Bacillus

Annu Rev Phytopathol, 1996, 34, 275 - 97
Molecular biology of rice tungro viruses; Hull R; Rice tungro, the most important virus disease of rice in South and Southeast Asia, is caused by a complex of two viruses, rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV) . RTBV is a plant pararetrovirus with bacilliform particles, the structure of which is based on T = 3 icosahedral symmetry cut across the threefold axis.The particles encapsidate a circular double-stranded DNA of 8 kbp that encodes four proteins . The current information on the properties, functions, and expression of these proteins is discussed, as is the evidence for replication by reverse transcription . Two major strains of RTBV have been recognized, one from the Indian subcontinent and the other from Southeast Asia . RTSV particles contain a single-stranded RNA genome of 12 kb that encodes a large polyprotein and possibly one or two smaller proteins . The properties and processing of the polyprotein are described and the resemblance to picornaviruses noted.

Annu Rev Phytopathol, 1996, 34, 249 - 74
Biology and epidemiology of rice viruses; Hibino H; The 15 known viruses that occur in rice are rice black-streaked dwarf, rice bunchy stunt, rice dwarf, rice gall dwarf, rice giallume, rice grassy stunt, rice hoja blanca, rice necrosis mosaic, rice ragged stunt, rice stripe necrosis, rice stripe, rice transitory yellowing, rice tungro bacilliform, rice tungro spherical, and rice yellow mottle viruses . This paper describes their geographical distribution, relation to vectors, infection cycles, field dispersal, and development, and lists recorded outbreaks of the viruses . Many rice viruses have become serious problems since rice cultivation has been intensified . Double-cropping of rice using improved, photo-insensitive cultivars of short growth duration has significantly influenced the incidence of these viruses.

Annu Rev Entomol, 1998, 43, 701 - 26
Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology; Gould F; This review examines potential impacts of transgenic cultivars on insect population dynamics and evolution . Experience with classically bred, insecticidal cultivars has demonstrated that a solid understanding of both the target insect's ecology and the cultivar's performance under varied field conditions will be essential for predicting area-wide effects of transgenic cultivars on pest and natural enemy dynamics . This experience has also demonstrated the evolutionary capacity of pests for adaptive response to insecticidal traits in crops . Biochemical and genetic studies of insect adaptation to the Bacillus thuringiensis (Bt) toxins expressed by currently marketed transgenic cultivars indicate a high risk for rapid adaptation if these cultivars are misused . Theoretical and practical issues involved in implementing strategies to delay pest adaptation to insecticidal cultivars are reviewed . Emphasis is placed on examining the "high dose"/refuge strategy that has become the goal of industry and regulatory authorities.

Annu Rev Entomol, 1998, 43, 395 - 419
Ecology and management of hazelnut pests; AliNiazee MT; Insect and mite pests cause serious damage to the hazelnut crop worldwide . The control strategies used against these pests include application of insecticides, classical and augmentative biological control, utilization of resistant varieties, and use of Bacillus thuringiensis-based preparations . In the United States, extensive research has been directed toward elucidating ecological interactions among different pests and natural enemies and understanding the role of abiotic factors in pest population dynamics . Differences exist worldwide regarding the understanding of pests and natural enemy biologies and, to a limited extent, control practices . An integrated pest management approach based on utilizing effective sampling and monitoring techniques and a near complete reliance on biological control and "soft" pesticides, including insect growth regulators, is currently under development both in North America and Europe/Turkey . Hazelnuts are on the verge of becoming one of the first crops in the United States that could possibly be produced commercially without the use of any broad-spectrum organic insecticides.

Annu Rev Entomol, 1996, 41, 451 - 72
Bacillus sphaericus toxins: molecular biology and mode of action; Charles JF et al.; Bacillus sphaericus is a spore-forming aerobic bacterium, several strains of which are pathogenic for mosquito larvae . During sporulation, the most active strains produce a crystal toxin with a high degree of larvicidal activity . The toxin is composed of two proteins of 51.4 and 41.9 kDa, which are encoded by highly conserved chromosomal genes . After B . sphaericus is ingested, these proteins are released in the larva's midgut, and, in susceptible mosquito species, bind to a specific receptor present on midgut brush-border membranes . The resulting damages to the midgut cells leads to the mosquitoes' death . During vegetative growth, some B . sphaericus strains also synthesize mosquito larvicidal proteins of 100 and 30.8 kDa (Mtx toxins), the mode of action of which is still unknown . The mechanism of acquisition of the recessive mosquito resistance to the crystal toxin varies with selection conditions.

Seizure, 2004 Apr, 13(3), 191 - 5
Epilepsia partialis continua in cat scratch disease; Puligheddu M et al.; Cat scratch disease (CSD) is a world-wide, diffuse, non-epidemic infection caused by the Gram-negative bacillus Bartonella henselae . The occurrence of encephalopathy represents an infrequent and atypical complication, whose manifestations include ischemic strokes, transverse myelitis and epileptic seizures . Status epilepticus has been described as the most frequent emergency in CSD encephalopathy . In this report, we describe a case of CSD complicated by an epilepsia partialis continua (EPC) manifested as rhythmic movements of the flexor muscles of the left hand . Although CSD is a benign, self-limited disease and a complete neurological recovery usually occurs, in the present case the EPC resulted in a partial epilepsy . Magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT) and back-averaged EEG data recorded during myoclonic activity document this CSD complication.

Med Vet Entomol, 2004 Mar, 18(1), 50 - 6
The naturally derived insecticide spinosad is highly toxic to Aedes and Anopheles mosquito larvae; Bond JG et al.; Spinosad is a naturally derived biorational insecticide with an environmentally favourable toxicity profile, so we investigated its potency against mosquito larvae (Diptera: Culicidae) . By laboratory bioassays of a suspension concentrate formulation of spinosad (Tracer), the 24 h lethal concentration (LC50) against Aedes aegypti (L.) third and fourth instars was estimated at 0.025 p.p.m . following logit regression . The concentration-mortality response of third- and fourth-instar Anopheles albimanus Weidemann did not conform to a logit model . The LC50 value of spinosad in Anopheles albimanus was 0.024 p.p.m . by quadratic linear regression . A field trial in southern Mexico demonstrated that spinosad 1 p.p.m . compared with the standard temephos (Abate) 1% granules 100 g/m3 water prevented Ae . aegypti breeding in plastic containers of water for 8 weeks; at 10 p.p.m . spinosad prevented breeding for > 22 weeks . In another field trial, spinosad at 5 p.p.m . and temephos both completely eliminated reproduction of Ae . aegypti for 13 weeks . In contrast, the bacterial insecticide Bacillus thuringiensis var . israelensis (Bti, Vectobac) AS) performed poorly with just 2 weeks of complete inhibition of Ae . aegypti breeding . Spinosad also effectively prevented breeding of Culex mosquitoes and chironomids in both trials to a degree similar to that of temephos . We conclude that spinosad merits evaluation as a replacement for organophosphate or Bti treatment of domestic water tanks in Mesoamerica . We also predict that spinosad is likely to be an effective larvicide for treatment of mosquito breeding sites.

BJU Int, 2004 Mar, 93(4), 491 - 4
Intravesical administration of gemcitabine in superficial bladder cancer: a phase I study with pharmacodynamic evaluation; De Berardinis E et al.; OBJECTIVE: To determine, in a phase I trial, the local and systemic toxicity and pharmacodynamics of intravesical gemcitabine in patients with superficial bladder cancer . PATIENTS AND METHODS: Twelve patients with histologically confirmed carcinoma localized to the bladder wall (stage T1 or Ta) resistant to previous administration of anticancer drugs and/or of bacille Calmette-Guerin were enrolled . They initially received intravesical gemcitabine starting at 500 mg and increased in 500 mg increments to 2000 mg . Three patients were treated at each dose level . RESULTS: There was no evidence of systemic toxicity and local toxicity was minimal . A pharmacological evaluation showed that gemcitabine was undetectable in plasma and its inactive metabolite (2',2'-difluorodeoxyuridine) was present at a mean (SD) concentration of 1.39 (1.05) mumol/L Deoxycytidine kinase was present in tumour tissue samples, and its activity was 27.3 (12.6) pmol/h/mg tissue; deoxycytidine deaminase activity varied from undetectable to 616 pmol/h/mg tissue . CONCLUSION: Intravesical gemcitabine appears to be well tolerated with no systemic and minimal local toxicity even at the highest dose (2000 mg) . A phase II trial of intravesical gemcitabine at 2000 mg given weekly for six consecutive weeks is now in progress in patients with superficial bladder cancer.

BJU Int, 2004 Mar, 93(4), 485 - 90
Intravesical bacillus Calmette-Guérin is superior to mitomycin C in reducing tumour recurrence in high-risk superficial bladder cancer: a meta-analysis of randomized trials; Shelley MD et al.; OBJECTIVE: To assess, in a systematic review and meta-analysis, the relative effectiveness of intravesical mitomycin C and bacillus Calmette-Guerin (BCG) for tumour recurrence, disease progression and overall survival in patients with medium- to high-risk Ta and T1 bladder cancer . METHODS: The major medical databases were searched comprehensively up to June 2003, and relevant journals hand-searched for randomized controlled trials, in any language, that compared intravesical mitomycin C with BCG in medium- to high-risk patients with Ta or T1 bladder cancer . RESULTS: Twenty-five articles were identified but only seven were considered eligible for the analysis . This represented 1901 evaluable patients in all, 820 randomized to mitomycin C and 1081 to BCG . Six trials had sufficient data for meta-analysis and included 1527 patients, 693 in the mitomycin and 834 in the BCG arm . There was no significant difference between mitomycin C and BCG for tumour recurrence in the six trials, with a weighted mean log hazard ratio, LHR, (variance) of -0.022 (0.005) . However, there was significant heterogeneity between trials (P = 0.001) . A subgroup analysis of three trials that included only high-risk Ta and T1 patients indicated no heterogeneity (P = 0.25) and a LHR for recurrence of -0.371 (0.012) . With mitomycin C used as the control in the meta-analysis, a negative ratio is in favour of BCG and, in this case, was highly significant (P < 0.001) . The seventh trial (in abstract form only) used BCG in low doses for two arms of the trial (27 mg and 13.5 mg) compared with a standard dose of mitomycin C (30 mg), and reported a significantly lower recurrence rate with BCG (27 mg) than for mitomycin C (P = 0.001) . Only two trials included sufficient data to analyse disease progression and survival, representing 681 patients (338 randomized to BCG and 343 to mitomycin C) . There was no significant difference between mitomycin C and BCG for disease progression, with a LHR of 0.044 (0.04) (P = 0.16), or survival, at -0.112 (0.03) (P = 0.50) . Adverse events were slightly more frequent with BCG . Local toxicity (dysuria, cystitis, frequency and haematuria) were associated with both mitomycin C (30%) and BCG (44%) . Systemic toxicity, e.g . chills, fever and malaise, occurred with both agents (12% and 19%, respectively) although skin rash was more common with mitomycin C . CONCLUSION: Tumour recurrence was significantly lower with intravesical BCG than with mitomycin C only in those patients at high risk of tumour recurrence . However, there was no difference in disease progression or survival, and the decision to use either agent might be based on adverse events and cost.

Appl Environ Microbiol, 2004 Mar, 70(3), 1514 - 21
Construction of a functional S-layer fusion protein comprising an immunoglobulin G-binding domain for development of specific adsorbents for extracorporeal blood purification; Vollenkle C et al.; The chimeric gene encoding a C-terminally-truncated form of the S-layer protein SbpA from Bacillus sphaericus CCM 2177 and two copies of the Fc-binding Z-domain was constructed, cloned, and heterologously expressed in Escherichia coli HMS174(DE3) . The Z-domain is a synthetic analogue of the B-domain of protein A, capable of binding the Fc part of immunoglobulin G (IgG) . The S-layer fusion protein rSbpA(31-1068)/ZZ retained the specific properties of the S-layer protein moiety to self-assemble in suspension and to recrystallize on supports precoated with secondary cell wall polymer (SCWP), which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall . Due to the construction principle of the S-layer fusion protein, the ZZ-domains remained exposed on the outermost surface of the protein lattice . The binding capacity of the native or cross-linked monolayer for human IgG was determined by surface plasmon resonance measurements . For batch adsorption experiments, 3-microm-diameter, biocompatible cellulose-based, SCWP-coated microbeads were used for recrystallization of the S-layer fusion protein . In the case of the native monolayer, the binding capacity for human IgG was 5.1 ng/mm(2), whereas after cross-linking with dimethyl pimelimidate, 4.4 ng of IgG/mm(2) was bound . This corresponded to 78 and 65% of the theoretical saturation capacity of a planar surface for IgGs aligned in the upright position, respectively . Compared to commercial particles used as immunoadsorbents to remove autoantibodies from sera of patients suffering from an autoimmune disease, the IgG binding capacity of the S-layer fusion protein-coated microbeads was at least 20 times higher . For that reason, this novel type of microbeads should find application in the microsphere-based detoxification system.

