blooms are increasing because of pollution of coastal waters and worldwide shipping . This mini-review deals with the marine biotoxin domoic acid, also known as amnesic shellfish poison, and its main producing pennate diatom genus Pseudo-nitzschia (Bacillariophyceae) . Besides contamination of seafood, these organisms have also been involved in human and marine wildlife mortality . The article aims to give an overview of all biological and environmental factors that should be considered when trying to evaluate a possible increase in toxic blooms of Pseudo-nitzschia spp . Pseudo-nitzschia blooms characteristically occur in a low light regime, at a time when the temperature is falling and at a wide range of salinities . Laboratory studies have shown that the production of domoic acid, a water-soluble amino acid, is related to silicon, phosphorus, nitrogen and trace metal (mainly iron) availability . Domoic acid has no known function in defence or primary metabolism; a role in excretion of excess photosynthetic energy or as a binding ligand for trace metals is suggested . The variability in domoic acid production by different Pseudo-nitzschia spp., or the presence of toxic and non-toxic strains of the same species, cannot be explained . The conclusion is drawn that an increase in toxic blooms of Pseudo-nitzschia spp . might be possible, especially because of the expected increase in nutrient availability from pollution and desert dust . Global warming may have an influence as well by lengthening the growth period for Pseudo-nitzschia, enlarging their global distribution and increasing the dust load through desertification.

FEMS Microbiol Lett, 2001 Feb 5, 195(1), 67 - 72
Identification of thermostabilizing residues in a Bacillus alkaline cellulase by construction of chimeras from mesophilic and thermostable enzymes and site-directed mutagenesis; Hakamada Y et al.; An alkaliphilic Bacillus sp . strain, KSM-64, produces a mesophilic alkaline endo-1,4-beta-glucanase that is suitable for use in detergents . The deduced amino acid sequence of the enzyme showed very high homology to that of a thermostable alkaline enzyme from alkaliphilic Bacillus sp . strain KSM-S237 . Analysis of chimeric enzymes produced from the genes encoding the mesophilic and thermostable enzymes suggested that the lysine residues at positions 137, 179, and 194 are responsible for their thermal stabilization . Replacing the corresponding Glu137, Asn179, and/or Asp194 with lysine by site-directed mutagenesis made the mesophilic enzyme more thermostable . Analyses of the hydrophilicity of deduced amino acid sequences and isoelectric focusing of the modified enzymes suggested that these three specific lysine residues and their replacements are all located on the surface of the enzyme molecule . This fact further suggested that specific ionic interaction is involved in the thermal stabilization of the enzyme.

FEMS Microbiol Lett, 2001 Feb 5, 195(1), 1 - 8
Why Bacillus thuringiensis insecticidal toxins are so effective: unique features of their mode of action; Aronson AI et al.; The spore-forming bacterium Bacillus thuringiensis produces intracellular inclusions comprised of protoxins active on several orders of insects . These highly effective and specific toxins have great potential in agriculture and for the control of disease-related insect vectors . Inclusions ingested by larvae are solubilized and converted to active toxins in the midgut . There are two major classes, the cytolytic toxins and the delta-endotoxins . The former are produced by B . thuringiensis subspecies active on Diptera . The latter, which will be the focus of this review, are more prevalent and active on at least three orders of insects . They have a three-domain structure with extensive functional interactions among the domains . The initial reversible binding to receptors on larval midgut cells is largely dependent upon domains II and III . Subsequent steps involve toxin insertion into the membrane and aggregation, leading to the formation of gated, cation-selective channels . The channels are comprised of certain amphipathic helices in domain I, but the three processes of insertion, aggregation and the formation of functional channels are probably dependent upon all three domains . Lethality is believed to be due to destruction of the transmembrane potential, with the subsequent osmotic lysis of cells lining the midgut . In this review, the mode of action of these delta-endotoxins will be discussed with emphasis on unique features.

Enzyme Microb Technol, 2001 Feb 1, 28(2-3), 161 - 167
trans-Sialidase catalyzed sialylation of beta-galactosyldisaccharide with an introduction of beta-galactosidase; Lee S et al.; Introduction of beta-galactosidase into a trans-sialidase reaction, i.e . sialic acid transfer reaction from a donor substrate (alpha2,3-sialyllactose) to an acceptor substrate (beta-galactosyldisaccharide), could improve the yield of desired sialylated trisaccharide by hydrolyzing lactose, a byproduct from the donor . When trans-sialidase reaction was performed with stoichiometric amounts (2 mM) of alpha2,3-sialyllactose and Galbeta(1,3)GlcNAc, the yield of NeuAcalpha(2,3)Galbeta(1,3)GlcNAc increased from 45% to 75% by the coupling of Escherichia coli beta-galactosidase . Furthermore, by changing the substrate ratio in the coupled reaction, i.e . two-fold excess of alpha2,3-sialyllactose to Galbeta(1,3)GlcNAc, above 95% of yield was achieved based on the amount of Galbeta(1,3)GlcNAc . However, two-fold excess of Galbeta(1,3)GlcNAc to alpha2,3-sialyllactose in this reaction was more desirable for the purification of NeuAcalpha(2,3)Galbeta(1,3)GlcNAc, since complete consumption of alpha2,3-sialyllactose was achieved . Efficiency of the coupled reaction was affected by the specificity of beta-galactosidase for acceptor substrate . When Galbeta(1,6)GlcNAc was used as the acceptor, E . coli beta-galactosidase hydrolyzed Galbeta(1,6)GlcNAc as well as lactose in the coupled reaction, resulting in a significant decrease in the yield of desired sialylated trisaccharide . The conversion yield of the sialylation of Galbeta(1,6)GlcNAc could be improved by employing Bacillus circulans beta-galactosidase.

Neth J Med, 2001 Feb, 58(2), 71 - 5
BCG immunotherapy: be cautious of granulomas . Disseminated BCG infection and mycotic aneurysm as late complications of intravesical BCG instillations; Kamphuis JT et al.; We describe a 65-year-old man with a granulomatous hepatitis and a progressive mycotic aneurysm of the abdominal aorta . One year before he received intravesical bacillus Calmette--Guerin (BCG) for carcinoma of the bladder without any complaints . Only post-mortem investigations could confirm that he suffered from a systemic BCG infection . Literature is reviewed for this rare complication.

Toxicology, 2001 Jan 2, 156(2-3), 101 - 7
Bacillus intermedius ribonuclease as inhibitor of cell proliferation and membrane current; Ilinskaya O et al.; The antiproliferative action of the guanine-specific ribonuclease secreted by Bacillus intermedius (binase) was studied in different chicken and mouse cell lines . The proliferation rate of chicken embryo fibroblasts, either normal or Rous sarcoma virus-transformed, was significantly reduced by binase treatment . Among mouse fibroblasts, v-ras-transformed NIH3T3 cells were sensitive to binase, whereas the growth of non-transformed, v-src-transformed or v-fms-transformed NIH3T3 cells was not affected . A 48 h treatment with binase inhibited the Ca2+-dependent K+ current of v-ras-transformed NIH3T3 cells but had no effect on this membrane current in non-transformed and in v-src- or v-fms-transformed NIH3T3 cells . Our results suggest that mammalian cells expressing the ras-oncogene are a potential target for the antiproliferative action of binase.

J Biotechnol, 2000 Dec 28, 84(3), 249 - 57
P450 in biotechnology: zinc driven omega-hydroxylation of p-nitrophenoxydodecanoic acid using P450 BM-3 F87A as a catalyst; Schwaneberg U et al.; Cytochrome P450 enzymes require the delivery of two electrons to the heme protein for their enzymatic function . NADPH or NADH are usually used as reduction equivalents . In the absence of a substrate, NADPH may inactivate P450 enzymes . Furthermore, it is expensive, making it unsuitable for the preparative synthesis of fine chemicals . Approaches for replacing NADPH with an electrochemically generated reduction by using platinum-electrodes and different mediators are known . In the present study, NADPH was substituted by the mediator cobalt(III)sepulchrate and zinc dust that serves as an electron source . The mutated fatty acid hydroxylase P450 BM-3 F87A from Bacillus megaterium was chosen as a catalyst, since it shows a three-fold higher sensitivity and a nearly five-fold higher activity for p-nitrophenoxydodecanoic acid (12-pNCA) than the wild-type enzyme . The formation of p-nitrophenolate can easily be monitored using a photometer at 410 nm . The turnover rate of the zinc/cobalt(III)sepulchrate system reaches 20% of the NADPH activity . Compared to the electrochemical approaches the activity is at least 77% higher (turnover 125 eq min-1) . The presented alternative cofactor system can be used instead of NADPH or expensive electrochemical devices (platinum electrodes) for fine chemical synthesis.

Vaccine, 2001 Jan 8, 19(11-12), 1460 - 6
Mucosal immunization against hepatitis B virus by intranasal co-administration of recombinant hepatitis B surface antigen and recombinant cholera toxin B subunit as an adjuvant; Isaka M et al.; Recombinant cholera toxin B subunit (rCTB) produced by Bacillus brevis carrying pNU212-CTB has been previously found to be a potent mucosal adjuvant to aluminium-non-adsorbed tetanus toxoid (nTT) and diphtheria toxoid (nDT) co-administered intranasally, and the possibility of needle-free inoculation of these vaccines with rCTB has been suggested . In this paper we examined the potentiality of rCTB as a mucosal adjuvant to aluminium-non-adsorbed yeast-derived recombinant hepatitis B surface antigen (rHBs) being a particulate antigen when administered intranasally with rCTB . In-house ELISA showed that a mixture of rHBs (1 or 5 microg) and rCTB (10 microg) elevated not only systemic responses but also mucosal immune responses at the nasal cavity, the lung, the saliva, the small intestine and the vagina against rHBs, and these could be further increased with higher doses of antigen . With antibody isotypes of IgG, there were equally high levels of serum HBs-specific IgG1, IgG2a and IgG2b antibodies and induction of mixed Th1- and Th2-type responses was considered to occur in combination of rHBs and rCTB . Serum anti-HBs titres in almost all mice obtained from sandwich EIA using a commercial kit were higher than 1000 milli-international units ml(-1) (mIU ml(-1)) . These results show that rCTB is also very effective as a mucosal adjuvant for a particulate antigen like rHBs, as well as soluble antigens like nTT and nDT reported previously, suggesting the possibility of intranasal immunization with rHBs plus rCTB in humans.

Vaccine, 2001 Jan 8, 19(11-12), 1391 - 6
DNA encoding a single mycobacterial antigen protects against leprosy infection; Martin E et al.; The continuing incidence of leprosy infection around the world and the inability of Mycobacterium bovis bacille Calmette-Guerin (BCG) to protect certain populations clearly indicates that an improved vaccine against leprosy is needed . The immuno dominant 35 kDa protein, shared by Mycobacterium leprae and Mycobacterium avium, but not Mycobacterium tuberculosis or BCG, is recognised by >90% of leprosy patients, making it an ideal candidate antigen for a subunit vaccine . Immunization of outbred Swiss Albino mice with a DNA-35 vaccine stimulated specific T cell activation and IFN-gamma production . DNA-35 immunization induced significant levels of protection against M . leprae footpad infection, comparable to that produced by BCG . Therefore, DNA immunization with the 35 kDa antigen is effective against M . leprae infection and genetic immunization with a combination of antigens holds the potential for an improved vaccine against leprosy.

Mol Immunol, 2000 Jun, 37(9), 527 - 36
Co-expression of interleukin-2 and green fluorescent protein reporter in mycobacteria: in vivo application for monitoring antimycobacterial immunity; Luo Y et al.; Recombinant Mycobacterium bovis bacillus Calmette-Guerin expressing green fluorescent protein (rBCG-GFP), driven by the mycobacterial heat shock protein 70 (HSP 70) promoter from an autonomously replicating plasmid, was genetically engineered to co-express mouse interleukin-2 (IL-2) by introduction of an independent HSP 60 promoter . To monitor host antimycobacterial immunity, C57BL/6 mice were intravenously infected with IL-2 expressing and non-expressing GFP rBCGs . Both rBCGs were clearly imaged and easily quantified with ultraviolet microscopy of tissue sections and whole organ suspensions . Enhanced mycobacterial clearance from the spleens of mice infected with the rBCG-IL-2/GFP strain was apparent by both diminished bacterial counts and spleen weights during the first 6 weeks post-infection relative to rBCG-GFP . T helper type 1 (TH1) cytokine production and proliferative response to BCG restimulation was also elevated from in vitro splenocyte cultures taken from the rBCG-IL-2/GFP-infected group . Taken together, these results suggest that IL-2 expression from rBCG augmented host protective immunity to mycobacterial infection via an enhanced TH1 immune response . Mycobacterial expression vectors that allow simultaneous but independent production of reporter proteins and bioactive substances provide an ideal means for monitoring the in vivo fate of recombinant mycobacteria.

Mol Cell Endocrinol, 2000 Dec 22, 170(1-2), 163 - 74
Single-column purification and bio-characterization of recombinant human parathyroid hormone-related protein (1-139); Wu C et al.; Recombinant human parathyroid hormone-related protein (hPTHrP) (1-139) was expressed using the IMPACT T7 (intein-mediated purification with an affinity chitin-binding tag) system, allowing purification of free recombinant peptide in a single chromatographic step . This system utilizes an intein, which is a protein splicing element from the Saccharomyces cerevisiae VMA1 gene . The intein has been modified so that it undergoes a self-cleavage reaction at its N-terminus at low temperatures in the presence of 1,4-dithiothreitol (DTT) . The cDNA encoding hPTHrP (1-139) was cloned into the pTYB1 vector to create an in-frame fusion at the N-terminus of the intein gene . The cDNA for the chitin-binding domain from Bacillus circulans is present at the C-terminus of intein for affinity purification of the three-part fusion protein on a chitin column . The recombinant plasmid was transfected into E . coli ER2566 cells and synthesis of the PTHrP fusion protein was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG) . This system produced pure hPTHrP (1-139) and an N-terminally truncated analogue, hPTHrP (27-139), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blot analysis, N-terminal sequence analysis and mass spectroscopy . hPTHrP (1-139) stimulated cAMP accumulation in ROS 17/2.8 osteoblastic bone cells, whereas hPTHrP (27-139) failed to elicit a response . hPTHrP (1-139) also inhibited the growth of the breast cancer cell line MDA-MB-231; the magnitude of the response was comparable with that of synthetic hPTHrP (1-34) and (1-86) . Neutralization of endogenous PTHrP and added hPTHrP (1-139) and N-terminal species with an anti-PTHrP antiserum completely abolished the growth inhibitory effects . These results indicate that the added peptides modulate cell growth by acting at the cell surface . Availability of recombinant hPTHrP (1-139) will allow further study of its biological function, as well as its structure.

Biochem Biophys Res Commun, 2001 Feb 2, 280(4), 1177 - 82
Fragmentary form of thermostable leucine dehydrogenase of Bacillus stearothermophilus: its construction and reconstitution of active fragmentary enzyme; Oikawa T et al.; X-ray crystallographic studies revealed that various amino acid dehydrogenases fold into two domains in each subunit, a substrate-binding domain and an NAD(P)(+)-binding domain (Baker, P . J., Turnbull, A . P., Sedelnikova, S . E., Stillman, T . J., and Rice, D . W . (1995) Structure 3, 693-705) . To elucidate the function and folding process of these two domains, we have genetically constructed a fragmentary form of thermostable leucine dehydrogenase of Bacillus stearothermophilus consisting of an N-terminal polypeptide fragment corresponding to the substrate-binding domain including an N-terminus, and a C-terminal fragment corresponding to the NAD(+)-binding domain . The two peptide fragments were expressed in separate host cells and purified . When both fragments were mixed, the leucine dehydrogenase activity with a specific activity of 1.4% of that of the wild-type enzyme appeared . This suggests that both peptide fragments mutually recognize each other, associate and fold correctly to be catalytically active, although the activity is low . However, the fragmentary form of enzyme produced catalyzed the oxidative deamination of l-leucine, l-isoleucine, and l-valine with broad substrate specificity compared to that of the wild-type enzyme . The fragmentary enzyme retained more than 75% of the initial activity after heating at 50 degrees C for 60 min . The fragmentary enzyme was more stable on heating than separate peptide fragments . These results suggest that the two domains of leucine dehydrogenase probably fold independently, and the two peptide fragments interact and associate with each other to form a functional active site .

Nucleic Acids Res, 2001 Feb 15, 29(4), 895 - 903
Characterization of BseMII, a new type IV restriction-modification system, which recognizes the pentanucleotide sequence 5'-CTCAG(N)(10/8)/; Jurenaite-Urbanaviciene S et al.; We report the properties of the new BseMII restriction and modification enzymes from Bacillus stearothermophilus Isl 15-111, which recognize the 5'-CTCAG sequence, and the nucleotide sequence of the genes encoding them . The restriction endonuclease R.BseMII makes a staggered cut at the tenth base pair downstream of the recognition sequence on the upper strand, producing a two base 3'-protruding end . Magnesium ions and S:-adenosyl-L-methionine (AdoMet) are required for cleavage . S:-adenosylhomocysteine and sinefungin can replace AdoMet in the cleavage reaction . The BseMII methyltransferase modifies unique adenine residues in both strands of the target sequence 5'-CTCAG-3'/5'-CTGAG-3' . Monomeric R.BseMII in addition to endonucleolytic activity also possesses methyltransferase activity that modifies the A base only within the 5'-CTCAG strand of the target duplex . The deduced amino acid sequence of the restriction endonuclease contains conserved motifs of DNA N6-adenine methylases involved in S-adenosyl-L-methionine binding and catalysis . According to its structure and enzymatic properties, R.BseMII may be regarded as a representative of the type IV restriction endonucleases.

J Bacteriol, 2001 Mar, 183(5), 1672 - 9
S-layer variation in Bacillus stearothermophilus PV72 is based on DNA rearrangements between the chromosome and the naturally occurring megaplasmids; Scholz HC et al.; Bacillus stearothermophilus PV72 expresses different S-layer genes (sbsA and sbsB) under different growth conditions . No stretches of significant sequence identity between sbsA and sbsB were detected . In order to investigate S-layer gene regulation in B . stearothermophilus PV72, we characterized the upstream regulatory region of sbsA and sbsB by sequencing and primer extension analysis . Both genes are transcribed from unique but different promoters, independently of the growth phase . Localization of sbsB in the sbsA-expressing strain PV72/p6 revealed that the coding region of the second S-layer gene sbsB is located not on the chromosome but on a natural megaplasmid of the strain, whereas the upstream regulatory region of sbsB was exclusively detected on the chromosome of PV72/p6 . For sbsB expression, the coding region has to be integrated into the chromosomally located expression site . After the switch to sbsB expression, the sbsA coding region was removed from the chromosome but could still be detected on the plasmid of the sbsB-expressing strain PV72/p2 . The sbsA upstream regulatory region, however, remained on the chromosome . This is the first report of S-layer variation not caused by intrachromosomal DNA rearrangements, but where variant formation depends on recombinational events between the plasmid and the chromosome.

Appl Environ Microbiol, 2001 Feb, 67(2), 995 - 1000
Molecular characterization of cycloinulooligosaccharide fructanotransferase from Bacillus macerans; Kim HY et al.; Cycloinulooligosaccharide fructanotransferase (CFTase) converts inulin into cyclooligosaccharides of beta-(2-->1)-linked D-fructofuranose by catalyzing an intramolecular transfructosylation reaction . The CFTase gene was cloned and characterized from Bacillus macerans CFC1 . The CFTase gene encoded a polypeptide of 1,333 amino acids with a calculated Mr of 149,563 . Western blot and zymography analyses revealed that the CFTase with a molecular mass of 150 kDa (CFT150) was processed (between Ser389 and Phe390 residue) to form a 107-kDa protein (CFT107) in the B . macerans CFC1 cells . The processed CFT107 was similar in its mass to the previously purified CFTase from B . macerans CFC1 . The CFT107 enzyme was produced by B . macerans CFC1 but was not detected from the recombinant Escherichia coli cells, indicating that the processing event occurred in a host-specific manner . The two CFTases (CFT150 and CFT107) exhibited the same enzymatic properties, such as influences of pH and temperature on the enzyme activity, the intermolecular transfructosylation ability, and the ability of hydrolysis of cycloinulooligosaccharides produced by the cyclization reaction . However, the thermal stability of CFT107 was slightly higher than that of CFT150 . The most striking difference between the two enzymes was observed in their Km values; the value for CFT150 (1.56 mM) was threefold lower than that for CFT107 (4.76 mM) . Thus, the specificity constant (kcat/Km) of CFT150 was about fourfold higher than that of CFT107 . These results indicated that the N-terminal 358-residue region of CFT150 played a role in increasing the enzyme's binding affinity to the inulin substrate.

Appl Environ Microbiol, 2001 Feb, 67(2), 979 - 81
Mannose phosphate isomerase isoenzymes in Plutella xylostella support common genetic bases of resistance to Bacillus thuringiensis toxins in Llpidopteran species; Herrero S et al.; A strong correlation between two mannose phosphate isomerase (MPI) isoenzymes and resistance to Cry1A toxins from Bacillus thuringiensis has been found in a Plutella xylostella population . MPI linkage to Cry1A resistance had previously been reported for a Heliothis virescens population . The fact that the two populations share similar biochemical, genetic, and cross-resistance profiles of resistance suggests the occurrence of homologous resistance loci in both species.

Appl Environ Microbiol, 2001 Feb, 67(2), 872 - 9
Binding analyses of Bacillus thuringiensis Cry delta-endotoxins using brush border membrane vesicles of Ostrinia nubilalis; Hua G et al.; Transgenic corn expressing the Bacillus thuringiensis Cry1Ab gene is highly insecticidal to Ostrinia nubilalis (European corn borer) larvae . We ascertained whether Cry1F, Cry9C, or Cry9E recognizes the Cry1Ab binding site on the O . nubilalis brush border by three approaches . An optical biosensor technology based on surface plasmon resonance measured binding of brush border membrane vesicles (BBMV) injected over a surface of immobilized Cry toxin . Preincubation with Cry1Ab reduced BBMV binding to immobilized Cry1Ab, whereas preincubation with Cry1F, Cry9C, or Cry9E did not inhibit BBMV binding . BBMV binding to a Cry1F-coated surface was reduced when vesicles were preincubated in Cry1F or Cry1Ab but not Cry9C or Cry9E . A radioligand approach measured 125I-Cry1Ab toxin binding to BBMV in the presence of homologous (Cry1Ab) and heterologous (Cry1Ac, Cry1F, Cry9C, or Cry9E) toxins . Unlabeled Cry1Ac effectively competed for 125I-Cry1Ab binding in a manner comparable to Cry1Ab itself . Unlabeled Cry9C and Cry9E toxins did not inhibit (125)I-Cry1Ab binding to BBMV . Cry1F inhibited (125)I-Cry1Ab binding at concentrations greater than 500 nM . Cry1F had low-level affinity for the Cry1Ab binding site . Ligand blot analysis identified Cry1Ab, Cry1Ac, and Cry1F binding proteins in BBMV . The major Cry1Ab signals on ligand blots were at 145 kDa and 154 kDa, but a strong signal was present at 220 kDa and a weak signal was present at 167 kDa . Cry1Ac and Cry1F binding proteins were detected at 220 and 154 kDa . Anti-Manduca sexta aminopeptidase serum recognized proteins of 145, 154, and 167 kDa, and anti-cadherin serum recognized the 220 kDa protein . We speculate that isoforms of aminopeptidase and cadherin in the brush border membrane serve as Cry1Ab, Cry1Ac, and Cry1F binding proteins.

Appl Environ Microbiol, 2001 Feb, 67(2), 713 - 20
Polysaccharide lyase: molecular cloning, sequencing, and overexpression of the xanthan lyase gene of Bacillus sp . strain GL1; Hashimoto W et al.; When grown on xanthan as a carbon source, the bacterium Bacillus sp . strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H . Nankai, W . Hashimoto, H . Miki, S . Kawai, and K . Murata, Appl . Environ . Microbiol . 65:2520-2526, 1999) . A gene for the lyase was cloned, and its nucleotide sequence was determined . The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308 . The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of Streptomyces griseus (identity score, 37.7%) . Escherichia coli transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa . During storage at 4 degrees C, the purified enzyme (97 kDa) from E . coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) from Bacillus sp . strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action . Logarithmically growing cells of Bacillus sp . strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa) . These results suggest that, in Bacillus sp . strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.

Curr Microbiol, 2000 Jul, 41(1), 65 - 9
Cloning of a new Bacillus thuringiensis cry1I-type crystal protein gene; Choi SK et al.; A new cry1I-type gene, cry1Id1, was cloned from a B . thuringiensis isolate, and its nucleotide sequence was determined . The deduced amino acid sequence of Cry1Id1 is 89.7%, 87.2%, and 83.4% identical to the Cry1Ia, Cry1Ib, and Cry1Ic proteins, respectively . The upstream sequence of the cry1Id1 structural gene was not functional as promoter in B . subtilis . The Cry1Id1 protein, purified from recombinant E . coli cells, had a toxicity comparable to that of Cry1Ia against Plutella xylostella, but it was significantly less active than Cry1Ia against Bombyx mori . Cry1Id1 was not active against the coleopteran insect, Agelastica coerulea.

Folia Biol (Krakow), 1999, 47(3-4), 143 - 8
Biocidal activity of Bacillus species for Anopheles larvae; Shakoori AR et al.; It is generally accepted that Bacillus thuringiensis (B.t.) is specifically toxic to some insects but does not pose any threat to the environment, operators, or consumers . There are several other Bacillus species which can be used as effective bioinsecticides . In this study four different species of Bacillus, i.e., B . coagulans, B . megaterium, B . brevis, and B . sphaericus were isolated from soil samples collected from Kala Shah Kakoo and Kasur areas, in the suburbs of Lahore . Isolated Bacillus species were administered to mosquito larvae to evaluate their biocidal activity . B . coagulans I from Kala Shah Kakoo showed 93% mortality, while B . coagulans III from Kasur showed 70% mortality . Bacterial isolates most toxic to Anopheles larvae showed optimum growth at 37 degrees C and pH 7 . These isolates have a great potency to controlling anopheline population.

Urology . 1999 Jun;53(6):1228.
Granulomatous nephritis as a complication of intrarenal bacille Calmette-Guérin therapy; Soda T et al.; A case of granulomatous nephritis after intrarenal bacille Calmette-Guerin (BCG) therapy is reported . High fever greater than 38.5 degrees C lasted for 1 month, without response to conservative therapy . Standard nephroureterectomy was subsequently carried out . Histopathologic findings from the surgical specimen were compatible with BCG-induced granulomatous nephritis . The use of a syringe pump for retrograde instillation of BCG was thought to be the major cause of this severe complication.

Arch Virol, 2000, 145(2), 275 - 89
Inter- and intra-site genetic diversity of natural field populations of rice tungro bacilliform virus in the Philippines; Arboleda M et al.; The genetic structure of rice tungro bacilliform virus (RTBV) populations within and between growing sites was analyzed in a collection of natural field isolates from different rice varieties grown in eight tungro-endemic sites of the Philippines . Total DNA extracts from 345 isolates were digested with EcoRV restriction enzyme and hybridized with a full-length probe of RTBV, a procedure shown in preliminary experiments capable of revealing high levels of polymorphism in RTBV field isolates . In the total population, 17 distinct EcoRV-based genome profiles (genotypes) were identified and used as indicators for virus diversity . Distinct sets of genotypes occurred in Isabela and North Cotabato provinces suggesting a geographic isolation of virus populations . However, among the sites in each province, there were few significant differences in the genotype compositions of virus populations . The number of genotypes detected at a site varied from two to nine with a few genotypes dominating . In general the isolates at a site persisted from season to season indicating a genetic stability for the local virus population . Over the sampling time, IRRI rice varieties, which have green leafhopper resistance genes, supported similar virus populations to those supported by other varieties, indicating that the variety of the host exerted no apparent selection pressures . Insect transmission experiments on selected RTBV field isolates showed that dramatic shifts in genotype and phenotype distributions can occur in response to host/environmental shifts.

Cancer Immunol Immunother, 2000 Mar, 48(12), 703 - 13
Observed localization of the long-term cultured rat adherent natural killer cells in mammary tumor tissues; Gu S et al.; Adherent natural killer (A-NK) cells were isolated from splenic lymphocytes and treated with long-term culture in the presence of recombinant interleukin-2 (rIL-2) . Immunocytochemical and flow-cytometric analysis revealed that most of the A-NK cells strongly expressed lymphocyte-function-associated antigen 1 (LFA-1alpha, and LFA-1beta) throughout the incubation . All A-NK cells from 8- to 150-day cultures, particularly those cultured for 8 days, showed significant cytolytic activity against all targets . Analysis of the tissue distribution of the injected {3H} uridine-labeled A-NK cells demonstrated that, in the first 3 h, most (over 60%) cells localized in the lungs, and that most cells remained temporally within the cavities of blood capillaries of the lungs and moved gradually into lymphoid and other tissues . Peritumoral injection of various kinds of adjuvant, particularly Freund's complete adjuvant (FCA) plus bacillus Calmetee-Guerin (BCG), resulted in a marked accumulation of {3H}A-NK cells in mammary tumor tissues 24 h after injection, and simultaneously in the formation of vessels resembling high-endothelium venules (HEV), infiltration of LFA-1+ lymphocytes and expression of the ICAM-1 molecule on the tumor cells in the sites of tumor tissues . When 30 x 10(6) A-NK cells were intravenously administered, significant retardation of tumor growth and prolongation of survival of tumor-bearing rats were observed in the groups that received the prior injection of adjuvants, especially FCA + BCG and Freund's incomplete adjuvant (FIA) + BCG . The suppressive effect of combination therapy on tumor growth was blocked effectively by the injection of anti-ICAM-1 mAb . These results indicate that the prior injection of proper adjuvant into the peritumoral region is effective for the selective accumulation or infiltration of A-NK cells into the sites of tumor tissues, and results in the marked retardation of tumor growth.

Arq Neuropsiquiatr, 1999 Sep, 57(3B), 723 - 34
Muscle involvement in leprosy . Study of the anterior tibial muscle in 40 patients; Werneck LC et al.; The involvement of skeletal striated muscle in leprosy is considered secondary due to peripheral neuropathy, but some studies point it to a primary muscle lesion . In order to investigate the muscle involvement in leprosy, we studied 40 patients (lepromatous 23, tuberculoid 13, borderline 2 and indeterminate 2) . The motor nerve conduction of the peroneal nerves had a reduction of the velocity, decreased compound muscle action potential and sometimes absence of potentials . The electromyographic study of the anterior tibial muscle showed signs of recent and chronic denervation in 77.5% of the cases and no myopathic potentials . The anterior tibial muscle biopsy revealed denervation in 45% of the cases, interstitial inflammatory myopathy in 30% and mixed (myopathic and neuropathic) pattern in 12.5% . Acid fast bacillus was detected in 25% of the cases, always in the interstitial tissue . Inflammatory reaction was present in the interstitial space and in patients with the lepromatous type . The histological findings clearly defined the presence of the so-called "Leprous Interstitial Myositis" on the top of denervation signs.