Appl Environ Microbiol, 2004 Mar, 70(3), 1378 - 84
Interaction of Bacillus thuringiensis toxins with larval midgut binding sites of Helicoverpa armigera (Lepidoptera: Noctuidae); Estela A et al.; In 1996, Bt-cotton (cotton expressing a Bacillus thuringiensis toxin gene) expressing the Cry1Ac protein was commercially introduced to control cotton pests . A threat to this first generation of transgenic cotton is the evolution of resistance by the insects . Second-generation Bt-cotton has been developed with either new B . thuringiensis genes or with a combination of cry genes . However, one requirement for the "stacked" gene strategy to work is that the stacked toxins bind to different binding sites . In the present study, the binding of (125)I-labeled Cry1Ab protein ((125)I-Cry1Ab) and (125)I-Cry1Ac to brush border membrane vesicles (BBMV) of Helicoverpa armigera was analyzed in competition experiments with 11 nonlabeled Cry proteins . The results indicate that Cry1Aa, Cry1Ab, and Cry1Ac competed for common binding sites . No other Cry proteins tested competed for either (125)I-Cry1Ab or (125)I-Cry1Ac binding, except Cry1Ja, which competed only at the highest concentrations used . Furthermore, BBMV from four H . armigera populations were also tested with (125)I-Cry1Ac and Cry1Ab to check the influence of the insect population on the binding results . Finally, the inhibitory effect of selected sugars and lectins was also determined . (125)I-Cry1Ac binding was strongly inhibited by N-acetylgalactosamine, sialic acid, and concanavalin A and moderately inhibited by soybean agglutinin . In contrast, (125)I-Cry1Ab binding was only significantly inhibited by concanavalin A . These results show that Cry1Ac and Cry1Ab use different epitopes for binding to BBMV.

Clin Exp Allergy, 2004 Mar, 34(3), 398 - 405
Bacillus Calmette-Guérin-induced interleukin-12 did not additionally improve clinical and immunologic parameters in asthmatic children treated with sublingual immunotherapy; Arikan C et al.; OBJECTIVE: To evaluate the effect of bacillus Calmette-Guerin (BCG) as an adjuvant to specific sublingual immunotherapy (SLIT) on the cytokine profile of peripheral blood mononuclear cells (PBMCs) and clinical outcome . METHODS: Thirty-two children with asthma and rhinitis allergic to house dust mite (HDM) with negative purified protein derivative (PPD) skin test response were enrolled . After a run-in period of 8 weeks, patients were randomized to receive either SLIT only (n=16) or one dose of BCG immunization before initiation of SLIT (n=16) with a standardized Dermatophagoides pteronyssinus (D . pteronyssinus)+D . farinea 50/50 extract . PPD-negative asthmatics (n=5) allergic to HDM receiving inhaled therapy only were included for comparison of cytokine levels in PBMC cultures . Efficacy was assessed both at the end of run-in and 6 months of treatment periods with criteria including symptom, medication and quality-of-life (QoL) scores, IgE levels, lung function, provocation concentration (PC20), eosinophil count and skin prick tests . IL-4, IL-5, IL-10, IL-12, IL-13 and IFN-gamma levels were determined in antigen specifically and polyclonally stimulated PBMC cultures . RESULTS: Both treatment groups showed significant improvement at the end of 6 months for asthma and rhinitis scores and QoL, number of asthma attacks, amount of beta2-agonists, inhaled and intranasal steroids, blood eosinophil counts and PC20 . Interestingly, phytohaemagglutinin (PHA)-stimulated IL-12 and D . pteronyssinus-stimulated IFN-gamma in PBMC were significantly higher in the treatment groups than controls . In addition, IL-12 levels in response to D . pteronyssinus and PHA stimulation were significantly higher in the SLIT+BCG group than the SLIT alone group and controls . CONCLUSION: The present study demonstrates that successful SLIT is parallel to increased IFN-gamma production by PBMC . Although simultaneous BCG vaccination enhanced IL-12 production, it did not additionally improve the clinical outcome.

Biochemistry, 2004 Mar 16, 43(10), 2784 - 91
The crystal structure of glutamyl endopeptidase from Bacillus intermedius reveals a structural link between zymogen activation and charge compensation; Meijers R et al.; Extracellular glutamyl endopeptidase from Bacillus intermedius (BIEP) is a chymotrypsin-like serine protease which cleaves the peptide bond on the carboxyl side of glutamic acid . Its three-dimensional structure was determined for C222(1) and C2 crystal forms of BIEP to 1.5 and 1.75 A resolution, respectively . The topology of BIEP diverges from the most common chymotrypsin architecture, because one of the domains consists of a beta-sandwich consisting of two antiparallel beta-sheets and two helices . In the C2 crystals, a 2-methyl-2,4-pentanediol (MPD) molecule was found in the substrate binding site, mimicking a glutamic acid . This enabled the identification of the residues involved in the substrate recognition . The presence of the MPD molecule causes a change in the active site; the interaction between two catalytic residues (His47 and Ser171) is disrupted . The N-terminal end of the enzyme is involved in the formation of the substrate binding pocket . This indicates a direct relation between zymogen activation and substrate charge compensation.

Biochim Biophys Acta, 2004 Mar 9, 1661(2), 154 - 65
Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy; Gufler PC et al.; In the present work, S-layer supported lipid membranes formed by a modified Langmuir-Blodgett technique were investigated by electrochemical impedance spectroscopy (EIS) . Basically two intermediate hydrophilic supports for phospholipid- (DPhyPC) and bipolar tetraetherlipid- (MPL from Thermoplasma acidophilum) membranes have been applied: first, the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177 recrystallized onto a gold electrode; and second, as a reference support, an S-layer ultrafiltration membrane (SUM), which consists of a microfiltration membrane (MFM) with deposited S-layer carrying cell wall fragments . The electrochemical properties and the stability of DPhyPC and MPL membranes were found to depend on the used support . The specific capacitances were 0.53 and 0.69 microF/cm(2) for DPhyPC bilayers and 0.75 and 0.77 microF/cm(2) for MPL monolayers resting on SbpA and SUM, respectively . Membrane resistances of up to 80 mega Ohm cm(2) were observed for DPhyPC bilayers on SbpA . In addition, membranes supported by SbpA exhibited a remarkable long-term robustness of up to 2 days . The membrane functionality could be demonstrated by reconstitution of membrane-active peptides such as valinomycin and alamethicin . The present results recommend S-layer-supported lipid membranes as promising structures for membrane protein-based biosensor technology.

J Microbiol Methods, 2004 Apr, 57(1), 1 - 7
Evaluation of the MicroFoss system for the detection of Listeria species in environmental samples; Odumeru JA et al.; The MicroFoss system was evaluated for its ability to detect Listeria species in environmental samples . The sensitivity and specificity of the MicroFoss were determined in relation to a standard culture method for Listeria detection . The sensitivities of both the MicroFoss and standard culture methods were similar (88.4%-MicroFoss, 90.7%-Culture) based on the total number of positive results obtained by both methods . The MicroFoss system detected Listeria spp . in 12 samples, which were not detected by culture, and the culture method detected Listeria spp . in 15 samples, which were not detected by the MicroFoss method . This was likely due to uneven distribution of low levels of Listeria organisms in the split sponge samples used to assess the performance of these test methods . The specificity value determined for the MicroFoss system was 92.7% . The majority of microbes causing false positive results in the MicroFoss system were Bacillus species, which were readily distinguishable from Listeria species by a simple Gram stain and morphological features . Listeria monocytogenes (89.4%-MicroFoss, 88.0%-Culture) and Listeria innocua (8.8%-MicroFoss, 7.7%-Culture) were the most common isolates of Listeria detected by the two test methods, with L . monocytogenes being the most predominant isolate detected . The highly comparable results and rapid nature of the MicroFoss system demonstrate its effectiveness as a detection system for species of Listeria in environmental samples . The fact that the sensitivity of the MicroFoss system was similar to that of the culture method and the Listeria results were obtained within 48 h of testing, support the use of the MicroFoss as an alternative rapid method for screening large numbers of environmental samples for Listeria spp.

Appl Spectrosc, 2004 Feb, 58(2), 203 - 11
Identification of sporulated and vegetative bacteria using statistical analysis of fourier transform mid-infrared transmission data; Foster NS et al.; A combined mid-infrared spectroscopic/statistical modeling approach for the discrimination and identification, at the strain level, of both sporulated and vegetative bacterial samples is presented . Transmission mode spectra of bacteria dried on ZnSe windows were collected using a Fourier transform mid-infrared (FT-IR) spectrometer . Five Bacillus bacterial strains (B . atrophaeus 49337, B . globigii Dugway, B . thuringiensis spp . kurstaki 35866, B . subtilis 49760, and B . subtilis 6051) were used to construct a reference spectral library and to parameterize a four-step statistical model for the systematic identification of bacteria . The statistical methods used in this initial feasibility study included principal component analysis (PCA), classification and regression trees (CART), and Mahalanobis distance calculations . Internal cross-validation studies successfully classified 100% of the samples into their correct physiological state (sporulated or vegetative) and identified 67% of the samples correctly as to their bacterial strain . Analysis of thirteen blind samples, which included reference and other bacteria, nonbiological materials, and mixtures of both nonbiological and bacterial samples, yielded comparable accuracy . The primary advantage of this approach is the accurate identification of unknown bacteria, including spores, in a matter of minutes.

Biochemistry (Mosc), 2004 Feb, 69(2), 181 - 7
Reconstruction of Bacillus thuringiensis ssp . israelensis Cry11A endotoxin from fragments corresponding to its N- and C-moieties restores its original biological activity; Revina LP et al.; Subtilisin hydrolyzes Cry11A endotoxin (of 70 kD) produced by Bacillus thuringiensis ssp . israelensis to fragments of 33- and 36-kD, which correspond to N- and C-terminal halves of the endotoxin molecule . Thermitase (a serine protease from Thermoactinomyces vulgaris) and insect gut proteases from Diptera and Lepidoptera exhibit the same hydrolytic effect on Cry11A . Hydrolyzates maintain high toxicity with respect to larvae of Aedes aegypti, Anopheles stephensi, and Culex pipiens . The 33- and 36-kD Cry11A endotoxin components purified by ion-exchange chromatography from the subtilisin hydrolyzate were inactive; however, equimolar mixture of these proteins exhibited almost the same activity as the initial hydrolyzate.

Biotechnol Lett, 2004 Jan, 26(2), 143 - 5
Bacillus thuringiensis var . israelensis production involving re-use of the supernatant; Luna CL et al.; The supernatant arising after biomass separation of Bacillus thuringiensis var . israelensis by flocculation/sedimentation was re-used after being supplemented with 25, 50 and 75% (w/v) of the original culture medium, based on corn steep liquor, glucose and mineral salts . Supplementation at 75% gave a spore concentration (1 x 10(10) c.f.u . ml(-1)) five times greater than that obtained with the other supplements.

Biotechnol Lett, 2004 Jan, 26(2), 115 - 9
Purification and characterization of a new peptide antibiotic produced by a thermotolerant Bacillus licheniformis strain; Mendo S et al.; A Bacillus licheniformis strain, 189, isolated from a hot spring environment in the Azores, Portugal, strongly inhibited growth of Gram-positive bacteria . It produced a peptide antibiotic at 50 degrees C . The antibiotic was purified and biochemically characterized . It was highly resistant to several proteolytic enzymes . Additionally, it retained its antimicrobial activity after incubation at pH values between 3.5 and 8; it was thermostable, retaining about 85% and 20% of its activity after 6 h at 50 degrees C and 100 degrees C, respectively . Its molecular mass determined by mass spectrometry was 3249.7 Da.

J Econ Entomol, 2004 Feb, 97(1), 51 - 8
Toxicity of VectoLex (Bacillus sphaericus) products to selected Australian mosquito and nontarget species; Brown ID et al.; Laboratory and field bioassay studies were conducted in southeast Queensland, Australia, on the efficacy of VectoLex Control Granule (CG; active ingredient {AI}:50 Bacillus sphaericus {B.s.} International Toxic Units {ITU}/mg) and VectoLex Water Dispersible Granule (WDG) (AI: 650 B.s . ITU/mg) formulations against third-instar larvae of Culex annulirostris Skuse, Culex quinquefasciatus Say, Culex sitiens Wiedemann, Ochlerotatus rigilax (Skuse), Ochlerotatus . notoscriptus (Skuse), and Aedes aegypti (L.) . The Gompertz model for log-linear mortality data were used to determine laboratory 48-h LC95 values . As with foreign evaluations, the B.s . formulations were most effective against Culex spp., with the WDG 10-100 times more effective than the CG on an ITU/mosquito basis . Consequently, Cx . annulirostris and Cx . quinquefasciatus were selected as target species for small-plot field evaluation of WDG efficacy over time . Weekly cohorts of caged third-instar Cx . annulirostris were exposed to replicated low (250 g/ha), medium (500 g/ha), and high (1,000 g/ha) dosages of WDG . Concurrent assessment of Cx . quinquefasciatus mortality outside the cages was also conducted . In water with high organic content, the low rate produced > 99% Cx . annulirostris mortality at 48 h, decreasing to 79% at week 3 and no control at week 4 . The medium and high rates resulted in 100% Cx . annulirostris mortality for 2 wk posttreatment, decreasing to 95% at week 3, and no control at week 4 . The WDG was equally effective against Cx . quinquefasciatus . Treatment did not affect water quality or nontarget shrimp and fish species survival.

Ying Yong Sheng Tai Xue Bao, 2003 Nov, 14(11), 2062 - 6
{Advances in safety studies of soil Bt toxin proteins released from transgenic Bt crops}; Bai Y et al.; Commercialized transgenic Bt (Bacillus thuringiensis) crops are permitted for field growth in a large scale, which leads to significant issues of ecological risk assessment in soil ecosystem . In this paper, some general safety problems involving in the soil Bt active toxins released from insect-resistant transgenic Bt crops in the forms of plant residues, root exudates and pollens were reviewed, including their adsorption by soil active-particles, their insecticidal activity, persistence, and biodegradation by soil microbes, and their effects on soil organisms.

J Biol Chem, 2004 May 7, 279(19), 20490 - 500 Epub 2004 Mar 02.
Cross-linking phosphatidylinositol-specific phospholipase C traps two activating phosphatidylcholine molecules on the enzyme; Zhang X et al.; Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), a bacterial model for the catalytic domain of mammalian PI-PLC enzymes, was cross-linked by 1-ethyl-3-{3-dimethylaminopropyl}carbodiimide hydrochloride to probe for the aggregation and/or conformational changes of PI-PLC when bound to activating phosphatidylcholine (PC) interfaces . Dimers and higher order multimers (up to 31% of the total protein when cross-linked at pH 7) were observed when the enzyme was cross-linked in the presence of PC vesicles . Aggregates were also detected with PI-PLC bound to diheptanoyl-PC (diC(7)PC) micelles, although the fraction of cross-linked multimers (19% at pH 7) was lower than when the enzyme was cross-linked in the presence of vesicles . PI-PLC cross-linked in the presence of a diC(7)PC interface exhibited an enhanced specific activity for PI cleavage . The extent of this cross-linking-enhanced activation was reduced in PI-PLC mutants lacking either tryptophan in the rim (W47A and W242A) of this (betaalpha)(8)-barrel protein . The higher activity of the native protein cross-linked in the presence of diC(7)PC correlated with an increased affinity of the protein for two diC(7)PC molecules as detected by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry . In contrast to wild type protein, W47A and W242A had only a single diC(7)PC tightly associated when cross-linked in the presence of that activator molecule . These results indicate that (i) each rim tryptophan residue is involved in binding a PC molecule at interfaces, (ii) the affinity of the enzyme for an activating PC molecule is enhanced when the protein is bound to a surface, and (iii) this conformation of the enzyme with at least two PC bound that is stabilized by chemical cross-linking interacts more effectively with activating interfaces, leading to higher observed specific activities for the phosphotransferase reaction.

Int J Food Microbiol, 2004 Mar 1, 91(2), 209 - 13
Effect of high pressure gaseous carbon dioxide on the germination of bacterial spores; Furukawa S et al.; Effect of high pressure gaseous carbon dioxide treatment (HGCT) at 6.5 MPa, 35 degrees C on the germination of bacterial spores was investigated . Germination of bacterial spores was estimated by the decrease of heat tolerance . Approximately, 40% of Bacillus coagulans and 70% of Bacillus licheniformis were germinated by HGCT for 120 min at 35 degrees C, respectively . Germination was confirmed by phase contrast microscopy . The effect of hydrostatic pressure treatment (HPT) at 6.5 MPa, 35 degrees C on the germination of B . coagulans and B . licheniformis spores were also investigated . Spores did not germinate by HPT alone at 6.5 MPa for 120 min.

Transfusion, 2004 Mar, 44(3), 337 - 42
The use of a bacteria detection system to evaluate bacterial contamination in PLT concentrates; Rock G et al.; BACKGROUND: Random-donor PLTs (RDPs) are functional at 7 days . Nevertheless, since the mid-1980s, concern for bacterial contamination has caused the storage period to be reduced to 5 days . The ability of a bacteria detection system (BDS, Pall) to determine bacterial contamination and permit extension of the PLT shelf life to 7 days was assessed . STUDY DESIGN AND METHODS: Blood was collected into CP2D and leukoreduced RDPs were prepared . Upon arrival at the hospital, a 2- to 3-mL aliquot was removed from each RDP and introduced into the Pall BDS pouch with a sterile docking device . The pouch was incubated at 37 degrees C for 24 hours and then the oxygen content was measured to determine bacterial contamination . Additionally, the RDPs were pooled and an aliquot was removed for culture with standard manual techniques . CCIs were calculated 1 hour after infusion . RESULTS: A total of 12,062 individual RDPs were tested . The Pall BDS detected bacteria in 5 units . All of these were positive on repeat sampling . Propionibacterium acnes, coagulase-negative Staphylococcus, and Bacillus species were confirmed by manual technique in 3 units, one could not be identified, and one was negative . Aliquots from PLT pools were positive in 80 of 2201 pools when tested by manual methods . Of these, 79 were false-positives and 1 unit contained coagulase-negative Staphylococcus . The Pall BDS was easy to use and required less than 5 minutes for all manipulations . After 7 days of storage, the PLTs gave an average CCI of 16 x 10(11)+/- 3.39 x 10(11) 1 hour after transfusion (n = 9) . CONCLUSIONS: The Pall BDS permits evaluation of RDPs for bacterial contamination . Culture-negative PLTs were successfully transfused in our institution up to and including 7 days after storage with good CCIs.

J Agric Food Chem, 2004 Mar 10, 52(5), 1390 - 7
Lower fumonisin mycotoxin levels in the grain of Bt corn grown in the United States in 2000-2002; Hammond BG et al.; Fumonisins were monitored in corn grain collected from Bt hybrids grown in 107 locations across the United States in 2000-2002 . Bt corn hybrids contain the Cry1Ab protein from Bacillus thuringiensis that controls European corn borers and other stalk-boring pests . Fumonisin levels were frequently lower in grain from Bt hybrids grown in field trials under conditions of natural (FACT trials) or manual insect infestation (university trials) . Over three years of FACT trials, there were 126/210 comparisons when fumonisin levels in grain from control hybrids were >2 ppm, exceeding U.S . FDA guidance levels of 2 ppm for human food . Grain from Bt hybrids was at or below 2 ppm of fumonisins for 58 of the 126 comparisons . The use of Bt hybrids can increase the percentage of corn grain that would be suitable for use in food and feed.

J Econ Entomol, 2003 Apr, 96(2), 396 - 400
Laboratory and field evaluation of tebufenozide, diflubenzuron, and Bacillus thurengiensis var . kurstaki for suppression of Douglas-fir tussock moth (Orgyia pseudotsugata (McDunnough)) in Idaho: a case study; Cook SP; The insect growth regulator tebufenozide (MIMIC 2LV) was tested to examine its impact on the Douglas-fir tussock moth, Orgyia pseudotsugata (McDunnough) (Lepidoptera: Lymantriidae) . Laboratory tests gave an estimated concentration of 1.26 ppm of the compound to achieve 50% population mortality (LC50) for second-instar O . pseudotsugata . At the highest concentration tested, tebufenozide resulted in significant larval mortality within 7 d with an estimated time to 50% population mortality (LT50) of 6.3 d . A field comparison oftebufenozide with diflubenzuron (Dimilin 4L) and Bacillus thurengiensis var . kurstaki (Btk, FORAY 48B) was also conducted . There was no significant difference in larval mortality within field plots that were treated with diflubenzuron (42.4%) or Btk (44.8%) . Larval mortality in the tebufenozide-treated plots (56.8%) was also similar to the mortality in the diflubenzuron and Btk treatments . All three treatments resulted in more larval mortality than that measured in untreated controlplots (11.2%) . Both tebufenozide and diflubenzuron treatments resulted in significantly more mortality (55.0% and 40.0%, respectively) to larvae-fed treated foliage 3 wk after application than was measured for larvae-fed foliage from untreated trees (11.0%) . There was no significant difference among the treatments in the percentage of host trees in the overstory that sustained >25% defoliation, and all three treatments resulted in less defoliation than was measured in the control plots . There was no significant difference among the treatments in the percentage of host trees in the understory that sustained >25% defoliation.

J Econ Entomol, 2003 Apr, 96(2), 388 - 95
Design and evaluation of an aerial spray trial with true replicates to test the efficacy of Bacillus thuringiensis insecticide in a boreal forest; Cadogan BL et al.; A field trial using true replicates was conducted successfully in a boreal forest in 1996 to evaluate the efficacy of two aerially applied Bacillus thuringiensis formulations, ABG 6429 and ABG 6430 . A complete randomized design with four replicates per treatment was chosen . Twelve to 15 balsam fir (Abies balsamea {L.} Mill.) per plot were randomly selected as sample trees . Interplot buffer zones, > or = 200 m wide, adequately prevented cross contamination from sprays that were atomized with four rotary atomizers (volume median diameters ranging from 64.6 to 139.4 microm) and released approximately 30 m above the ground . The B . thuringiensis formulations were not significantly different (P > 0.05) from each other in reducing spruce budworm (Choristoneura fumiferana {Clem.}) populations and protecting balsam trees from defoliation but both formulations were significantly more efficacious than the controls . The results suggest that true replicates are a feasible alternative to pseudoreplication in experimental forest aerial applications.

J Econ Entomol, 2003 Apr, 96(2), 292 - 9
Preparation of spray-dried wettable powder formulations of Bacillus thuringiensis-based biopesticides; Teera-Arunsiri A et al.; Bacillus thuringiensis is the most widely used biopesticide among many methods available to control insects . To make a saleable product, B . thuringiensis must be substantially concentrated by removal of water and formulated to improve longevity, efficacy, and ease of transport of the product . B . thuringiensis subsp . aizawai culture broth as an active ingredient was mixed with various adjuvants and then spray dried . The optimum conditions for spray drying were found to be an outlet temperature of 60-85 degrees C and an inlet temperature of 120-180 degrees C . Various adjuvants had different effects on physical and biological properties of the dried product . Gelatinized tapioca starch and milk powder improved suspensibility but adversely affected wettability of the dried formulated product . Vegetable oil and Tween 20 enhanced wettability but resulted in poor suspensibility . Silica fume was used to enhance flowability because it reduced clumping and caking of the powder resulting from the addition of vegetable oil . Formulation containing 10% wt:wt B . thuringiensis, 10% wt:wt gelatinized tapioca starch, 10% wt:wt sucrose, 38% wt:wt tapioca starch, 20% wt:wt milk powder, 10% wt:wt silica fume, 2% wt:wt polyvinyl alcohol, 5% vol:vol Tween 20, 1% vol:vol refined rice bran oil, and 1% vol:vol antifoam solution was found to be optimum in terms of the physical and biological properties of the dried product . This formulation had 55% suspensibility, 24 s for wetting time, and 5.69 x 10(4) CFU/ml of LC50 value against Spodoptera exigua larvae.