J Urol, 2000 May, 163(5), 1588 - 90
Induction of mycobacteremia by intravesical bacillus Calmette-Guerin instillation in an experimental animal model and detection with polymerase chain reaction; Aygun C et al.; PURPOSE: The aim of this study was to detect mycobacteremia by polymerase chain reaction (PCR), induced by the instillation of bacillus Calmette-Guerin (BCG) to guinea pig bladder . We also investigated the peak time and the effect of the dose of BCG in injured and non-injured bladder . The sensitivities of routine culture and PCR were also compared . MATERIALS AND METHODS: Five different doses (0, 0.069, 0.69, 6.9 and 69 mg.) of BCG were instilled into 5 injured and 5 non-injured bladders . Blood samples were collected at 0, 5, 15, 30 and 60 minutes following instillation for routine culture and PCR for each dose . A total of 50 female guinea pigs were used . RESULTS: Three of 5 samples (60%) obtained 30 minutes after the instillation of 69 mg . BCG into injured bladders were PCR positive . Furthermore, 4 of 5 samples (80%) were PCR positive when samples were obtained at the 60th minute following instillation . All the other samples were negative for PCR and routine culture . All the routine tuberculosis culture results were negative, including those which were PCR positive . CONCLUSIONS: Mycobacteremia was detected only in injured bladders and with high doses of BCG . PCR is a highly sensitive and rapid diagnostic method for detection of mycobacteremia.

J Urol, 2000 May, 163(5), 1553 - 9
Autocrine IL-6 production by human transitional carcinoma cells upregulates expression of the alpha5beta1 firbonectin receptor; Zhang GJ et al.; PURPOSE: Studies have demonstrated elevated expression and secretion of IL-6 by transitional cell carcinomas (TCC) following bacillus Calmette-Guerin (BCG) therapy . At present, the role of IL-6 on the biology of TCC is poorly understood . This study evaluated the influence of IL-6 expression on a critical variable regulating BCG-tumor interaction, the tumor expression of alpha5beta1 integrin . MATERIALS AND METHODS: A human TCC cell line (253J) was transfected with an expression vector containing the full-length IL-6 cDNA sequence . Overexpression of IL-6 mRNA and protein was confirmed by Northern analysis and ELISA, respectively . Clones found to overexpress IL-6 were then assayed for alpha5beta1 expression using Northern analysis and flow cytometry . The effect of alterations in alpha5beta1 expression on tumor adherence to fibronectin (FN), and BCG adherence to tumor cells was determined using specific adherence assays . RESULTS: mRNAs for both the alpha5 and beta1 subunits of the FN receptor were increased an average of 9.4 fold and 125.7 fold respectively in the IL-6 overexpressing transfectants relative to the parental 253J cells . Increased mRNA of alpha5 and beta1 was associated with increased cell surface expression of both proteins . Increased protein expression resulted in greater FN substrate binding affinity and increased adherence of BCG to tumor cells . CONCLUSIONS: Autocrine expression of IL-6 upregulates the expression of FN receptor subunits in TCC . Increased alpha5beta1 expression increases cellular adherence to FN, and BCG adherence to tumor cells . These results suggest a role for IL-6 in mediating the antitumor activity of BCG by influencing BCG's adherence to TCC.

J Biol Chem, 2001 Apr 6, 276(14), 10775 - 81 Epub 2001 Jan 08.
Cysteine-scanning mutagenesis reveals a highly amphipathic, pore-lining membrane-spanning helix in the glutamate transporter GltT; Slotboom DJ et al.; The carboxyl-terminal membrane-spanning segment 8 of the glutamate transporter GltT of Bacillus stearothermophilus was studied by cysteine-scanning mutagenesis . 21 single cysteine mutants were constructed in a stretch ranging from Gly-374 to Gln-404 . Two mutants were not expressed, four were inactive, and two showed severely reduced glutamate transport activity . Cysteine mutations at the other positions were well tolerated . Only the two most amino- and carboxyl-terminal mutants (G374C, I375C, S399C, and Q404C) could be labeled with the large thiol reagent fluorescein maleimide, indicating unrestricted access and a location in a loop structure outside the membrane . The labeling pattern of these mutants using membrane- permeable and -impermeable thiol reagents showed that the N and C termini of the mutated stretch are located extra- and intracellularly, respectively . Thus, the location of the membrane-spanning segment was confined to a stretch of 23 residues between Gly-374 and Ser-399 . Cysteine residues in three mutants in the central part of the segment (M381C, V388C, and N391C) could be labeled with the small and flexible reagent 2-aminoethyl methanethiosulfonate hydrobromide only, suggesting accessibility via a narrow aqueous pore . When the region was modeled as an alpha-helix, all positions at which cysteine mutations lead to inactive or severely impaired transporters cluster on one face of this helix . The inactive mutants showed neither proton motive force-driven uptake activity nor exchange activity nor glutamate binding . The results indicate that transmembrane segment 8 forms an amphipathic alpha-helix . The hydrophilic face of the helix lines an aqueous pore and contains many residues that are important for activity.

J Biol Chem, 2001 Mar 23, 276(12), 9059 - 65 Epub 2000 Dec 27.
Molecular cloning and characterization of the gene coding for azoreductase from Bacillus sp . OY1-2 isolated from soil; Suzuki Y et al.; Azo dyes are regarded as pollutants because they are not readily reduced under aerobic conditions . Bacillus sp . OY1-2 transforms azo dyes into colorless compounds, and this reduction is mediated by a reductase activity for the azo group in the presence of NADPH . A 1.2-kbp EcoRI fragment containing the gene that encodes azoreductase was cloned by screening the genomic library of Bacillus sp . OY1-2 with digoxigenin-labeled probe designed from the N-terminal amino acid sequence of the purified enzyme . An open reading frame encoding the azoreductase, consisting of 178 amino acids, was predicted from the nucleotide sequence . In addition, because only a Bacillus subtillis hypothetical protein was discovered in the public databases (with an amino acid identity of 52.8%), the gene encoding the azoreductase cloned in this study was predicted to be a member of a novel family of reductases . Southern blot analysis revealed that the azoreductase gene exists as a single copy gene on a chromosome . Escherichia coli-expressing recombinant azoreductase gave a ten times greater reducing activity toward azo dyes than the original Bacillus sp . OY1-2 . In addition, the expressed azoreductase purified from the recombinant E . coli lysate by Red-Sepharose affinity chromatography showed a similar activity and specificity as the native enzyme . This is the first report describing the sequencing and characterization of a gene encoding the azo dye-reducing enzyme, azoreductase, from aerobic bacteria and its expression in E . coli.

Int J Syst Evol Microbiol, 2000 Nov, 50 Pt 6, 2181 - 7
Bacillus siralis sp . nov., a novel species from silage with a higher order structural attribute in the 16S rRNA genes; Pettersson B et al.; A novel bacterial strain (171544T) was recently isolated from silage and was classified in the genus Bacillus by 16S rDNA sequence analysis . Additional silage samples have been investigated in the present study and four organisms resembling strain 171544T were isolated . Phenotypic and genotypic characterization of these bacteria showed that they constitute a new species of the genus Bacillus . This taxon was positioned in the family Bacillaceae on the basis of evolutionary distance trees using 16S rDNA sequences . Bacillus circulans, Bacillus firmus and Bacillus benzoevorans were the most closely related species with 165 rDNA similarities of 97.2, 96.3 and 95.9%, respectively . All five silage isolates shared a higher order structural feature in the 3' region of the 16S rRNA gene comprising an extension to helix 49 of 24 bp and highly similar random amplified polymorphic DNA patterns that distinguished them from the type strains of B . circulans and B . firmus . Moreover, they possessed a unique pattern of phenotypic features including subterminally or terminally located endospores which distinctly swelled the sporangium, strictly aerobic metabolism but with the ability to utilize nitrate as a terminal electron acceptor under anaerobic conditions, and hydrolysis of casein but not starch . The name Bacillus siralis is therefore proposed for this new taxon . The type strain of B . siralis is strain 171544T (= NCIMB 13601T = CIP 106295T).

Acta Virol, 2000 Jun-Aug, 44(3), 189 - 92
Mating in Bacillus thuringiensis can induce plasmid integrative prophage J7W-1; Kanda K et al.; Bacillus thuringiensis serovar israelensis, a bacterium which possesses plasmid transfer ability after mating, has been lysogenized by plasmid integrative phage J7W-1 . The induction of phage in this J7W-1 lysogen was observed after mating with phage-insensitive strains, such as B . thuringiensis serovar thuringiensis, B . cereus and B . subtilis, as well as the phage-sensitive strain serovar israelensis . The phage induction was not observed after mating with B . thuringiensis strains AF101, serovar dendrolimus and serovar indiana . Because these strains are naturally associated with J7W-1 or its related phage, the data strongly suggest a constitutive expression of the repressor encoded by the prophage in these strains . However, the phage induction was observed in B . thuringiensis serovar aizawai, although it contained the J7W-1 DNA homologous region(s).

Acta Virol, 2000 Jun-Aug, 44(3), 183 - 7
Temperature influences induction of a J7W-1-related phage in Bacillus thuringiensis serovar indiana; Kanda K et al.; Induction of a plasmid-integrative J7W-1-related phage in Bacillus thuringiensis serovar indiana by ethidium bromide was influenced by the temperature at which the host cells were cultured . Under optimal growth conditions, the maximum titer of the phage produced by the serovar indiana reached 1.2 x 10(6) PFU/ml at 37 degrees C while at 27 degrees C it was lower by an order of magnitude (1.3 x 10(5) PFU/ml) . The temperature-sensitive period was estimated to occur early during the phage induction . However, the temperature effect observed with the serovar indiana did not occur with the serovar israelensis . In the latter case, the phage induction was the same at 37 degrees C or 27 degrees C . Thus we assume that the temperaturesensitive phage induction observed with the serovar indiana as host was not a phenomenon caused by the phage genome but rather by product(s) encoded by certain host gene(s).

J Appl Microbiol, 2001 Jan, 90(1), 115 - 22
Phylogenetic analysis of Bacillus thuringiensis serovars based on 16S rRNA gene restriction fragment length polymorphisms; Joung KB et al.; AIMS: To determine the 16S rRNA gene fingerprints of Bacillus thuringiensis strains to reveal phylogenetic relationships among them . METHODS AND RESULTS: Using 16S rRNA gene restriction fragment length polymorphisms generated by HindIII and EcoRI, 86 Bacillus thuringiensis strains were classified . This includes 80 B . thuringiensis serovars and five more strains, kurstaki HD-1, subtoxicus, dendrolimus, tenebrionis and sandiego, to assess not only interserovar DNA relatedness but also intraserovar DNA relatedness, and the non-motile strain, hence non-serotypeable, B . thuringiensis var . wuhanensis . All 86 B . thuringiensis strains tested showed distinct ribotypes . The dendrogram resulting from the numerical analysis of the distance matrix shows four distinct phylogenetic groups and two ungrouped serovars, finitimus and bolivia, at the 92.5% DNA relatedness rate . CONCLUSION: 16S rRNA gene fingerprinting cannot only be used for the classification of B . thuringiensis strains amenable or not to serotyping, but can also reveal phylogenetic relationships between strains . SIGNIFICANCE AND IMPACT OF THE STUDY: In future screening programmes, 16S rRNA gene restriction pattern analysis could be determined for novel B . thuringiensis strains, allowing them not only to be grouped but also to be positioned on the phylogenetic tree.

J Appl Microbiol, 2001 Jan, 90(1), 106 - 14
The application of a fermented food ingredient containing 'variacin', a novel antimicrobial produced by Kocuria varians, to control the growth of Bacillus cereus in chilled dairy products; O'Mahony T et al.; AIMS: The feasibility of applying variacin, a lantibiotic produced by Kocuria varians in the form of a spray-dried fermented ingredient to control the growth of psychrotrophic Bacillus cereus strains in chilled dairy foods, was evaluated . METHODS AND RESULTS: A range of chilled dairy food formulations modelling commercially-available products were fabricated, to which were added varying amounts of active ingredient . These were subsequently challenged with a B . cereus spore cocktail over a range of abuse temperatures . This work was validated by the inclusion of the fermented ingredient to commercial products . CONCLUSION: Results demonstrate the functionality of the bacteriocin at refrigeration abuse temperatures, and indicate the robust nature of the proteinaceous antimicrobial agent with regard to processing . SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates the applicability of fermented food ingredients containing naturally-occurring antimicrobials as additional hurdles in food preservation.

Biol Chem, 2000 Nov, 381(11), 1123 - 5
Characterization of the specific DNA nicking activity of restriction endonuclease N.BstNBI; Morgan RD et al.; N.BstNBI is a unique restriction endonuclease isolated from Bacillus stearothermophilus . We have characterized the recognition sequence and the cleavage site of N.BstNBI . Mapping of cleavage sites of N.BstNBI showed that it recognizes an asymmetric sequence, 5' GAGTC 3', and cleaves only on the top strand 4 base pairs away from its recognition sequence . To verify the nicking activity of N . BstNBI, we have constructed two plasmids containing a single recognition sequence (pNB1) or no recognition site (pNB0) . When pNB1 and pNB0 were incubated with the enzyme, N.BstNBI nicked only the plasmid pNB1, suggesting that N.BstNBI is a specific nicking endonuclease.

J Biomed Mater Res, 2001, 58(1), 127 - 35
Effects of steam sterilization on thermogelling chitosan-based gels; Jarry C et al.; A new thermogelling chitosan-glycerophosphate system has been recently proposed for biomedical applications such as drug and cell delivery . The objectives of this work were to characterize the effect of steam sterilization on the in vitro and in vivo end performances of the gel and to develop a filtration-based method to assess its sterility . Autoclaving 2% (w/v) chitosan solutions for as short as 10 min resulted in a 30% decrease in molecular weight, 3-5-fold decrease in dynamic viscosity, and substantial loss of mechanical properties of the resulting gel . However, sterilization did not impair the ability of the system to form a gel at 37 degrees C . The antimicrobial activity of chitosan against several microorganisms was evaluated after inoculation of chitosan solutions and removal of the cells by filtration . It was found that, although chitosan was bacteriostatic against the heat sterilization bioindicator Bacillus stearothermophilus, the bacteria could rapidly grow after separation from the chitosan solution by filtration . This indicated that B . stearothermophilus is an adequate strain to validate a heat sterilization method on chitosan preparations, and accordingly this strain was used to assess the sterility of chitosan solution following a 10 min autoclaving time .

Anal Biochem, 2001 Jan 15, 288(2), 149 - 55
Investigation of the interactions between beta-lactams and a metallo-beta-lactamase from bacillus cereus using a monoclonal antibody; Chambers SJ et al.; A monoclonal antibody recognizing the active site of a beta-lactamase from Bacillus cereus was identified and characterized . The binding of the monoclonal antibody to the active site was quantitatively inhibited by a broad spectrum of beta-lactam antibiotics . The levels of inhibition were found to be associated with particular structural features of the antibiotics and their ability to form stable enzyme/substrate complexes . A novel, broad specificity assay for beta-lactams was developed based on the inhibition of antibody binding of all the beta-lactams studied . The assay is applicable to detection of beta-lactams at or close to the MRL level and would be complementary to existing receptor-based assays . The approach described is relevant to the study of kinetic aspects of beta-lactamases and could prove a useful tool in future drug development .

Biochim Biophys Acta, 2000 Dec 29, 1543(2), 253 - 274
Protein engineering of bacterial alpha-amylases; Nielsen JE et al.; alpha-Amylases constitute a very diverse family of glycosyl hydrolases that cleave alpha1-->4 linkages in amylose and related polymers . Recent structural and mutagenic studies of archeael, mammalian and bacterial alpha-amylases have resulted in a wealth of information on the catalytic mechanism and on the structural features of this enzyme class . Because of their high thermo-stability, the Bacillus alpha-amylases have found widespread use in industrial processes, and much attention has been devoted to optimising these enzymes for the very harsh conditions encountered there . Stability has been a major area of focus in this respect, and several remarkably stable bacterial alpha-amylases have been produced by bioengineering techniques . Protein engineering studies of pH-activity profiles and of substrate specificities have also been initiated, although without much success . In the coming years it is likely, however, that the focus of alpha-amylase engineering will shift from engineering stability to these new areas.

Biochem, Eng . J. . 2001 Jan, 7(1), 35 - 39
Cultivation of Bacillus thuringiensis in an airlift reactor with wire mesh draft tubes; Huang T et al.; An aeration strategy was proposed for foam control in an airlift reactor with double wire mesh draft tubes . The airlift reactor was employed in the cultivation of Bacillus thuringiensis for thuringiensin production . The aeration strategy involved two situations . If the foam rose and touched the foam probe, the air flow rate was dropped to a low value for a certain period . However, if the DO value was already below 10% of the saturation when the air flow rate was dropped, the conventional foam control was employed . The production of thuringiensin based on the proposed strategy was up to 70% higher than that of using the conventional cultivation method with addition of antifoam agents for foam control.

Pediatr Nephrol, 2000 Dec, 15(3-4), 186 - 7
Leclercia adecarboxylata peritonitis in a child receiving chronic peritoneal dialysis; Fattal O et al.; A 5 year old boy with end-stage renal disease presented with clinical and laboratory findings of peritonitis . Peritoneal fluid revealed infection with Leclercia adecarboxylata . This is a motile, gram-negative bacillus, formerly designated enteric group 41 and Escherichia adecarboxylata . To our knowledge, this is the first reported case of peritonitis due to this organism.

Curr Opin Rheumatol, 2001 Jan, 13(1), 74 - 9
Whipple disease and arthritis; Puechal X; Whipple disease is a chronic, multisystem, curable, bacterial infection that usually affects middle-aged men and has a wide range of clinical manifestations . The most common symptoms are weight loss and diarrhea, preceded in three quarters of cases by arthritis for a mean of 6 years . In most patients, periodic acid-Schiff staining of proximal small bowel biopsy specimens reveals inclusions within the macrophages, corresponding to bacterial structures . However, patients with various manifestations of the disease may have no gastrointestinal symptoms and negative jejunum biopsy results . Before the onset of gastrointestinal symptoms, a strong index of clinical suspicion is the key to diagnosis . The classic setting is long-term, unexplained, seronegative oligoarthritis or polyarthritis with a palindromic or relapsing course, although chronic destructive polyarthritis and spondyloarthropathy have been repeatedly reported . Identification of the Whipple bacterium, Tropheryma whippelii, has led to the development of polymerase chain reaction as a diagnostic tool in patients in the early stages of the disease or with atypical Whipple disease . This technique can be used to detect the bacterium in many tissues and fluids, including synovial tissue and fluid . The recent cultivation of the Whipple bacillus should lead to the development of serologic tests, further facilitating diagnosis . These recent major advances may show that the infection is more frequent than previously suspected and may expand the clinical spectrum of the disease . It may also allow earlier diagnosis, thereby preventing the development of the severe systemic and sometimes fatal forms of the disease.

Dermatology, 2000, 201(4), 326 - 31
Bacillary angiomatosis in HIV-infected patients--an epidemiological and clinical study; Plettenberg A et al.; BACKGROUND: No data were available on the epidemiological and clinical characteristics of bacillary angiomatosis (BA) in Germany . OBJECTIVE: To determine epidemiological and clinical data on HIV-associated BA . METHODS: A chart review of all BA cases between 1990 and 1998 was performed in 23 German AIDS treatment units . RESULTS: A total of 21 cases of BA was diagnosed . During this period, the participating HIV centers treated about 17,000 HIV-infected patients . As a result, a BA prevalence of 1.2 cases/1,000 patients can be assumed . 19 BA were localized in the skin; in 5 cases bones and in 4 cases the liver were involved . Out of 20 patients who received antibiotic therapy, 13 had complete remission . The median time of duration up to complete remission was 32 days (9-82) . During the follow-up of the 20 patients, 7 relapses were observed . CONCLUSION: BA is a rare HIV-associated disease with a prevalence of 1,2 cases/1,000 patients in the presented study .

J Immunol, 2001 Jan 15, 166(2), 1352 - 9
Th1 to Th2 cytokine shifts in nonobese diabetic mice: sometimes an outcome, rather than the cause, of diabetes resistance elicited by immunostimulation; Serreze DV et al.; Numerous immunostimulatory protocols inhibit the development of T cell-mediated autoimmune insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetic (NOD) mouse model . Many of these protocols, including treatment with the nonspecific immunostimulatory agents CFA or bacillus Calmette-Guerin (BCG) vaccine, have been reported to mediate protection by skewing the pattern of cytokines produced by pancreatic beta-cell autoreactive T cells from a Th1 (IFN-gamma) to a Th2 (IL-4 and IL-10) profile . However, most of these studies have documented associations between such cytokine shifts and disease protection rather than a cause/effect relationship . To partially address this issue we produced NOD mice genetically deficient in IFN-gamma, IL-4, or IL-10 . Elimination of any of these cytokines did not significantly alter the rate of spontaneous IDDM development . Additional experiments using these mice confirmed that CFA- or BCG-elicited diabetes protection is associated with a decreased IFN-gamma to IL-4 mRNA ratio within T cell-infiltrated pancreatic islets, but this is a secondary consequence rather than the cause of disease resistance . Unexpectedly, we also found that the ability of BCG and, to a lesser extent, CFA to inhibit IDDM development in standard NOD mice is actually dependent upon the presence of the Th1 cytokine, IFN-gamma . Collectively, our studies demonstrate that while Th1 and Th2 cytokine shifts may occur among beta-cell autoreactive T cells of NOD mice protected from overt IDDM by various immunomodulatory therapies, it cannot automatically be assumed that this is the cause of their disease resistance.

Spectrochim Acta A Mol Biomol Spectrosc, 2000 Dec, 56(14), 2637 - 44
Surface-enhanced raman spectra of some anti-tubercle bacillus drugs; Wang Y et al.; Surface-enhance Raman (SER) spectra of pyrazinamide (PZA), isoniazid (INAZ), and isonicotiamide (INCT) in silver sols have been studied over a range of solution concentration and pH . A linear calibration curve has been obtained for each of the sample molecules, and the lower limits of detection are estimated to be 5 ng for PZA and INAZ, and 10 ng for INCA . The variation of SER intensity with the sample solution pH is explained in terms of the charge of the sample species and the stability of the sols . The normal Raman and infrared spectra for the pure drug samples have also been collected, from which some vibration assignments for the molecules are given . The absorption behaviors of the molecules on the silver particle surfaces are also suggested.

Arthritis Rheum, 2000 Dec, 43(12), 2843 - 7
Chronic destructive oligoarthritis associated with Propionibacterium acnes in a female patient with acne vulgaris: septic-reactive arthritis?
Delyle LG, Vittecoq O, Bourdel A, Duparc F, Michot C, Le Loet X.
Propionibacterium acnes is an anaerobic bacillus implicated in certain chronic arthritides . This report describes an HLA-B27+ 17-year-old woman with acne vulgaris who presented with rapidly destructive arthritis in the left shoulder as well as an evolving left subclavicular adenopathy . One year later, arthritis was detected in the left knee; the inflammatory synovial fluid was sterile . Growth of P acnes was observed in cultures of the shoulder synovium and lymph nodes, but polymerase chain reaction was negative for Borrelia, Chlamydia, and Ureaplasma DNA . Three months of treatment with amoxicillin and rifampicin led to clinical disappearance of the oligoarthritis, but arthritis recurred in the left knee after discontinuation of therapy . On biopsy, bacteria were undetectable in the knee synovium, but chronic arthritis was evident histologically . Antibiotics were reintroduced for 12 months and were again effective against the clinical symptoms . Although the asymmetry, histologic features, arthritis-acne association, and genetic predisposition of this chronic destructive oligoarthritis would seem to indicate a reactive arthropathy, the isolation of P acnes from 2 distinct specimens prompted us to propose calling this a case of septic-reactive arthritis, which is further supported by the absence of progression after antibiotic therapy and the persistence of the rheumatism . To our knowledge, this is the first demonstration of the efficacy of prolonged antibiotic therapy on the joint manifestations of chronic rheumatism associated with acne.

Int J Tuberc Lung Dis, 2000 Dec, 4(12), 1126 - 32
Bacillary disease and health seeking behavior among Filipinos with symptoms of tuberculosis: implications for control; Tupasi TE et al.; SETTING: Urban and rural communities and urban poor settlements in the Philippines . OBJECTIVE: To determine bacillary disease and action taking among individuals with symptoms of tuberculosis (TB), and to analyze their implications for TB control . STUDY DESIGN AND METHOD: Subjects aged 20 years and older were interviewed in the 1997 nationwide stratified multi-cluster survey . Sputum acid-fast smears and cultures were done in subjects with abnormal screening chest radiographs . RESULTS: Individuals with TB symptoms comprised 18.1% of the population studied . The prevalence of bacillary disease was 39/1000 in symptomatic subjects compared to 13/1000 in asymptomatic subjects . Symptom screening had a 14.3% positive predictive value and a 91.4% negative predictive value for bacillary disease . Significantly more symptomatic than asymptomatic subjects attended chest radiographic screening during the survey . However, in response to their symptoms, the majority (43.0%) took no action or self medicated (31.6%), while 11.8% consulted a private practitioner, 7.5% a public health center, 4.4% a hospital, and 1.7% a traditional healer . CONCLUSION: Sputum smear examination after symptom screening was acceptable for case finding . The health seeking behavior of subjects with TB symptoms was inappropriate . A health education program and public-private collaboration in directly observed therapy, short course (DOTS) are essential for TB control in the Philippines.

Mem Inst Oswaldo Cruz, 2000, 95 Suppl 1, 211 - 4
Simulium spp . control program in Rio Grande do Sul, Brazil; Mardini LB et al.; Insects of the Simuliidae family have been the object of control in Rio Grande do Sul since the 70s . Their constant attacks became a social-economical problem as well as a problem of Public Health, with serious consequences to men and to the economy of the areas in which the insects develop . At first, the control was done with a chemical larvicide Themephos ABATE 500 E, but an imperfect measuring of outflow to determine the quantity of the product made Simulium spp . resistant to it . From 1983 on, following a study of a new method for the outflow measuring, we started to use a biological larvicide Bacillus thuringiensis serovar israelensis based . The biological control uses the new method in 36.4% of the state area, assisting about 3,500,000 inhabitants.

Mem Inst Oswaldo Cruz, 2000, 95 Suppl 1, 207 - 10
The use of bacterial larvicides in mosquito and black fly control programmes in Brazil; Regis L et al.; Bacillus spp . based larvicides are increasingly replacing, with numerous advantages, chemical insecticides in programmes for controlling black fly and mosquito populations . Brazil was among the pioneers in adopting Bacillus thuringiensis israelensis (B.t.i) to control black flies . However, the major current mosquito control programme in Brazil, the Programme for Eradication of Aedes aegypti launched in 1997, only recently decided to replace temephos by B.t.i based larvicides, in the State of Rio de Janeiro . In the last decade, works developed by research groups in Brazilian institutions have generated a significant contribution to this subject through the isolation of B . sphaericus new strains, the development of new products and the implementation of field trials of Bacillus efficacy against mosquito species under different environmental conditions.

J Econ Entomol, 2000 Dec, 93(6), 1773 - 8
Variation in performance on cry1Ab-transformed and nontransgenic rice varieties among populations of Scirpophaga incertulas (Lepidoptera: Pyralidae) from Luzon Island, Philippines; Bentur JS et al.; We quantified variation in performance under greenhouse conditions among seven populations of Scirpophaga incertulas (Walker) from Luzon Island, Philippines, on three rice varieties: 'IR58' transformed with the cry1Ab gene from Bacillus thuringiensis Berliner, and nontransgenic IR58 and IR62 . On IR62, S . incertutas performance did not differ among provinces for any of the 10 parameters measured, but there was a significant effect of town within province for one parameter, 20-d-old larval weight . Larval survival after 48 h on cy1Ab-transformed IR58 did not differ significantly among provinces, but did differ significantly among towns within a province . There was no geographic variation in larval survival after 48 h on control plants of IR58 . Surviving insects from the cry1Ab-transformed IR58 were transferred to IR62 to complete development . There was no geographic variation in the percentage of insects completing development to adult emergence and the time required by the transferred female insects to complete development . However, there was variation among provinces in male developmental time . The absence of geographic variation on nontransgenic IR58 and the very limited variation on IR62 indicated that there was little variation in general vigor among the S . incertulas populations and thus that the variation in performance oil cry1Ab-transformed IR58 was probably attributable to differences in susceptibility to Cry1Ab.

J Econ Entomol, 2000 Dec, 93(6), 1708 - 13
Efficacy tests and determination of optimal spray timing values to control nantucket pine tip moth (Lepidoptera: Tortricidae) infestations; Nowak JT et al.; The Nantucket pine tip moth, Rhyaciona frustrana (Comstock), a common regeneration pest of loblolly pine, Pinus taeda L., has been shown to reduce tree volume yields through larval feeding . Chemical applications can be effective in protecting trees from the growth losses associated with this feeding, and optimum spray timing values are commonly used to reduce the number of necessary applications and to increase insecticide efficacy . Optimal spray timing values for the Georgia Piedmont were obtained for the following four insecticides available for use in loblolly pine plantations: permethrin (Pounce), lambda-cyhalothrin (Warrior T), spinosad (SpinTor 2 SC), and Bacillus thuringiensis variety kurstaki Berliner (Foray 48B) . Optimal timing values were similar between the first and second generations for each of these compounds . All of the insecticides used in this study significantly reduced tip moth damage below the control treatment levels . Lambda-cyhalothrin was the most efficacious and had the longest spray timing window . B . thuringiensis was the least effective and had the shortest timing window . Spinosad and permethrin were similar in efficacy and spray timing values . This information is applicable to regions where there are three tip moth generations per year, as found in the southern Piedmont region and the coastal plain of Virginia and most of North Carolina.

J Econ Entomol, 2000 Dec, 93(6), 1669 - 79
Indirect reduction of ear molds and associated mycotoxins in Bacillus thuringiensis corn under controlled and open field conditions: utility and limitations; Dowd PF; In 1995, ears of a experimental inbred (CG59-2) containing a synthetic Bacillus thuringiensis Cry IA (b) gene driven by PEPC, pith and pollen promoters and artificially infested with Ostrinia nubilalis (Hubner) larvae in small plot studies were free from insect damage, whereas 40-50% of the corresponding non-Bt inbred ears were damaged . Bt inbred ears that were inoculated with Aspergillus flavus Link and Fusarium proliferatum T . Matsushima (Nirenberg) or exposed to natural mold inoculum after infestation with O . nubilalis were free of visible signs of mold, as compared with approximately 30-40% of the non-Bt ears similarly treated . Results in 1996 using the same inbred with a single allele dose of the Bt gene showed similar trends . Mean total fumonisin levels for non-Bt versus Bt inbred ears were not significantly different (2.8 versus 0.8 ppm, respectively) in 1996 . In paired hybrid studies run in 0.4-ha (1-acre) fields, an event 176 Bt hybrid had significantly lower amounts of damage and signs of Fusarium spp . mold, but not fumonisin, compared with a corresponding non-Bt hybrid from 1996 to 1998 . However, two hybrid pairs that contained either MON810 or Bt11 constructs examined in similar fields at the same site had lower levels of fumonisin in both 1997 (30- to 40-fold) and 1998 . High intrafield variability in insect infestation and presence of Helicoverpa zea (Boddie) in Bt hybrids was apparently responsible for fewer significant differences in fumonisin levels in 1998 . Similar trends for all three hybrid pairs were noted in small plot trials at another site . Incidence of other ear pests or insect predators varied as much among non-Bt hybrids as they did for Bt/non-Bt hybrid pairs.