J Econ Entomol, 2003 Apr, 96(2), 280 - 6
Lethal and sublethal effects of single and double applications of Bacillus thuringiensis variety kurstaki on spruce budworm (Lepidoptera: Tortricidae) larvae; Moreau G et al.; We conducted laboratory experiments to examine the effects of single versus double exposures of spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae) female larvae to various concentrations of a Bacillus thuringiensis variety kurstaki (Btk) commercial formulation (Foray 48B) . Our main objective was to document the vulnerability to Btk and the sublethal responses of fifth-instar larvae that survived from a first ingestion of Btk during their fourth stadium and to compare them with insects treated either during their fifth or fourth stadium only . As reported in the literature, fifth-instar larvae were more vulnerable than fourth-instar larvae, but only at low and medium concentrations . Fifth-instar larvae that had survived Btk ingestion during their fourth stadium were more vulnerable to a high concentration of Btk and had a shorter feeding inhibition period than those that had not been exposed during their fourth stadium . Compared with a single treatment at the fourth stadium, a double exposure to Btk further reduced the population by 20-30%, depending on the concentration applied . The second treatment also induced another feeding inhibition period and increased larval development time by 14% . The impact of the different treatments on pupal weight depended on whether treated insects exhibited supernumerary instars . In the absence of developmental polymorphism, a higher concentration, a late, or a double exposure to Btk significantly reduced pupal weight.

Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 586 - 7 Epub 2004 Feb 25.
Crystallization and preliminary X-ray study of gamma-type cyclodextrin glycosyltransferase from Bacillus clarkii; Akita M et al.; A gamma-cyclodextrin glycosyltransferase (EC 2.4.1.19) from Bacillus clarkii was crystallized using the hanging-drop vapour-diffusion method at 293 K . X-ray diffraction data were collected to 2.2 A . The crystal belongs to space group R3, with unit-cell parameters a = b = 211.6, c = 52.7 A . The asymmetric unit contains one protein molecule, with a corresponding V(M) of 3.03 A(3) Da(-1) and a solvent content of 59.4% . Molecular replacement was successfully carried out using a homology model based on the three-dimensional structure of the CGTase from Thermonanaerobacterium thermosulfurigenes EM1 as a search model.

Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 564 - 6 Epub 2004 Feb 25.
Crystallization and preliminary X-ray crystallographic analysis of the C-terminal domain of ParC protein from Bacillus stearothermophilus; Hsieh TJ et al.; Type IIA topoisomerases are multidomain enzymes composed of four major domains: the ATPase domain, the TOPRIM domain, the DNA-cleavage/religation domain and the C-terminal domain (CTD) . Although crystal structures of the first three domains are available, the three-dimensional structure of the less-conserved CTD has yet to be determined . In order to provide a three-dimensional structure of this structurally uncharacterized region, the 36 kDa CTD of ParC protein, the DNA-cleavage/religation subunit of topoisomerase IV, from Bacillus stearothermophilus has been cloned, purified and crystallized . The crystals belonged to the trigonal space group P3(1) (or P3(2)), with unit-cell parameters a = b = 83.5, c = 45.1 A . The asymmetric unit contains one molecule and the solvent content is 51.2% . A 98.9% complete native data set has been collected from a frozen crystal to 2.0 A resolution with an overall R(merge) of 6.5%.

Am J Trop Med Hyg, 2004 Feb, 70(2), 197 - 200
Parallel assessment of 24 monthly doses of rifampin, ofloxacin, and minocycline versus two years of World Health Organization multi-drug therapy for multi-bacillary leprosy; Villahermosa LG et al.; Monthly doses of rifampin, ofloxacin, and minocycline (ROM) are expected to be effective treatment for multi-bacillary leprosy . Patients with MB leprosy received ROM (n = 10) or World Health Organization multi-drug therapy (MDT) (n = 11) . Treatment with ROM was given as 24 consecutive monthly observed doses of rifampin (600 mg), ofloxacin (400 mg), and minocycline (100 mg) . Treatment with MDT was given as 24 consecutive monthly observed doses of rifampin (600 mg) and clofazimine (300 mg), and unobserved daily dapsone (100 mg) and clofazimine (50 mg) . Twenty patients completed the 24-month regimens with > 99% compliance . Treatments with ROM and MDT were safe, tolerable, and caused similar improvements in lesions, bacterial indices, and histology . All MDT recipients developed clofazimine-induced pigmentation . Six ROM and nine MDT recipients assessed at five or more years after completion of treatment had no evidence of relapse . Twenty-four months of treatment with ROM is a safe, well-tolerated, and convenient regimen that may provide an alternate therapy to MDT for MB leprosy . Larger trials with sufficient follow-up would better define the role of ROM.

J Med Entomol, 2004 Jan, 41(1), 58 - 64
Inheritance and mechanism of resistance to Bacillus sphaericus in Culex quinquefasciatus (Diptera: Culicidae) from China and Brazil; Oliveira CM et al.; Investigations on the inheritance and mechanism of resistance to Bacillus sphaericus Neide in Culex quinquefasciatus Say colonies, selected with strains C3-41 (RLCq1/C3-41) and 2362 (CqRL1/2362), were performed in China and Brazil, respectively . The progeny of reciprocal F1 crosses (susceptible female x resistant male and vice versa) from both resistant colonies responded alike in bioassays, indicating recessive inheritance . Data on larvae susceptibility from the backcross offspring between F1 and their respective susceptible and resistant parental colonies are consistent with a monofactorial and autosomal mode of inheritance . In vitro binding assays between 125I binary (Bin2) toxin and the brush border membrane fractions (BBMF) from CqRL1/2362 and RLCq1/C3-41 larvae showed that resistance, in both colonies, is caused by a failure in the binding step of the B . sphaericus Bin2 toxin to its specific midgut receptor . The specific and saturable binding of Bin2 toxin to BBMF from F1 larvae (CqRL1/2362 X susceptible counterpart) confirms the recessive inheritance of the resistance gene . Further studies are needed to advance understanding of B . sphaericus resistance.

FEMS Microbiol Lett, 2004 Feb 16, 231(2), 191 - 6
The Erp protein is anchored at the surface by a carboxy-terminal hydrophobic domain and is important for cell-wall structure in Mycobacterium smegmatis; Kocincova D et al.; Erp (Exported Repetitive Protein), also known as P36, Pirg and Rv3810, is a member of a mycobacteria-specific family of extracellular proteins . In pathogenic species, the erp gene has been described as a virulence factor . The Erp proteins comprise three domains . The N- and C-terminal domains are similar in all mycobacterial species, while the central domain consists of a repeated module that differs considerably between species . Here we show that the Erp protein is loosely attached to the surface and that the carboxy-terminal domain, which displays hydrophobic features, anchors Erp at the surface of the bacillus . The hydrophobic region is not necessary for the complementation of the altered colony morphology of a Mycobacterium smegmatis erp- mutant but proved to be necessary to achieve resistance to detergent at wild-type levels.

Clin Exp Allergy, 2004 Feb, 34(2), 207 - 12
Effect of repeated intradermal injections of heat-inactivated Mycobacterium bovis bacillus Calmette-Guérin in adult asthma; Shirtcliffe PM et al.; BACKGROUND: There are a number of reports in the Chinese medical literature from the last 30 years regarding the efficacy of repeated doses of heat-inactivated bacillus Calmette-Guerin (BCG) in established asthma . There is also epidemiological and experimental evidence that exposure to mycobacteria has the potential to suppress the development of asthma/atopy . METHODS: Thirty-one Mantoux-negative adults with stable moderately severe asthma who were skin prick test positive to house dust mite were randomized to receive one injection (0.1 mL) a week for 4 weeks of heat-inactivated BCG or normal saline . Markers of asthma severity (including peak flow, forced expiratory volume in 1 s, major and minor exacerbations, symptom scores and beta-agonist use), blood eosinophil and IgE levels were monitored for 3 months . RESULTS: There were no statistically significant differences between the treatment group and placebo for any of the outcome variables . The recruitment to the trial was halted early and the number of injections reduced in a number of patients due to excessive local reactions to BCG . CONCLUSIONS: In addition to the lack of efficacy of repeated heat-inactivated BCG injections, the occurrence of severe local reactions will limit the therapeutic application of this approach in asthma.

Transpl Infect Dis, 2003 Dec, 5(4), 195 - 8
Oerskovia xanthineolytica endocarditis in a renal transplant patient: case report and review of the literature; Urbina BY et al.; Oerskovia species were, until recently, only rarely associated with human disease . This gram-positive bacillus can be easily misidentified as a diphtheroid, a common contaminant in blood cultures . There have been 17 reports of invasive Oerskovia infection in immunocompromised hosts . We report a case of Oerskovia xanthineolytica endocarditis in a renal transplant patient and review the microbiologic and clinical characteristics of this potential pathogen.

Prescrire Int, 2003 Dec, 12(68), 226 - 9
Intradermal BCG: partial protection against tuberculosis in children: unproven efficacy of multipuncture administration; The immunological aspects of latency in tuberculosis; Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA . jchan@aecom.yu.edu

A unique feature of Mycobacterium tuberculosis is its ability to exist in the granuloma of an asymptomatic host in a latent state that can subsequently reactivate to cause active disease . The latent state of infection poses a major obstacle to eradicating tuberculosis . In latent tuberculosis, the host immune response is capable of controlling the infection and yet falls short of eradicating the pathogen . That the host immune response contributes to the maintenance of latent tuberculous infection is supported by the observation that certain immunodeficient states, including those associated with the human immunodeficiency virus and tumor necrosis factor neutralization therapy, are associated with increased risks for developing reactivation disease . Latent tuberculosis is the product of a complex set of interactions between M . tuberculosis and the host immune response . The molecular basis for the persistence phenotype of M . Tuberculosis and the pertinent host immune mechanisms that contribute to the maintenance of tuberculous latency are just beginning to be understood . This review discusses the interactions between M . tuberculosis and the macrophage, the primary host cell that the tubercle bacillus parasitizes.

J Int Assoc Physicians AIDS Care (Chic Ill), 2003 Oct-Dec, 2(4), 133 - 9
Clinical vignette in antiretroviral therapy: jaundice; Zell SC; HIV caregivers face many challenges following initiation of ART . The development of jaundice is uncommon but worrisome . In this case, two distinct and contrasting episodes of jaundice were observed . In the first instance, isolated elevation of the indirect bilirubin without elevation of the alkaline phosphatase was noted . The normal PT and serum aminotransferase levels indicate the absence of intrinsic liver dysfunction . Elevations in the indirect bilirubin may result from either impaired uptake/conjugation or excess production . The latter, usually from acquired hemolysis, may be a complication of an occult NHL . A work-up for this AIDS-related malignancy was not initiated since the caregivers recognized jaundice as a complication of IDV, which inhibits UDP-glucuronyl transferase and produces a Gilbert's-like syndrome . Physicians can expect to encounter this syndrome even more frequently with ATV . Experienced patients given RTV-boosted ATV have experienced elevations of unconjugated hyper-bilirubinemia in up to 45 percent of cases in clinical trials . However, such elevations do not reflect liver dysfunction and symptomatic jaundice requiring dosage reduction that occurred infrequently (7 to 8 percent of study patients) . Counseling patients about this syndrome may promote adherence and prevent self-directed interruptions of ATV that compromise efficacy . The second case of jaundice provides a more formidable diagnostic challenge . The triad of LFT abnormalities (mild elevation of aminotransferases, normal PT, and marked cholestatic jaundice) implies an acute process that is mildly toxic to hepatocytes without affecting their synthetic function . The subacute nature of the patient's cholestatic jaundice suggests either intrahepatic infiltrative disease of the liver or extrahepatic obstruction of the biliary tree, most likely due to the patient's relatively modest level of pain and lack of fever . Despite LFT abnormalities occurring 17 months after a switch in his ART, cumulative drug-related toxicities must still be considered . Ritonavir can produce significant elevations in the AST/ALT, especially with pre-existing chronic liver disease as with hepatitis C virus coinfection . The NRTIs can produce hepatic steatosis, a result of mitochondrial toxicity and impaired fatty acid oxidation . However, jaundice and cholestasis are not typical of the latter syndrome . With a negative contrast CT that excludes parenchymal liver disease, investigation of the biliary tree to assess the presence of AIDS-related cholangitis was the next step . Performing a sphincterotomy or stent placement, and obtaining brushings or biopsy specimens to determine the extent of extrahepatic obstruction may help define a pathogen and be life-saving . The negative results of the ERCP justify the final diagnostic step, a liver biopsy to evaluate microscopic infiltrative disease that might not have been detected on contrast abdominal CT . Examples might include granulomatous disease (MAC), fungal etiologies (histoplasmosis), carcinomatosis (lymphoma, hepatoma, cholangiocarcinoma), and microvascular disease (bacillary angiomatosis) . The failure to observe granulomatous inflammation in the liver does not exclude MAC infection, as MAC may involve other peri-aortic or mesenteric lymph nodes . This form of IRIS is unlikely given the abdominal CT findings, lack of systemic complaints, and extended persistence of liver aminotransferases . The nonspecific results of the liver biopsy are a common outcome in advanced AIDS patients with elevated alkaline phosphatase levels . Despite not having identified a pathogen, the biopsy establishes chronic liver disease and prompts re-evaluation and change of treatment to NFV . The subsequent normalization of the patient's aminotransferase levels suggests a prior adverse effect of LPV/r in the setting of unexplained, chronic liver disease . Most importantly, this case highlights the importance of HIV caregivers to review ART for safety when noting chronic liver dysfunction . Patients need to be counseled to minimize acetaminophen use, to consume alcohol in moderation, and to avoid behavior with risk for hepatitis C . Finally, all HIV patients should receive appropriate vaccination against hepatitis A and B if serology shows lack of protective immunity.

J Ind Microbiol Biotechnol, 2004 Feb, 31(2), 83 - 7 Epub 2004 Feb 19.
Compatibility of alkaline xylanases from an alkaliphilic Bacillus NCL (87-6-10) with commercial detergents and proteases; Kamal Kumar B et al.; Alkaline xylanases from alkaliphilic Bacillus strains NCL (87-6-10) and Sam III were compared with the commercial xylanases Pulpzyme HC and Biopulp for their compatibility with detergents and proteases for laundry applications . Among the four xylanases evaluated, the enzyme from the alkaliphilic Bacillus strain NCL (87-6-10) was the most compatible . The enzyme retained its full activity (40 degrees C for 1 h) in the presence of detergents, whereas Pulpzyme HC and Sam III showed only 30% and 50% of their initial activity, respectively . Biopulp, though stable to detergents, had only marginal activity (5%)at pH 10 . However, all four enzymes retained significant activity (80%) for 60 min in the presence of the proteases Alcalase and Conidiobolus protease . Supplementation of the enzyme enhanced the cleaning ability of the detergents.

Protein Eng Des Sel, 2004 Jan, 17(1), 85 - 93
Fusion of the antiferritin antibody VL domain to barnase results in enhanced solubility and altered pH stability; Martsev SP et al.; Chimeric immunotoxins that combine antigen recognition domains of antibodies and cytotoxic RNases have attracted much attention in recent years as potential targeted agents for cancer immunotherapy . In an attempt to obtain a structurally minimized immunofusion for folding/stability studies, we constructed the chimeric protein VL-barnase . The chimera comprises a small cytotoxic enzyme barnase, ribonuclease from Bacillus amyloliquefaciens, fused to the C-terminus of the light chain variable domain (VL) of the anti-human ferritin monoclonal antibody F11 . While the individual VL domain was expressed in Escherichia coli as insoluble protein packed into inclusion bodies, its fusion to barnase resulted in a significant ( approximately 70%) fraction of soluble protein, with only a minor insoluble fraction ( approximately 30%) packed into inclusion bodies . The in vivo solubilizing effect of barnase was also observed in vitro and suggests a chaperone-like role that barnase exerted with regard to the N-terminal VL domain . Cytoplasmic VL-barnase was analyzed for structural and functional properties . The dimeric state of the chimeric protein was demonstrated by size-exclusion chromatography, thus indicating that fusion to barnase did not abrogate the intrinsic dimerization propensity of the VL domain . Ferritin-binding affinity and specificity in terms of constants of association with isoferritins were identical for the isolated VL domain and its barnase fusion, and RNase activity remained unchanged after the fusion . Intrinsic fluorescence spectra showed a fully compact tertiary structure of the fusion protein . However, significantly altered pH stability of the fusion protein versus individual VL and barnase was shown by the pH-induced changes in both intrinsic fluorescence and binding of ANS . Together, the results indicate that VL-barnase retained the antigen-binding affinity, specificity and RNase activity pertinent to the two individual constituents, and that their fusion into a single-chain chimeric protein resulted in an altered tertiary fold and pH stability.

Arch Biochem Biophys, 2004 Jan 15, 421(2), 227 - 35
Antiamylase-pullulanase enzyme monoclonals which specifically inhibit amylase or pullulanase activity; Kim CH et al.; Monoclonal antibodies against amylase-pullulanase enzyme from Bacillus circulans F-2 have been produced to locate and characterize the catalytic sites of the enzyme . The antibodies have been examined for inhibition of both enzyme activities of amylase and pullulanase and then classified into four types: Type I which inhibited amylase activity, Type II which inhibited pullulanase activity, Type III which inhibited both enzyme activities, and Type IV which had no effect on either enzyme activity . Only two monoclonal antibodies (MAP-12 and MAP-17) as Type I and two antibodies (MAP-3 and MAP-5) as Type II were isolated . The inhibitory activities of the antibodies were characterized and compared . In Type II antibodies, the maximal demonstrated inhibition on the pullulanase activity was 88% for MAP-3 with 1 microg of antibody and 90% for MAP-5 with 2 microg of antibody, but did not inhibit the amylase activity . In Type I antibodies, in contrast, the maximal demonstrated inhibition on the amylase activity was 94% for MAP-12 and 97% for MAP-17 with 1 microg of antibody, respectively, but no inhibition of the pullulanase was noted . MAP-12 recognized sequential epitope, while MAP-17 recognized conformation-dependent epitope of amylase activity-related regions . However, both MAP-3 and MAP-5 recognized the conformation-dependent epitope of the pullulanase activity-related region . Furthermore, the antibodies of MAP-3, MAP-5, MAP-12, and MAP-17 did not compete with one another for binding to the enzyme, indicating that they have different target epitopes on the enzyme . Antibody binding of MAP-12 and MAP-17 to the enzyme was not specifically affected by any of the antiamylase compounds tested: (a) nojirimycin; and (b) 1-deoxynojirimycin . Kinetic analysis of their effects provides evidence that both antibodies of MAP-12 and MAP-17 decrease the catalytic rate of enzyme activity and have little or no effect on substrate binding.

Antimicrob Agents Chemother, 2004 Mar, 48(3), 1058 - 60
Inhibition of beta-lactamase II of Bacillus cereus by penamaldic derivatives of penicillins; Navarro PG et al.; The penamaldic derivatives of amoxicillin, ampicillin, and penicillins G and V, stabilized with Zn(2+), were obtained from a methanolic medium . The enzymatic kinetic results show that the these derivatives elicit reversible inhibition of the enzyme metallo-beta-lactamase from Bacillus cereus, with inhibition constant values determined at pH 7.0 and 25 degrees C.

Semin Oncol, 2004 Feb, 31(1 Suppl 1), 112 - 6
Vaccine therapy for small cell lung cancer; Krug LM; One novel approach to the treatment of lethal residual disease in patients with small cell lung cancer (SCLC) relies on the induction of a host-immune response to attack chemoresistant tumor cells . Because of its neuroectodermal origin, SCLC has a number of specific antigens that could be capitalized on as immune targets . This article reviews two vaccine strategies currently in clinical study . The anti-idiotype vaccine to the GD3 ganglioside, BEC-2, has recently been tested in a phase III trial . In this trial, patients with SCLC who had completed initial chemotherapy were randomized to observation or vaccination with BEC-2 plus bacillus Calmette-Guerin adjuvant . A series of other trials have established the immunogenicity of several keyhole limpet hemocyanin conjugate vaccines relevant to SCLC, including GM2, Globo H, fucosyl GM1, and polysialic acid . To optimize an immune response against a broad range of tumor phenotypes, these components will be combined into a polyvalent vaccine . A randomized phase II trial of this polyvalent vaccine is planned to start in 2004.

Rev Med Liege, 2003 Dec, 58(12), 757 - 60
{Gallbladder tuberculosis associated with cholelithiasis}; Rouas L et al.; Tuberculosis of the gallbladder is rare, even in our country known for being an endemic area . The positive diagnosis depends on suspicion of tuberculosis, peroperative findings and histological examination . From a review of the literature, the physiopathology of this infection is discussed, emphasizing the role of lithiasis in the development of tuberculous lesions . The authors report a case of gallbladder tuberculosis in a female patient who presented with a clinical picture of chronic cholelithiasis . The diagnosis of gallbladder tuberculosis was reached only after surgery and proven by histopathology . In our case, the presence of stones associated with non specific inflammatory alterations and possibly low resistance against tubercle bacillus, is believed to have been of importance for the development of the tuberculous infection.

Proc Natl Acad Sci U S A, 2004 Mar 2, 101(9), 2696 - 9 Epub 2004 Feb 20.
Induction and transmission of Bacillus thuringiensis tolerance in the flour moth Ephestia kuehniella; Rahman MM et al.; The use of Bacillus thuringiensis (Bt) endotoxins to control insect vectors of human diseases and agricultural pests is threatened by the possible evolution of resistance in major pest species . In addition to high levels of resistance produced by receptor insensitivity (5, 16, 17), several cases of tolerance to low to medium levels of toxin have been reported in laboratory colonies of lepidopteran species (3, 18) . Because the molecular basis of some of these cases of tolerance to the toxin are not known, we explored alternative mechanisms . Here, we present evidence that tolerance to a Bt formulation in a laboratory colony of the flour moth Ephestia kuehniella can be induced by preexposure to a low concentration of the Bt formulation and that the tolerance correlates with an elevated immune response . The data also indicate that both immune induction and Bt tolerance can be transmitted to offspring by a maternal effect and that their magnitudes are determined by more than one gene.

J Immunol, 2004 Mar 1, 172(5), 2935 - 43
Posttranscriptional inhibition of gene expression by Mycobacterium tuberculosis offsets transcriptional synergism with IFN-gamma and posttranscriptional up-regulation by IFN-gamma; Qiao Y et al.; Host defense against Mycobacterium tuberculosis requires the cytokine IFN-gamma and IFN regulatory factor 1 (IRF-1), a transcription factor that is induced to high levels by IFN-gamma . Therefore, we chose to study regulation of IRF-1 expression as a model for effects of M . tuberculosis on response to IFN-gamma . We found that IRF-1 mRNA abundance increased far more than transcription rate in human monocytic THP-1 cells stimulated by IFN-gamma, but less than transcription rate in cells infected by M . tuberculosis . IFN-gamma stimulation of infected cells caused a synergistic increase in IRF-1 transcription, yet IRF-1 mRNA abundance was similar in uninfected and infected cells stimulated by IFN-gamma, as was the IRF-1 protein level . Comparable infection by Mycobacterium bovis bacillus Calmette-Guerin failed to induce IRF-1 expression and had no effect on the response to IFN-gamma . We also examined the kinetics of transcription, the mRNA t(1/2), and the distribution of IRF-1 transcripts among total nuclear RNA, poly(A) nuclear RNA, and poly(A) cytoplasmic RNA pools in cells that were infected by M . tuberculosis and/or stimulated by IFN-gamma . Our data suggest that infection by M . tuberculosis inhibits RNA export from the nucleus . Moreover, the results indicate that regulated entry of nascent transcripts into the pool of total nuclear RNA affects IRF-1 expression and that this process is stimulated by IFN-gamma and inhibited by M . tuberculosis . The ability of infection by M . tuberculosis to limit the increase in IRF-1 mRNA expression that typically follows transcriptional synergism may contribute to the pathogenicity of M . tuberculosis.

Infect Immun, 2004 Mar, 72(3), 1504 - 11
Gammadelta T cells in immunity induced by Mycobacterium bovis bacillus Calmette-Guérin vaccination; Lee J et al.; Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccination is efficacious for newborns or adults with no previous exposure to environmental mycobacteria . To determine the relative contribution and the nature of gammadelta T-cell receptor-positive T cells in newborns, compared to CD4(+) T cells, in immunity induced by M . bovis BCG vaccination, 4-week-old specific-pathogen-free pigs were vaccinated with M . bovis BCG and monitored by following the gammadelta T-cell immune responses . A flow cytometry-based proliferation assay and intracellular staining for gamma interferon (IFN-gamma) were used to examine gammadelta T-cell responses . Pigs were found to mount Th1-like responses to M . bovis BCG vaccination as determined by immunoproliferation and IFN-gamma production . The gammadelta T-cell lymphoproliferation and IFN-gamma production to stimulation with mycobacterial antigens were significantly enhanced by M . bovis BCG vaccination . The relative number of proliferating gammadelta T cells after stimulating peripheral blood mononuclear cells with Mycobacterium tuberculosis H37Rv culture filtrate protein was higher than that of CD4(+) T cells at an early time point after M . bovis BCG vaccination, but CD4(+) T cells were found to be more abundant at a later time point . Although the gammadelta T-cell responses were dependent on the presence of CD4(+) T cells for the cytokine interleukin-2, the enhanced gammadelta T cells were due to the intrinsic changes of gammadelta T cells caused by M . bovis BCG vaccination rather than being due solely to help from CD4(+) T cells . Our study shows that gammadelta T cells from pigs at early ages are functionally enhanced by M . bovis BCG vaccination and suggests an important role for this T-cell subset in acquired immunity conferred by M . bovis BCG vaccination.