J Econ Entomol, 2000 Dec, 93(6), 1657 - 61
Biological control of Bactocera oleae (Diptera: Tephritidae) using a Greek Bacillus thuringiensis isolate; Navrozidis EI et al.; Bacillus thuringiensis, isolate 114A, was used in toxicity experiments against the wild population ofthe olive pest Bactroceria oleae (Gmelin) . In laboratory experiments, spores and crystals of the B.t . were delivered to the insects with the food . Longevity, oviposition period, number of eggs produced, and percent hatch were recorded . Olive fruits from the oviposition test were dipped into a suspension containing spores and crystals of B . thuringiensis 114A after the eggs were deposited . In field experiments, four to six sprayings per year of B.t . 114A isolate were applied for three successive years . It was found that, in addition to the longevity of B . oleae, the oviposition period, number of eggs and percent egg hatch decreased . Also, the percentage of pupation and emergence was reduced when olive fruits with eggs in their mesocarp were dipped in the solution of spores and crystals . Field applications with the toxins of 114A isolate of B . thuringiensis have resulted in significant protection of the olive production.

J Econ Entomol, 2000 Dec, 93(6), 1588 - 95
Insecticide resistance and dominance levels; Bourguet D et al.; Dominance has been assessed in different ways in insecticide resistance studies, based on three phenotypic traits: the insecticide concentration required to give a particular mortality (DLC), mortality at a particular insecticide dose (DML), and fitness in treated areas (DWT) . We propose a general formula for estimating dominance on a scale of 0 to 1 (0 = complete recessivity and 1 = complete dominance) . DLC, DML, and DWT are not directly related and their values depend on genetic background and environmental conditions . We also show that pest management strategies can have the consequence to increase DWT via the selection of dominance modifiers . Studies on resistance to Bacillus thuringiensis toxins provide the ultimate example of the complexity of the definition of the concept of dominance . Almost all studies have focused on calculation of DLC, which provides little information about the efficiency of pest management programs . For instance, one assumption of the high dose/refuge strategy is that Bacillus thuringiensis resistance must be effectively recessive (i.e., DML must be close to zero) . However, DWT, rather than DML, is relevant to the resistance management strategy . Therefore, we strongly suggest that the time has come to focus on fitness dominance levels in the presence and absence of insecticide.

Curr Opin Ophthalmol, 2000 Dec, 11(6), 443 - 8
Ocular tuberculosis; Bodaghi B et al.; Despite the use of highly sensitive molecular tools, such as polymerase chain reaction, for the detection of Mycobacterium tuberculosis, ocular tuberculosis remains a subject of controversy . The diagnosis is often presumptive in the absence of ocular biopsies . Choroiditis is the most common ocular manifestation in patients with pulmonary and systemic tuberculosis . Indocyanine green angiography seems to be an interesting method to determine choroidal involvement . PCR technology is proposed to evaluate the presence of the tubercule bacillus DNA in ocular fluids and tissues when conventional microbiologic methods fail to confirm a bacterial etiology . Most of the presumed cases of ocular tuberculosis should be treated with associations of antituberculous drug, especially when a systemic steroid regimen is required.

J Nat Prod, 2000 Dec, 63(12), 1653 - 7
Microbial transformations of the antimelanoma agent betulinic acid; Kouzi SA et al.; Microbial transformation studies of the antimelanoma agent betulinic acid (1) were conducted . Screening experiments showed a number of microorganisms capable of biotransforming 1 . Three of these cultures, Bacillus megaterium ATCC 14581, Cunninghamella elegans ATCC 9244, and Mucor mucedo UI-4605, were selected for preparative scale transformation . Bioconversion of 1 with resting-cell suspensions of phenobarbital-induced B . megaterium ATCC 14581 resulted in the production of the known betulonic acid (2) and two new metabolites: 3beta,7beta-dihydroxy-lup-20(29)-en-28-oic acid (3) and 3beta,6alpha, 7beta-trihydroxy-lup-20(29)-en-28-oic acid (4) . Biotransformation of 1 with growing cultures of C . elegans ATCC 9244 produced one new metabolite characterized as 1beta,3beta, 7beta-trihydroxy-lup-20(29)-en-28-oic acid (5) . Incubation of 1 with growing cultures of M . mucedo UI-4605 afforded metabolite 3 . Structure elucidation of all metabolites was based on NMR and HRMS analyses . In addition, the antimelanoma activity of metabolites 2-5 was evaluated against two human melanoma cell lines, Mel-1 (lymph node) and Mel-2 (pleural fluid).

J Dermatol, 2000 Nov, 27(11), 717 - 23
Erythema induratum associated with tuberculous lymphadenitis: analysis of a case using polymerase chain reactions with different primer pairs to differentiate bacille Calmette-Guérin (BCG) from virulent strains of Mycobacterium tuberculosis complex; Wang TC et al.; We retrospectively analyzed a bacille Calmette-Guerin (BCG) vaccinated female patient who developed erythema induratum in association with tuberculous lymphadenitis . The polymerase chain reaction (PCR) with two pairs of primers was performed . Only one pair was able to detect the myocobacterial DNA from the paraffin-embedded specimen of the erythema induratum (EI) lesion . However, both pairs showed positive results with the tuberculous lymphadenitis specimen . Sequence analysis of the PCR products excluded the possibility of BCG as the potential pathogen . Both lesions responded favorably to antituberculous therapy . Our study attests to the tuberculous nature of EI and demonstrates that BCG vaccination per se does not induce the lesion or interfere with the PCR result . Finally, using at least two pairs of primers targeting different genomic segments and possessing species-identification capability may raise the detection rates and solve some controversies about the nature of tuberculids.

Virus Res, 2000 Nov, 71(1-2), 49 - 62
Extreme resistance to Potato leafroll virus in potato cv . Russet Burbank mediated by the viral replicase gene; Thomas PE et al.; High levels of field resistance to Potato leafroll virus (PLRV; Genus: Polerovirus; Family: Luteoviridae) were achieved by expression of the unmodified, full-length PLRV replicase gene in potato plants cv . Russet Burbank . A high degree of resistance was also achieved, but less frequently, by expression of a truncated construct of the replicase gene . In limited testing, neither miss-frame nor antisense constructs of the replicase gene conferred resistance . The degree of resistance expressed among different transformant lines ranged from near immunity to full susceptibility . Resistance to the Colorado potato beetle (Leptinotarsa decemlineata Say) was combined with resistance to PLRV by expression of the cry3A insect control protein gene from Bacillus thuringiensis var . tenebrionis in combination with the unmodified, full-length, viral replicase gene . Resistance was expressed as a reduced incidence of infection detectable by foliage symptoms or serological tests . Reduced incidence of infection was not associated with a decrease in virus antigen concentration in the few plants of resistant lines that became infected . Virus was not detected in the foliage of symptomless plants but was detected in progeny plants produced from the tubers of inoculated but symptomless test plants of some resistant lines . The resistance was effective under natural exposure and against plant-to-plant spread of PLRV by the aphid vector, Myzus persicae Sulzer . Three of the resistant lines selected in these studies were released and are now in commercial production.

J Nutr Biochem, 2000 Nov, 11(11-12), 555 - 561
Fiber-rich diets alter rat intestinal leukocytes metabolism; Cavaglieri CR et al.; This study addressed the following question: What is the effect of fermentable and nonfermentable fiber-rich diets on intestinal immune cells' function and metabolism? For this purpose, weaning rats received, for 8 weeks, two types of fiber-enriched (30%) diets with different fermentable/nonfermentable fiber ratios, that is, oat bran (0.3) and wheat bran (0.14) . The results of these two experimental groups were compared with those of the low-fiber control group having a 0.22 fermentable/nonfermentable fiber ratio . The total number and proportion of leukocytes in plasma, total number of cells in the lymphoid organs, lymphocyte proliferative activity and capacity of phagocytosis, hydrogen peroxide production, and adherence of macrophages were investigated . The activities of key enzymes of glycolysis and glutaminolysis, and of the Krebs cycle of lymphocytes from the mesenteric lymph nodes and macrophages from the intraperitoneal cavity were determined . The metabolic response of lymphocytes and macrophages from rats fed the three diets to Bacillus Calmette-Guerin-stimulus was also investigated . The number of lymphocytes in the mesenteric lymph nodes was lower in both fiber-rich diets than in the control but did not have any difference in the remaining lymphoid organs . Wheat bran caused a significant reduction in the phagocytosis capacity and adherence index of macrophages, whereas oat bran did not have a significant effect . The response of glucose and glutamine metabolism to Bacillus Calmette-Guerin-stimulus was not altered by the diets in lymphocytes, whereas in macrophages, the increase in glutaminase and hexokinase activities was abolished.

Curr Microbiol, 2001 Feb, 42(2), 129 - 33
Two delta-endotoxin genes, cry9Da and a novel related gene, commonly occurring in Lepidoptera-specific Bacillus thuringiensis Japanese isolates that produce spherical parasporal inclusions; Wasano N et al.; Six Lepidoptera-specific Bacillus thuringiensis isolates, which belong to the four H serovars (sotto, fukuokaensis, canadensis, and galleriae) and produce spherical parasporal inclusions, were examined for assignment of the classes of the delta-endotoxin genes . Gene analysis was conducted by PCR technique with primers designed to probe the genes cry9Ca and cry9Da . The data revealed that the delta-endotoxin of a serovar canadensis isolate is encoded by the gene cry9Da, while those of the five other strains are encoded by an undescribed delta-endotoxin gene . DNA fragments from five strains had an identical 1917-bp nucleotide sequence, covering the four conserved regions and a partial sequence of the block 5 region . The deduced amino acid sequence exhibited a 70.6% homology to that of the corresponding region of the Cry9Ea delta-endotoxin protein which is active on the order Lepidoptera, and a 63.1% homology to the Cry9Ca protein highly toxic to the noctuid lepidopterans . The results showed that Japanese isolates of B . thuringiensis producing spherical parasporal inclusions with Lepidoptera-specific activity are categorized into two groups: one produces the class Cry9Da protein and the other a novel delta-endotoxin allied to the class Cry9 . It also appeared that heterogeneous multiple H serovars are involved in each group.

Curr Microbiol, 2001 Feb, 42(2), 96 - 9
PCR analysis of cry7 genes in Bacillus thuringiensis by the five conserved blocks of toxins; Ben-Dov E et al.; An alternative PCR analysis to screen for cry7 genes is proposed, based on the five conserved blocks of amino acids of Bacillus thuringiensis toxins and their encoding DNA sequences . A complete set of five primers was constructed, four direct and one reverse, yielding four specific amplicons . Modified profiles can identify new cry genes.

Acta Crystallogr D Biol Crystallogr, 2001 Jan, 57(Pt 1), 165 - 7
Purification, crystallization and quaternary structure analysis of a glycerol dehydrogenase S305C mutant from Bacillus stearothermophilus; Burke J et al.; Bacillus stearothermophilus glycerol dehydrogenase (GlyDH) is a 39.5 kDa molecular weight metalloenzyme which catalyzes the oxidation of glycerol to dihydroxyacetone with the concomitant reduction of NAD(+) to NADH . Despite its classification as a member of the 'iron-containing' polyol dehydrogenase family, studies on recombinant B . stearothermophilus GlyDH have shown this enzyme to be Zn(2+)-dependent . Crystals of a S305C GlyDH mutant were obtained by the hanging-drop vapour-diffusion method, using ammonium sulfate and PEG 400 as precipitating agents, in the presence and absence of NAD(+) . The crystals belong to space group I422, with approximate unit-cell parameters a = b = 105, c = 149 A and one subunit in the asymmetric unit, corresponding to a packing density of 2.6 A(3) Da(-1) . The crystals diffract X-rays to at least 1.8 A resolution on a synchrotron-radiation source . Determination of the structure will provide insights into the key determinations of catalytic activity of this class of enzymes, for which no structures are currently available.

J Bacteriol, 2001 Jan, 183(2), 476 - 82
GerN, an antiporter homologue important in germination of Bacillus cereus endospores; Thackray PD et al.; A homologue of the grmA spore germination gene of Bacillus megaterium and of a NaH-antiporter gene (napA) of Enterococcus hirae has been identified in Bacillus cereus 569 (ATCC 10876) . The putative protein product has 58 and 43% amino acid identity with GrmA and NapA, respectively . Insertional inactivation of this B . cereus gene, named gerN, did not affect vegetative growth or sporulation . The null mutant spores were 30-fold slower to germinate in inosine (5 mM) but germinated almost normally in response to L-alanine (10 mM) . The null mutant spores germinated after several hours with inosine as the sole germinant, but germination was asynchronous and the normal order of germination events was perturbed . At a suboptimal germinant concentration (50 microM), inosine germination was completely blocked in the mutant, while the rate of germination in 50 microM L-alanine was reduced to one-third of that of the wild type . The requirement for GerN function in the response to a particular germinant suggests that a germination receptor may have a specifically associated antiporter, which is required at the initiation of germination and which, in the case of the inosine receptor, is GerN . Since germination in suboptimal concentrations of L-alanine shows a delay, additional germination transporters may be required for optimal response at low germinant concentrations.

Appl Environ Microbiol, 2001 Jan, 67(1), 462 - 3
Cyt1A from Bacillus thuringiensis lacks toxicity to susceptible and resistant larvae of diamondback moth (Plutella xylostella) and pink bollworm (Pectinophora gossypiella); Meyer SK et al.; We tested Cyt1Aa, a cytolytic endotoxin of Bacillus thuringiensis, against susceptible and Cry1A-resistant larvae of two lepidopteran pests, diamondback moth (Plutella xylostella) and pink bollworm (Pectinophora gossypiella) . Unlike previous results obtained with mosquito and beetle larvae, Cyt1Aa alone or in combination with Cry toxins was not highly toxic to the lepidopteran larvae that we examined.

Appl Environ Microbiol, 2001 Jan, 67(1), 330 - 8
Plasmid transfer between Bacillus thuringiensis subsp . israelensis strains in laboratory culture, river water, and dipteran larvae; Thomas DJ et al.; Plasmid transfer between strains of Bacillus thuringiensis subsp . israelensis was studied under a range of environmentally relevant laboratory conditions in vitro, in river water, and in mosquito larvae . Mobilization of pBC16 was detected in vitro at a range of temperatures, pH values, and available water conditions, and the maximum transfer ratio was 10(-3) transconjugant per recipient under optimal conditions . Transfer of conjugative plasmid pXO16::Tn5401 was also detected under this range of conditions . However, a maximum transfer ratio of 1.0 transconjugant per recipient was attained, and every recipient became a transconjugant . In river water, transfer of pBC16 was not detected, probably as a result of the low transfer frequency for this plasmid and the formation of spores by the introduced donor and recipient strains . In contrast, transfer of plasmid pXO16::Tn5401 was detected in water, but at a lower transfer ratio (ca . 10(-2) transconjugant per donor) . The number of transconjugants increased over the first 7 days, probably as a result of new transfer events between cells, since growth of both donor and recipient cells in water was not detected . Mobilization of pBC16 was not detected in killed mosquito larvae, but transfer of plasmid pXO16::Tn5401 was evident, with a maximum rate of 10(-3) transconjugant per donor . The reduced transfer rate in insects compared to broth cultures may be accounted for by competition from the background bacterial population present in the mosquito gut and diet or by the maintenance of a large population of B . thuringiensis spores in the insects.

Appl Environ Microbiol, 2001 Jan, 67(1), 323 - 9
Importance of Cry1 delta-endotoxin domain II loops for binding specificity in Heliothis virescens (L.); Jurat-Fuentes JL et al.; We constructed a model for Bacillus thuringiensis Cry1 toxin binding to midgut membrane vesicles from Heliothis virescens . Brush border membrane vesicle binding assays were performed with five Cry1 toxins that share homologies in domain II loops . Cry1Ab, Cry1Ac, Cry1Ja, and Cry1Fa competed with (125)I-Cry1Aa, evidence that each toxin binds to the Cry1Aa binding site in H . virescens . Cry1Ac competed with high affinity (competition constant {K(com)} = 1.1 nM) for (125)I-Cry1Ab binding sites . Cry1Aa, Cry1Fa, and Cry1Ja also competed for (125)I-Cry1Ab binding sites, though the K(com) values ranged from 179 to 304 nM . Cry1Ab competed for (125)I-Cry1Ac binding sites (K(com) = 73.6 nM) with higher affinity than Cry1Aa, Cry1Fa, or Cry1Ja . Neither Cry1Ea nor Cry2Aa competed with any of the (125)I-Cry1A toxins . Ligand blots prepared from membrane vesicles were probed with Cry1 toxins to expand the model of Cry1 receptors in H . virescens . Three Cry1A toxins, Cry1Fa, and Cry1Ja recognized 170- and 110-kDa proteins that are probably aminopeptidases . Cry1Ab and Cry1Ac, and to some extent Cry1Fa, also recognized a 130-kDa molecule . Our vesicle binding and ligand blotting results support a determinant role for domain II loops in Cry toxin specificity for H . virescens . The shared binding properties for these Cry1 toxins correlate with observed cross-resistance in H . virescens.

Appl Environ Microbiol, 2001 Jan, 67(1), 317 - 22
Effect of water activities of heating and recovery media on apparent heat resistance of Bacillus cereus spores; Coroller L et al.; Spores of Bacillus cereus were heated and recovered in order to investigate the effect of water activity of media on the estimated heat resistance (i.e., the D value) of spores . The water activity (ranging from 0.9 to 1) of the heating medium was first successively controlled with three solutes (glycerol, glucose, and sucrose), while the water activity of the recovery medium was kept near 1 . Reciprocally, the water activity of the heating medium was then kept at 1, while the water activity of the recovery medium was controlled from 0.9 to 1 with the same depressors . Lastly, in a third set of experiments, the heating medium and the recovery medium were adjusted to the same activity . As expected, added depressors caused an increase of the heat resistance of spores with a greater efficiency of sucrose with respect to glycerol and glucose . In contrast, when solutes were added to the recovery medium, under an optimal water activity close to 0.98, a decrease of water activity caused a decrease in the estimated D values . This effect was more pronounced when sucrose was used as a depressor instead of glycerol or glucose . When the heating and the recovery media were adjusted to the same water activity, a balancing effect was observed between the protective influence of the solutes during heat treatment and their negative effect during the recovery of injured cells, so that the overall effect of water activity was reduced, with an optimal value near 0.96 . The difference between the efficiency of depressors was also less pronounced . It may then be concluded that the overall protective effect of a decrease in water activity is generally overestimated.

Appl Environ Microbiol, 2001 Jan, 67(1), 185 - 9
Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis; Hansen BM et al.; Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen B . thuringiensis has also been involved in outbreaks of diarrhea . The diarrheal types of diseases are attributed to enterotoxins . Two different enterotoxic protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE), and an enterotoxic protein, enterotoxin T, have been characterized, and the genes have been sequenced . PCR primers for the detection of these genes were deduced and used to detect the genes in 22 B . cereus and 41 B . thuringiensis strains . At least one gene of each of the two protein complexes HBL and NHE was detected in all of the B . thuringiensis strains, while six B . cereus strains were devoid of all three HBL genes, three lacked at least two of the three NHE genes, and one lacked all three . Five different sets of primers were used for detection of the gene (bceT) encoding enterotoxin T . The results obtained with these primer sets indicate that bceT is widely distributed among B . cereus and B . thuringiensis strains and that the gene varies in sequence among different strains . PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously detected the bceT gene, as confirmed by Southern analysis . The occurrence of the genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of the bceT gene is significantly associated in the 63 strains . We suggest an approach for detection of enterotoxin-encoding genes in B . cereus and B . thuringiensis based on PCR analysis with the six primer sets for the detection of genes in the HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection of bceT . PCR analysis of the 16S-23S rRNA gene internal transcribed spacer region revealed identical patterns for all strains studied.

Appl Environ Microbiol, 2001 Jan, 67(1), 172 - 8
Analysis of bacterial communities in the rhizosphere of chrysanthemum via denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA as well as DNA fragments coding for 16S rRNA; Duineveld BM et al.; The effect of developing chrysanthemum roots on the presence and activity of bacterial populations in the rhizosphere was examined by using culture-independent methods . Nucleic acids were extracted from rhizosphere soil samples associated with the bases of roots or root tips of plants harvested at different stages of development . PCR and reverse transcriptase (RT) PCR were used to amplify 16S ribosomal DNA (rDNA) and 16S rRNA, respectively, and the products were subjected to denaturing gradient gel electrophoresis (DGGE) . Prominent DGGE bands were excised and sequenced to gain insight into the identities of predominantly present (PCR) and predominantly active (RT-PCR) bacterial populations . The majority of DGGE band sequences were related to bacterial genera previously associated with the rhizosphere, such as Pseudomonas, Comamonas, Variovorax, and Acetobacter, or typical of root-free soil environments, such as Bacillus and Arthrobacter . The PCR-DGGE patterns observed for bulk soil were somewhat more complex than those obtained from rhizosphere samples, and the latter contained a subset of the bands present in bulk soil . DGGE analysis of RT-PCR products detected a subset of bands visible in the rDNA-based analysis, indicating that some dominantly detected bacterial populations did not have high levels of metabolic activity . The sequences detected by the RT-PCR approach were, however, derived from a wide taxonomic range, suggesting that activity in the rhizosphere was not determined at broad taxonomic levels but rather was a strain- or species-specific phenomenon . Comparative analysis of DGGE profiles grouped all DNA-derived root tip samples together in a cluster, and within this cluster the root tip samples from young plants formed a separate subcluster . Comparison of rRNA-derived bacterial profiles showed no grouping of root tip samples versus root base samples . Rather, all profiles derived from 2-week-old plant rhizosphere soils grouped together regardless of location along the root.

J Infect Dis, 2001 Feb 1, 183(3), 469 - 77 Epub 2000 Dec 21.
Enumeration of T cells specific for RD1-encoded antigens suggests a high prevalence of latent Mycobacterium tuberculosis infection in healthy urban Indians; Lalvani A et al.; Knowledge of the prevalence of latent Mycobacterium tuberculosis infection is crucial for effective tuberculosis control, but tuberculin skin test surveys have major limitations, including poor specificity because of the broad antigenic cross-reactivity of tuberculin . The M . tuberculosis RD1 genomic segment encodes proteins, such as early secretory antigenic target (ESAT)-6, that are absent from M . bovis bacille Calmette-Guerin (BCG) and most environmental mycobacteria . We recently identified circulating ESAT-6-specific T cells as an accurate marker of M . tuberculosis infection . Here, interferon-gamma-secreting T cells specific for peptides derived from ESAT-6 and a second RD1 gene product, CFP10, were enumerated in 100 prospectively recruited healthy adults in Bombay (Mumbai), India . Eighty percent responded to >/=1 antigen, and many donors had high frequencies of T cells that were specific for certain immunodominant peptides . In contrast, of 40 mostly BCG-vaccinated, United Kingdom-resident healthy adults, none responded to either antigen . This study suggests an 80% prevalence of latent M . tuberculosis infection in urban India.

Yi Chuan Xue Bao, 2000, 27(9), 839 - 44
{The characteristics of Bacillus thuringiensis strain YBT833 and its transformants that containing different ICP genes}; Lu SQ et al.; Four different transformants were selected by transferring cry1C into Bacillus thuringiensis strain YBT833 . Southern blot and Plasmid profiles results all proved that cry1C was transferred into strain YBT833 . However, it was found by PCR analysis that transformant YBT833-1 kept all indigenous ICP(insecticidal crystal protein) genes of strain YBT833 while transformant YBT833-2 lost cry1Ab, and transformant YBT833-3 lost all ICP genes . SDS-PAGE showed that transformants of YBT833-1, YBT833-2 and YBT833 all had 130 kDa and 65 kDa peptide bands, but transformant YBT833-3 did not have 65 kDa peptide band . Bioassay results showed that the toxicities of all transformants to Spodoptera exigua, Heliothis armigera and Plutella xylostella were lower than that of strain YBT833, except the toxicity of transformant YBT833-2 to P . xylostella which was obviously higher than that of YBT833.

Indian J Pediatr, 1999, 66(1 Suppl), S87 - 8
Inflammatory bowel disease in children: Indian perspective; Mehta S; The magnitude of inflammatory bowel disease in childhood in India is largely unknown . At the Pediatric Gastroenterology section of PGIMER, Chandigarh, 15 out of 294 children (5%) admitted for colonic disorders were diagnosed to have ulcerative colitis . Diagnosis is suspected on the basis of clinical presentation and established by sigmoidoscopy, rectal biopsy and double contrast barium enema . Widely prevalent bacillary dysentery, acute amoebic colitis and antibiotic induced colitis need to be kept in differential diagnosis of acute presentation of ulcerative colitis . Chronic or recurrent colitis needs to be differentiated from tuberculous colitis . Inflammatory bowel disease deserves clinical suspicion in all unusual cases of colitis which do not respond to treatment of common infective agents.

Trans R Soc Trop Med Hyg, 2000 Sep-Oct, 94(5), 488 - 92
Efficacy of Bacillus sphaericus in control of the filariasis vector Culex quinquefasciatus in an urban area of Olinda, Brazil; Regis L et al.; The efficacy of Bacillus sphaericus 2362 against Culex quinquefasciatus was tested in 1991-94 in a major Brazilian endemic zone for bancroftian filariasis . Continuous selection pressure against the mosquito population was sustained for 18 months through treatment of 2500 potential breeding sites occurring within a 5.7-km2 urban area in the Metropolitan Region of Recife . The impact of this control intervention was evaluated by comparing entomological indices with those from an untreated area . Application of the larvicide kept the Cx . quinquefasciatus population density significantly lower when compared to the untreated area, despite some operational difficulties . Adult densities remained lower for at least 5 months after spraying ceased . Pre-trial microfilaria rates from the untreated and operational area were 13.1% and 7.2%, respectively . A 60% reduction in human exposure to infective bites was recorded as a consequence of this vector population control.

Mol Biotechnol, 2000 Oct, 16(2), 97 - 107
Domain I plays an important role in the crystallization of Cry3A in Bacillus thuringiensis; Park HW et al.; The insecticidal bacterium Bacillus thuringiensis synthesizes endotoxin Cry proteins of two size classes, 135 and 70 kDa, and both form crystalline inclusions in cells after synthesis . Crystallization of 135-kDa proteins is due to intermolecular attraction of regions in the C-terminal half of the molecule, and the N-terminal half fails to crystallize when synthesized in vivo . Alternatively, endotoxins of the 70-kDa class such as Cry2A and Cry3A, which correspond to the N-terminal half of 135-kDa molecules, crystallize readily after synthesis . Cry molecules of this size class consist of three principal domains, but the domains responsible for crystallization are not known . To identify these domains, chimeric proteins were constructed in which Cry3A Domains I or III, or I and III were substituted for the corresponding domains in truncated Cry1C molecules . Cry1C molecules with only Cry3A Domain III did not crystallize, whereas when Cry3A Domains I and III, or Domain I alone, were substituted, large inclusions were obtained . Except for the chimera consisting of Cry3A Domains I and III and Cry1C Domain II, most chimeras were not as stable as wild-type Cry3A or truncated Cry1C . These results show that Cry3A Domain I plays an important role in its crystallization in vivo.

Z Gerontol Geriatr, 2000 Oct, 33(5), 374 - 80
Tuberculosis in the elderly; Rajagopalan S et al.; Tuberculosis (TB) today remains one of the world's most lethal infectious diseases . An estimated one-third of the world's population is infected with the tubercle bacillus-Mycobacterium tuberculosis (Mtb), and 7 to 8 million people develop TB disease each year (27) . For purpose of clarity, TB infection (latent TB) is defined as harboring Mtb without evidence of active infection, and TB disease is active infection without Mtb based on clinical and laboratory findings . Recognizing that TB has been one of the most neglected international health problems and that the TB epidemic is rampant in many parts of the world, the World Health Organization (WHO) declared TB to be a global health emergency in 1993 (23) . Despite the steady decline in TB cases since this time resulting from the overall implementation of more effective infection control practices, directly observed therapy (DOT), and efforts to control the human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) epidemic, preventive and control strategies among other high-risk populations such as the elderly evidently remain a clinical and epidemiological challenge . The geriatric population among all ethnic groups and both genders, represent the largest reservoir of TB infection, particularly in developed nations (9) . Clinical features of TB in older adults may be atypical, non-specific, and confused with concomitant age-related diseases (28) . Underlying acute or chronic diseases, malnutrition, and the biological changes with aging, can disrupt integumental barriers, impair microbial clearance mechanisms, and contribute to the expected age-associated decline in cellular immune responses to infecting agents such as Mtb . Diagnosis of TB can be difficult and consequently overlooked; this treatable infection may unfortunately be recognized only at autopsy . Furthermore, therapy of TB in the elderly is challenging because of the increased incidence of adverse drug reactions . Optimal treatment of associated chronic diseases, minimization of invasive procedures, limitation of polypharmacy, and adequate nutritional support are essential for this vulnerable population . The institutionalized elderly in addition are especially at high risk for reactivation of latent TB as well as susceptible to new TB infection . This article will discuss the global epidemiology, pathogenesis and immunologic aspects, unique clinical consideration, treatment and prevention of TB, briefly inclusive of the recent published guidelines for targeted tuberculin testing and treatment of latent TB infection as it pertains to the elderly.

Indian J Pediatr, 2000 Feb, 67(2 Suppl), S9 - 13
Tuberculin and BCG tests; Rajajee S; The tuberculin test is widely used for the diagnosis of tuberculosis in children as it is the only one that provides evidence of infection with M . tuberculosis . Of the tuberculins that are available, the most widely used are PPDS and PPDRT 23, in various strengths . A positive test indicates prior infection with the tubercle bacillus but not necessarily active disease . A positive test may also result from BCG vaccination though the response is usually less than 10 mm and tends to wane with time . In areas with a high prevalence of atypical mycobacteria in the environment, positive reactions may also be due to cross-reactivity . BCG has been recommended by some workers as a diagnostic test but suffers from the disadvantages of low specificity.

Indian J Pediatr, 2000 Feb, 67(2 Suppl), S3 - 8
Diagnosis of tuberculosis; Lodha R et al.; Diagnosis of tuberculosis in children is usually based on clinical signs and symptoms, chest roentgenogram, tuberculin testing and history of contact with adult patients . The diagnostic tests for tuberculosis can be broadly divided into 2 groups: demonstration/isolation of Mycobacterium tuberculosis or one of its components {Ziehl Neelson (ZN) staining, special stains, cultures, polymerase chain reaction}; demonstration of host's response to exposure to M . tuberculosis (Mantoux test, serodiagnosis) . The yield of tests used for demonstration of mycobacterium or its components is poor because of pauci-bacillary nature of disease in children . Diagnostic use of PCR is still experimental . Serodiagnosis does not have acceptable sensitivity and specificity hence, at present, does not have any role in diagnosis of tuberculosis in children . The diagnosis is suspected on history and a combination of imaging studies and Mantoux test still remains the most widely used investigation.

Biosci Biotechnol Biochem, 2000 Oct, 64(10), 2266 - 8
Trypsin inhibitory activity of bovine fetuin de-O-glycosylated by endo-alpha-N-acetylgalactosaminidase; Ashida H et al.; The effects of bovine fetuin O-glycans on its trypsin inhibitory activity were examined . De-sialylated (asialo-) and de-O-glycosylated fetuin were prepared from native fetuin using Arthrobacter neuraminidase and the mixture of it and Bacillus endo-alpha-N-acetylgalactosaminidase, respectively . De-sialylation and de-O-glycosylation from fetuin were confirmed with SDS-PAGE followed by western blotting using anti-human Thomsen-Friedenreich antigen (T antigen) antibody which recognizes O-linked galactosyl beta1,3 N-acetylgalactosamine (Gal beta1-->3GalNAc) . Native fetuin completely inhibited the trypsin activity at about a 1:1 molar ratio . In contrast, the trypsin inhibitory activity of asialo- and de-O-glycosylated fetuin decreased about a half and one-third of that of native fetuin, respectively.

J Mol Biol, 2001 Jan 12, 305(2), 259 - 67
Multiple display of peptides and proteins on a macromolecular scaffold derived from a multienzyme complex; Domingo GJ et al.; The acyltransferase components (E2) from the family of 2-oxo acid dehydrogenase multienzyme complexes form large protein scaffolds, to which multiple copies of peripheral enzymes bind tightly but non-covalently . Sixty copies of the E2 polypeptide from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus assemble to form a pentagonal dodecahedral scaffold with icosahedral symmetry . This protein scaffold can be modified to present foreign peptides and proteins on its surface . We show that it is possible to display two epitopes (MAL1 and MAL2) from the circumsporozoite CS proteins of Plasmodium falciparum and Plasmodium berghei, respectively, and a green fluorescent protein (EGFP), on the E2 surface . Immunization with an E2 scaffold displaying the MAL1 epitope elicited MAL1-specific antibodies in rabbits . EGFP (25 kDa) displayed as an N-terminal fusion in each of the 60 copies of the E2 chain folded into its active form, as judged by its fluorescence and detection in localized foci in Escherichia coli cells in vivo . Simultaneous display of a hexahistidine affinity tag, the MAL1 epitope and the green fluorescent protein, all on the same E2 scaffold, could be achieved by reversible denaturation and reassembly of mixtures of appropriately modified E2 chains . This new methodology offers several important advantages over other current display technologies, not least in the size of insert that can be accommodated and the multiplicity of display that can be achieved .

Lett Appl Microbiol, 2000 Dec, 31(6), 416 - 20
Nisin depletes ATP and proton motive force in mycobacteria; Chung HJ et al.; This study examined the inhibitory effect of nisin and its mode of action against Mycobacterium smegmatis, a non-pathogenic species of mycobacteria, and M . bovis-Bacill Carmette Guerin (BCG), a vaccine strain of pathogenic M . bovis . In agar diffusion assays, 2.5 mg ml(-1) nisin was required to inhibit M . bovis-BCG . Nisin caused a slow, gradual, time- and concentration-dependent decrease in internal ATP levels in M . bovis-BCG, but no ATP efflux was detected . In mycobacteria, nisin decreased both components of proton motive force (membrane potential, Delta Psi and Delta pH) in a time- and concentration-dependent manner . However, mycobacteria maintained their intracellular ATP levels during the initial time period of Delta Psi and Delta pH dissipation . These data suggest that the mechanism of nisin in mycobacteria is similar to that in food-borne pathogens.

Int J Dermatol, 2000 Nov, 39(11), 856 - 8
Tuberculosis verrucosa cutis associated with tuberculous lymphadenitis; Pereira MB et al.; A 34-year-old man presented with a large cutaneous lesion on his left thigh that had started as a small papule when he was 13 years of age . The lesion had enlarged slowly over the last 21 years . The patient had received bacillus Calmette-Guerin (BCG) vaccination in childhood . The family history was significant for tuberculosis . Clinical examination revealed a large, purplish-red, indurated plaque measuring 30 x 29 cm on the left thigh, extending to the buttock area . The edges of the lesion had a serpiginous contour with an involuted center (Fig . 1) . A left inguinal lymph node was palpated . Chest X-ray and blood cell count were normal . No other focus of disease was identified . Laboratory testing for human immunodeficiency virus (HIV) infection was negative . Purified protein derivative (PPD) intradermal injection disclosed a 19-mm skin induration . Both the cutaneous lesion and the inguinal lymph node were biopsied . Histopathologic sections of the skin fragment showed epidermal hyperkeratosis, neovascular proliferation, and a dense dermal lymphocytic infiltrate . The histopathology of the lymph node demonstrated few granulomas with focal areas of central necrosis . Staining for fungus was negative . Ziehl-Neelsen staining was negative on both the skin and lymph node specimens . Culture for fungus and Leishmania sp . was negative . Tissue culture on Lowenstein-Jensen medium from skin and lymph node was positive for Mycobacterium colonies after 5 and 7 weeks, respectively . Multidrug therapy was instituted with rifampin 600 mg/day, isoniazid 400 mg/day, and pyrazinamide 2 g/day for 2 months, and then rifampin 600 mg/day and isoniazid 400 mg/day alone for the next 4 months . An excellent response was obtained at the end of treatment (Fig . 2).

J Immunol, 2001 Jan 1, 166(1), 447 - 57
Lipoprotein access to MHC class I presentation during infection of murine macrophages with live mycobacteria; Neyrolles O et al.; Following uptake by macrophages, live mycobacteria initially reside within an immature phagosome that resists acidification and retains access to recycling endosomes . Glycolipids are exported from the mycobacterial phagosome and become available for immune recognition by CD1-restricted T cells . The aim of this study was to explore the possibility that lipoproteins might similarly escape from the phagosome and act as immune targets in cells infected with live mycobacteria . We have focused on a 19-kDa lipoprotein from Mycobacterium tuberculosis that was previously shown to be recognized by CD8(+) T cells . The 19-kDa Ag was found to traffic separately from live mycobacteria within infected macrophages by a pathway that was dependent on acylation of the protein . When expressed as a recombinant protein in rapid-growing mycobacteria, the 19-kDa Ag was able to deliver peptides for recognition by MHC class I-restricted T cells by a TAP-independent mechanism . Entry into the class I pathway was rapid, dependent on acylation, and could be blocked by killing the mycobacteria by heating before infection . Although the pattern of 19-kDa trafficking was similar with different mycobacterial species, preliminary experiments suggest that class I presentation is more efficient during infection with rapid-growing mycobacteria than with the slow-growing bacillus Calmette-Guerin vaccine strain.

J Immunol, 2001 Jan 1, 166(1), 439 - 46
Classically restricted human CD8+ T lymphocytes derived from Mycobacterium tuberculosis-infected cells: definition of antigenic specificity; Lewinsohn DM et al.; Previous studies in murine and human models have suggested an important role for HLA Ia-restricted CD8(+) T cells in host defense to Mycobacterium tuberculosis (Mtb) . Therefore, understanding the Ags presented via HLA-Ia will be important in understanding the host response to Mtb and in rational vaccine design . We have used monocyte-derived dendritic cells in a limiting dilution analysis to generate Mtb-specific CD8(+) T cells . Two HLA-Ia-restricted CD8(+) T cell clones derived by this method were selected for detailed analysis . One was HLA-B44 restricted, and the other was HLA-B14 restricted . Both were found to react with Mtb-infected, but not bacillus Calmette-Guerin-infected, targets . For both these clones, the Ag was identified as culture filtrate protein 10 (CFP10)/Mtb11, a 10.8-kDa protein not expressed by bacillus Calmette-Guerin . Both clones were inhibited by the anti-class I Ab and anti-HLA-B,C Abs . Using a panel of CFP10/Mtb11-derived 15-aa peptides overlapping by 11 aa, the region containing the epitopes for both clones has been defined . Minimal 10-aa epitopes were defined for both clones . CD8(+) effector cells specific for these two epitopes are present at high frequency in the circulating pool . Moreover, the CD8(+) T cell response to CFP10/Mtb11 can be largely accounted for by the two epitopes defined herein, suggesting that this is the immunodominant response for this purified protein derivative-positive donor . This study represents the first time CD8(+) T cells generated against Mtb-infected APC have been used to elucidate an Mtb-specific CD8(+) T cell Ag.

J Intern Med, 2000 Nov, 248(5), 415 - 24
Amplification of IS6110 sequence for detection of Mycobacterium tuberculosis complex in HIV-negative patients with fever of unknown origin (FUO) and evidence of extrapulmonary disease; Ritis K et al.; OBJECTIVES: Extrapulmonary tuberculosis (TB) constitutes the main cause of classic fever of unknown origin (FUO) in many populations . The aim of this study was to improve the diagnostic field of the disease using a nested polymerase chain reaction (PCR) assay, specific for the IS6110 insertion element of Mycobacterium tuberculosis complex, in order to achieve a more timely diagnosis and treatment . SETTING: Twenty-four, HIV-negative classic FUO patients who were admitted to the Regional Hospital of Alexandroupolis between April 1997 and July 1999 . SUBJECTS AND DESIGN: The above patients were considered as putative extrapulmonary TB after 3 weeks of in-patient investigation and underwent exhaustive examination for diagnosis of the disease . For this purpose, specimens were obtained from peripheral blood and bone marrow from these patients, as well as from damaged tissues, and analysed by both PCR and conventional methods . Anti-tuberculous treatment was initiated in 16 out of 24 patients and the response to this regimen was considered as the final criterion for diagnosis of tuberculosis . RESULTS: Extrapulmonary TB was established in 11 patients . The PCR-based methodology, when applied to samples derived from bone marrow aspirations and suspected damaged tissues, was able to diagnose 10 of them, whereas the conventional methods were able to detect only two . CONCLUSIONS: Our results confirm the diagnostic value of molecular detection of M . tuberculosis in cases of FUO, thus supporting the application of PCR in tissue samples suspected of bacillus infection . Furthermore, our studies demonstrate that bone marrow aspiration specimens constitute an alternative, easy, safe and reliable source for such PCR analysis.

Can J Urol, 2000 Feb, 7(1), 944 - 8
Long term follow-up of intravesical Bacillus Calmette-Guerin for the treatment of bladder transitional cell carcinoma; Leblanc B et al.; OBJECTIVE: To review the long-term follow-up, in terms of recurrence and progression, of transitional cell carcinoma of the bladder treated with intravesical BCG with the following indications: CIS, Ta and T1 . MATERIALS AND METHODS: Ninety-two patients who had received complete course of BCG between 1987 and 1993 were included in the study and followed for an average of 59 months (range 12 to 102) . RESULTS: The recurrence and progression were looked at . Patients treated with BCG for Carcinoma in situ, 11 of 19 (53%) remained tumor-free after 1 or 2 courses of BCG for the duration of the follow-up (mean 4.9 years, range 1.5 to 8.5 years) . For patients treated for recurring tumors, 17 of 50 (34%) had no recurrences after 1 or 2 courses of BCG with the same follow-up . When facing multiple tumors, 10 of 23 (43%) patients did not experience recurrences . Therefore, in the 92 patients treated, 38 presented no recurrences after 1 or 2 courses of BCG, for a success rate of 41% . In terms of progression, of the 19 patients treated with BCG for CIS, 4 (21%) went on to develop muscle invasive disease . Of the 50 patients treated for recurrent tumors, 2 (4%) eventually developed lamina propria invasion (initial lesion was a Ta tumor), 4 (8%) carcinoma in situ and 7 (14%) muscle invasive disease, for an overall progression rate of 26% in this group . Of the 25 patients treated for multiple tumors, 1 (4%) developed CIS and 3 (12%) presented with muscle invasive disease, for an overall progression rate of 16% for the duration of the follow-up . Therefore, 21 of 92 (23%) patients had progression of their disease following BCG therapy . No prognostic factors for recurrence or progression could be identified in these tumors . CONCLUSION: When indications warrant its use, BCG is effective in reducing recurrences and limiting progression in TCC of the bladder . Recurrence within 2 years of treatment is, however, a sign of poor prognosis and other therapeutic options should be sought.

Can Respir J, 2000 Nov-Dec, 7(6), 481 - 5
Thoracic complications of Lemierre syndrome; Gowan RT et al.; Lemierre syndrome is a severe, septicemic illness most commonly caused by the anaerobic Gram-negative bacillus Fusobacterium necrophorum . It is characterized by an acute oropharyngeal infection, with secondary septic thrombophlebitis of the internal jugular vein and frequent metastatic infections . This report of a patient with the Lemierre syndrome is complemented by a review of the literature on the subject.

J Immunol, 2000 Dec 15, 165(12), 7140 - 9
T cell expression cloning of a Mycobacterium tuberculosis gene encoding a protective antigen associated with the early control of infection; Skeiky YA et al.; Infection of C57BL/6 mice with Mycobacterium tuberculosis results in the development of a progressive disease during the first 2 wk after challenge . Thereafter, the disease is controlled by the emergence of protective T cells . We have used this infection model in conjunction with direct T cell expression cloning to identify Ags involved with the early control of the disease . A protective M . tuberculosis-specific CD4 T cell line derived from mice at 3 wk postchallenge was used to directly screen an M . tuberculosis genomic expression library . This screen resulted in the identification of a genomic clone comprising two putative adjacent genes with predicted open reading frames of 10 and 41 kDa, MTB10 and MTB41, respectively (the products of Rv0916c and Rv0915c, respectively, in the TubercuList H37Rv database) . MTB10 and MTB41 belong to the PE and PPE family of proteins recently identified to comprise 10% of the M . tuberculosis genome . Evaluation of the recombinant proteins revealed that MTB41, but not MTB10, is the Ag recognized by the cell line and by M . tuberculosis-sensitized human PBMC . Moreover, C57BL/6 mice immunized with MTB41 DNA developed both CD4- (predominantly Th1) and CD8-specific T cell responses to rMTB41 protein . More importantly, immunization of C57BL/6 mice with MTB41 DNA induced protection against infection with M . tuberculosis comparable to that induced by bacillus Calmette-Guerin . Thus, the use of a proven protective T cell line in conjunction with the T cell expression cloning approach resulted in the identification of a candidate Ag for a subunit vaccine against tuberculosis.

Biochem Biophys Res Commun, 2000 Dec 20, 279(2), 653 - 6
Recombinant Cry3Aa has insecticidal activity against the Andean potato weevil, Premnotrypes vorax; Gomez S et al.; The Andean potato weevil, Premnotrypes vorax, an insect of the order Coleoptera, is a major cause of damage to potato crops in the Andean regions of South America . The insecticidal Cry proteins from Bacillus thuringiensis are useful biological pesticides, and some are toxic to Coleopteran insects . We overexpressed recombinant, histidine-tagged Cry3Aa protein in Escherichia coli host cells . The recombinant protein was solubilized at high pH with urea, purified using Ni(2+)-nitrilo-triacetic acid affinity resin, and dialysed to lower pH and remove urea . Bioassays were performed with an insect media whose surface was spread with 70 microgram/mL purified native or recombinant toxins . First instar larvae exposed to toxin treated media for 5 days exhibited mortalities from 57% (native Cry3Aa) to 52% (recombinant Cry3Aa) . Purified native and recombinant Cry3Aa proteins appeared to be equally toxic to the Andean potato weevil .

Biochem Biophys Res Commun, 2000 Dec 20, 279(2), 313 - 9
Novel oxidatively stable subtilisin-like serine proteases from alkaliphilic Bacillus spp.: enzymatic properties, sequences, and evolutionary relationships; Saeki K et al.; The genes for five subtilisin-like serine proteases from alkaliphilic strains of Bacillus exhibiting resistance to oxidative inactivation were cloned and sequenced . The deduced amino acid sequences of the enzymes were highly homologous (greater than 88% identity) . They were composed of 638 or 639 amino acids, including a possible approximately 200-amino acid prepro-peptide, and unique stretches of approximately 160 amino acids were found in the C-terminal regions . The molecular masses of mature enzymes (433 or 434 amino acids) were approximately 45 kDa for all . Amino acid sequence comparison and phylogenetic analysis indicated that these enzymes are far removed from other known subtilisins in the line of molecular evolution . We propose that these novel proteases be categorized as a new class of subtilisins, named oxidatively stable, alkaline protease .

Carbohydr Res, 2000 Nov 3, 329(2), 301 - 8
New syntheses of 1D- and 1L-1,2-anhydro-myo-inositol and assessment of their glycosidase inhibitory activities; Falshaw A et al.; The 1D and 1L enantiomers of 1,2-anhydro-myo-inositol (conduritol B epoxide) were synthesised from 1D-pinitol and 1L-quebrachitol, respectively, and their activities were compared in selected glycosidase inhibition assays . The 1D enantiomer was found to be the active isomer, functioning as an irreversible inhibitor of sweet almond beta-D-glucosidase . Neither isomer was active against the alpha-D-glucosidase from Bacillus stearothermophilus or the beta-D-galactosidase from Aspergillus oryzae.

Vaccine, 2000 Nov 22, 19(7-8), 779 - 87
Mycobacterium bovis bacillus calmette-guérin induces protective immunity against infection by Plasmodium yoelii at blood-stage depending on shifting immunity toward Th1 type and inducing protective IgG2a after the parasite infection; Matsumoto S et al.; Bacillus calmette-guerin (BCG)-vaccination raised dramatically the survival rates of A/J mice from infection by Plasmodium yoelii 17XL at blood-stage . The analysis of the immune response of spleen cells indicated that BCG vaccination biased the immune response toward Th1 type . Neutralization of IFN-gamma and nitric oxide abrogated the protection . The kinetics of Ab production in the course of P . yoelii 17XL infection was monitored . Surprisingly, larger amounts of parasite-specific Abs were produced in BCG-vaccinated mice than in the placebo control . The vast majority of the produced IgG against parasites in BCG-vaccinated mice was IgG2a, which was observed hardly in placebo controls . The peak of IgG2a production coincided with the clearance of infection . The naive mice transferred adoptively with IgG2a from self-cured mice survived the lethal challenge from the parasite . These data indicated that BCG vaccination protected A/J mouse from P . yoelii 17XL infection by biasing immunity toward Th1-type after parasite infection and enhancing production of IgG2a, which ultimately played a major role in protection.

Biotechnol Appl Biochem, 2000 Dec, 32 ( Pt 3), 189 - 95
Site-specific and random immobilization of thermolysin-like proteases reflected in the thermal inactivation kinetics; Mansfeld J et al.; Immobilization of proteins usually leads to random orientation of the molecules on the surface of the carrier material, whereby mechanistic interpretations of changes in properties, such as thermal stability, become very difficult . Recently, we have prepared several mutant enzymes of the thermolysin-like neutral protease from Bacillus stearothermophilus, containing cysteine residues in different positions on the surface of the protein molecule . These enzymes allowed site-specific immobilization to Activated Thiol-Sepharose and showed that stabilization effects strongly depend on the position of attachment {Mansfeld, Vriend, Van den Burg, Eijsink and Ulbrich-Hofmann (1999) Biochemistry 38, 8240-8245} . The greatest stabilization was achieved after immobilization of the mutant enzymes S65C and T56C/S65C within the structural region (positions 56-69) where unfolding is initiated . In this study thermal inactivation kinetics of these two mutant enzymes, as well as those of the pseudo-wild-type enzyme and thermolysin, were compared for different types of immobilization . Besides site-specific immobilization via thiol groups, the enzymes were bound randomly via their amino groups or by mixed-type binding . The basic matrix was Sepharose 4B in all carriers . Whereas the enzymes bound site-specifically to Activated Thiol-Sepharose showed clear first-order inactivation kinetics like the soluble enzymes, the other immobilized enzyme preparations were characterized by distinct biphasic inactivation kinetics reflecting the heterogeneity of enzyme molecules on the carrier with respect to thermal unfolding . Site-specific binding resulted in stronger stabilization than the mixed binding type . However, immobilization to a highly functionalized carrier via amino groups increased stability further, suggesting that multiple fixation outside of the unfolding region 56-65 is able to increase stability of the enzyme molecules additionally.

FEBS Lett, 2000 Dec 8, 486(2), 173 - 7
Rational re-design of the substrate binding site of flavocytochrome P450 BM3; Ost TW et al.; Bacillus megaterium P450 BM3 is a fatty acid hydroxylase with selectivity for long chain substrates (C(12)-C(20)) . Binding or activity with substrates of chain length <C(12) has not been reported . Rational mutagenesis was used to re-design the enzyme to encourage binding of short chain fatty acids (C(4)-C(10)) . We show that wild-type P450 BM3 has activity and weak affinity for substrates as short as butyrate (C(4)) . However, turnover/binding of short chain substrates is dramatically increased by introducing a novel substrate carboxylate binding site close to the heme . Mutant L181K shows catalytic efficiency (k(cat)/K(M)) increased >13-fold with butyrate, while the L75T/L181K double mutant has k(cat)/K(M) increased >15-fold with hexanoate and binding (K(d)) improved >28-fold for butyrate . Removing the arginine 47/lysine 51 carboxylate binding motif at the mouth of the active site disfavours binding of all fatty acids, indicating its importance in the initial recognition of substrates.

Microbes Infect, 2000 Nov, 2(13), 1553 - 7
Genetic heterogeneity of Mendelian susceptibility to mycobacterial infection; Doffinger R et al.; Mendelian susceptibility to poorly virulent mycobacterial species, such as bacillus Calmette-Guerin (BCG) and environmental nontuberculous mycobacteria (NTM), is a phenotypically heterogeneous syndrome . It has therefore long been suspected to be genetically heterogeneous . In the past 5 years, this prediction has been confirmed and different types of mutations (dominant or recessive, nonfunctional or hypofunctional) in four genes (IFNGR1, IFNGR2, IL12B, IL12RB1) have revealed both allelic and nonallelic heterogeneity . The eight disorders resulting from these mutations are genetically different but immunologically related, as impaired IFN-gamma-mediated immunity is the common pathogenic mechanism accounting for mycobacterial infection in all patients . The severity of the phenotype depends on the genotype . Complete IFN-gammaR1 and IFN-gammaR2 deficiencies predispose patients to a more severe clinical course than partial IFN-gammaR1 and IFN-gammaR2 deficiencies and complete IL-12 p40 and IL-12Rbeta1 deficiencies.

Urol Oncol, 2000 Dec 15, 6(1), 16 - 19
Bacillus Calmette-Guerin . Is monthly maintenance an option for transitional cell carcinoma of the bladder?
Flanigan RC, DeLaurentis DA, Waters WB, Kunz K.
Purpose: Recently, a Southwest Oncology Group study (SWOG 8507) demonstrated increased efficacy for a bacillus Calmette-Guerin (BCG) maintenance (mtce) program (3 weekly treatments at 3 months, 6 months, and every 6 months thereafter for 3 years) following 6 weekly instillations with BCG as compared to no mtce ("6+3" protocol) . The remarkable results from the mtce arm were unfortunately accompanied by grade 3 or 4 toxicity in 26% . In fact; only 16% of the patients in the maintenance arm received BCG at each of the 7 prescribed courses . Herein, we report on a series of 37 patients with high risk (rapidly recurring grade 2 or 3) Ta, T1 transitional cell carcinoma (TCC) or carcinoma in situ (TIS) of the bladder who received 6 weekly BCG treatments followed by monthly mtce for one year . Patients and Methods: This was a prospective, non-randomized trial of 37 patients with high risk superficial transitional cell carcinoma (TCC) who received one or two 6-week induction courses of intravesical Tice BCG, followed by monthly mtce for 12 months . Entry criteria were identical to those of SWOG 8507 . The mean follow-up interval was 40.7 months . Results: Twenty eight of thirty-seven patients (75.7%) remained free of disease recurrence at a median of 40.7 (range 13-101) months . Only one patient progressed to muscle invasive disease . Only 1 of 37 (2.7%) patient experienced grade 3/4 toxicity . Conclusion: In this single institution, monthly maintenance protocol, freedom from recurrence was significant with dramatically less grade 3 or 4 toxicity than reported in SWOG 8507.

J Invertebr Pathol, 2000 Nov, 76(4), 270 - 7
Toxicity of chitinase-producing Bacillus thuringiensis ssp . kurstaki HD-1 (G) toward Plutella xylostella; Wiwat C et al.; One-hundred fifty isolates of Bacillus thuringiensis were tested for their ability to produce chitinase using colloidal chitin agar as the primary plating medium . Of 14 strains that produced chitinase, B . thuringiensis ssp . kurstaki HD-1(G) was identified as the highest chitinase producer and selected for further study . This bacterium produced the highest amount of chitinase (19.3 mU/ml) when it was cultivated in nutrient broth supplemented with 0.3% colloidal chitin on a rotary shaker (200 rpm) at 30 degrees C for 2 days . The toxicities of B . thuringiensis ssp . kurstaki HD-1(G) and B . thuringiensis ssp . kurstaki wa-p-2, a chitinase nonproducer, were assayed toward Plutella xylostella (diamondback moth) larvae, resulting in LC(50)'s of 4.93 x 10(4) and 1.32 x 10(5) spores/ml, respectively . If the culture broth from B . thuringiensis ssp . kurstaki HD-1(G) was used as the suspending liquid instead of phosphate buffer, their LC(50)'s were reduced to 6.23 x 10(3) and 7.60 x 10(4) spores/ml, respectively . The histopathological changes of the midgut epithelial cells of diamondback moth larvae were compared after feeding on B . thuringiensis ssp . kurstaki HD-1(G) with and without the presence of supernatant containing chitinase under light microscopy and transmission electron microscopy . The midgut epithelial cells of larvae fed for 30 min in the presence of chitinase, with or without spores and endotoxin crystals, appeared more elongated and swollen than those of the control larvae . A number of different cellular changes such as extensive cellular disintegration and appearance of numerous vacuoles were observed from the larvae fed on B . thuringiensis ssp . kurstaki HD-1(G) supplemented with supernatant containing chitinase . Thus increased toxicity and changes in epithelial cells were correlated with the presence of chitinase but this was not distinguished from the possible presence of vegetative-stage insecticidal proteins.

Int J Med Microbiol, 2000 Oct, 290(4-5), 497 - 502
The pathogenesis of clostridial myonecrosis; Stevens DL; These pieces of evidence can be assimilated into a molecular and cellular model of pathogenesis which is initiated by direct toxin effects upon venous capillary endothelial cell function, leading to expression of pro-inflammatory mediators and adhesion molecules, and initiation of platelet aggregation . Toxin-induced hyperadhesion of leukocytes (see above section) with enhanced respiratory burst activity (due to toxins directly or to toxin-induced IL-8 or PAF synthesis by host cells) and toxin-induced chemotaxis deficits could result in neutrophil-mediated vascular injury . Direct toxin-induced cytopathic effects on EC may also contribute to vascular abnormalities associated with gas gangrene . Over prolonged incubation periods, PLC at sublytic concentrations causes EC to undergo profound shape changes similar to those described following prolonged TNF or interferon gamma exposure . In vivo, conversion of EC to this fibroblastoid morphology could contribute to the localized vascular leakage and massive swelling observed clinically with this infection . Similarly, the direct cytotoxicity of PFO could disrupt endothelial integrity and contribute to progressive edema both locally and systemically . Thus, via the mechanisms outlined above, both PLC and PFO may cause local, regional and systemic vascular dysfunction . For instance, local absorption of exotoxins within the capillary beds could affect the physiological function of the endothelium lining the postcapillary venules, resulting in impairment of phagocyte delivery at the site of infection . Toxin-induced endothelial dysfunction and microvascular injury could also cause loss of albumin, electrolytes, and water into the interstitial space resulting in marked localized edema . These events, combined with intravascular platelet aggregation and leukostasis, would increase venous pressures and favor further loss of fluid and protein in the distal capillary bed . Ultimately, a reduced arteriolar flow would impair oxygen delivery thereby attenuating phagocyte oxidative killing and facilitating anaerobic glycolysis of muscle tissue . The resultant drop in tissue pH, together with reduced oxygen tension, might further decrease the redox potential of viable tissues to a point suitable for growth of this anaerobic bacillus . As infection progresses and additional toxin is absorbed, larger venous channels would become affected, causing regional vascular compromise, increased compartment pressures and rapid anoxic necrosis of large muscle groups . When toxins reach arterial circulation, systemic shock and multiorgan failure rapidly ensue, and death is common.

Int J Med Microbiol, 2000 Oct, 290(4-5), 463 - 9
Bacillus thuringiensis and its use in transgenic insect control technologies; Van Rie J; The insecticidal activity of Bacillus thuringiensis (Bt) is mainly due to the production of crystals containing insecticidal crystal proteins (ICPs) . These proteins are very selectively active against certain insect species, including some agronomically important pest species . Some ICP genes have been used for bioengineered crop protection, resulting in transgenic crop plants with excellent insect protection.

Int J Med Microbiol, 2000 Oct, 290(4-5), 295 - 9
Regulation of toxin and virulence gene transcription in Bacillus thuringiensis; Lereclus D et al.; Bacillus thuringiensis is a spore-forming bacterium well known for its insecticidal properties and its ability to produce a crystal inclusion during sporulation . The specific activity of B . thuringiensis against insect larvae is due to the crystal proteins (Cry proteins) . Two different transcriptional mechanisms (dependent and independent of sporulation) are responsible for cry gene transcription during the stationary phase . In addition to these specific insecticidal toxins, B . thuringiensis produces potential virulence factors including haemolysins, degradative enzymes and enterotoxins . A pleiotropic regulator (PlcR) that activates the transcription of various genes encoding such extracellular proteins has been identified . Its expression at the onset of the stationary phase is dependent on the growth medium and is controlled by the transition state regulator, SpoOA.

Can J Microbiol, 2000 Nov, 46(11), 1004 - 11
Subtilisins of Bacillus spp . hydrolyze keratin and allow growth on feathers; Evans KL et al.; Keratinase is a serine protease produced by Bacillus licheniformis PWD-1 that effectively degrades keratin and confers the ability to grow on feathers to a protease-deficient B . subtilis strain . Studies presented herein demonstrate that B . licheniformis Carlsberg strain NCIMB 6816, which produces the well-characterized serine protease subtilisin Carlsberg, also degrades and grows on feathers . The PWD-1 and Carlsberg strains showed a similar time-course of enzyme production, and the purified serine proteases have similar enzymatic properties on insoluble azokeratin and soluble FITC-casein . Kinetic analysis of both enzymes demonstrated that they have high specificity for aromatic and hydrophobic amino acids in the P1 substrate position, although keratinase discriminates more than subtilisin Carlsberg against charged residues at this site . Nucleotide sequence analysis of the serine protease genes from B . licheniformis strains PWD-1, Carlsberg NCIMB 6816, ATCC 12759, and NCIMB 10689 showed that the kerA-encoded protease of PWD-1 differs from the others only by having V222, rather than A222, near the active site serine S220 . Further, high-level expression of subE-encoded subtilisin from B . subtilis (78% similar to subtilisin Carlsberg) also confers growth on feathers on a protease-deficient B . subtilis strain . While strain PWD-1 and the kerA protease efficiently degrade keratin, keratin hydrolysis and growth on feathers is a property that can be conferred by appropriate expression of the major subtilisins, including the industrially produced enzymes.

Rev Cubana Med Trop, 2000 Jan-Apr, 52(1), 37 - 43
{Introduction of Bacillus sphaericus strain-2362 (GRISELESF) for biological control of malaria vectors in Guatemala}; Blanco Castro SD et al.; Malaria continues to be an important health problem in a number of countries of Central and South America where it is considered as a highly prevent endemic disease . The objective of this paper is to assess the entomo-epidemiological impact of a pilot program for the biological control of malaria-transmitting vectors, which was implemented in 1998 in Escuintla, Republic of Guatemala . This program was based on the use of 20,000 L of biolarvicide Bacillus sphaericus- strain-2362 (GRISELESF) which was applied in the 46 localities of highest epidemiological risk at a rate of 10 mL/m2 of effective area of breeding . The entomologic effectiveness of this biolarvicide was monitored from the first 72 hours to 4 months after the application . There was a total larval reduction of 94.57 in the maturity stage of the water phase of Anopheles albimanus vector . The epidemiological analysis was carried out by comparing the rate of malaria prevalence (per 1000 pop) during 1997 and 1998 . The five treated municipalities showed a statistically significant reduction of 50% (p 0.01) . The results obtained in this paper coincided with those reported by comparable studies, so, this allowed us to recommend the use of the biolarvicide Bacillus sphaericus (strain-2362) as part of a comprehensive program of malaria-transmitting vector control in the Republic of Guatemala and other countries of the region.