Biometals, 2004 Feb, 17(1), 45 - 52
Qualitative and quantitative composition of pigments in Phaeodactylum tricornutum (Bacillariophyceae) stressed by iron; Kosakowska A et al.; The effect of Fe(III) deficiency on qualitative and quantitative changes in pigment composition in Phaeodactylum tricornutum Bohlin was demonstrated by HPLC and AAS . Maximum content of pigments showed the diatom cells incubated at the optimum iron concentration, i.e., 10 microM . The contents of chlorophyll a, chlorophyll c1 + c2, fucoxanthin, diadinoxanthin and beta,beta-carotene were 109.99, 20.16, 40.39, 1.29 and 1.48 fg per cell, respectively . The results obtained showed that Fe(III) affected qualitative and quantitative pigment composition in P . tricornutum . The content of individual pigments, proportions between accompanying pigments and their ratios to chlorophyll a were important indicators of phytoplankton response to iron stress . The strong reduction in beta,beta-carotene content, several times (2-5) increase in diadinoxanthin level as compared to beta,beta-carotene, and high amount of diadinoxanthin in relation to chlorophyll a were observed in algae growing at very low Fe(III) concentrations, 0.001 and 0.01 microM . The data suggested that phytoplankton pigments could be a potential physiological marker.

J Econ Entomol, 2003 Dec, 96(6), 1838 - 42
Geographic variation in susceptibility of Chilo suppressalis (Lepidoptera: Pyralidae) to Bacillus thuringiensis toxins in China; Meng F et al.; Geographic variation in the susceptibility of the striped stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), in China to Bacillus thuringiensis (Bt) insecticidal crystal proteins Cry1Ac and Cry1Ab was studied to establish baseline information for comparing the future response of populations with increased exposure to Bt products . Rice is the major host of C . suppressalis, and Bt rice ma) be released in China in the near future . Twelve populations of the pest were collected from the major rice-growing regions of China . LC50 estimates were determined for all populations for Cry1Ac and for eight populations for Cry1Ab . The bioassay results indicated that the range of LC50 in neonate larvae to Cry1Ac and Cry1Ab was from approximately 15 to approximately 157 mg (AI)/L and approximately 2 to approximately 34 mg (AI)/L, respectively . LC50 values were lower for Cry1Ab than for Cry1Ac, and there was a significant positive correlation between the two toxins tested.

J Econ Entomol, 2003 Dec, 96(6), 1786 - 91
Efficacy of VectoBac (Bacillus thuringiensis variety israelensis) formulations for mosquito control in Australia; Russell TL et al.; Laboratory bioassays were conducted on the efficacy of a water-dispersible granule (WG) formulation of Bacillus thuringiensis variety israelensis (VectoBac WG; active ingredient {AI}: 3,000 Bti international toxic units {ITU}/mg) against third instars of six common Australian mosquito species, Aedes aegypti (L.), Ochlerotatus vigilax (Skuse), Ochlerotatus notoscriptus (Skuse), Culex sitiens Wiedemann, Culex annulirostris Skuse, and Culex quinquefasciatus Say . The normal model for log-linear mortality data was used to determine laboratory 48-h LC50 and LC95 values . The target mosquito species tested were extremely sensitive to the VectoBac WG formulation, with the most sensitive species (Cx . annulirostris and Cx . quinquefasciatus, LC95 value of 0.019 ppm) being twice as susceptible as the most tolerant (Oc . notoscriptus, LC95 value of 0.037 ppm) . Cx . annulirostris was selected as a target species for a small-plot evaluation of VectoBac WG and VectoBac 12 aqueous solution (AS) ({AI}: 1,200 Bti ITU/mg) efficacy over time, in freshwater in southeastern Queensland, Australia . Replicated cohorts of caged third instars were exposed weekly to six concentrations of WG formulation (0.004-0.13 ppm) and three concentrations of the 12AS formulation (0.04-0.13 ppm) . In water with high organic content, treatment concentrations of 0.008 ppm WG and 0.04 ppm 12AS and above produced significant larval control (> or = 96%) at 48 h posttreatment, with no residual control at week 1 . Water quality was not affected by treatment with either formulation.

J Econ Entomol, 2003 Dec, 96(6), 1662 - 7
Increased mortality of gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae) exposed to gypsy moth nuclear polyhedrosis virus in combination with the phenolic gycoside salicin; Cook SP et al.; Second instar gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), larvae suffered significantly greater mortality from aerially applied gypsy moth nuclear polyhedrosis virus (Gypchek) when the virus was consumed on quaking aspen, Populus tremuloides Michx., versus red oak, Quercus spp . L., foliage . Laboratory assays in which various doses of Gypchek and salicin (a phenolic glycoside present in aspen foliage) were tested in combination demonstrated that salicin significantly increased total larval mortality and lowered the LD50 estimates (dose of Gypchek that resulted in 50% population mortality) for the virus, although not significantly . While salicin did not impact larval survival in the absence of Gypcek, it did act to significantly deter feeding when it was present in high concentrations (up to 5.0%) within the treatment formulations . The enhanced activity of Gypchek in the presence of salicin is similar to prior reports of enhanced activity of the bacterial pathogen Bacillus thuringiensis when consumed concurrently with phenolic glycosides commonly present in aspen foliage . The enhancement of viral activity is in contrast to the inhibitory effects on the virus reported for another common group of phenolic compounds, tannins.

J Econ Entomol, 2003 Dec, 96(6), 1653 - 61
Evaluation of oils and microbial pathogens for control of lepidopteran pests of sweet corn in New England; Hazzard RV et al.; Vegetable and mineral oil, Beauveria bassiana (Balsamo) and Bacillus thuringiensis subsp . kurstaki Berliner were evaluated for control of Ostrinia nubilalis (Hubner) (Lepidoptera: Pyralidae), Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) and Spodoptera frugiperda (J . E . Smith) (Lepidoptera: Noctuidae) in sweet corn (Zea mays L.) . Field experiments in Maine and Massachusetts during 1993 and 1994 evaluated oils and pathogens singly or in combinations, using a single application directly to the top of the silk channel, immediately after pollination . Mineral oil alone provided equal (1993) or better (1994) control compared with corn oil . In both years, mineral or corn oil plus B . thuringiensis resulted in 93-98% marketable ears, compared with 48-52% marketable ears in untreated plots . In three factorial experiments with B . bassiana, B . thuringiensis and corn oil, B . bassiana at 5 x 10(7) conidia per ear provided little or no control while B . thuringiensis and corn oil provided significant though not always consistent control of all three species . The combination of B . thuringiensis and corn oil provided the largest and most consistent reduction in numbers of larvae and feeding damage to ears.

Insect Biochem Mol Biol, 2004 Jan, 34(1), 101 - 12
Analysis of glycan structures on the 120 kDa aminopeptidase N of Manduca sexta and their interactions with Bacillus thuringiensis Cry1Ac toxin; Knight PJ et al.; The Bacillus thuringiensis Cry1Ac toxin specifically binds to a 120 kDa aminopeptidase N (APN) receptor in Manduca sexta . The binding interaction is mediated by GalNAc, presumably covalently attached to the APN as part of an undefined glycan structure . Here we detail a simple, rapid and specific chemical deglycosylation technique, applicable to glycoproteins immobilized on Western blots . We used the technique to directly and unambiguously demonstrate that carbohydrates attached to 120 kDA APN are in fact binding epitopes for Cry1Ac toxin . This technique is generally applicable to all putative Cry toxin/receptor combinations . We analyzed the various glycans on the 120 kDA APN using carbohydrate compositional analysis and lectin binding . The data indicate that in the average APN molecule, 2 of 4 possible N-glycosylation sites are occupied with fucosylated paucimannose {Man(2-3)(Fuc(1-2)GlcNAc(2)-peptide} type N-glycans . Additionally, we identified 13 probable O-glycosylation sites, 10 of which are located in the Thr/Pro rich C-terminal "stalk" region of the protein . It is likely that 5-6 of the 13 sites are occupied, probably with simple {GalNAc-peptide} type O-glycans . This O-glycosylated C-terminal stalk, being GalNAc-rich, is the most likely binding site for Cry1Ac.

J Infect Dis, 2004 Mar 1, 189(5), 812 - 9 Epub 2004 Feb 13.
Mapping immune reactivity toward Rv2653 and Rv2654: two novel low-molecular-mass antigens found specifically in the Mycobacterium tuberculosis complex; Aagaard C et al.; New tools are urgently needed for the detection of latent tuberculosis (TB) . We evaluated the diagnostic potential of 2 novel Mycobacterium tuberculosis complex-specific candidate antigens (Rv2653 and Rv2654) and investigated T cell recognition during natural infection in humans and experimental infection in guinea pigs . Peripheral blood mononuclear cells stimulated with peptide pools covering the full length of Rv2654 induced interferon- gamma release in 10 of 19 patients with TB . Neither Rv2654 single peptides nor Rv2654 pools were recognized by bacille Calmette-Guerin-vaccinated donors . However, peptides from Rv2653 were recognized by both patients group . The cross-reactive epitope(s) in Rv2653 were located in a 36-amino acid stretch in the center of the molecule . Rv2654 also induced M . tuberculosis-specific skin-test responses in 3 of 4 aerosol-infected guinea pigs . Rv2654 is a strongly recognized T cell antigen that is highly specific for TB and has potential as a novel cell-mediated immunity-based TB diagnostic agent.

Int J Tuberc Lung Dis, 2004 Jan, 8(1), 45 - 51
Tuberculin reactivity and tuberculosis epidemiology in the Pakaanóva (Wari') Indians of Rondônia, south-western Brazilian Amazon; Escobar AL et al.; OBJECTIVE: To investigate the characteristics of tuberculin skin test reactivity in the Pakaanova Indians, in Amazonia, Brazil, after revaccination of all study participants with bacille Calmette-Guerin (BCG) . METHODS: The investigation was designed as a post-BCG vaccination purified protein derivative (PPD) survey . Data included PPD readings, age, sex, nutritional status, place of residence, previous tuberculosis, physical examinations and BCG status . Bivariate and multivariate logistic regression analyses were conducted . RESULTS: About 90% (n = 505) of the total population participated . One third (32.1%) of the subjects presented induration > or = 10 mm at 72 h . Induration sizes showed weak linear correlation with age; differences between sexes were not observed . Skin reaction was not associated with nutritional status . Individuals with a history of tuberculosis were six times more likely to test positive . History of tuberculosis, age, and previous BCG vaccination were significantly associated with PPD reactivity in the multivariate analyses . CONCLUSION: The Pakaanova showed a high proportion (58.4%) of non-reactors, even with a recent BCG booster . Sex differences in PPD reactivity were either not present or could not be demonstrated . The association between age and PPD reactivity resembles that observed in other Amazonian populations . The authors discuss the potential of PPD testing as a screening tool to enhance tuberculosis detection, especially in indigenous populations in Amazonia with limited access to health services.

Crit Care Nurs Q, 2004 Jan-Mar, 27(1), 87 - 91
A case study in Hansen's disease acquired after heart transplant; Launius BK et al.; Hansen's disease, leprosy, is a chronic infectious disease caused by the acid-fast bacillus Mycobacterium leprae . There are multiple forms of the disease ranging from the relatively benign to the progressive, malignant lepromatous leprosy . There is effective antimicrobial treatment available that is capable of curing the disease . We report the case of a post heart transplant patient acquiring Hansen's disease.