J Soc Biol, 2000, 194(1), 25 - 8
{Mendelian predisposition to mycobacterial infections in humans}; Casanova JL; Selective susceptibility to poorly pathogenic mycobacteria, such as bacille Calmette-Guerin (BCG) vaccine and environmental non-tuberculous mycobacteria (NTM), bas long been suspected to be a mendelian disorder but its molecular basis has remained elusive . Recently, recessive mutations in the interferon gamma receptor ligand-binding chain (IFN gamma R1), interferon gamma receptor signalling chain (IFN gamma R2), interleukin 12 p40 subunit (IL-12 p40), and interleukin 12 receptor beta 1 chain (IL-12R beta 1) genes have been identified in a number of patients with disseminated BCG or NTM infection . Although genetically distinct, these conditions are immunologically related and highlight the essential role of interferon gamma-mediated immunity in the control of mycobacteria in man.

Biochemistry, 2000 Dec 12, 39(49), 15217 - 24
Calculation of z-coordinates and orientational restraints using a metal binding tag; Gaponenko V et al.; We introduce a new simple methodology allowing the measurement of (1)H-(15)N residual dipolar couplings, dipolar shifts, and unpaired electron-amide proton distances . This method utilizes a zinc finger tag fused at either the N- or the C-terminus of a protein . We have demonstrated this fusion strategy by incorporating the zinc finger of the retroviral gag protein onto the C-terminus of barnase, a ribonuclease produced by Bacillus amiloliquifaciance . We show that this tag can be substituted with cobalt and manganese . Binding of cobalt to the gag zinc finger-barnase fusion protein introduced sufficient anisotropic paramagnetic susceptibility for orientation of the molecule in the magnetic field . Partial alignment permitted measurement of (1)J(HN) scalar couplings along with dipolar couplings . Replacement of bound cobalt with diamagnetic zinc removes the paramagnetic-induced orientation of barnase, permitting the measurement of only (1)J(HN) scalar couplings . Dipolar couplings, ranging from -0.9 to 0.6 Hz, were easily measured from the difference in splitting frequencies in the presence of cobalt and zinc . The observed paramagnetic anisotropy induced by cobalt binding to the metal binding tag also permitted measurement of dipolar shifts . Substitution of manganese into the metal binding tag permitted the measurement of unpaired electron-amide proton distances using paramagnetic relaxation enhancement methodology . The availability of both amide proton dipolar shifts and unpaired electron to amide proton distances permitted the direct calculation of z-coordinates for individual amide protons . This approach is robust and will prove powerful for global fold determination of proteins identified in genome initiatives.

Eur J Biochem, 2000 Dec, 267(24), 7158 - 69
Sites of limited proteolysis in the pyruvate decarboxylase component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus and their role in catalysis; Chauhan HJ et al.; The E1 component (pyruvate decarboxylase) of the pyruvate dehydrogenase complex of Bacillus stearothermophilus is a heterotetramer (alpha2beta2) of E1alpha and E1beta polypeptide chains . The domain structure of the E1alpha and E1beta chains, and the protein-protein interactions involved in assembly, have been studied by means of limited proteolysis . It appears that there may be two conformers of E1alpha in the E1 heterotetramer, one being more susceptible to proteolysis than the other . A highly conserved region in E1alpha, part of a surface loop at the entrance to the active site, is the most susceptible to cleavage in E1 (alpha2beta2) . As a result, the oxidative decarboxylation of pyruvate catalysed by E1 in the presence of dichlorophenol indophenol as an artificial electron acceptor is markedly enhanced, but the reductive acetylation of a free lipoyl domain is unchanged . The parameters of the interaction between cleaved E1 and the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase E2 component are identical to those of the wild-type E1 . However, a pyruvate dehydrogenase complex assembled in vitro with cleaved E1p exhibits a markedly lower overall catalytic activity than that assembled with untreated E1 . This implies that active site coupling between the E1 and E2 components has been impaired . This has important implications for the way in which a tethered lipoyl domain can interact with E1 in the assembled complex.

J Gastroenterol Hepatol, 2000 Oct, 15(10), 1223 - 6
Clinical relapse in Whipple's disease despite maintenance therapy; Garas G et al.; Whipple's disease is a multisystem disorder that was first reported just over 100 years ago . Only recently, the bacillus responsible for the condition was identified and subsequently cultured . However, differences of opinion remain regarding the best antibiotic regimen and duration of therapy at primary diagnosis and there is also great uncertainty about the management of disease relapse . We report a case of clinical relapse of Whipple's disease in a man who was on a prolonged therapy with trimethoprim-sulfamethoxazole . We describe his management and review the literature on the treatment of this condition, with particular reference to the recurrence of the disease.

Lepr Rev, 2000 Sep, 71(3), 325 - 31
Relapses after fixed duration multiple drug therapy: the AMFES cohort; Gebre S et al.; Relapse rates after multiple-drug therapy (MDT) have been low, although there remains a concern about the possibility of late relapse in those with an initially high bacterial load . In all, 502 patients in the AMFES cohort completed fixed-duration MDT and are included in this report . There have been no confirmed relapses in the AMFES cohort, in a follow-up period of up to 8 years after completion of treatment, even in the 57 cases with an initial average bacillary index of > or = 4.0, 20 of whom have been followed for more than 5 years after ceasing MDT . Methods of diagnosing a relapse are discussed.

Lepr Rev, 2000 Sep, 71(3), 318 - 24
ENL reactions in the multibacillary cases of the AMFES cohort in central Ethiopia: incidence and risk factors; Saunderson P et al.; Erythema nodosum leprosum (ENL), or type 2 leprosy reactions are an important complication of multibacillary leprosy . The AMFES cohort includes 300 new multibacillary cases that have been followed for up to 10 years from the start of treatment, in central Ethiopia . Sixteen (5.3%) patients had ENL reactions . The incidence of ENL was maximal in the second and third years after the start of treatment, reaching 6.9 episodes per 100 person years at risk . Factors associated with being lepromatous {LL classification and a high bacillary index (BI)} gave an increased risk of developing ENL; in the univariate analysis, LL classification gave a relative risk of 3.6 (95% CI 1.3-10) and a BI of 6 gave a relative risk of 8.6 (95% CI 2.3-32) for the development of ENL . HIV co-infection was found to be a risk factor in this cohort, but as the numbers involved are small (only two HIV positive patients had ENL), this finding must be confirmed in larger studies . Ten of the 16 cases had recurrent episodes and five had at least five episodes occurring over a period of more than 2 years . The management and prognosis of ENL reactions are discussed.

Biochem J, 2000 Dec 15, 352 Pt 3, 623 - 8
Analysis of myo-inositol hexakisphosphate hydrolysis by Bacillus phytase: indication of a novel reaction mechanism; Kerovuo J et al.; Phytic acid (myo-inositol hexakisphosphate, InsP(6)) hydrolysis by Bacillus phytase (PhyC) was studied . The enzyme hydrolyses only three phosphates from phytic acid . Moreover, the enzyme seems to prefer the hydrolysis of every second phosphate over that of adjacent ones . Furthermore, it is very likely that the enzyme has two alternative pathways for the hydrolysis of phytic acid, resulting in two different myo-inositol trisphosphate end products: Ins(2,4,6)P(3) and Ins(1,3,5)P(3) . These results, together with inhibition studies with fluoride, vanadate, substrate and a substrate analogue, indicate a reaction mechanism different from that of other phytases . By combining the data presented in this study with (1) structural information obtained from the crystal structure of Bacillus amyloliquefaciens phytase {Ha, Oh, Shin, Kim, Oh, Kim, Choi and Oh (2000) Nat . Struct . Biol . 7, 147-153}, and (2) computer-modelling analyses of enzyme-substrate complexes, a novel mode of phytic acid hydrolysis is proposed.

Microbiology, 2000 Dec, 146 Pt 12, 3033 - 9
Cooperative, synergistic and antagonistic haemolytic interactions between haemolysin BL, phosphatidylcholine phospholipase C and sphingomyelinase from Bacillus cereus; Beecher DJ et al.; Haemolysis of erythrocytes from different species (sheep, bovine, swine and human), caused by various combinations of phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC), sphingomyelinase (SMase) and the three-component, pore-forming toxin haemolysin BL (HBL) from Bacillus cereus was analysed . The lytic potency of HBL did not correlate with phospholipid (PL) content, but lysis by the individual or combined enzymes did . SMase alone lysed ruminant erythrocytes, which contain 46-53% sphingomyelin (SM) . The cooperative action of PC-PLC and SMase was needed to lyse swine and human erythrocytes (22-31% PC and 28-25% SM) . SMase synergistically enhanced haemolysis caused by HBL for all erythrocytes tested, which all contained >25% SM . PC-PLC enhanced HBL haemolysis only in cells containing significant amounts of PC (swine, 22% PC; human, 31% PC) . Unexpectedly, PC-PLC inhibited HBL lysis of sheep erythrocytes (<2% PC) and enhanced the discontinuous haemolysis pattern that is characteristic of HBL in sheep blood agar . Inhibition and pattern enhancement was abolished by washing PC-PLC-treated erythrocytes or by adding EDTA, suggesting that enzymic alteration of the membrane is not involved, but that zinc in the active site is required, perhaps to facilitate binding . These observations highlight the potential for cooperative and synergistic interactions among virulence factors in B . cereus infections and dependence of these effects on tissue composition.

J Clin Microbiol, 2000 Dec, 38(12), 4373 - 81
Results of multiple diagnostic tests for Mycobacterium avium subsp . paratuberculosis in patients with inflammatory bowel disease and in controls; Collins MT et al.; Mycobacterium avium subsp . paratuberculosis has been incriminated as a cause of Crohn's disease (CD); however, studies to date have been relatively small and generally only used a single diagnostic assay . The objective of the study was to reexamine the association of M . avium subsp . paratuberculosis and CD using multiple diagnostic tests . Five methods were used to detect M . avium subsp . paratuberculosis infections in 439 inflammatory bowel disease (IBD) patients and 324 control subjects in the United States and Denmark . Most assays were adaptations of diagnostic tests for this infection performed routinely on animals . PCR for IS900, a genetic element unique to M . avium subsp . paratuberculosis, was positive significantly more often on resected bowel and lymph node tissues from CD patients (19.0%) and ulcerative colitis (UC) patients (26.2%) than from controls (6 . 3%) (P < 0.05) . Positive IS900 PCR results occurred more often in U . S . than in Danish IBD patients, 32.0 versus 13.3% (P = 0.025) . The majority of Danish patients were bacillus Calmette-Guerin (Mycobacterium bovis BCG) vaccinated (CD, 77.5%; UC, 86.6%; controls, 83.0%) whereas none of the U.S . patients with IBD and only 2% of U . S . controls were vaccinated . Among Danish IBD patients, positive PCR findings were four times more common among subjects who were not BCG vaccinated (33.3%) than among BCG vaccinates (8.8%, P = 0.02) . Culture of the same tissues tested by PCR using modified BACTEC 12B medium failed to grow M . avium subsp . paratuberculosis from patients or controls . U.S . CD patients had the highest serological evidence (enzyme-linked immunosorbent assay {ELISA} for serum antibodies) of M . avium subsp . paratuberculosis infection (20.7% of patients positive) which was higher than for all UC patients studied (6.1%) or healthy controls (3.8%, P < 0.005) . Among Danish patients alone, however, no significant differences in rates of ELISA-positive results among CD, UC, or control patients were found . For 181 study subjects, both IS900 PCR and ELISA were performed . Although 11 were ELISA positive and 36 were PCR positive, in no instance was a patient positive by both tests, suggesting that these states are mutually exclusive . Evaluation of cytokine-mediated immune responses of IBD patients was complicated by the influence of immunosuppressive therapy given most IBD patients . Gamma interferon (IFN-gamma) release by peripheral blood leukocytes after M . avium purified protein derivative PPD antigen stimulation showed significantly lower responses in CD patients than in UC patients or controls in both U.S . (by ex vivo assay) and Danish (by in vitro assay) populations (P < 0.05) . Interleukin-5 responses were not different among CD, UC, or control groups . Collectively, the PCR, ELISA, and IFN-gamma tests for M . avium subsp . paratuberculosis together with the unexpected observation that BCG vaccination influenced M . avium subsp . paratuberculosis detection, lead us to conclude that M . avium subsp . paratuberculosis, or some similarly fastidious mycobacterial species, infects at least a subset of IBD patients . Whether the infection is primary (causal) or secondary, it may contribute to the etiopathogenesis of IBD.

Int J Epidemiol, 2000 Dec, 29(6), 1085 - 91
BCG vaccine effectiveness in preventing tuberculosis and its interaction with human immunodeficiency virus infection; Arbelaez MP et al.; BACKGROUND: To explore Bacillus Calmette-Guerin vaccine (BCG) as a protective factor against tuberculosis (TB) and how human immunodeficiency virus (HIV) infection modifies the effect of BCG on TB . METHODS: Two matched case-control studies were conducted . One study compared TB cases and controls who were HIV positive . The second compared TB cases and controls who were HIV negative . The study population consisted of 88 TB cases and 88 controls among HIV-positive individuals and 314 TB cases and 310 controls among HIV-negative individuals . Cases were new TB diagnoses, confirmed by either bacteriology, pathology, radiology or clinical response to treatment; controls were selected from people without TB symptoms and who sought medical attention in the same institution where a case was enrolled . BCG was assessed by the presence of a typical scar . RESULTS: The level of protection against all clinical forms of TB was 22% among HIV positive individuals (odds ratio {OR} = 0.78, 95% CI : 0.48-1.26) and 26% among HIV negatives (OR = 0.74, 95% CI : 0.52-1.05) . There was a significant difference (P = 0.002) in the level of protection against extrapulmonary TB (ETB) between HIV-negative (OR = 0.54, 95% CI : 0.32-0.93) and HIV-positive individuals (OR = 1.36, 95% CI : 0.72-2.57) . CONCLUSION: BCG has a modest protective effect against all forms of TB independent of HIV status, and BCG confers protection against extrapulmonary TB among HIV-negative individuals . However, HIV infection seems to abrogate the protective effect of BCG against extrapulmonary TB . Our data support the public health importance of BCG vaccine in the prevention of extrapulmonary TB among immunocompetent individuals.

Semin Urol Oncol, 2000 Nov, 18(4), 280 - 8
Intravesical therapy for superficial bladder cancer; Malkowicz SB; Intravesical therapy is a classic approach to the treatment of superficial bladder cancer . Bacillus Calmette-Guerin (BCG) has assumed the role of the most commonly administered agent, and clearly the most effective form of therapy for carcinoma in situ . Recent data quantify the advantage of "booster" treatments over a 3-year interval but suggest that current regimens are not easily tolerated by most patients . Initial studies suggest a role for intravesical interferon in potentiating the effect of BCG . Classic intravesical chemotherapy can decrease tumor recurrence by 14% to 17%, but no data exist to demonstrate any positive effect on decreasing tumor progression . Recent studies suggest a growing rationale for the intravesical administration of a single dose of a chemotherapeutic agent to decrease early tumor recurrence.

Appl Environ Microbiol, 2000 Dec, 66(12), 5509 - 13
Effect of calcium in assay medium on D value of Bacillus stearothermophilus ATCC 7953 spores; Sasaki K et al.; The D value of commercial biological indicator spore strips using Bacillus stearothermophilus ATCC 7953 was increased by higher calcium concentrations in assay media . The calcium concentration in assay media varied among the manufacturers . The calcium concentration in assay media is an important factor to consider to minimize the variation of D value.

Appl Environ Microbiol, 2000 Dec, 66(12), 5174 - 81
Role of proteolysis in determining potency of Bacillus thuringiensis Cry1Ac delta-endotoxin; Lightwood DJ et al.; Bacillus thuringiensis protein delta-endotoxins are toxic to a variety of different insect species . Larvicidal potency depends on the completion of a number of steps in the mode of action of the toxin . Here, we investigated the role of proteolytic processing in determining the potency of the B . thuringiensis Cry1Ac delta-endotoxin towards Pieris brassicae (family: Pieridae) and Mamestra brassicae (family: Noctuidae) . In bioassays, Cry1Ac was over 2,000 times more active against P . brassicae than against M . brassicae larvae . Using gut juice purified from both insects, we processed Cry1Ac to soluble forms that had the same N terminus and the same apparent molecular weight . However, extended proteolysis of Cry1Ac in vitro with proteases from both insects resulted in the formation of an insoluble aggregate . With proteases from P . brassicae, the Cry1Ac-susceptible insect, Cry1Ac was processed to an insoluble product with a molecular mass of approximately 56 kDa, whereas proteases from M . brassicae, the non-susceptible insect, generated products with molecular masses of approximately 58, approximately 40, and approximately 20 kDa . N-terminal sequencing of the insoluble products revealed that both insects cleaved Cry1Ac within domain I, but M . brassicae proteases also cleaved the toxin at Arg423 in domain II . A similar pattern of processing was observed in vivo . When Arg423 was replaced with Gln or Ser, the resulting mutant toxins resisted degradation by M . brassicae proteases . However, this mutation had little effect on toxicity to M . brassicae . Differential processing of membrane-bound Cry1Ac was also observed in qualitative binding experiments performed with brush border membrane vesicles from the two insects and in midguts isolated from toxin-treated insects.

FEMS Microbiol Lett, 2000 Dec 1, 193(1), 7 - 11
Mutagenesis of the glucoamylase signal peptide of Saccharomyces diastaticus and functional analysis in Saccharomyces cerevisiae; Lee JW et al.; To improve the efficiency of the glucoamylase signal peptide (GSP) of Saccharomyces diastaticus for the secretion of foreign proteins, hybrid plasmids containing one of four types of GSP mutant (m1, Pro(-18)-->Leu(-18); m2, Tyr(-13)-->Leu(-13); m3, Ser(-9)-->Leu(-9); m4, Asn(-5)-->Pro(-5)) were constructed and evaluated in Saccharomyces cerevisiae using Bacillus endo-1,4-beta-D-glucanase (CMCase) as a reporter gene . CMCase secretion by m1, m2 and m3 GSP mutants was increased, likely resulting from a higher probability of the modified GSP to assume an alpha-helical structure . Especially in the case of m3, the substitution of Leu for a polar residue, Ser(-9), in the hydrophobic region resulted in approximately a twofold increase in extracellular CMCase activity . In mutant 4, which disrupts the alpha-helix of GSP, CMCase was less efficiently secreted.

Curr Infect Dis Rep, 2000 Apr, 2(2), 141 - 146
Recent Advances in Diagnosis and Treatment of Cat Scratch Disease; Margileth AM; The cause of cat scratch disease (CSD), first described in France in 1950 and in the United States in 1951, was unknown until 1983 when the bacterium in lymph nodes was detected using a Warthin-Starry silver stain . Afipia felis has been an infrequent cause of CSD since1988, when this gram-negative bacterium was first isolated from 10 patients with CSD . In 1992 Bartonella organisms were isolated from immunocompetent and immunocompromised patients . An indirect fluorescent antibody test to detect bartonella-specific serum immunoglobulins was developed in 1992 . Since then multiple studies have shown that three Bartonella species may produce either CSD in humans, usually Bartonella henselae or Bartonella clarridgeiae, or bacteremia in healthy cats . Also, these two bacteria and Bartonella quintana cause bacillary angiomatosis, bacillary peliosis, or relapsing bacteremia in humans . Cats are healthy carriers of Bartonella organisms and may be bacteremic for months to years . Cat-to-cat transmission of Bartonella organisms involves the cat flea in absence of direct contact transmission . CSD is the most common cause of regional lymphadenitis in children and adolescents . Present knowledge on the etiology, clinical features, epidemiology, pathogenesis, diagnosis, and management of CSD are presented . Also, brief comments about the etiology, clinical presentation, and treatment of bacillary angiomatosis and bacillary peliosis are provided.

Appl Microbiol Biotechnol, 2000 Oct, 54(4), 528 - 34
Bioconversion of lutein to products with aroma; Sanchez-Contreras A et al.; A residual mud sample from the marigold flower dehydration process was screened and 19 putative colonies were isolated for their ability to degrade lutein in a chemically defined medium supplemented with marigold flower flour as a carbon source . Among the colonies isolated, two generated volatile compounds in fermentation and one was chosen for further study for its ability to produce a strong tobacco smell . This colony contained two microorganisms, identified as Geotrichum sp . and Bacillus sp . The aroma production requires the presence of both microorganisms and lutein . Using gas chromatography coupled to mass spectrometry (GC/ MS), four compounds were identified: 7,8-dihydrobeta-ionol, beta-ionone, 7,8-dihydro-beta-ionone, and 3-hydroxy-beta-ionone, in proportions of 84.2%, 9.4%, 3.5%, and 2.9%, respectively.

Microbios, 2000, 103(405), 85 - 96
Effects of macromolecular growth substrates on production of extracellular enzymes by Bacillus species in continuous culture; Mahmood AU et al.; Bacillus species 11089 and alkalophilic Bacillus species 11203 were both capable of growth in continuous culture on macromolecular substrates, starch, soybean flour, casein, pectin, polypectate, polygalacturonate and carboxymethylcellulose (CMC) when these were used as the carbon-energy source in a mineral salts basal medium . High maximum specific growth rate (micronmax) and biomass values occurred when cells were grown on starch, soybean flour and casein, and low values on pectin, polypectate, polygalacturonic acid and CMC . Hydrolytic enzymes (protease, amylase, polygalacturonate lyase and alkaline phosphatase) were subject to regulation (induction and/or repression) depending on the nature of the growth substrate utilized . In general, high levels of enzymes were produced on soybean flour, casein and starch but low levels on CMC, pectin, polypectate and polygalacturonate.

Teratology, 2000 Dec, 62(6), 413 - 9
Nonspecific stimulation of the maternal immune system . I . Effects On teratogen-induced fetal malformations; Holladay SD et al.; BACKGROUND: Maternal immune stimulation has reported, but unconfirmed, efficacy for reducing chemical-induced morphologic defects in mice . METHODS: Teratogenic chemicals (2,3,7, 8-tetrachlorodibenzo-p-dioxin {TCDD}, ethyl carbamate {urethane}, methylnitrosourea {MNU}, or valproic acid {VA}) were given to pregnant mice to induce cleft palate (TCDD, urethane), digital defects (urethane, MNU), or exencephaly (VA) . Before teratogen administration, the immune system of female mice was stimulated by intraperitoneal (IP) administration of pyran copolymer or attenuated bacillus Calmette Guerin (BCG), or by footpad injection with Freund's complete adjuvant (FCA) . RESULTS: Fetal defects caused by all four chemicals studied were reduced by maternal immunostimulation, sometimes dramatically . In addition to reducing VA-induced exencephaly, immunostimulation with FCA resulted in fetal mice displaying anury (absence of tails) . Activated maternal immune cells could not be detected in fetal circulation using flow cytometry and a fluorescent cell-tracking probe . CONCLUSIONS: For the chemicals tested, maternal immune stimulation has efficacy in reducing fetal defects . Immune protection against teratogenesis may be an indirect effect of maternal immune cell activation .

J Biotechnol, 2001 Nov 30, 84(2), 127 - 32
The ddcA gene from Streptomyces fradiae encodes an extracellular beta-lactamase with penicillinase and cephalosporinase activities; Fouces R et al.; The ddcA gene from Streptomyces fradiae, which is located adjacent to the left edge of the tylosin biosynthetic cluster, has been cloned and sequenced . DNA sequence analysis revealed an ORF of 1194 bp that encodes a product of 42.6 kDa . This protein showed significant similarity to the extracellular endopeptidase with beta-lactamase activity encoded by the adp gene from Bacillus cereus and to PBPs (DD-carboxypeptidases and DD-endopeptidases) and beta-lactamases . Moreover, it contains three characteristic motifs conserved in PBPs and beta-lactamases, including an essential serine residue in the active centre and a putative leader peptide . Heterologous expression of the ddcA gene in Streptomyces lividans demonstrated the presence in the transformants of an extracellular beta-lactamase active against penicillin G, ampicillin and the chromogenic cephalosporin nitrocefin.

Proc Natl Acad Sci U S A, 2000 Nov 21, 97(24), 12980 - 4
Frequency of resistance to Bacillus thuringiensis in field populations of pink bollworm; Tabashnik BE et al.; Strategies for delaying pest resistance to genetically modified crops that produce Bacillus thuringiensis (Bt) toxins are based primarily on theoretical models . One key assumption of such models is that genes conferring resistance are rare . Previous estimates for lepidopteran pests targeted by Bt crops seem to meet this assumption . We report here that the estimated frequency of a recessive allele conferring resistance to Bt toxin Cry1Ac was 0.16 (95% confidence interval = 0.05-0.26) in strains of pink bollworm (Pectinophora gossypiella) derived from 10 Arizona cotton fields during 1997 . Unexpectedly, the estimated resistance allele frequency did not increase from 1997 to 1999 and Bt cotton remained extremely effective against pink bollworm . These results demonstrate that the assumptions and predictions of resistance management models must be reexamined.

J Nat Prod, 2000 Nov, 63(11), 1492 - 6
Kurstakins: a new class of lipopeptides isolated from Bacillus thuringiensis; Hathout Y et al.; A novel class of lipopeptides was isolated from Bacillus thuringiensis kurstaki HD-1 . Four compounds (1-4) were separated by high-performance liquid chromatography and their primary structures determined using a combination of chemical reactions and mass spectrometry . The four lipopeptides were found to have the same amino acid sequence, Thr-Gly-Ala-Ser-His-Gln-Gln, but different fatty acids . The fatty acyl chain is linked to the N-terminal amino acid residue via an amide bond . Each lipopeptide has a lactone linkage between the carboxyl terminal amino acid and the hydroxyl group in the side chain of the serine residue . Antifungal activity was demonstrated against Stachybotrys charatum.

Carbohydr Res, 2000 Oct 20, 329(1), 97 - 107
Purification and characterisation of a malto-oligosaccharide-forming amylase active at high pH from Bacillus clausii BT-21; Duedahl-Olesen L et al.; Bacillus clausii BT-21 produced an extracellular malto-oligosaccharide-forming amylase active at high pH when grown on starch substrates . The enzyme was purified to homogeneity by affinity and anion-exchange chromatography . The molecular weight of the enzyme estimated by sodium dodecyl sulfate polyacrylamide electrophoresis was 101 kDa . The enzyme showed an optimum of activity at pH 9.5 and 55 degrees C . Maltohexaose was detected as the main initially formed starch hydrolysis product . Maltotetraose and maltose were the main products obtained after hydrolysis of starch by the enzyme for an extended period of time and were not further degraded . The enzyme readily hydrolysed soluble starch, amylopectin and amylose, while cyclodextrins, pullulan or dextran were not degraded . The mode of action during hydrolysis of starch indicated an exo-acting type of amylolytic enzyme mainly producing maltohexaose and maltotetraose . Amino acid sequencing of the enzyme revealed high homology with the maltohexaose-forming amylase from Bacillus sp . H-167.

J Gen Virol, 2000 Dec, 81(Pt 12), 3073 - 82
Characterization of repetitive DNA regions and methylated DNA in ascovirus genomes; Bigot Y et al.; The accompanying phylogenetic study of large double-stranded DNA viruses based on their delta DNA polymerase genes suggests that ascoviruses (family ASCOVIRIDAE:) and iridoviruses (family IRIDOVIRIDAE:) are closely related and may share a common ancestor . This relationship was unexpected because of marked differences between these viruses . Iridoviruses produce icosahedral virions and occur broadly among vertebrates and invertebrates, whereas ascoviruses typically produce reniform or bacilliform virions and are restricted to insect hosts, primarily lepidopterans . Detailed comparisons of these two virus types are not possible because fundamental information on the properties of the virions and their genomes is lacking, especially for ascoviruses . To facilitate further investigation of the putative evolutionary relationship between ascoviruses and iridoviruses, the genomes of representative viruses from each family were compared with respect to physical configuration, presence of DNA repeats and degree of DNA methylation . Genomes from Spodoptera frugiperda (SfAV1), Heliothis virescens (HvAV3) and Diadromus pulchellus (DpAV4) ascoviruses were all found to be circular and partially superhelical and to contain large interspersed repeats of 1-3 kbp . Mosquito (IV type 3), lepidopteran (IV type 6) and isopod (IV type 31) iridovirus genomes were all linear and lacked large regions of repetitive DNA . Ascovirus and iridovirus genomes were methylated and one, DpAV4, had the highest degree of methylation of any reported animal DNA virus . The major differences in the physical and biochemical characteristics of ascoviruses and iridoviruses reported here provide a foundation for further studies of their relatedness while making their possible close relationship and divergence during evolution of even greater interest.

J Immunol, 2000 Dec 1, 165(11), 6463 - 71
Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans; Geluk A et al.; CD8(+) T cells are thought to play an important role in protective immunity to tuberculosis . Although several nonprotein ligands have been identified for CD1-restricted CD8(+) CTLs, epitopes for classical MHC class I-restricted CD8(+) T cells, which most likely represent a majority among CD8(+) T cells, have remained ill defined . HLA-A*0201 is one of the most prevalent class I alleles, with a frequency of over 30% in most populations . HLA-A2/K(b) transgenic mice were shown to provide a powerful model for studying induction of HLA-A*0201-restricted immune responses in vivo . The Ag85 complex, a major component of secreted Mycobacterium tuberculosis proteins, induces strong CD4(+) T cell responses in M . tuberculosis-infected individuals, and protection against tuberculosis in Ag85-DNA-immunized animals . In this study, we demonstrate the presence of HLA class I-restricted, CD8(+) T cells against Ag85B of M . tuberculosis in HLA-A2/K(b) transgenic mice and HLA-A*0201(+) humans . Moreover, two immunodominant Ag85 peptide epitopes for HLA-A*0201-restricted, M . tuberculosis-reactive CD8(+) CTLs were identified . These CD8(+) T cells produced IFN-gamma and TNF-alpha and recognized Ag-pulsed or bacillus Calmette-Guerin-infected, HLA-A*0201-positive, but not HLA-A*0201-negative or uninfected human macrophages . This CTL-mediated killing was blocked by anti-CD8 or anti-HLA class I mAb . Using fluorescent peptide/HLA-A*0201 tetramers, Ag85-specific CD8(+) T cells could be visualized in bacillus Calmette-Guerin-responsive, HLA-A*0201(+) individuals . Collectively, our results demonstrate the presence of HLA class I-restricted CD8(+) CTL against a major Ag of M . tuberculosis and identify Ag85B epitopes that are strongly recognized by HLA-A*0201-restricted CD8(+) T cells in humans and mice . These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.

Ann Intern Med, 2000 Nov 21, 133(10), 779 - 89
Hospital ventilation and risk for tuberculous infection in canadian health care workers . Canadian Collaborative Group in Nosocomial Transmission of TB; Menzies D et al.; BACKGROUND: The risk for and determinants of transmission of tuberculosis in hospitals caring for moderate numbers of patients with tuberculosis remain uncertain . OBJECTIVE: To study the association of tuberculin conversion among health care workers with ventilation of patient care areas . DESIGN: Cross-sectional observational survey . SETTING: 17 acute-care community or university hospitals . PARTICIPANTS: All personnel who worked at least 2 days per week in the respiratory and physiotherapy departments or in selected nursing units . MEASUREMENTS: Participating workers underwent tuberculin skin testing and completed self-administered questionnaires . Previous tuberculin tests and bacille Calmette-Guerin vaccinations were verified . Records of patients with tuberculosis who were hospitalized in the 3 years preceding the study were reviewed . Air exchanges per hour in patient care areas were measured by using a tracer gas technique . Multivariate proportional hazards regression was used to estimate the effect of occupational factors on documented tuberculin conversion, after adjustment for nonoccupational factors, among participants with at least one previous negative result on tuberculin skin testing . RESULTS: Tuberculin conversion was associated with ventilation of general or nonisolation patient rooms of less than 2 air exchanges per hour (adjusted hazard ratio, 3.4 {95% CI, 2.1 to 5.8}); with work in moderate- to high-risk hospitals (adjusted hazard ratio, 2.2 {CI, 1.3 to 3.5}); and with work in the nursing (adjusted hazard ratio, 4.3 {CI, 2.7 to 6.9}), respiratory therapy (adjusted hazard ratio, 6.1 {CI, 3.1 to 12.0}), and physiotherapy (adjusted hazard ratio, 3.3 {CI, 1.5 to 7.2}) departments or housekeeping (adjusted hazard ratio, 4.2 {CI, 2.3 to 7.6}) . Conversion was not associated with inadequate ventilation of respiratory isolation rooms (adjusted hazard ratio, 1.0 {CI, 0.8 to 1.3}) . CONCLUSION: Tuberculin conversion among health care workers was strongly associated with inadequate ventilation in general patient rooms and with type and duration of work, but not with ventilation of respiratory isolation rooms.

Pediatr Dermatol, 2000 Sep-Oct, 17(5), 373 - 6
Lichen scrofulosorum; Torrelo A et al.; A 12-month-old boy with pulmonary tuberculosis developed a papular lichenoid eruption which showed epithelioid granulomas on histology, consistent with lichen scrofulosorum . Stains and cultures for mycobacteria in the skin were negative, and a polymerase chain reaction (PCR) analysis failed to detect the DNA of Mycobacterium tuberculosis in a skin biopsy specimen, thus making lichen scrofulosorum one of the remaining manifestations of M . tuberculosis infection in which evidence of the bacillus has not been found to date . Lichen scrofulosorum is now considered a rare form of tuberculid but should not be neglected.

Infect Immun, 2000 Dec, 68(12), 7094 - 9
Identification of a Mycobacterium bovis BCG auxotrophic mutant that protects guinea pigs against M . bovis and hematogenous spread of Mycobacterium tuberculosis without sensitization to tuberculin; Chambers MA et al.; Tuberculosis remains one of the most significant diseases of humans and animals . The only currently available vaccine against this disease is a live, attenuated vaccine, bacillus Calmette-Guerin (BCG), which was originally derived from Mycobacterium bovis and despite its variable efficacy is the most widely administered vaccine in the world . With the advent of the human immunodeficiency virus-AIDS pandemic concern has been raised over the safety of BCG . Moreover, since BCG sensitizes vaccinated individuals to the tuberculin test, vaccination with BCG prevents diagnosis of infection in vaccinated individuals . Recently, auxotrophic strains of BCG have been generated by insertional mutagenesis which have been shown to be safer than the parent BCG strain following administration to mice with severe combined immunodeficiency disease . These strains have also been shown to give comparable protection against intravenous and intratracheal challenge of BALB/c mice with M . tuberculosis relative to conventional BCG . Here we report that one of these mutants, a leucine auxotroph of BCG, conferred significant protection of the lungs and spleens of guinea pigs infected with M . bovis and protection of the spleens of guinea pigs infected with M . tuberculosis in the absence of a cutaneous hypersensitivity reaction to tuberculin . Therefore, protective immunity to tuberculosis may, at least in part, be achieved without sensitization to the tuberculin skin test . These results indicate that it may be possible to develop a new generation of vaccines based on BCG that are protective, are safe for use in the immunocompromised, and do not preclude the use of the tuberculin skin test in both humans and animals.

Infect Immun, 2000 Dec, 68(12), 6954 - 61
Immunopathologic effects of tumor necrosis factor alpha in murine mycobacterial infection are dose dependent; Bekker LG et al.; In experimental mycobacterial infection, tumor necrosis factor alpha (TNF-alpha) is required for control of bacillary growth and the protective granulomatous response, but may cause immunopathology . To directly examine the positive and detrimental effects of this cytokine, a murine model was used in which different amounts of TNF-alpha were delivered to the site of infection . Mice with a disruption in the TNF-alpha gene (TNF-KO) or wild-type mice were infected with low or high doses of recombinant Mycobacterium bovis BCG that secreted murine TNF-alpha (BCG-TNF) . Infection of TNF-KO mice with BCG containing the vector (BCG-vector) at a low dose led to increased bacillary load in all organs and an extensive granulomatous response in the lungs and spleen . The mice succumbed to the infection by approximately 40 days . However, when TNF-KO mice were infected with low doses of BCG-TNF, bacillary growth was controlled, granulomas were small and well differentiated, the spleen was not enlarged, and the mice survived . Infection with high inocula of BCG-TNF resulted in bacterial clearance, but was accompanied by severe inflammation in the lungs and spleen and earlier death compared to the results from the mice infected with high inocula of BCG-vector . Wild-type mice controlled infection with either recombinant strain, but showed decreased survival following high-dose BCG-TNF infection . The effects of TNF-alpha required signaling through an intact receptor, since the differential effects were not observed when TNF-alpha receptor-deficient mice were infected . The results suggest that the relative amount of TNF-alpha at the site of infection determines whether the cytokine is protective or destructive.

Infect Immun, 2000 Dec, 68(12), 6883 - 90
Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guérin: involvement of toll-like receptors; Tsuji S et al.; The constituents of mycobacteria are an effective immune adjuvant, as observed with complete Freund's adjuvant . In this study, we demonstrated that the cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guerin (BCG-CWS), a purified noninfectious material consisting of peptidoglycan, arabinogalactan, and mycolic acids, induces maturation of human dendritic cells (DC) . Surface expression of CD40, CD80, CD83, and CD86 was increased by BCG-CWS on human immature DC, and the effect was similar to those of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), heat-killed BCG, and viable BCG . BCG-CWS induced the secretion of TNF-alpha, IL-6, and IL-12 p40 . CD83 expression was increased by a soluble factor secreted from BCG-CWS-treated DC and was completely inhibited by monoclonal antibodies against TNF-alpha . BCG-CWS-treated DC stimulated extensive allogeneic mixed lymphocyte reactions . The level of TNF-alpha secreted through BCG-CWS was partially suppressed in murine macrophages with no Toll-like receptor 2 (TLR 2) or TLR4 and was completely lost in TLR2 and TLR4 double-deficient macrophages . These results suggest that the BCG-CWS induces TNF-alpha secretion from DC via TLR2 and TLR4 and that the secreted TNF-alpha induces the maturation of DC per se.

Infect Immun, 2000 Dec, 68(12), 6750 - 7
Hemin-binding surface protein from Bartonella quintana; Carroll JA et al.; Bartonella quintana, the agent of trench fever and a cause of endocarditis and bacillary angiomatosis in humans, has the highest reported in vitro hemin requirement for any bacterium . We determined that eight membrane-associated proteins from B . quintana bind hemin and that a approximately 25-kDa protein (HbpA) was the dominant hemin-binding protein . Like many outer membrane proteins, HbpA partitions to the detergent phase of a Triton X-114 extract of the cell and is heat modifiable, displaying an apparent molecular mass shift from approximately 25 to 30 kDa when solubilized at 100 degrees C . Immunoblots of purified outer and inner membranes and immunoelectron microscopy with whole cells show that HbpA is strictly located in the outer membrane and surface exposed, respectively . The N-terminal sequence of mature HbpA was determined and used to clone the HbpA-encoding gene (hbpA) from a lambda genomic library . The hbpA gene is 816 bp in length, encoding a predicted immature protein of approximately 29.3 kDa and a mature protein of 27.1 kDa . A Fur box homolog with 53% identity to the Escherichia coli Fur consensus is located upstream of hbpA and may be involved in regulating expression . BLAST searches indicate that the closest homologs to HbpA include the Bartonella henselae phage-associated membrane protein, Pap31 (58.4% identity), and the OMP31 porin from Brucella melitensis (31.7% identity) . High-stringency Southern blots indicate that all five pathogenic Bartonella spp . possess hbpA homologs . Recombinant HbpA can bind hemin in vitro; however, it does not confer a hemin-binding phenotype upon E . coli . Intact B . quintana treated with purified anti-HbpA Fab fragments show a significant (P < 0.004) dose-dependent decrease in hemin binding relative to controls, suggesting that HbpA plays an active role in hemin acquisition and therefore pathogenesis . HbpA is the first potential virulence determinant characterized from B . quintana.

J Neurooncol, 2000 Jun, 48(2), 113 - 20
A pilot study of autologous cancer cell vaccination and cellular immunotherapy using anti-CD3 stimulated lymphocytes in patients with recurrent grade III/IV astrocytoma; Wood GW et al.; The study objectives were to determine; (1) whether activated T cells could be generated from peripheral blood of patients immunized with their own cancer cells, (2) whether adoptive transfer of the activated T cells to patients had toxic effects and (3) whether the infused cells produced clinical responses . Study patients had recurrent, surgically accessible grade III/IV astrocytomas . The patients were tapered off steroids after total surgical resection and immunized with autologous cancer cells plus Bacillus, Calmette and Guerin (BCG) . Peripheral blood mononuclear cells were activated with anti-CD3, expanded with interleukin-2 (IL-2) and reinfused to patients . The number of activated T cells that was given back to patients varied between 10(10) and 10(11) . Side effects that were observed following immunization and adoptive cell transfer included mainly transient flu-like symptoms . One patient's tumor partially regressed, but there was no effect on survival . Two other patients' tumors regressed, and the patients are apparently disease-free more than 5 and 4 years later . The other six patients' tumors were apparently unaffected by the treatment . Patient age, tumor grade and CD4/CD8 composition of infused cells were positively correlated with clinical responses . Cellular immunotherapy is feasible and is associated with minimal toxicity . Additional appropriately controlled studies will be required to determine whether cellular immunotherapy could be used as a treatment for central nervous system malignancy . Additional studies also will be required to determine the underlying immunological mechanisms.

Yakugaku Zasshi, 2000 Oct, 120(10), 839 - 48
{Tracing the past half century of my microbial transformation studies}; Naito A; In the middle of 1950's, microbial transformation technology was introduced into the field of synthetic chemistry as a new methodology . There was a sudden interest in research on the problems of producing steroid hormones by microbial transformation . At that time, the first project entitled "The Study for Microbial Transformation of Steroids", the "Tsuda Project", was established in the Institute of Applied Microbiology (IAM), University of Tokyo, in the spring 1956, in which I took part . This paper summarizes a number of results of our microbial transformation reactions not only in the synthesis of steroidal compounds, but also more broadly for other organic compounds, such as pravastatin, etc . The results are divided into five categories: 1) Microbial transformation of steroids, 2) Correlation between isolation sources of Pseudomonas spp . and their transformation activities, 3) Fermentation Production of prednisolone by Bacillus pulvifaciens SANK 71760, 4) Microbial transformation of siccanin, and 5) Development and fermentation production of pravastatin . About 30 years later, almost at the end of my microbial transformation studies, I had the opportunity to find some microbial strains having superior hydroxylation ability of ML-236BNa to pravastatin . Fortunately, Streptomyces carbophilus SANK 62585 was finally selected as a potent microbial converter with the formation of a lesser amount of by-products . With the view of industrial production of pravastatin, many studies and improvements were made to the culturing conditions to obtain productivity available commercially.

Arch Microbiol, 2000 Oct, 174(4), 233 - 8
Adaptation of the food-borne pathogen Bacillus cereus to carvacrol; Ultee A et al.; Carvacrol, a natural antimicrobial compound present in the essential oil fraction of oregano and thyme, is bactericidal towards Bacillus cereus . A decrease of the sensitivity of B . cereus towards carvacrol was observed after growth in the presence of non-lethal carvacrol concentrations . A decrease of the melting temperature (Tm) of membranes from 20.5 degrees C to 12.6 degrees C was the immediate effect of the addition of carvacrol . Cells adapted to 0.4 mM carvacrol showed a lower membrane fluidity than nonadapted cells . Adaptation of 0.4 mM carvacrol increased the Tm from 20.5 degrees C to 28.3 degrees C . The addition of carvacrol to cell suspensions of adapted B . cereus cells decreased Tm again to 19.5 degrees C, approximately the same value as for the non-adapted cells in the absence of carvacrol . During adaptation, changes in the fatty acid composition were observed . The relative amount of iso-C13:0, C14:0, and iso-C15:0 increased and cis-C16:1 and C18:0 decreased . The head-group composition also changed, two additional phospholipids were formed and one phospholipid was lacking in the adapted cells . It could be concluded that B . cereus adapts to carvacrol when present at non-lethal concentrations in the growth medium by lowering its membrane fluidity by changing the fatty acid and headgroup composition.

J Am Mosq Control Assoc, 2000 Sep, 16(3), 229 - 33
Effectiveness of aerial- and ground-applied Bacillus formulations against Anopheles quadrimaculatus larvae in Arkansas rice plots; Dennett JA et al.; Experimental Bacillus larvicides designed to float on or near the water surface were compared to labeled standard Bacillus corn-cob-based larvicides using sentinel Anopheles quadrimaculatus larvae in Arkansas rice plots during the 1998 growing season . Experimental floating formulations of Bacillus thuringiensis israelensis applied at 5.58 and 11.18 kg/ha provided up to 100% control of 3rd- and 4th-stage Anopheles larvae within 24-48 h, whereas the water-dispersible granule formulations containing Bacillus sphaericus required 48-72 h to yield >75% mortality in 0.16-ha plots at 11.18 kg/ha . Detecting and targeting the smaller developmental stages (1st- and 2nd-stage larvae) could increase the effectiveness of the tested compounds against An . quadrimaculatus in Arkansas and other rice-growing regions.

Curr Microbiol, 2000 Dec, 41(6), 430 - 40
Pesticidal and receptor binding properties of Bacillus thuringiensis Cry1Ab and Cry1Ac delta-endotoxin mutants to Pectinophora gossypiella and Helicoverpa zea; Karim S et al.; Bacillus thuringiensis produces several larvicidal crystalline inclusions during sporulation . An understanding of their mechanisms of action is commercially important . In this study, two toxins, Cry1Ab and Cry1Ac, were compared that showed 98% amino acid identity in domain I and II, but differed significantly in domain III . Using site-directed mutagenesis techniques, two conserved loop 2 Arg's ((368)RR(369)) of Cry1Ab and Cry1Ac toxins were replaced with Ala ((368)AR(369), (368)RA(369), (368)AA(369)), Glu ((368)EE(369)), Phe ((368)FF(369)), His ((368)HH(369)), and Lys ((368)KK(369)) . The effect of these mutants on structural stability, larvicidal potency, receptor binding, and ionic permeability towards two important cotton pests, pink bollworm (Pectinophora gossypiella) and bollworm (Helicoverpa zea) were analyzed . All seven mutants of Cry1Ab, excluding (368)AR(369), produced a stable protoxin, whereas for Cry1Ac all seven mutants yielded stable protoxin . Results showed that all the stable mutants behaved similarly to the wild type on incubation with trypsin and gut extract of both insect larvae . The Cry1Ab mutants, (368)AR(369), (368)AA(369), (368)FF(369), and (368)HH(369), lost toxicity; (368)EE(369) had reduced toxicity; whereas the more conserved change (368)KK(369) retained the toxicity similar to the wild type towards P . gossypiella . Double mutants of Cry1Ac, (368)AA(369) and (368)FF(369), abolished the toxicity . Double mutant (368)KK(369) of Cry1Ac retained its toxicity against P . gossypiella, whereas single mutants (368)AR(369), (368)RA(369), and (368)HH(369) retained only reduced toxicity . All the mutants of Cry1Ab lost their toxicity against H . zea except (368)KK(369) . In Cry1Ac single mutants, (368)AR(369) and (368)RA(369), reduction in the toxicity was observed . A double mutant of Cry1Ac, (368)KK(369), also retained reduced toxicity . All the other double mutants lost their toxicity . Voltage clamping experiments on H . zea midguts provided an additional evidence about the insecticidal property and inhibition of I(sc) across the transepithelial membrane of the insect midgut.

Curr Microbiol, 2000 Dec, 41(6), 396 - 401
Characterization of INTA 51-3, a new atypical strain of Bacillus thuringiensis from Argentina; Benintende GB et al.; Several isolates of Bacillus thuringiensis native to Argentina obtained in a nationwide screening program showed atypical crystal morphology . One of these strains, INTA 51-3, was further characterized in order to determine other features like protein composition of its parasporal crystal, plasmid pattern, identification of cry genes and toxicological properties . B . thuringiensis INTA 51-3 (serovar tohokuensis) had an amorphous inclusion containing a major protein component of ca . 130 kDa . After trypsin digestion of solubilized crystals, SDS-PAGE resolved a unique protease-resistant peptide of ca . 90 kDa . The plasmid pattern from INTA 51-3 resembled that of the standard strain HD-1 . However, Southern analysis showed no hybridization to fragments of cry1Aa, cry2Aa, cry3A, and cry11A genes . Degenerate primers were used for identification of the cry1 genes by PCR . Nevertheless, the presence of cry1 type gene(s) in B . thuringiensis INTA 51-3 was confirmed . Highly concentrated crystal suspensions showed to be weakly toxic only to lepidopteran species.

Curr Microbiol, 2000 Dec, 41(6), 388 - 91
Peroxide reductase activity of NADH dehydrogenase in the presence of an endogenous 20-kDa component of an alkaliphilic Bacillus; Koitabashi T et al.; The membrane-bound NADH dehydrogenase of an alkaliphilic Bacillus YN-1 involved in the respiratory chain exhibits reductase activity for hydrogen peroxide and cumene hydroperoxide in the presence of the 22-kDa component (AhpC) from Amphibacillus xylanus (Koyama et al . Biochem . Biophys . Res . Commun . 247, 659-662) . In this study, AhpC-like polypeptide with an apparent molecular mass of 20 kDa was isolated from the cell-free extract of YN-1 . The NADH dehydrogenase exhibited reductase activity for cumene hydroperoxide in the presence of the purified AhpC-like component from YN-1 . It is likely that the NADH dehydrogenase is not only involved in the respiratory chain, but also functions for scavenging peroxide in the presence of its own endogenous AhpC component . The enzyme expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST) showed the NADH dehydrogenase activity as high as the native enzyme from YN-1 . While the fusion protein was unable to reduce cumene hydroperoxide in the presence of AhpC-like protein from YN-1, the protein obtained by the cleavage treatment of the fusion protein to release GST exhibited the reductase activity as much as the native enzyme.

J Food Prot, 2000 Nov, 63(11), 1534 - 7
Ultraviolet spectrophotometric characterization and bactericidal properties of electrolyzed oxidizing water as influenced by amperage and pH; Len SV et al.; To identify the primary component responsible in electrolyzed oxidizing (EO) water for inactivation, this study determined the concentrations of hypochlorous acid (HOCl) and hypochlorite ions (OCl-) and related those concentrations to the microbicidal activity of the water . The ultraviolet absorption spectra were used to determine the concentrations of HOCl and OCl- in EO water and the chemical equilibrium of these species with change in pH and amperage . EO water generated at higher amperage contained a higher chlorine concentration . The maximum concentration of HOCl was observed around pH 4 where the maximum log reduction (2.3 log10 CFU/ml) of Bacillus cereus F4431/73 vegetative cells also occurred . The high correlation (r = 0.95) between HOCl concentrations and bactericidal effectiveness of EO water supports HOCl's role as the primary inactivation agent . Caution should be taken with standard titrimetric methods for measurement of chlorine as they cannot differentiate the levels of HOCl present in EO water of varying pHs.

J Food Prot, 2000 Nov, 63(11), 1496 - 502
Bacillus cereus group strains, their hemolysin BL activity, and their detection in foods using a 16S RNA and hemolysin BL gene-targeted multiplex polymerase chain reaction system; Tsen HY et al.; Hemolysin BL (HBL) is a major virulence factor for Bacillus cereus group strains . It is also a target enterotoxin for the most commonly used B . cereus detection kit, i.e., the B . cereus enterotoxin (diarrheal type) reversed passive latex agglutination (BCET-RPLA) test kit . A survey of the HBL activities and the cytotoxicities to the Chinese hamster ovary (CHO) cells for the B . cereus group strains, however, showed that although only part of the B . cereus group strains are HBL active, all strains show cytotoxicity to the CHO cells . Thus, methods that allow the detection of not only the HBL but also of the B . cereus group strains are important . In this study, by comparison of the gene sequences of the 16S rRNA for B . cereus group and other bacteria strains, we designed primers B16S1 and B16S2 specific to all the B . cereus group strains . In addition, because HBL is a major enterotoxin, we also designed HBL gene-specific polymerase chain reaction (PCR) primers, i.e., Hm1 and Hm2, that generated the same results as those of the hemolysis and BCET-RPLA assays . Primers B16S1/B16S2 and Hm1/Hm2 could be combined into a multiplex PCR system for the simultaneous detection of B . cereus group cells and the possible presence of their HBL enterotoxins . Also, all these PCR systems allowed the detection of n x 10(0) CFU B . cereus cells per g of food sample if an 8-h enrichment step was performed prior to the PCR.

Plasmid, 2000 Nov, 44(3), 280 - 4
Molecular analysis of two rolling-circle replicating cryptic plasmids, pBMYdx and pBMY1, from the soil gram-positive Bacillus mycoides; Di Franco C et al.; Two cryptic plasmids of two environmental strains of the soil Bacillus mycoides were cloned and sequenced . They are of a small size (3377 and 3476 bp) and carry regions homologous to double- and single-strand origins of replication of rolling-circle replication modules . In addition, both plasmids have ORFs with homologies with Mob and Rep proteins, in the same relative position and orientation . While dso- and sso-like sequences are similar in pBMY1 and pBMYdx, the putative Mob and Rep proteins are not homologous between the two but show similarity with Mob and Rep proteins of different bacterial plasmids .

Int J Food Microbiol, 2000 Nov 1, 61(2-3), 193 - 7
Acid and bile tolerance of spore-forming lactic acid bacteria; Hyronimus B et al.; Criteria for screening probiotics such as bile tolerance and resistance to acids were studied with 13 spore-forming lactic acid producing bacteria . Different strains of Sporolactobacillus, Bacillus laevolacticus, Bacillus racemilacticus and Bacillus coagulans grown in MRS broth were subjected to low pH conditions (2, 2.5 and 3) and increasing bile concentrations . Among these microorganisms, Bacillus laevolacticus DSM 6475 and all Sporolactobacillus strains tested except Sporolactobacillus racemicus IAM 12395, were resistant to pH 3 . Only Bacillus racemilacticus and Bacillus coagulans strains were tolerant to bile concentrations over 0.3% (w/v).

Occup Environ Med, 2000 Dec, 57(12), 840 - 2
Occupational asthma caused by bacillary amylase used in the detergent industry; Hole AM et al.; Four cases are reported of occupational asthma due to amylase derived from Bacillus licheniformis, used in detergent washing powders . It is thought that these are the first reported cases of asthma due to this enzyme in the detergent industry . All four employees (men) were from the same factory and none had a history of asthma or atopy . All developed symptoms of wheeze at work after an initial symptom free period . Symptoms improved during periods away from work . All undertook serial peak flow recordings (not diagnostic) and underwent skin prick tests, radio allergosorbent test (RAST) measurement, and specific bronchial provocation testing . The bronchial provocation testing was performed by a dust tipping method in a single blind manner, with lactose as an inert control and powdered amylase, provided by the employer, as an active agent . Serial measurements of forced expiratory volume in 1 second (FEV(1)) were recorded and histamine provocative concentration causing a 20% fall in FEV(1) (PC(20)) tests were determined before and 24 hours after each challenge . Patient 1 developed an isolated early reaction, patient 2 an isolated late reaction, and patients 3 and 4 developed dual reactions . All showed an increased non-specific bronchial responsiveness after active challenge . The introduction of encapsulated enzymes in the detergent industry was followed by a reduction in the incidence of respiratory sensitisation . These patients developed occupational asthma despite working only with encapsulated enzymes . This highlights the importance of careful surveillance after the introduction of new agents in the workplace.

J Mol Microbiol Biotechnol, 2000 Oct, 2(4), 587 - 92
Sugar uptake and carbon catabolite repression in Bacillus megaterium strains with inactivated ptsHI; Wagner A et al.; We have determined the role played by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in carbon catabolite repression (CCR) of xylose utilization in Bacillus megaterium . For that purpose we have cloned, sequenced and inactivated the genes ptsH and ptsl of B . megaterium, encoding HPr and EI of the PTS, respectively . While glucose uptake of a ptsHI mutant is not affected at 12.5 mM of glucose, CCR of the xyl operon is reduced in this mutant from 16-fold to 3-fold . This may be attributed to the loss of the corepressor of CcpA, HPr(Ser-P), or could result from the slower growth rate of the mutant . In contrast, CCR exerted by fructose or mannitol is completely abolished . We conclude that glucose triggers additional mechanisms of CCR than fructose or mannitol . The remaining 3-fold glucose repression is relieved in a strain in which ptsHI and glk, encoding glucokinase, are inactivated . This result indicates that glucose metabolism is necessary for CCR . The ability of the ptsHI mutant to take up glucose suggests the existence of a second, non-PTS glucose uptake system . The Km and vmax values of this transporter ranged between 2 and 5 mM and 154 to 219 nmol/{(mg protein)*min}, respectively.

Z Gastroenterol, 2000 Sep, 38(9), 785 - 9
{Bacillary angiomatosis of the liver, a suspected ultrasound diagnosis?}; Braden B et al.; Infections with Bartonella henselae or Bartonella quintana present with vasoproliferative lesions in skin and parenchymatous organs in immunocompromised patients . A case report of a 38-year-old patient with HIV infection and hepatitis B surface antigen status is described . The dominant clinical symptoms in our patient were fever and icterus . Ultrasonography of the abdomen showed a picture similar to that of liver cirrhosis . Irregular (echorich) nodes with hyper-vascularization were suspected to be hepatocellular carcinoma . Ultrasound guided puncture of one of these lesions and histological examination revealed the diagnosis of bartonella infection . Under antibiotic treatment with clarithromycin and doxycycline the fever and the hyperbilirubinemia decreased . The sonographically detectable lesions reduced in size . Vasoproliferative lesions in immunodeficient patients caused by bartonella infection show a characteristic slightly hyperechogenic irregular pattern at ultrasound . Typically, circumscribed hypervascularization might be shown by color Doppler imaging . Liver cirrhosis and diffuse tumor infiltration should be excluded.

Mol Microbiol, 2000 Oct, 38(2), 289 - 98
Role of two arginine residues in domain II, loop 2 of Cry1Ab and Cry1Ac Bacillus thuringiensis delta-endotoxin in toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N; Lee MK et al.; Two arginine residues (368-369) of Cry1Ab and Cry1Ac were mutated to alanine, glutamic acid and lysine by site-directed mutagenesis . Insecticidal activities of the mutant toxins on Manduca sexta and Lymantria dispar larvae were examined . Cry1Ac mutant toxins (c)RR-AA and (c)RR-EE and Cry1Ab mutant toxins (b)RR-AA and (b)RR-EE showed great reductions in toxicity against both insects . In contrast, conservatively changed (c)RR-KK and (b)RR-KK mutants did not alter toxicity to either insect . Binding assays with brush border membrane vesicles (BBMVs) prepared from L . dispar midguts demonstrated that (c)RR-AA, (c)RR-EE, (b)RR-AA and (b)RR-EE bound with lower affinities compared with their respective wild-type toxins . To M . sexta BBMVs, (c)RR-AA and (c)RR-EE showed great reductions in BBMV binding . However, (b)RR-AA and (b)RR-EE did not alter BBMV competition patterns, despite their reduced toxicity . Further binding assays were performed with aminopeptidase N (APN) purified from L . dispar and M . sexta BBMVs using surface plasmon resonance (BIAcore) . Direct correlation between toxicity and APN binding was observed for the mutant toxins using this technique . The inconsistency between BBMV and APN binding data with Cry1Ab to M . sexta suggests the possibility of a different Cry1Ab toxin-binding mechanism or the importance of another receptor in M . sexta.

Mol Microbiol, 2000 Oct, 38(2), 198 - 212
The trans-activation domain of the sporulation response regulator Spo0A revealed by X-ray crystallography; Lewis RJ et al.; Sporulation in Bacillus involves the induction of scores of genes in a temporally and spatially co-ordinated programme of cell development . Its initiation is under the control of an expanded two-component signal transduction system termed a phosphorelay . The master control element in the decision to sporulate is the response regulator, Spo0A, which comprises a receiver or phosphoacceptor domain and an effector or transcription activation domain . The receiver domain of Spo0A shares sequence similarity with numerous response regulators, and its structure has been determined in phosphorylated and unphosphorylated forms . However, the effector domain (C-Spo0A) has no detectable sequence similarity to any other protein, and this lack of structural information is an obstacle to understanding how DNA binding and transcription activation are controlled by phosphorylation in Spo0A . Here, we report the crystal structure of C-Spo0A from Bacillus stearothermophilus revealing a single alpha-helical domain comprising six alpha-helices in an unprecedented fold . The structure contains a helix-turn-helix as part of a three alpha-helical bundle reminiscent of the catabolite gene activator protein (CAP), suggesting a mechanism for DNA binding . The residues implicated in forming the sigmaA-activating region clearly cluster in a flexible segment of the polypeptide on the opposite side of the structure from that predicted to interact with DNA . The structural results are discussed in the context of the rich array of existing mutational data.

Lett Appl Microbiol, 2000 Nov, 31(5), 385 - 9
Bacillus cereus produces most emetic toxin at lower temperatures; Finlay WJ et al.; Seven emetic toxin-producing strains of Bacillus cereus were examined for toxin production in Skim Milk Medium at incubation temperatures ranging from 10 to 50 degrees C . Minimum and maximum growth temperatures were found to be 12 and 46 degrees C, respectively . At 12 and 15 degrees C, levels of toxin production were significantly higher (P < 0.01) than that observed at 30 degrees C, while no toxin was produced above 37 degrees C . Increased levels of sporulation were observed at increased temperatures, and no correlation was found between levels of sporulation and toxin production (R(2) = 0.086).

J Infect Dis, 2000 Dec, 182(6), 1800 - 3 Epub 2000 Nov 08.
Lower hookworm incidence, prevalence, and intensity of infection in children with a Bacillus Calmette-Guérin vaccination scar; Barreto ML et al.; Bacillus Calmette-Guerin (BCG), the most common vaccine worldwide, has broad effects on the immune system . Hookworm infections are a major source of morbidity . In response to a preliminary report of BCG vaccination protection against nematodes in human immunodeficiency virus-infected adults, data from an ongoing prospective study were analyzed to determine the intensity (eggs per gram of stool), prevalence, and incidence of different helminths in children with and without a BCG vaccination scar . Adjusted prevalence and incidence ratios were estimated by using logistic regression . Children with a BCG vaccination scar were found to have statistically significantly lower hookworm prevalence (41%), incidence (37%), and mean egg counts (39%), after controlling for age, sex, and socioeconomic factors . There was no BCG association with incidence, prevalence, or intensity of infection with Schistosoma mansoni, Ascaris lumbricoides, or Trichuris trichiura . Such protection would have implications for public health and for research on mechanisms behind human immunological responses to hookworm.

Can J Microbiol, 2000 Oct, 46(10), 913 - 9
Characterization of the anti-cancer-cell parasporal proteins of a Bacillus thuringiensis isolate; Yamashita S et al.; An unusual activity, associated with non-insecticidal and non-haemolytic parasporal inclusion proteins of a Bacillus thuringiensis soil isolate, designated 89-T-26-17, was characterized . The parasporal inclusion of this isolate was bipyramidal, rounded at both ends, containing proteins of 180, 150, 120, 100, and 88 kDa . No homologies with the Cry and Cyt proteins of B . thuringiensis were detected based on N-terminal sequences . Proteolytic processing of the inclusion proteins by proteinase K, trypsin, and chymotrypsin produced a major protein of 64 kDa exhibiting cytocidal activity against human leukaemic T cells and uterus cervix cancer (HeLa) cells . The protease-activated proteins showed no cytotoxicity to normal T cells.