Cancer Gene Ther, 2004 Mar, 11(3), 194 - 207
Treatment of bladder carcinomas using recombinant BCG DNA vaccines and electroporative gene immunotherapy; Lee CF et al.; Intravesical immunotherapy with live Mycobacterium bovis bacillus Calmette-Guerin (BCG) is the treatment of choice for superficial bladder cancers . Nevertheless, a significant proportion of patients do not respond to this therapy, and adverse effects are common . Here, we report the cloning of recombinant mycobacterial DNA vaccines and demonstrate the ability of multicomponent and multisubunit DNA vaccines to enhance Th1-polarized cytokine-mediated responses as well as effector cell responses . Splenocytes from immunized groups of mice were restimulated in vitro and examined for cytotoxicity against murine bladder tumur (MBT-2) cells . We used four combined recombinant BCG DNA vaccines (poly-rBCG) for electroporative gene immunotherapy (EPGIT) in vivo, and found that tumor growth was significantly inhibited and mouse survival was prolonged . Increased immune cell infiltration and induction of apoptosis were noted after treatment with poly-rBCG alone, with the murine interleukin-12 (mIL-12) vaccine alone, and-most significantly-with the poly-rBCG+mIL-12 vaccine combination . Electroporation of poly-rBCG+mIL-12 resulted in complete tumor eradication in seven of eight mice (P<.01) within 28 days . Thus, EPGIT using multicomponent multisubunit BCG is highly effective in the treatment of bladder cancer . This approach presents new possibilities for the treatment of bladder cancer using recombinant BCG DNA vaccines.

J Bacteriol, 2004 Mar, 186(5), 1229 - 38
Plasmid-dependent methylotrophy in thermotolerant Bacillus methanolicus; Brautaset T et al.; Bacillus methanolicus can efficiently utilize methanol as a sole carbon source and has an optimum growth temperature of 50 degrees C . With the exception of mannitol, no sugars have been reported to support rapid growth of this organism, which is classified as a restrictive methylotroph . Here we describe the DNA sequence and characterization of a 19,167-bp circular plasmid, designated pBM19, isolated from B . methanolicus MGA3 . Sequence analysis of pBM19 demonstrated the presence of the methanol dehydrogenase gene, mdh, which is crucial for methanol consumption in this bacterium . In addition, five genes (pfk, encoding phosphofructokinase; rpe, encoding ribulose-5-phosphate 3-epimerase; tkt, encoding transketolase; glpX, encoding fructose-1,6-bisphosphatase; and fba, encoding fructose-1,6-bisphosphate aldolase) with deduced roles in methanol assimilation via the ribulose monophosphate pathway are encoded by pBM19 . A shuttle vector, pTB1.9, harboring the pBM19 minimal replicon (repB and ori) was constructed and used to transform MGA3 . Analysis of the resulting recombinant strain demonstrated that it was cured of pBM19 and was not able to grow on methanol . A pTB1.9 derivative harboring the complete mdh gene could not restore growth on methanol when it was introduced into the pBM19-cured strain, suggesting that additional pBM19 genes are required for consumption of this carbon source . Screening of 13 thermotolerant B . methanolicus wild-type strains showed that they all harbor plasmids similar to pBM19, and this is the first report describing plasmid-linked methylotrophy in any microorganism . Our findings should have an effect on future genetic manipulations of this organism, and they contribute to a new understanding of the biology of methylotrophs.

Onderstepoort J Vet Res, 2003 Dec, 70(4), 307 - 16
Review of blackfly (Diptera: Simuliidae) control in South Africa; Myburgh E et al.; The medical, veterinary and economic importance of blackflies in South Africa, and the historical development of blackfly control programmes in various South African rivers, are reviewed in this paper . In 1996 it was estimated that blackflies can cause more than R 88 million damages per annum along the middle and lower Orange River where Simulium chutteri is considered the main pest species . A clear link between the construction of dams and the spread of the blackfly problem was shown . Four phases characterize the development of blackfly control in South Africa: (1) during the 1960s blackflies in the Vaal River were controlled with DDT; (2), during the 1970s and into the 1980s blackflies were controlled using water-flow manipulation; (3) when used at strategic times, water-flow manipulation could be used to enhance the effect of natural predator populations; and (4) during the 1990s the organophosphate temephos and toxins produced by the bacterium Bacillus thuringiensis var . israelensis were tested for their efficacy against blackflies . The larvicides temephos and B . thuringiensis proved to be effective and are still used in several control programmes . The latest research focuses on the factors that influence adult blackfly survival and annoyance, as well as the development of methods that can be used to protect sheep from blackfly attacks.

Onderstepoort J Vet Res, 2003 Dec, 70(4), 255 - 63
Leishmania donovani-derived lipophosphoglycan plus BCG induces a Th1 type immune response but does not protect Syrian golden hamsters (Mesocricetus auratus) and BALB/c mice against Leishmania donovani; Tonui WK et al.; The efficacy of Leishmania donovani-derived lipophosphoglycan (LPG) plus Mycobacterium bovis Bacille Calmette-Guerin (BCG) as a vaccine candidate against visceral leishmaniosis in susceptible BALB/c mouse and Syrian golden hamster (Mesocricetus auratus) models was investigated . Following a triple vaccination with a total dose of 150 microl BCG plus 60 microg or 30 microg of LPG for hamsters and BALB/c mice respectively, there were no noticeable side effects both locally and systemically; implying that the molecule was safe at this dosage level . Vaccinated animals demonstrated an activation of both the humoral as well as cell-mediated responses to LPG, which correlated with resistance against the disease . Protection by LPG plus BCG, was however, poor as the remaining immunized animals showed disease progression leading to severity of the disease as illustrated by emaciation, mass loss and heavy splenic parasitaemia in hamsters . These data nevertheless suggest that it may be rewarding to further evaluate the potential of LPG as a vaccine candidate in leishmaniosis using other adjuvants, which may enhance its immunogenicity.

Pest Manag Sci, 2004 Feb, 60(2), 167 - 72
Long-term selection for resistance to transgenic cotton expressing Bacillus thuringiensis toxin in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae); Meng F et al.; Selection experiments for resistance to transgenic Bt cotton expressing Cry1Ac toxin in Helicoverpa armigera (Hubner) were conducted using a leaf-feeding method with 42 selection episodes over 45 generations . The cotton bollworm developed resistance to transgenic Bt cotton after 12 generations (F12) of selection . The survival rate of F12 neonates feeding on leaves of seedling stage for 4 days and boll-opening stage for 5 days of R19 line were ca 34 and 72%, respectively, compared with ca 0 and 40% for a non-selected sister strain (NYCS), but lower than or similar to that of F12 feeding on leaves of non-Bt cotton, Sumian 12 (ca 87 or 76%) . Resistance to B thuringiensis HD-1 Dipel in neonates (F12) was ca 6-fold . After 42 generations of selection, the strain developed a very high level of resistance to Cry1Ac protoxin, 210 g kg(-1) MVPII wettable powder and 200 g litre(-1) MVPII liquid formulation, the resistance ratios being 1680-, 1780- and ca 1200-fold, respectively, compared with a laboratory susceptible strain (HZS) . When compared with the non-selected NYCS, the resistance ratios to the above toxins were ca 540-, 580- and 510-fold, respectively, but to Dipel only ca 16-fold . The results indicated that it is very important to develop and implement effective resistance-management strategies and to detect early resistance to Bt cotton in field populations.

J Food Prot, 2004 Feb, 67(2), 399 - 402
Factors contributing to the occurrence of antimicrobial drug residues in Kenyan milk; Shitandi A et al.; The study investigated factors contributing to the occurrence of antimicrobial drug residues in milk within four major milk production districts in Kenya . The frequency of contamination was studied among small- and large-scale dairy producers to determine if there were differences between the two types of producers . Field samples (n = 1,600) were analyzed with the improved Dutch tube diffusion test, a microbial inhibitor test (Bacillus stearothermophilus) . In total, 144 and 64 samples from small- and large-scale producers, respectively, were found to contain beta-lactam antibiotics at levels exceeding the established Codex maximum residue level for penicillin G (4 microg/kg) . The difference in results between the two categories of producers was found to be significant (P < 0.001) . To explain the higher frequency of antibiotic contamination of milk from small-scale producers, a questionnaire was constructed and used with 220 randomly selected smallholders in the selected districts . The results suggested (i) lack of understanding of risks related to antibiotic contamination of food, (ii) poor or no treatment records, and (iii) lack of a monitoring system as major risks for contamination . It was concluded that intensification of the education among small-scale dairy producers would greatly reduce the occurrence of antimicrobial residues in milk.

J Food Prot, 2004 Feb, 67(2), 387 - 90
A colony blot immunoassay for the rapid identification of Bacillus cereus; Chen CH et al.; A colony blot immunoassay was developed for the rapid identification of Bacillus cereus using antibodies against the 28.5-kDa cell-surface antigen of B . cereus . Suspect colonies from plates were blotted onto a Whatman #541 membrane, dried, and fixed by UV irradiation . The membrane was then immersed in an anti-B . cereus antibody-horseradish peroxidase conjugate for 60 min . After washing and reacting with 4-chloro-1-naphthol and H2O2, the appearance of purple spots indicated the presence of B . cereus . This assay effectively identified 61 of 62 B . cereus strains tested . Among 38 non-B . cereus strains, which were other Bacillus spp . (19 genera), 36 gave true-negative results, and 2 showed false-positive results . The sensitivity and specificity for B . cereus were 98.4 and 94.7%, respectively . The present assay is easy to use, and the rapid identification of B . cereus can be completed in 2.5 h.

Rev Chir Orthop Reparatrice Appar Mot, 2004 Feb, 90(1), 75 - 8
{Muscular tuberculosis: an unusual focus of Koch bacillus}; Dendane A et al.; We report an exceptional case of tuberculosis in an 8-year-old girl . The focus of infection was situated in the anterior muscle compartment of the left thigh . Diagnosis was established on the basis of histological findings . A 12-month antituberculosis regimen was given . Outcome was good with no general or functional sequelae . Based on this case and data reported in the literature, we discuss the routes of dissemination of the Koch bacillus explaining this unusual sporadic localization.

Biochemistry, 2004 Feb 24, 43(7), 2080 - 90
Phosphatidylinositol-specific phospholipase C forms different complexes with monodisperse and micellar phosphatidylcholine; Berg OG et al.; Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus forms a premicellar complex E(#) with monodisperse diheptanoylphosphatidylcholine (DC(7)PC) that is distinguishable from the E complex formed with micelles . Results are interpreted with the assumption that in both cases amphiphiles bind to the interfacial binding surface (i-face) of PI-PLC but not to the active site . Isothermal calorimetry and fluorescence titration results for the binding of monodisperse DC(7)PC give an apparent dissociation constant of K(2) = 0.2 mM with Hill coefficient of 2 . The gel-permeation, spectroscopic, and probe partitioning behaviors of E(#) are distinct from those of the E complex . The aggregation and partitioning behaviors suggest that the acyl chains in E(#) but not in E remain exposed to the aqueous phase . The free (E) and complexed (E(#) and E) forms of PI-PLC, each with distinct spectroscopic signatures, readily equilibrate with changing DC(7)PC concentration . The underlying equilibria are modeled and their significance for the states of the PI-PLC under monomer kinetic conditions is discussed to suggest that the Michaelis-Menten complex formed with monodisperse DC(7)PC is likely to be E(#)S or its aggregate rather than the classical monodisperse ES complex.

Zhejiang Da Xue Xue Bao Yi Xue Ban, 2004 Jan, 33(1), 65 - 7
{Experimental study on attachment of bacillus Calmette-Guerin to bladder wall with different types of injury}; Ding GQ et al.; OBJECTIVE: To investigate whether different types of injury on bladder wall can influence bacillus Calmette-Guerin (BCG) attachment . METHODS: The bladder mucosa of 24 rabbits were treated by electrocautery,cryocautery and incision on left lateral wall, right lateral wall and posterior wall, respectively . Then radiolabeled BCG ((3)H-BCG) was instilled into bladder . Two hours latter, the injured bladder wall with different methods and non-injured wall (anterior wall of bladder) were surgically removed and digested . The quantity of BCG of each specimen was determined by liquid scintillation counter . RESULT: The quantity of BCG attachment to bladder wall with different injuries was significantly higher than that of non-injured wall (P<0.001), meanwhile there was no statistically difference among the BCG levels of different injury types (P>0.05) . CONCLUSION: BCG attachment is not influenced by different types of injury on the bladder wall.