Regul Toxicol Pharmacol, 2000 Oct, 32(2), 156 - 73
Safety and advantages of Bacillus thuringiensis-protected plants to control insect pests; Betz FS et al.; Plants modified to express insecticidal proteins from Bacillus thuringiensis (referred to as Bt-protected plants) provide a safe and highly effective method of insect control . Bt-protected corn, cotton, and potato were introduced into the United States in 1995/1996 and grown on a total of approximately 10 million acres in 1997, 20 million acres in 1998, and 29 million acres globally in 1999 . The extremely rapid adoption of these Bt-protected crops demonstrates the outstanding grower satisfaction of the performance and value of these products . These crops provide highly effective control of major insect pests such as the European corn borer, southwestern corn borer, tobacco budworm, cotton bollworm, pink bollworm, and Colorado potato beetle and reduce reliance on conventional chemical pesticides . They have provided notably higher yields in cotton and corn . The estimated total net savings to the grower using Bt-protected cotton in the United States was approximately $92 million in 1998 . Other benefits of these crops include reduced levels of the fungal toxin fumonisin in corn and the opportunity for supplemental pest control by beneficial insects due to the reduced use of broad-spectrum insecticides . Insect resistance management plans are being implemented to ensure the prolonged effectiveness of these products . Extensive testing of Bt-protected crops has been conducted which establishes the safety of these products to humans, animals, and the environment . Acute, subchronic, and chronic toxicology studies conducted over the past 40 years establish the safety of the microbial Bt products, including their expressed insecticidal (Cry) proteins, which are fully approved for marketing . Mammalian toxicology and digestive fate studies, which have been conducted with the proteins produced in the currently approved Bt-protected plant products, have confirmed that these Cry proteins are nontoxic to humans and pose no significant concern for allergenicity . Food and feed derived from Bt-protected crops which have been fully approved by regulatory agencies have been shown to be substantially equivalent to the food and feed derived from conventional crops . Nontarget organisms exposed to high levels of Cry protein are virtually unaffected, except for certain insects that are closely related to the target pests . Because the Cry protein is contained within the plant (in microgram quantities), the potential for exposure to farm workers and nontarget organisms is extremely low . The Cry proteins produced in Bt-protected crops have been shown to rapidly degrade when crop residue is incorporated into the soil . Thus the environmental impact of these crops is negligible . The human and environmental safety of Bt-protected crops is further supported by the long history of safe use for Bt microbial pesticides around the world .

Microbiology, 2000 Nov, 146 ( Pt 11), 2825 - 32
The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects; Salamitou S et al.; Bacillus thuringiensis has been widely used for 40 years as a safe biopesticide for controlling agricultural pests and mosquitoes because it produces insecticidal crystal proteins . However, spores have also been shown to contribute to overall entomopathogenicity . Here, the opportunistic properties of acrystalliferous B . thuringiensis Cry(-) and Bacillus cereus strains were investigated in an insect species, Galleria mellonella, and in a mammal, BALB/c mice . In both animal models, the pathogenicity of the two bacterial species was similar . Mutant strains were constructed in which the plcR gene, encoding a pleiotropic regulator of extracellular factors, was disrupted . In larvae, co-ingestion of 10(6) spores of the parental strain with a sublethal concentration of Cry1C toxin caused 70% mortality whereas only 7% mortality was recorded if spores of the DeltaplcR mutant strain were used . In mice, nasal instillation of 10(8) spores of the parental strain caused 100% mortality whereas instillation with the same number of DeltaplcR strain spores caused much lower or no mortality . Similar effects were obtained if vegetative cells were used instead of spores . The cause of death is unknown and is unlikely to be due to actual growth of the bacteria in mice . The lesions caused by B . thuringiensis supernatant in infected mice suggested that haemolytic toxins were involved . The cytolytic properties of strains of B . thuringiensis and B . cereus, using sheep, horse and human erythrocytes and G . mellonella haemocytes, were therefore investigated . The level of cytolytic activity is highly reduced in DeltaplcR strains . Together, the results indicate that the pathogenicity of B . thuringiensis strain 407 and B . cereus strain ATCC 14579 is controlled by PlcR.

J Clin Pathol, 2000 Oct, 53(10), 750 - 5
Whipple's disease revisited; Misbah SA et al.; Whipple's disease has traditionally been considered to be a rare multisystem disorder dominated by malabsorption . The recent identification of the Whipple's disease bacillus has, using polymerase chain reaction based assays, fueled advances in the investigation, diagnosis, and management of this disease . This leader reviews the aetiology, clinical manifestations, investigation, and treatment of Whipple's disease in the light of this new information.

FEMS Immunol Med Microbiol, 2000 Nov, 29(3), 177 - 81
Super-infection by Bacillus thuringiensis H34 or 3a3b can lead to death in mice infected with the influenza A virus; Hernandez E et al.; Bacterial super-infections are the main cause of complication and mortality after influenza virus (IAV) infection . Since Bacillus thuringiensis (Bt) is considered non-pathogenic for humans and is widely sprayed in urban areas, the aim of this work was to evaluate the potential pathogenicity of a combined infection Bt-IAV in a mouse model of pneumonia . Bacteria used for super-infections were Bt serotype H34 isolated from human infection and the insecticidal strain 3a3b obtained from a commercial source . Virus strain was A/Scotland/20/74 (H3N2) adapted to BALB/c mice by serial lung passage . Combined infection with 4% of the viral lethal dose 50% (LD(50)) and 10(2) spores of Bt H34 killed 40% of the mice . Mortality rates increased up to 55% and 100% when combined infections were done with respectively 10(4) and 10(7) spores . The insecticidal strain Bt 3a3b was less pathogenic than Bt H34 . A dose of 10(4) spores associated with 4% of IAV LD(50) killed 50% of the mice . This inoculum must be compared with the doses usually sprayed in agriculture: 10(11) spores m(-2) . Total protection against super-infection was obtained when mice were treated with amantadine . Even if only a few cases of Bt human infection have been reported, these results suggest a possible risk for workers spraying Bt-based biopesticides during flu outbreaks.

FEMS Microbiol Lett, 2000 Nov 15, 192(2), 179 - 83
Progesterone side-chain cleavage by Bacillus sphaericus; Wadhwa L et al.; The side-chain of progesterone was cleaved by Bacillus sphaericus to produce two C-19 keto androstene steroids . The structures of these metabolites were androstenedione and 1-dehydroandrostenedione . High concentrations of glucose in the culture medium inhibited conversion of progesterone to these two metabolites.

Immunol Lett, 2000 Nov 1, 74(3), 183 - 8
Systemic or local co-administration of lactoferrin with sensitizing dose of antigen enhances delayed type hypersensitivity in mice; Zimecki M et al.; Lactoferrin (LF), a major defense protein synthesized and stored in granulocytes has been implicated in maintaining immune homeostasis during an insult-induced metabolic imbalance . In this study, we demonstrated that lactoferrin augments the delayed type hypersensitivity (DTH) response to specific antigens in mice . Lactoferrin (LF) was given to mice orally or intraperitoneally (i.p . ) at the time of immunization, or subcutaneously (s.c.) in a mixture with the immunizing doses of the following antigens, sheep red blood cells (SRBC), Calmette-Guerin bacillus (BCG) or ovalbumin (OVA) . A DTH reaction was determined 24 h after administration of an eliciting dose of antigen as a specific increase in foot pad swelling . Lactoferrin enhanced DTH reaction to all studied antigens in a dose-dependent manner . Lactoferrin (LF) given to mice in conjunction with antigen administered in an incomplete Freund's adjuvant induced the DTH response at the level of control mice given antigen in a complete Freund's adjuvant . In addition, LF remarkably increased DTH response to a very small, otherwise non-immunogenic SRBC dose . The increase in DTH response was less pronounced for orally administered LF than for any other routes of administration, however, statistically significant augmentation was demonstrated for each antigen studied . Although the costimulatory action of LF was accompanied by the appearance of bovine lactoferrin-specific cellular responses in mice, it is very unlikely that such responses will be generated in humans, since bovine lactoferrin is a dietary antigen to which a tolerance has been acquired . Considering the involvement of LF in generation of stimulatory signals during the induction phase of an antigen specific immune responses, we suggest that LF may be useful for development of safer and more efficacious vaccination protocols.

Enzyme Microb Technol, 2000 Nov 15, 27(9), 704 - 708
Addition of polar organic solvents can improve the product selectivity of cyclodextrin glycosyltransferase . Solvent effects on cgtase; Blackwood AD et al.; Cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) is an enzyme that produces cyclodextrins from starch via an intramolecular transglycosylation reaction . Addition of small amounts (10% v/v) of polar organic solvents can affect both the overall production yield and the type of cyclodextrin produced from a maltodextrin substrate under simulated industrial process conditions . Using CGTase from Thermoanaerobacter sp . all solvents produced an increase in cyclodextrin yield when compared with a control, the greatest increase being obtained with addition of ethanol (26%) . In addition product selectivity was affected by the nature of the organic solvent used: beta-cyclodextrin was favoured in the absence of any solvent and on the addition of dimethylsulphoxide, t-butanol and dimethylformanide while alpha-cyclodextrin was favoured by addition of acetonitrile, ethanol and tetrahydrofuran . With CGTase from Bacillus circulans strain 251 relatively smaller increases in overall cyclodextrin production were achieved (between 5-10%) . Addition of t-butanol to a B . circulans catalysed reaction however did produce the largest selectivity for beta-cyclodextrin of any solvent-enzyme combination (82%) . The effect of solvent addition was shown not to be related to the product inhibition of CGTase, but may be related to reduced competition from the intermolecular transglycosylation reaction that causes degradation of cyclodextrin products . This rate of this reaction was shown to be dependent on the nature of the organic solvent used.

Microbiol Res, 2000 Sep, 155(3), 205 - 7
Growth and survival of phosphate-solubilizing bacteria in calcium alginate; Viveganandan G et al.; Calcium alginate was superior to conventional charcoal-soil (3:1) carrier for phosphate-solubilizing bacteria (PSB) . High populations of Pseudomonas striata (27) and Bacillus polymyxa (H-5) could be maintained in this polymer during storage . Incorporation of charcoal-soil (3:1) adversely affected the initial loading of these organisms in alginate gel . Alginate alone supported maximum survival of these organisms at elevated storage temperature (40 degrees C).

J Lipid Res, 2000 Nov, 41(11), 1832 - 40
Kinetic steps for the hydrolysis of sphingomyelin by Bacillus cereus sphingomyelinase in lipid monolayers; Fanani ML et al.; The sphingomyelinase (Sphmase) activity degrading sphingomyelin (Sphm) monolayers shows a slow-reaction latency period before exhibiting constant rate catalysis . These two kinetic regions are regulated independently by the lateral surface pressure and by lipids that are biomodulators of cell function such as ceramide, glycosphingolipids, fatty acids, and lysophospholipids . Knowledge of the interfacial adsorption of Sphmase, precatalytic activation, initiation of effective catalysis, and the corresponding kinetic parameters is necessary for studying the level at which different lipids modulate the activity . We dissected some kinetic steps and determined the rate constants for degradation of Sphm, under controlled intermolecular organization, by Sphmase . Six models, adapted to two dimensions, were used to elucidate possible mechanisms for the interfacial activation of Sphmase during the lag time . The models consider enzyme binding to the substrate monolayer and a subsequent, essentially irreversible interfacial activation; this is supported experimentally by monolayer transfer experiments . Some mechanisms involve enzyme-substrate binding and associated states of the enzyme in the bulk subphase or at the interface, prior to complete activation . The activity of Sphmase is consistent with kinetics involving enzyme partitioning into the interface followed by substrate association, and by a process that proceeds with bimolecular kinetic dependence on the interfacial Sphmase concentration, and a subsequent slow step of activation . A possible equilibrium between the apparent monomolecular and bimolecular activated states of the interfacial enzyme, coupled to a slow activation, constitute rate-limiting steps that can explain the existence of lag time and the achievement of a maximum constant rate of degradation of Sphm monolayers by Sphmase.

J Clin Microbiol, 2000 Nov, 38(11), 4285 - 7
Value of examining three acid-fast bacillus sputum smears for removal of patients suspected of having tuberculosis from the "airborne precautions" category; Craft DW et al.; We examined the potential risk of tuberculosis transmission if we modified our policy for release of patients from the "airborne precautions" category from three negative acid-fast bacillus (AFB) smears to two, or even one . Over a 4-year period, respiratory cultures from 42 patients grew Mycobacterium tuberculosis . Of these, 36 patients (81%) had a positive AFB smear result on the first submitted specimen . One additional patient (2%) had a first smear-positive finding on the second submitted specimen, and no patients had a first smear-positive result on the third submitted specimen . Respiratory cultures from five patients (12%) grew M . tuberculosis without ever having a positive AFB smear result . These data indicate that in our institution, reducing the number of negative smears required before removal of patients from the airborne precautions category would pose little, if any, increase in the risk of spreading tuberculosis.

J Clin Microbiol, 2000 Nov, 38(11), 4269 - 71
Serodiagnosis of Bartonella bacilliformis infection by indirect fluorescence antibody assay: test development and application to a population in an area of bartonellosis endemicity; Chamberlin J et al.; Bartonella bacilliformis causes bartonellosis, a potentially life-threatening emerging infectious disease seen in the Andes Mountains of South America . There are no generally accepted serologic tests to confirm the disease . We developed an indirect fluorescence antibody (IFA) test for the detection of antibodies to B . bacilliformis and then tested its performance as an aid in the diagnosis of acute bartonellosis . The IFA is 82% sensitive in detecting B . bacilliformis antibodies in acute-phase blood samples of laboratory-confirmed bartonellosis patients . When used to examine convalescent-phase sera, the IFA is positive in 93% of bartonellosis cases . The positive predictive value of the test is 89% in an area of Peru where B . bacilliformis is endemic and where the point prevalence of infection is 45%.

J Clin Microbiol, 2000 Nov, 38(11), 4131 - 6
Outbreak of Bacillus cereus infections in a neonatal intensive care unit traced to balloons used in manual ventilation; Van Der Zwet WC et al.; In 1998, an outbreak of systemic infections caused by Bacillus cereus occurred in the Neonatal Intensive Care Unit of the University Hospital Vrije Universiteit, Amsterdam, The Netherlands . Three neonates developed sepsis with positive blood cultures . One neonate died, and the other two neonates recovered . An environmental survey, a prospective surveillance study of neonates, and a case control study were performed, in combination with molecular typing, in order to identify potential sources and transmission routes of infection . Genotypic fingerprinting by amplified-fragment length polymorphism (AFLP) showed that the three infections were caused by a single clonal type of B . cereus . The same strain was found in trachea aspirate specimens of 35 other neonates . The case control study showed mechanical ventilation with a Sensormedics ventilation machine to be a risk factor for colonization and/or infection (odds ratio, 9.8; 95% confidence interval, 1.1 to 88.2) . Prospective surveillance showed that colonization with B . cereus occurred exclusively in the respiratory tract of mechanically ventilated neonates . The epidemic strain of B . cereus was found on the hands of nursing staff and in balloons used for manual ventilation . Sterilization of these balloons ended the outbreak . We conclude that B . cereus can cause outbreaks of severe opportunistic infection in neonates . Typing by AFLP proved very useful in the identification of the outbreak and in the analysis of strains recovered from the environment to trace the cause of the epidemic.

Sheng Wu Gong Cheng Xue Bao, 2000 May, 16(3), 392 - 5
{Study on continuous synthesis of galacto-oligosaccharide by immobilized Bacillus stearothermophilus}; Wei DZ et al.; The galacto-oligosaccharide was synthesized continuously by immobilized Bacillus stearothermophilu producing beta-galactosidase in fibrous bed reactor . The effect of substrate concentration, pH, reaction temperature and retention time on production of GOS was investigated . The optimal reaction conditions were determined . Substrate concentration were 450 g/L; Reaction temperature was 55 degrees C; pH was 7.0; Residence time was 100 min . The product yield reached up to 50.7% . GOS synthesis was promoted by feeding 1.5% D-glactose after 24 h . The immobilized cell reactor can work stably for 120 h.

J Econ Entomol, 2000 Oct, 93(5), 1515 - 21
Frequency of alleles conferring resistance to a Bacillus thuringiensis toxin in a Philippine population of Scirpophaga incertulas (Lepidoptera: Pyralidae); Bentur JS et al.; Using the F2 screen methodology, we estimated the frequency of alleles conferring resistance to the Cry1Ab toxin of Bacillus thuringiensis Berliner in a Philippine population of the stem borer Scirpophaga incertulas (Walker) . Evaluation of >450 isofemale lines for survival of F2 larvae on cry1Ab plants did not detect the presence of an allele conferring a high level of resistance . The frequency of such an allele in the sampled population was conservatively estimated to be <3.6 x 10(-3) with 95% confidence and a detection probability of 94% . However, there was evidence of the presence of alleles conferring partial resistance to Cry1Ab . The frequency of alleles for partial resistance was estimated as 4.8 x 10(-3) with a 95% CI between 1.3 x 10(-3) and 1.04 x 10(-2) and a detection probability of 94% . Our results suggest that the frequency of alleles conferring resistance to Cry1Ab in the population of S . incertulas sampled is not too high to preclude successful implementation of the high dose/refuge resistance management strategy.

Nature, 2000 Oct 19, 407(6806), 897 - 900
Isolation of a 250 million-year-old halotolerant bacterium from a primary salt crystal; Vreeland RH et al.; Bacteria have been found associated with a variety of ancient samples, however few studies are generally accepted due to questions about sample quality and contamination . When Cano and Borucki isolated a strain of Bacillus sphaericus from an extinct bee trapped in 25-30 million-year-old amber, careful sample selection and stringent sterilization techniques were the keys to acceptance . Here we report the isolation and growth of a previously unrecognized spore-forming bacterium (Bacillus species, designated 2-9-3) from a brine inclusion within a 250 million-year-old salt crystal from the Permian Salado Formation . Complete gene sequences of the 16S ribosomal DNA show that the organism is part of the lineage of Bacillus marismortui and Virgibacillus pantothenticus . Delicate crystal structures and sedimentary features indicate the salt has not recrystallized since formation . Samples were rejected if brine inclusions showed physical signs of possible contamination . Surfaces of salt crystal samples were sterilized with strong alkali and acid before extracting brines from inclusions . Sterilization procedures reduce the probability of contamination to less than 1 in 10(9).

Chemosphere, 2000 Aug, 41(3), 447 - 52
Removal of 2,4-dichlorophenol by suspended and immobilized Bacillus insolitus; Wang CC et al.; In this experiment, Bacillus insolitus was isolated and selected from a mixed culture that have been acclimated to chlorophenols . Decomposition of chlorophenolic compounds will be studied using this pure culture in both suspended and immobilized form . The results are: at lower initial concentrations of 2,4-dichlorophenol (10-50 mg/l), immobilized Bacillus insolitus shows a higher removal of 2,4-dichlorophenol than Bacillus insolitus in suspended growth . When the 2,4-dichlorophenol concentration becomes higher (50-200 mg/l), both immobilized and suspended Bacillus insolitus have approximately the same efficiency for removal of 2,4-dichlorophenol . Higher concentrations of 2,4-dichlorophenol are inhibitive to the growth of either suspended or immobilized Bacillus insolitus . At lower concentrations of 2,4-dichlorophenol, immobilized mixed culture may have the same removal efficiency of 2,4-dichlorophenol as immobilized pure culture of Bacillus insolitus . But with regard to the overall 2,4-dichlorophenol removal efficiency, immobilized pure culture is considered to be superior to immobilized mixed culture.

PDA J Pharm Sci Technol, 2000 Sep-Oct, 54(5), 398 - 412
The effect of media composition on the thermal resistance of Bacillus stearothermophilus; Penna TC et al.; Spores of Bacillus stearothermophilus ATCC 7953 were developed at 62 degrees C on 32 media composed of various amounts of 11 components: D-glucose, L-glutamic acid, yeast extract, peptone, sodium chloride, magnesium sulfate, ammonium phosphate, potassium phosphate, calcium chloride, ferrous sulfate and manganese sulfate . Statistical models were used and demonstrated a strong interaction of yeast/peptone/ammonium phosphate, contributing positively to the best sporulation yield (6-7 log10 spores) . The most influential medium components on the thermal resistance (at 121 degrees C) of spores in suspension (calcium acetate, pH 9.7) were yeast extract (positively) and potassium phosphate (negatively), both creating the positive interaction, for spores from a 6-day incubation period . However, the strong negative effect of sodium chloride decreased the D-value from 1.81 min to 0.57 min upon increasing the incubation period (62 degrees C) from 3 days to 6 days . The D-glucose and peptone exhibited greater effects than the yeast extract and potassium phosphate interaction on D-values for 3-day spores on strip, just as the highly joint-positive peptone/sodium chloride effect maintained the thermal resistance of 6-day spores on strips . The spores on strip system showed less stability than the spores in suspension . The most stable spore system confirmed D-values at 121 degrees C at a range between 1.5 min and 1.9 min, which were obtained by keeping sodium chloride and potassium phosphate at minimum concentrations and yeast extract and peptone at maximum concentrations, regardless of the 3- to 6-day sporulation.

Int J Tuberc Lung Dis, 2000 Oct, 4(10), 925 - 30
Tuberculosis infection in an Aboriginal (First Nations) population of Canada; Smeja C et al.; BACKGROUND: The incidence of active tuberculosis (TB) among the Cree, an Aboriginal population of Canada, is dropping, but it remains three times that of the general population . We analyzed data from tuberculin skin test (TST) surveys to determine estimates of prevalence of infection and annual risk of infection (ARI) in this population . METHODS: TST surveys targeting 12-year-old students were conducted annually from 1993 to 1998 . Students with no record of previous positive TST (> or = 10 mm) were offered TST (5 TU PPD-T) . Data collected included result of previous TST reading for all students, readings of TSTs performed (mm induration) and BCG (bacille Calmette-Guerin) vaccination status for those positive on TST . RESULTS: A total of 1274 children were screened (participation rate 94%) . TST reaction size frequency distribution plots a bimodal curve . The prevalence of infection among 12 year olds was 15.3% over this period . ARI estimates range from 0.6 to 2.4% (average ARI 1.4%) . A significant downward linear trend in ARI was observed over the period (P < 0.001) . DISCUSSION: Calculated ARI may be over-estimated due to prior BCG vaccination; however, the trend in ARI confirms decreasing transmission of TB infection . Better knowledge of human immunodeficiency virus seroprevalence among pregnant women is needed to complete the evaluation of the BCG program.

Pediatr Infect Dis J, 2000 Oct, 19(10), 968 - 72
Evaluation of Mycobacterium tuberculosis transmission from a pediatrician and initial compliance to prophylaxis of contacts in an outpatient pediatric clinic; Brassard P et al.; OBJECTIVE: The risk that latent infection will progress to active tuberculosis is greater in infants and children than for most other age groups . We set out to determine the rate of transmission of Mycobacterium tuberculosis to pediatric patients exposed to a pediatrician with smear-negative and culture-positive pulmonary tuberculosis . We also explored factors associated with compliance to prophylaxis . METHODS: Clinic and hospital billing records were used to identify patients age 5 or less who were seen during the pediatrician's potential contagious period . Patient were notified by registered mail of their putative exposure and were offered a tuberculin skin test screening with 5 tuberculin units of purified protein derivative (Tubersol, Connaught) and chest radiography of children with a tuberculin skin test > or =5 mm . RESULTS: A total of 456 patients were identified as exposed; 140 contacts never responded for evaluation and 93 letters were not delivered because of incorrect mailing addresses . Of the 223 who completed screening 1 (0,4%) had a initial skin test result of 8 mm . The remaining 222 contacts had repeated negative test results . The only positive child (15 months old) was born in Honduras and had received Calmette-Guerin bacillus at birth . No active tuberculosis cases were identified in the 456 contacts up to 2 years after exposure . Compliance with prophylaxis was associated with having two or less children in the household (odds ratio, 2.5; 95% confidence interval, 1.1 to 5.9) . CONCLUSION: We found no evidence of transmission of M . tuberculosis in an outpatient pediatric setting . Only 43% of exposed children completed screening, and 38% of those offered prophylaxis completed their initial 3 months of therapy.

Biosci Biotechnol Biochem, 2000 Sep, 64(9), 2018 - 20
The aman6 gene encoding a yeast mannan backbone degrading 1,6-alpha-D-mannanase in Bacillus circulans: cloning, sequence analysis, and expression; Maruyama Y et al.; A gene (aman6) encoding endo-1,6-alpha-D-mannanase, a yeast mannan backbone degrading enzyme from Bacillus circulans was cloned . The putative aman6 was 1,767 base pairs long and encoded a mature 1,6-alpha-D-mannanase protein of 589 amino acids and a signal peptide of 36 amino acids . The purified mature 1,6-alpha-D-mannanase from the Escherichia coli transformant showed 61-kDa protein, and N-terminal amino acid sequence and other general properties of the recombinant enzyme were identical to those of 1,6-alpha-D-mannanase from Bacillus circulans TN-31.

Biosci Biotechnol Biochem, 2000 Sep, 64(9), 1923 - 9
Thermally induced disintegration of the Bacillus stearothermophilus dihydrolipoamide dehydrogenase; Hiromasa Y et al.; Upon heat treatment of the pyruvate dehydrogenase complex from Bacillus stearothermophilus, the most thermostable component is a dihydrolipoamide dehydrogenase (E3c) . To understand this stability, the thermal disintegration of E3 dissociated from the complex (E3d) was examined, comparing with that of E3c . Judging from residual activity and inactivation rate, E3d was less thermostable than E3c; E3d and E3c lost half of their original activities upon incubations for 30 min at 79 degrees C and 90 degrees C, respectively . Heat treatment of E3d raised the fluorescence intensities of Trp residue, intrinsic FAD, and extrinsic 8-anilinonaphthalene-1-sulfonate . E3d lost FAD, and inactive E3d polypeptides were aggregated . The sulfonate bound to the aggregate became notably fluorescent . The thermal disintegration of E3d was speculated to be a consecutive reaction that was different from the concurrent disintegration reaction of the complex . Some interactions with other component polypeptides was suggested to improve the thermostability of E3c.

Acta Crystallogr D Biol Crystallogr, 2000 Nov, 56 ( Pt 11), 1443 - 5
Crystallization and preliminary X-ray analysis of glucose dehydrogenase from Bacillus megaterium IWG3; Yamamoto K et al.; Glucose dehydrogenase from Bacillus megaterium IWG3 has been crystallized in the presence of NAD(+) using the hanging-drop vapour-diffusion method with PEG 2000 as the precipitant . Crystals belong to space group C2 and have unit-cell parameters a = 120.8 (1), b = 66.7 (1), c = 119.6 (1) A, beta = 93.25 (3) degrees with standard deviations in parentheses . Assumption of four subunits in the asymmetric unit gave the most probable Matthews coefficient V(M) of 2.1 A(3) Da(-1) (solvent content 41.7% by volume) . X-ray diffraction data were collected to 1.7 A on a synchrotron-radiation source.

Biochemistry, 2000 Oct 31, 39(43), 13136 - 43
Ancient adaptation of the active site of tryptophanyl-tRNA synthetase for tryptophan binding; Praetorius-Ibba M et al.; The amino acid binding domains of the tryptophanyl (TrpRS)- and tyrosyl-tRNA synthetases (TyrRS) of Bacillus stearothermophilus are highly homologous . These similarities suggest that conserved residues in TrpRS may be responsible for both determining tryptophan recognition and discrimination against tyrosine . This was investigated by the systematic mutation of TrpRS residues based upon the identity of homologous positions in TyrRS . Of the four residues which interact directly with the aromatic side chain of tryptophan (Phe5, Met129, Asp132, and Val141) replacements of Asp132 led to significant changes in the catalytic efficiency of Trp aminoacylation (200-1250-fold reduction in k(cat)/K(M)) and substitution of Val141 by the larger Glu side chain reduced k(cat)/K(M) by 300-fold . Mutation of Pro127, which determines the position of active-site residues, did not significantly effect Trp binding . Of the mutants tested, D132N TrpRS also showed a significant reduction in discrimination against Tyr, with Tyr acting as a competitive inhibitor but not a substrate . The analogous residue in B . stearothermophilusTyrRS (Asp176) has also been implicated as a determinant of amino acid specificity in earlier studies {de Prat Gay, G., Duckworth, H . W., and Fersht, A . R . (1993) FEBS Lett . 318, 167-171} . This striking similarity in the function of a highly conserved residue found in both TrpRS and TyrRS provides mechanistic support for a common origin of the two enzymes.

Crit Rev Microbiol, 2000, 26(3), 133 - 45
Immune aspects of Bartonella; Karem KL; Bartonella species have been recognized as important human pathogens only recently . Until the early 1990s, this genus was represented by one species, Bartonella bacilliformis . The recent identification of other Bartonella species as the agents of cat-scratch disease and bacillary angiomatosis has left little doubt of their emerging importance as opportunistic human pathogens . Over the last decade, extensive research has been performed on Bartonella species, resulting in an explosion in our knowledge of the genetic diversity of this genus . Unusual aspects of disease sequelae have fueled worldwide interest in defining the natural history, pathology, and molecular biology of Bartonella species . While much information about these interests has been presented, the advancement of immunological knowledge regarding Bartonella species has been slow . This review discusses immunological data on Bartonella species, focusing on the three primary human pathogens of this genus: B . bacilliformis, B . quintana, and B . henselae.

Sheng Wu Gong Cheng Xue Bao, 2000 Jul, 16(4), 485 - 9
{Studies on submerged fermentation of alkaline beta-1,4-glycanases by Bacillus pumilus A-30}; Chen SC et al.; The effects of stirring rate, pH control and feed time of (NH4)2SO4 on beta-1,4-glycanases were investigated . The operating conditions of batch fermentation and fed-batch culture were optimized . The results showed there were a large amount of bacterial cells adsorbing on the surface of wheat bran, and stirring rate had a pronounced influence on the adsorption and the production of beta-1,4-glycanases . The uncontrolled pH could promote the induction of beta-1,4-glycanases . When (NH4)2SO4 was fed at a constant rate in the earlier age, and regulated later by determining the content of (NH4)2SO4, xylanase and CMCase activity could reach 616 IU/mL and 1.33 IU/mL respectively.

Drug Saf, 2000 Oct, 23(4), 279 - 93
Drug-induced rheumatic disorders: incidence, prevention and management; Vergne P et al.; The purpose of this article is to review the causes, the clinical manifestations and the management of the more frequent drug-induced rheumatic disorders . These include: (i) articular and periarticular manifestations induced by fluoroquinolones, nonsteroidal anti-inflammatory drugs, injections of corticosteroids, and retinoids; (ii) multisystemic manifestations such as drug-induced lupus and arthritis induced by vaccination, Bacillus Calmette-Guerin therapy and cytokines; (iii) drug-induced disorders of bone metabolism (corticosteroid-induced osteoporosis, drug-induced osteomalacia and osteonecrosis); and (iv) iatrogenic complex regional pain syndromes . Disorders caused by nonpharmacological and rarely used treatments have been deliberately excluded . Knowledge of these drug-induced clinical symptoms or syndromes allows an earlier diagnosis and treatment, and earlier drug withdrawal if necessary . With the introduction of new medications such as the recombinant cytokines and antiretroviral treatments, the number of drug-induced rheumatic disorders is likely to increase.

Br J Biomed Sci, 2000, 57(3), 234 - 40
The bovine tubercle bacillus; Collins CH; The bovine tubercle bacillus has always been eclipsed by the much higher incidence and the social and economic importance of its human cousin, as well as by the clinical unimportance of differentiating between the two . Nevertheless, in view of the resurgence of tuberculosis generally, the increase in the number of immunosuppressed individuals (i.e . the AIDS epidemic) and the 'great badger debate', there is renewed interest in it . Briefly, this review explores the history of Mycobacterium bovis and its potential for transmission between cattle and humans.

Environ Health Perspect, 2000 Oct, 108(10), 919 - 30
Human cell exposure assays of Bacillus thuringiensis commercial insecticides: production of Bacillus cereus-like cytolytic effects from outgrowth of spores; Tayabali AF et al.; Most contemporary bioinsecticides are derived from scaled-up cultures of Bacillus thuringiensis subspecies israelensis (Bti) and kurstaki (Btk), whose particulate fractions contain mostly B . thuringiensis spores (> 10(12)/L) and proteinaceous aggregates, including crystal-like parasporal inclusion bodies (PIB) . Based on concerns over relatedness to B . cereus-group pathogens, we conducted extensive testing of B . thuringiensis (BT) products and their subfractions using seven human cell types . The Bti/Btk products generated nonspecific cytotoxicities involving loss in bioreduction, cell rounding, blebbing and detachment, degradation of immunodetectable proteins, and cytolysis . Their threshold dose (Dt approximately equal.5 times 10(-14)% BT product/target cell) equated to a single spore and a target cell half-life (tLD(50)) of approximately 16 hr . At Dts > 10(4), the tLD(50) rapidly shifted to < 4 hr; with antibiotic present, no component, including PIB-related {delta}-endotoxins, was cytolytic up to an equivalent of approximately 10(9 )Dt . The cytolytic agent(s) within the Bti/Btk-vegetative cell exoprotein (VCP) pool is an early spore outgrowth product identical to that of B . cereus and acting possibly by arresting protein synthesis . No cytolytic effects were seen with VCP from B . subtilis and Escherichia coli . These data, including recent epidemiologic work indicate that spore-containing BT products have an inherent capacity to lyse human cells in free and interactive forms and may also act as immune sensitizers . To critically impact at the whole body level, the exposure outcome would have to be an uncontrolled infection arising from intake of Btk/Bti spores . For humans, such a condition would be rare, arising possibly in equally rare exposure scenarios involving large doses of spores and individuals with weak or impaired microbe-clearance capacities and/or immune response systems.

Protein Expr Purif, 2000 Nov, 20(2), 142 - 51
Purification and characterization of the highly thermostable proteases from Bacillus stearothermophilus TLS33; Sookkheo B et al.; Three thermostable proteases, designated S, N, and B, are extracellular enzymes produced by Bacillus stearothermophilus strain TLS33 . They were purified by lysine affinity chromatography, strong anion exchange Q HyperD chromatography, and Ultrogel AcA44 gel filtration . The molecular masses of the enzymes determined by SDS-PAGE and zymography were approximately 36, 53, and 71 kDa, respectively . Thermostable protease S bound strongly to the lysine affinity column and could be purified by this single step . The optimum pH values of proteases S, N, and B were shown to be 8.5, 7.5, and 7.0, respectively . The maximum activities for the enzymes were at 70, 85, and 90 degrees C, respectively . Proteases S, N, and B at pH 7.0 in the presence of 5 mM CaCl(2) retained half their activities after 30 min at 72, 78, and 90 degrees C, respectively . All three thermostable proteases were strongly inhibited by the metal chelators EDTA and 1,10-phenanthroline, and the proteolytic activities were restored by addition of ZnCl(2) . They can thus be classified as Zn(2+) metalloproteases . The cleavage specificities of proteases S, N, and B on a 30-residue synthetic peptide from pro-BPN' subtilisin were Tyr-Ile, Phe-Lys, and Gly-Phe, respectively .

J Biol Chem, 2001 Jan 26, 276(4), 2487 - 93 Epub 2000 Oct 20.
Interactions of a novel inhibitor from an extremophilic Bacillus sp . with HIV-1 protease: implications for the mechanism of inactivation; Dash C et al.; The active site cleft of the HIV-1 protease (PR) is bound by two identical conformationally mobile loops known as flaps, which are important for substrate binding and catalysis . The present article reports, for the first time, an HIV-1 PR inhibitor, ATBI, from an extremophilic Bacillus sp . The inhibitor is found to be a hydrophilic peptide with Mr of 1147, and an amino acid sequence of Ala-Gly-Lys-Lys-Asp-Asp-Asp-Asp-Pro-Pro-Glu . Sequence homology exhibited no similarity with the reported peptidic inhibitors of HIV-1 PR . Investigation of the kinetics of the enzyme-inhibitor interactions revealed that ATBI is a noncompetitive and tight binding inhibitor with the IC(50) and K(i) values 18.0 and 17.8 nm, respectively . The binding of the inhibitor with the enzyme and the subsequent induction of the localized conformational changes in the flap region of the HIV-1 PR were monitored by exploiting the intrinsic fluorescence of the surface exposed Trp-42 residues, which are present at the proximity of the flaps . We have demonstrated by fluorescence and circular dichroism studies that ATBI binds in the active site of the HIV-1 PR and thereby leads to the inactivation of the enzyme . Based on our results, we propose that the inactivation is due to the reorganization of the flaps impairing its flexibility leading toward inaccessibility of the substrate to the active site of the enzyme.

Proc Natl Acad Sci U S A, 1997 Apr 15, 94(8), 3519 - 3523
Initial frequency of alleles for resistance to Bacillus thuringiensis toxins in field populations of Heliothis virescens; Gould F et al.; The risk of rapid pest adaptation to an insecticide is highly dependent on the initial frequency of resistance alleles in field populations . Because we have lacked empirical estimates of these frequencies, population-genetic models of resistance evolution have relied on a wide range of theoretical estimates . The recent commercialization of genetically engineered cotton that constitutively produces an insecticidal protein derived from the biocontrol agent, Bacillus thuringiensis (Bt) has raised concern that we lack data needed to quantify the risk of insect pests such as Heliothis virescens rapidly adapting to this ecologically valuable class of toxins . By individually mating over 2,000 male H . virescens moths collected in four states to females of a Bt toxin-resistant laboratory strain, and screening F(1) and F(2) offspring for tolerance of the toxic protein, we were able to directly estimate the field frequency of alleles for resistance as 1.5 x 10(-3) . This high initial frequency underscores the need for caution in deploying transgenic cotton to control insect pests . Our single-pair mating technique greatly increases the efficiency of detecting recessive resistance alleles . Because alleles that decrease target site sensitivity to Bt toxins and other insecticides are often recessive, this technique could be useful in estimating resistance allele frequencies in other insects exposed to transgenic insecticidal crops or conventional insecticides.

J Leukoc Biol, 2000 Oct, 68(4), 538 - 44
A novel tumor necrosis factor (TNF) mimetic peptide prevents recrudescence of Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection in CD4+ T cell-depleted mice; Briscoe H et al.; Tumor necrosis factor (TNF) is required to control mycobacterial infections, but its therapeutic value is limited by its in vivo instability and toxicity . The efficacy of a nontoxic TNF-mimetic peptide (TNF70-80) was tested in mice infected with Mycobacterium bovis bacillus Callette-Guerin (BCG) . In vitro TNF70-80 and recombinant human TNF (hTNF) acted with interferon gamma (IFN-gamma) to reduce bacterial replication and to induce synthesis of bactericidal nitric oxide (NO) in BCG-infected, bone marrow-derived murine macrophages . The dose-dependent inhibitory effect on bacterial replication was blocked by neutralizing anti-IFN-gamma and anti-hTNF mAbs . Further, n-monomethyl-L-arginine (n-MMA) and a soluble TNF-receptor I (TNFRI-IgG) blocked bacterial growth and NO synthesis . Therefore, the peptide acted with IFN-gamma via induction of NO synthase and signaled through TNFRI receptors . Concomitant in vivo treatment with TNF70-80 or hTNF prevented reactivation of chronic BCG infection in mice depleted of CD4+ T cells by injecting anti-CD4 antibodies . Granuloma number and bacterial load were comparable in treated, T cell-depleted mice and in chronically infected, intact animals . Thus, TNF70-80 and hTNF can modulate recrudescent BCG infection in CD4+ T cell-deficient mice.

J Dairy Res, 2000 Aug, 67(3), 403 - 13
Standardized reaction times used to describe the mechanism of enzyme-induced gelation in whey protein systems; Ipsen R et al.; Whey protein isolate (WPI), either untreated or pretreated at 80 degrees C for 30 min, was incubated with a proteinase from Bacillus licheniformis until a gel was formed . Standardized reaction times, directly linked to the degree of hydrolysis, were obtained from plots of the relative amount of peptides released v . reaction time obtained under different conditions (enzyme concentration, temperature, pH, NaCl addition) . This provided a connection between the gelation profile and the degree of hydrolysis . In the case of untreated WPI, gelation occurred at lower degrees of proteolysis when the enzyme concentration was decreased, demonstrating that a rate-limiting aggregation process occurred at the same time as the proteolysis in a manner similar to the renneting of milk . This was not the case for preheated WPI, when gelation was found to take place at a constant degree of proteolysis, independent of the enzyme concentration . In this case, the mechanism could be described by assuming the thermally induced aggregates present in this substrate had progressively more stabilizing peptide segments shaved off, resulting in increased attraction between individual aggregates that ultimately led to gelation . Results obtained at 40-60 degrees C supported this, as we found no effect of temperature on the degree of proteolysis at gelation for the untreated WPI, whereas the degree of proteolysis decreased with increasing temperature when heated WPI was hydrolysed . The effect of pH and NaCl addition on the process was to reduce repulsion between the aggregating species so that gelation was induced at a decreased degree of proteolysis.

Res Microbiol, 2000 Sep, 151(7), 547 - 55
Construction of chromosomal integrants of Bacillus sphaericus 2362 by conjugation with Escherichia coli; Aquino de Muro M et al.; IncP-based plasmids conjugated between Escherichia coli and mosquitocidal strains of Bacillus sphaericus at frequencies of 10(-7) to 10(-9) per recipient . Plasmid transfer was most efficient when a restriction-deficient strain of B . sphaericus 2362 (serotype 5a5b) was used as recipient and was least efficient with recipients from serotypes 1a and 2a2b . A deleted version of the cryptic locus 'gene 80' from strain 2362 was cloned into the suicide vector pMTL30, which could not replicate in B . sphaericus to provide a site for chromosomal integration . Conjugational transfer from E . coli and integration into the B . sphaericus recipient chromosome was achieved with this construct . The coding region of the cry11A gene from Bacillus thuringiensis subsp . israelensis was PCR-amplified and fused to the promoter of the crystal protein (Bin) gene of B . sphaericus 2362 . This construct was cloned into the integrative vector, conjugated with B . sphaericus 2362 and chromosomal integrants were recovered which harboured the cry11A gene . The fusion gene was efficiently transcribed in the recombinant host, but cells failed to accumulate appreciable amounts of Cry11A toxin . This system offers a simple and efficient means of transferring plasmids into B . sphaericus and obtaining chromosomal integration for strain construction and gene analysis.

Bioorg Khim, 2000 Sep, 26(9), 672 - 8
{Ribonuclease from Bacillus thuringiensis var . subtoxicus . Gene structure and biosynthesis regulation}; Shul'ga AA et al.; The gene for extracellular guanyl-specific ribonuclease of Bacillus thuringiensis var . subtoxicus (RNase Bth), a close homologue of the B . intermedius RNase (binase), was completely sequenced . Analysis of nucleotide sequences in the regions adjoining RNase genes revealed an identical organization of the chromosomal loci of RNase Bth and binase . Growth characteristics of the Bacillus thuringiensis var . subtoxicus strain and its synthesis of RNase were studied . It was shown that the exogenous inorganic phosphate inhibits the biosynthesis of RNase . At the same time, actinomycin D in low doses stimulates the enzyme synthesis . Comparative analysis of the influence of inorganic phosphate and actinomycin D on the biosynthesis of RNAse Bth and binase suggests a possibility of coincidence of regulatory pathways of synthesis of these enzymes.

Bioorg Khim, 2000 Sep, 26(9), 662 - 71
{MALD-MS in the quantitative analysis of peptides and proteins}; Mirgorodskaia OA et al.; A modified method of isotope dilution was applied to the quantitative determination of peptides and proteins by MALDI MS at subpicomolar level . The essence of the method consists in the quantitative analysis of the enzymic hydrolysis products rather than the starting compounds . This allows the measurements to be performed at a higher resolution and makes the method independent of the molecular mass of oligopeptides and proteins examined . Fragments obtained by hydrolysis of the same oligopeptide or protein in a known concentration by the same enzyme and labeled with the stable 18O isotope are used as internal standards . The label is introduced by carrying out the hydrolysis in H(2)18O, and the oligopeptide concentration is calculated from the isotope distribution between the labeled and unlabeled hydrolysis products in the mass spectrum . This method was tested in the determination of concentrations of the angiotensinogen (1-14) fragment (oligopeptide), extracellular RNAase from Bacillus amyloliquefaciens (protein) and its protein inhibitor, barstar M . Usefulness of this method in kinetic studies was also demonstrated.

Curr Microbiol, 2000 Mar, 40(3), 205 - 9
Occurrence of Bacillus thuringiensis on cured tobacco leaves; Kaelin P et al.; A worldwide survey was conducted to evaluate the frequency and distribution of Bacillus thuringiensis populations on cured tobacco leaves during post-harvest storage . In total, 133 tobacco samples of different types and origins were analyzed . Nine percent of the samples showed the presence of B . thuringiensis, and 24 B . thuringiensis strains were isolated and characterized . The majority of the isolates produced bipyramidal crystals, and three fourths of them showed a second type of crystal protein (cuboidal or heterogeneous crystals) . Only three isolates showed the rhomboidal crystal morphology characteristic of the anti-coleopteran B . thuringiensis subsp . tenebrionis . PCR analysis with primers specific for cry1 and cry3 genes revealed eight distinct cry gene profiles . The results of this study indicate that B . thuringiensis is naturally present at low frequency on the phylloplane of cured tobacco leaves and that its distribution is worldwide.

Curr Microbiol, 2000 Mar, 40(3), 200 - 4
The crystal proteins from Bacillus thuringiensis subsp . thompsoni display a synergistic activity against the codling moth, Cydia pomonella; Rang C et al.; Crystal proteins from Bacillus thuringiensis subsp . thompsoni strain HnC are active against the codling moth, Cydia pomonella, a major pest of orchards . Inclusion bodies purified from strain HnC displayed an LC(50) of 3.34 x 10(-3) microgram/microliter . HnC-purified crystals were tenfold more active than Cry2Aa and Cry1Aa toxins, and 100-fold more toxic than Cry1Ab . The 34-kDa and 40-kDa proteins contained in HnC inclusion bodies were shown to act synergistically . The toxicity of crystal proteins produced by the recombinant B . thuringiensis strain BT-OP expressing the full-length native operon was about tenfold higher than that of the 34-kDa protein . When the gene encoding the non-insecticidal 40-kDa protein, which is not active, was introduced into the recombinant strain producing only the 34-kDa protein, the toxicity was raised tenfold and was similar to that of the strain BT-OP.

J Biol Chem, 2001 Jan 26, 276(4), 2644 - 51 Epub 2000 Oct 17.
Sequence-specific and methylation-dependent and -independent binding of rice nuclear proteins to a rice tungro bacilliform virus vascular bundle expression element; He X et al.; Nuclear proteins from rice (Oryza sativa) were identified that bind specifically to a rice tungro bacilliform virus promoter region containing a vascular bundle expression element (VBE) . One set of proteins of 29, 33, and 37 kDa, present in shoot and cell suspension extracts but hardly detectable in root extracts, bound to a site containing the sequence AGAAGGACCAGA within the VBE, which also contains two CpG and one CpNpG potential methylation motifs . Binding by these proteins was determined to be cytosine methylation-independent . However, a novel protein present in all analyzed extracts bound specifically to the methylated VBE . A region of at least 49 nucleotides overlapping the VBE and complete cytosine methylation of the three Cp(Np)G motifs was required for efficient binding of this methylated VBE-binding protein (MVBP).

J Biotechnol, 2001 Nov 17, 84(1), 67 - 72
Deletion of aprA and nprA genes for alkaline protease A and neutral protease A from bacillus thuringiensis: effect on insecticidal crystal proteins; Tan Y et al.; The aprA gene encoding alkaline protease A (AprA) was cloned from Bacillus thuringiensis subsp . kurstaki, and the cloned gene was used to construct aprA-deleted (aprA1) strains of B . thuringiensis . An aprA1 strain of B . thuringiensis that contained the wild-type gene for neutral protease A (nprA(+)) displayed levels of extracellular proteolytic activity that were similar to those of an aprA(+)nprA(+) strain . However, when EDTA was included in the protease assay to inhibit NprA activity the aprA1nprA(+) strain displayed only 2% of the extracellular proteolytic activity of the aprA(+)nprA(+) strain . A strain that was deleted for both aprA and nprA (aprA1nprA3 strain) failed to produce detectable levels of proteolytic activity either in the presence or absence of EDTA in the assay . Compared with the aprA(+)nprA(+) strain the aprA1nprA(+) strain yielded 10% more full-length Cry1Bb crystal protein and the aprA1nprA3 strain yielded 25% more full-length Cry1Bb protein . No significant differences were seen in the 50% lethal dose of Cry1Bb protein from aprA(+)nprA(+) and aprA1nprA3 strains against three species of lepidopteran insects . These results suggest that enhanced yield of certain crystal proteins can be obtained by deletion of the genes aprA and nprA which are the major extracellular proteases of B . thuringiensis.

Int J Syst Evol Microbiol, 2000 Sep, 50 Pt 5, 1715 - 22
Phylogeny of Bacillus sphaericus-like organisms; Nakamura LK; The mesophilic round-spored bacteria embrace four species, namely Bacillus sphaericus, Bacillus fusiformis, Bacillus silvestris and Bacillus pasteurii . Although not displayed by all strains, mosquito pathogenicity is a noteworthy characteristic of B . sphaericus sensu lato . Phylogenetic analysis based on 16S rDNA sequences from 58 strains identified as B . sphaericus was used to examine the genetic heterogeneity of the taxon . Results from sequence analysis were compared with whole-cell fatty acid profiles and other phenotypic determinations . The B . sphaericus-like strains segregated into seven distinct clusters in a phylogenetic tree generated from 16S sequences . One cluster represented B . sphaericus and another B . fusiformis . A third cluster containing all of the pathogenic strains was closely related to, or was possibly part of, the B . fusiformis group . The remaining four groups were distinct and represented unnamed taxa that were more closely related to B . sphaericus and B . fusiformis than to the psychrophilic round-spored species, Bacillus globisporus and Bacillus psychrophilus . Groups based on phenotypic analysis corresponded to the 16S rDNA phylogenetic clusters . Data showed that B . sphaericus was genetically and phenotypically a highly heterogeneous taxon including at least seven genetically distinct taxa . The pathogenic strains were members of a distinct group and not of the species B . sphaericus sensu stricto . This heterogeneity partially accounts for the apparent variability of mosquito pathogenicity among B . sphaericus strains.

FEMS Microbiol Lett, 2000 Sep 15, 190(2), 309 - 16
Recombinant BCG approach for development of vaccines: cloning and expression of immunodominant antigens of M . tuberculosis; Dhar N et al.; In spite of major advances in our understanding of the biology and immunology of tuberculosis, the incidence of the disease has not reduced in most parts of the world . In an attempt to improve the protective efficacy of Mycobacterium bovis bacille Calmette-Guerin (BCG), we have developed a generic vector system, pSD5, for expression of genes at varying levels in mycobacteria . In this study, we have cloned and overexpressed three immunodominant secretory antigens of M . tuberculosis, 85A, 85B and 85C, belonging to the antigen 85 complex . All the genes were cloned under the control of a battery of mycobacterial promoters of varying strength . The expression was analysed in the fast-growing strain M . smegmatis and the slow-growing vaccine strain M . bovis BCG . The recombinant BCG constructs were able to express the antigens at high levels and the majority of the expressed antigens was secreted into the medium . These results show that by using this strategy the recombinant BCG approach can be successfully used for the development of candidate vaccines against infections associated with mycobacteria as well as other pathogens.

Pediatr Hematol Oncol, 2000 Oct-Nov, 17(7), 541 - 50
Prevention of childhood leukemia by BCG vaccination in newborns? A population-based case-control study in Lower Saxony, Germany; von Kries R et al.; Data from a case-control study in Lower Saxony, Germany, were used to assess whether the risk for childhood cancer may be reduced by bacille Calmette-Guerin (BCG) vaccination in the neonatal period . There were 420 newly diagnosed childhood cancer cases from the German cancer registry and 613 controls eligible for this study . A mailed questionnaire was completed during a telephone interview with parents . Details on the perinatal history were abstracted from the birth charts by nurses blinded to the children's case-control status . Complete information was available for 259 cases and for 323 controls . A total of 85% of the controls had been BCG vaccinated in the newborn period . The adjusted odds ratios for BCG vaccination were 0.90 (95% confidence interval; 0.51-1.61) for leukemia and 0.61 (95% confidence interval; 0.25-1.50) for other cancers . Based on these data the probability of a 50% or more reduction of more reduction of the cancer risk by BCG vaccination in the newborn period is small . The statistical power of this study, however, was not high enough to rule out a smaller, still relevant reduction in cancer risk.

J Insect Physiol, 2001 Jan 1, 47(1), 1 - 10
Developmental expression and stress induction of glutathione S-transferase in the spruce budworm, Choristoneura fumiferana; Feng Q et al.; Developmental and stress-induced expression of Choristoneura fumiferana glutathione S-transferase (CfGST) mRNA and protein were examined using Northern blots and Western blots . High levels of CfGST mRNA and protein were detected in 1st instar larvae and diapausing 2nd instar larvae . Expression of CfGST gradually decreased during larval development from 3rd to 5th instar, after which the expression increased once again, reaching peak levels in 6th instar larvae . CfGST mRNA and protein were undetectable in the pupal stage . Exposure to low temperature did not induce an increase in CfGST expression . Feeding on balsam fir foliage resulted in an increase in the expression of CfGST as compared to larvae that fed on artificial diet . The bacterial insecticide, Bacillus thuringiensis delta-endotoxin (Bt), the non-steroidal ecdysone analog, tebufenozide, and the synthetic pyrethroid, permethrin, induced the expression of CfGST mRNA in 5th instar larvae, whereas the chitin synthesis inhibitor, diflubenzuron, did not have any such effect . These results suggest that CfGST plays an important role in detoxifying various allelochemicals and insecticides in the spruce budworm . The developmental expression pattern strongly suggests that in addition to detoxification, CfGST might be involved in other functions.

J Bacteriol, 2000 Nov, 182(21), 6114 - 22
Cloning and characterization of the str operon and elongation factor Tu expression in Bacillus stearothermophilus; Krasny L et al.; The complete primary structure of the str operon of Bacillus stearothermophilus was determined . It was established that the operon is a five-gene transcriptional unit: 5'-ybxF (unknown function; homology to eukaryotic ribosomal protein L30)-rpsL (S12)-rpsG (S7)-fus (elongation factor G {EF-G})-tuf (elongation factor Tu {EF-Tu})-3' . The main operon promoter (strp) was mapped upstream of ybxF, and its strength was compared with the strength of the tuf-specific promoter (tufp) located in the fus-tuf intergenic region . The strength of the tufp region to initiate transcription is about 20-fold higher than that of the strp region, as determined in chloramphenicol acetyltransferase assays . Deletion mapping experiments revealed that the different strengths of the promoters are the consequence of a combined effect of oppositely acting cis elements, identified upstream of strp (an inhibitory region) and tufp (a stimulatory A/T-rich block) . Our results suggest that the oppositely adjusted core promoters significantly contribute to the differential expression of the str operon genes, as monitored by the expression of EF-Tu and EF-G.

J Bacteriol, 2000 Nov, 182(21), 5969 - 81
Two-dimensional gel electrophoresis analyses of pH-dependent protein expression in facultatively alkaliphilic Bacillus pseudofirmus OF4 lead to characterization of an S-layer protein with a role in alkaliphily; Gilmour R et al.; The large majority of proteins of alkaliphilic Bacillus pseudofirmus OF4 grown at pH 7.5 and 10.5, as studied by two-dimensional gel electrophoresis analyses, did not exhibit significant pH-dependent variation . A new surface layer protein (SlpA) was identified in these studies . Although the prominence of some apparent breakdown products of SlpA in gels from pH 10.5-grown cells led to discovery of the alkaliphile S-layer, the largest and major SlpA forms were present in large amounts in gels from pH 7.5-grown cells as well . slpA RNA abundance was, moreover, unchanged by growth pH . SlpA was similar in size to homologues from nonalkaliphiles but contained fewer Arg and Lys residues . An slpA mutant strain (RG21) lacked an exterior S-layer that was identified in the wild type by electron microscopy . Electrophoretic analysis of whole-cell extracts further indicated the absence of a 90-kDa band in the mutant . This band was prominent in wild-type extracts from both pH 7.5- and 10.5-grown cells . The wild type grew with a shorter lag phase than RG21 at either pH 10.5 or 11 and under either Na(+)-replete or suboptimal Na(+) concentrations . The extent of the adaptation deficit increased with pH elevation and suboptimal Na(+) . By contrast, the mutant grew with a shorter lag and faster growth rate than the wild type at pH 7 . 5 under Na(+)-replete and suboptimal Na(+) conditions, respectively . Logarithmically growing cells of the two strains exhibited no significant differences in growth rate, cytoplasmic pH regulation, starch utilization, motility, Na(+)-dependent transport of alpha-aminoisobutyric acid, or H(+)-dependent synthesis of ATP . However, the capacity for Na(+)-dependent pH homeostasis was diminished in RG21 upon a sudden upward shift of external pH from 8 . 5 to 10.5 . The energy cost of retaining the SlpA layer at near-neutral pH is apparently adverse, but the constitutive presence of SlpA enhances the capacity of the extremophile to adjust to high pH.

Am J Respir Crit Care Med, 2000 Oct, 162(4 Pt 1), 1314 - 7
Risk of tuberculosis infection and tuberculous meningitis after discontinuation of Bacillus Calmette-Guerin in Beijing; Zhang LX et al.; In Beijing, the notification rate of smear-positive tuberculosis (TB) has been below 20 per 100,000 since 1986, and continues to decline . To accurately measure the risk of TB infection in a population in which the results of tuberculin skin testing were not confounded by vaccination with Bacillus Calmette-Guerin (BCG), BCG vaccination at birth was discontinued from 1988 in Shun-yi County . In 1995, the prevalence of TB infection among 12,836 primary school children aged 6 to 7 yr and without BCG scars was 1.4%, giving an estimated annual risk of infection of 0.19% (95% confidence interval: 0.16 to 0.22%) . The prevalence of TB infection in children aged 5 to 9 yr in Beijing in 1950 was 46% . The number of cases of tuberculous meningitis did not increase after discontinuation of BCG . We conclude that discontinuation of BCG had no detectable harmful effects, and that control of TB in Beijing has markedly reduced the prevalence of TB infection since 1950.

Biochem Biophys Res Commun, 2000 Oct 5, 276(3), 1048 - 55
Identification of the functional site in the mosquito larvicidal binary toxin of Bacillus sphaericus 1593M by site-directed mutagenesis; Elangovan G et al.; To study the mode of action of the binary toxin (51- and 42-kDa) of Bacillus sphaericus, amino acid residues were substituted at selected sites of the N- and C-terminal regions of both peptides . Bioassay results of the mutant binary toxins tested against mosquito larvae, Culex quinquefasciatus, revealed that most of the substitutions made on both peptides led to either decrease or total loss of the activity . Furthermore, receptor binding studies carried out for some of the mutants of the 42-kDa peptide showed mutations in N- and C-terminal regions of the 42-kDa peptide did not affect the binding of the binary toxin to brush border membrane vesicles of mosquito larvae . One of the mutants having a single amino acid substitution at the C-terminal region ((312)R) of the 42-kDa peptide completely abolished the biological activity, implicating the role of this residue in membrane pore formation . These results indicate the importance of the C-terminal region of the 42-kDa of binary toxin, in general, and particularly the residue (312)R for biological activity against mosquito larvae .

J Urol, 2000 Nov, 164(5), 1546 - 9
Elevated prostate specific antigen serum levels after intravesical instillation of bacillus Calmette-Guerin; Leibovici D et al.; PURPOSE: We determined whether intravesical bacillus Calmette-Guerin (BCG) instillation is associated with elevated prostate specific antigen (PSA) . MATERIALS AND METHODS: We treated 36 consecutive patients with bladder cancer with a 6-week course of BCG, followed by cystoscopy at 6 weeks . Blood samples for PSA determination were obtained before each BCG instillation and at cystoscopy with each patient also serving as a control . PSA elevation was defined as 2-fold the baseline level in at least 2 specimens and any PSA level greater than 4 ng./ml . was considered clinically significant . Digital rectal examination was done to identify firm nodules and prostate size . The prostate was examined histologically by transrectal ultrasound guided biopsy or after radical cystectomy . RESULTS: We observed elevated PSA in 27 men (75%) during BCG treatment, of whom 15 (41.6%) had a clinically significant elevation . Overall average PSA increased from 1.3 ng./ml . before BCG instillation to 3.8 during treatment (range 0.1 to 21.5, p <0.0001) . In those with a clinically significant elevation average PSA increased from 2.31 ng./ml . at baseline to 6.97 during treatment (p <0.0001) and returned to 3.86 ng./ml . 3 months after treatment . Palpation demonstrated prostatic findings in 10 patients, including firm nodules in 7, while there was significantly elevated PSA in 5 with firm nodules and 2 with diffuse prostatic enlargement . Histological examination of the prostate in 10 patients was diagnostic for granulomatous prostatitis, nonspecific inflammation and benign prostatic hyperplasia in 3, 3 and 4, respectively, of whom none had prostate cancer . CONCLUSIONS: Intravesical BCG therapy is associated with significantly elevated PSA in up to 40% of cases . This effect is self-limited and PSA reverts to normal in 3 months . Therefore, we suggest that prostate biopsy be withheld in such patients and PSA monitored.

FEMS Microbiol Lett, 2000 Oct 15, 191(2), 227 - 34
Regulation of anaerobic arginine catabolism in Bacillus licheniformis by a protein of the Crp/Fnr family; Maghnouj A et al.; Arginine anaerobic catabolism occurs in Bacillus licheniformis through the arginine deiminase pathway, encoded by the gene cluster arcABDC . We report here the involvement of a new protein, ArcR, in the regulation of the pathway . ArcR is a protein of the Crp/Fnr family encoded by a gene located 109 bp downstream from arcC . It binds to a palindromic sequence, very similar to an Escherichia coli Crp binding site, located upstream from arcA . Residues in the C-terminal domain of Crp that form the DNA binding motif, in particular residues Arg-180 and Glu-181 that make specific bonds with DNA, are conserved in ArcR, suggesting that the complexes formed with DNA by Crp and ArcR are similar . Moreover, the pattern of DNase I hypersensitivity sites induced by the binding of ArcR suggests that ArcR bends the DNA in the same way as Crp . From the absence of anaerobic induction following inactivation of arcR and from the existence of a binding site upstream of the arcA transcription start point, it can be inferred that ArcR is an activator of the arginine deiminase pathway.

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