Appl Microbiol Biotechnol, 2004 Aug, 65(2), 158 - 62 Epub 2004 Feb 14.
Influence of media and temperature on bacteriocin production by Bacillus cereus 8A during batch cultivation; Bizani D et al.; Cerein 8A is a bacteriocin produced by the soil bacterium Bacillus cereus 8A, isolated from native woodlands of Brazil . The influence of temperature and media on the growth of B . cereus 8A and the production of this bacteriocin was studied during batch cultivation . Maximum activity was detected by cultivation in brain/heart infusion broth, reaching 3200 activity units ml(-1) . Bacteriocin was also produced in peptone, MRS, Mueller-Hinton and nutrient broth, while no activity was observed during cultivation in thioglycollate or tryptic soy broth . Temperature had a strong influence on bacteriocin production, which was higher at 30 degrees C than at 25 degrees C . An important decrease in bacteriocin activity was observed at 37 degrees C . The relationship between growth and specific production rates, as a function of the temperature, showed different kinetics of production and there were several peaks in the specific production rates during growth . Bacteriocin was produced at the stationary phase, indicating it is synthesized as a secondary metabolite.

Transplantation, 2004 Feb 15, 77(3), 456 - 59
Macrophage colony-stimulating factor inhibits tumor necrosis factor production and prolongs skin graft survival; Nishina T et al.; BACKGROUND: Despite the availability of a variety of immunosuppressive agents, acute rejection and infection after organ transplantation remain serious problems . METHODS AND RESULTS: We examined the effect of macrophage colony-stimulating factor (M-CSF) on the production of tumor necrosis factor (TNF) in a Bacille de Calmette Guerin-lipopolysaccharide-challenged mouse model . Both serial and repeated injections of M-CSF inhibited TNF production in a dose-dependent manner . Electrophoretic mobility shift assay showed that M-CSF-induced inhibition of TNF production was a result of suppression of nuclear factor-kappaB . High-dose M-CSF significantly prolonged skin graft survival in mice with orthotopic transplantation compared with the control and low-dose M-CSF groups . The combined administration of low-dose M-CSF and cyclosporine also significantly prolonged graft survival compared with the control and low-dose single agent-treated groups . CONCLUSIONS: Our results indicate that M-CSF at a high dose is a potent inhibitor of cytokine production and can potentially be used as an immunosuppressive agent for allograft rejection.

J Egypt Soc Parasitol, 2003 Aug, 33(2), 517 - 30
Cytopathological action in mosquito larvae fed with Bacillus sphaericus (strain faiyoum) spore/crystal complex; Labib IM et al.; Ingestion of Bacillus sphaericus (strain faiyoum) spore/crystal complex by larvae of Anopheles pharoensis, Culex pipiens and Aedes caspius was rapidly followed by a dissolution of the protein crystalline inclusions inside the anterior stomach of the three species . During the first day of the, B . sphaericus spores germinated within the mid-gut lumen, and were in a vegetative stage between 36-48 hours after ingestion when larvae began to die . Ultrastructural observations focused on larval mid-gut showed alterations, which differ according to the mosquito species, being localized mainly in the gastric caecae and posterior stomach . With the bacterial concentration used, neither general cell swelling nor complete breakdown of the mid-gut epithelium was recorded before larval death . In An . pharoensis larval mid-gut epithelium large low-electron-density areas appeared, rough endoplasmic reticula formed numerous concentrical structures and mitochondria swelled . Large vacuoles (of unknown origin) appear early in the Cx . pipiens mid-gut cells and rough endoplasmic reticula broke into small vesicles . Mid-gut epithelial cells of Ae . caspius showed mitochondria swelling except in the anterior stomach and a vacuolization of smooth reticula: these aspects remained unchanged until the larvae died.

J Egypt Soc Parasitol, 2003 Aug, 33(2), 425 - 36
Laboratory evaluation of Bacillus sphaericus recycling in mosquito larvae; Labib IM et al.; After ingestion by Culex pipiens and Anopheles pharoensis 4th instar larvae, spores of Bacillus sphaericus strain faiyoum rapidly germinated inside live mosquito midgut . Bacterial counts and electron microscopic observations on intoxicated larvae revealed that the number of viable spores rapidly decreased during the first 12 h, with a maximum between 12 and 24 h . In cadavers, the number of heat-resistant spores quickly increased between the first and second day post-feeding . After one week, the number of spores inside dead larvae reached approximately 20 times the number of ingested spores for both mosquito species (4 x 16(5) spores/larva) . Ultrathin sections of recycled spores showed the presence of a crystalline inclusion identical to that initially present in spores before ingestion . Bioassay on Cx pipiens 4th instar larvae showed a similar toxicity between in vivo recycled spores (LC50 = 1.1 +/- 0.3 x 10(5) spores/ml after 24h exposure) and culture-medium-grown spores of B . sphaericus strain faiyoum (LC50 = 1.7 +/- 10(5) spores/ml).

J Egypt Soc Parasitol, 2003 Aug, 33(2), 331 - 40
Soil characteristics as factors governing the existence, recycling and persistence of Bacillus thuringiensis in Egypt; Merdan BA et al.; The simple correlation and regression analyses showed that the soil sand percentage and the available copper levels had significant negative and positive contributions, respectively, to the abundance and distribution of B . thuringiensis in the Egyptian agricultural soil . Among the B . thuringiensis isolates, only 11.26% of strains showed larvicidal activity against Culex pipiens . The larvicidal potency LD50 varied from 5.01X10(5) to 5.4X10(6) spores per milliliter among the isolates.

New Microbiol, 2004 Jan, 27(1), 29 - 35
Population dynamics in ageing Helicobacter pylori; Cellini L et al.; The aim of this work was to characterize population changes occurring in aged broth cultures of Helicobacter pylori . Experiments were performed using clinical strains cultured immediately after isolation and after multiple subcultures in solid medium . Morphological changes in the ageing bacteria during a 7-day broth culture were analysed by optical and electron microscopy . The expression of the virulence factor, CagA, together with the presence of the cell cycle regulator, cGMP, were also assessed . The transition from bacillary to coccoid forms was the main morphological change observed in freshly isolated bacteria, together with the increase in cGMP from 1 to 2.25 nmoles/mg of proteins within the first 7 days of broth culture . A similar trend of morphological and physiological changes was observed in cells after multiple subcultures in solid medium with a major presence of large cell clusters . The cagA gene product was always expressed in all experimental conditions evaluated . These data show a significant morphological and physiological diversity in fresh, ageing and aged cultures of H . pylori.

J Biol Chem, 2004 Apr 16, 279(16), 15779 - 86 Epub 2004 Feb 12.
The role of Bacillus thuringiensis Cry1C and Cry1E separate structural domains in the interaction with Spodoptera littoralis gut epithelial cells; Avisar D et al.; The Bacillus thuringiensis delta-endotoxins Cry1C and Cry1E share toxicity against several important lepidopteran species . Their combined use to delay development of resistance in target insects depends on their differential interaction with the gut epithelial cells . The three structural domains and combinations of two consecutive domains of Cry1C and Cry1E were separately expressed in Escherichia coli, and their interactions with the brush border membrane vesicles (BBMV) of Cry1E-tolerant and -susceptible Spodoptera littoralis larvae were studied . About 80% reduction in binding of Cry1E and each of its separate domains to BBMV of Cry1E-tolerant larvae was observed, whereas Cry1C was toxic to all larvae and bound equally to BBMV derived from both Cry1E-tolerant and -susceptible larvae . These results suggest differential interactions of the two toxins with BBMV encompassing all three domains . Comparable binding assays performed with fluorescent Cry1C and Cry1C domain II showed that Cry1C has higher Bmax and lower Kd than Cry1C domain II and further supported the existence of toxin multisite interactions . Competitive binding assays were used to estimate the sequence of interaction events . Cry1C domain II could compete with domain III binding, whereas domain III did not interfere with domain II binding, indicating sequential interactions of domain III and then domain II with the same membrane site . No competition between domain II of Cry1C and Cry1E was observed, confirming the existence of different domain II binding sites for the two toxins . Taken together, all three domains specifically interact with the epithelial cell membrane . The folding of the three-domain toxin probably dictates the sequence of interaction events.

Australas J Dermatol, 2004 Feb, 45(1), 51 - 4
Granulomas in common variable immunodeficiency: A diagnostic dilemma; Lun KR et al.; A 60-year-old man with common variable immunodeficiency presented with a 7-year history of violaceous plaques and papules on the thighs, arms and trunk . In the preceding 2 years he had developed new lesions on both hands . He had been previously diagnosed with sarcoidosis on the basis of skin and visceral histology, but subsequent opinion was that these were sarcoid-like granulomas rather than being representative of true sarcoidosis . Biopsy of the hand lesions showed necrotizing granulomas, and a single acid-fast bacillus (AFB) was identified on Wade-Fite stain . Subsequent repeat tissue biopsies for histology, culture and polymerase chain reaction testing failed to confirm the presence of mycobacterial organisms and it was felt that the organism was a contaminant introduced during tissue processing . The hand lesions responded well to intralesional injections of triamcinolone acetonide 10 mg/mL and oral tetracycline 500 mg b.d . was later introduced with a good clinical response . The diagnostic dilemma of finding granulomatous inflammation in a patient with common variable immunodeficiency, and the significance of a single AFB on histology are discussed . The treatment of sarcoid-like granulomas with tetracycline therapy is also commented on.

Mar Biotechnol (NY), 2002 Mar, 4(2), 179 - 88
Characterization of surfactin-like cyclic depsipeptides synthesized by Bacillus pumilus from ascidian Halocynthia aurantium; Kalinovskaya NI et al.; A marine bacterium (KMM 1364), identified as Bacillus pumilus, was isolated from the surface of ascidian Halocynthia aurantium . Structural analysis revealed that the strain KMM 1364 produced a mixture of lipopeptide surfactin analogs with major components with molecular masses of 1035, 1049, 1063, and 1077 . The variation in molecular weight represents changes in the number of methylene groups in the lipid and/or peptide portions of the compounds . Structurally, these lipopeptides differ from surfactin in the substitution of the valine residue in position 4 by leucine, and have been isolated as two carboxy-terminal variants, with valine or isoleucine in position 7 . As constituents of the lipophilic part of the peptides, only beta-hydroxy-C(15)-, beta-hydroxy-C(16)-, and a high amount of beta-hydroxy-C(17) fatty acid were determined.

Mar Biotechnol (NY), 2002 Mar, 4(2), 111 - 8
Antifouling potential of some marine organisms from India against species of Bacillus and Pseudomonas; Bhosale SH et al.; Crude methanolic extracts of 37 marine organisms (16 species of flora, 21 species of fauna) were screened for antibacterial properties against 5 strains of bacteria isolated from marine environments . Of these, 10 plant and 9 animal extracts exhibited antibacterial activity against at least one bacterial strain . The extracts of 6 species were active against all the strains: i.e., Stoechospermum marginatum (brown algae), Cymodocea rotundata (seagrass), Petrosia sp . and Psammaplysilla purpurea (sponges), Sinularia compressa (soft coral), and Cassiopeia sp . (jellyfish) . Among the plants, Padina tetrastromatica (brown algae) extract exhibited significant activity (9-11-mm inhibition zone at 500 microg per 6-mm disc) against Bacillus pumilus and Pseudomonas vesicularis, while the extracts of Petrosia, Psammaplysilla, and Cassiopeia were strongly active (11-13-mm inhibition zone at 500 microg per 6-mm disc) against B . circulans and P . putida . It was further confirmed that the attachment of bacterial strains on glass slides was inhibited remarkably with increasing concentrations of bioextracts of Petrosia sp . and Psammaplysilla purpurea . The present findings could form the basis for exploring the antibacterial potential of bioactive molecules from some of the marine organisms that exhibited moderate to strong antibacterial properties.

Appl Microbiol Biotechnol, 2004 Aug, 65(2), 193 - 9 Epub 2004 Feb 11.
Structural and functional characterization of the Bacillus megaterium uvrBA locus and generation of UV-sensitive mutants; Nahrstedt H et al.; The Bacillus megaterium genes uvrB and uvrA, encoding two subunits of the (A)BC excinuclease, which is responsible for nucleotide excision repair, were isolated and functionally characterized . RNA analyses revealed co-transcription of both genes probably forming a bicistronic operon . Expression of uvrB and uvrA was inducible by the DNA-damaging agent mitomycin C . This finding agrees with the presence of a potential DinR box within the uvrBA promoter . Single inactivation of uvrB or uvrA as well as the parallel knockout of both genes resulted in mutants highly sensitive to UV irradiation . Thus, this locus represents an attractive target for generating biologically safe containment strains of B . megaterium.

Insect Biochem Mol Biol, 2004 Mar, 34(3), 193 - 202
Fluorescent-based assays establish Manduca sexta Bt-R(1a) cadherin as a receptor for multiple Bacillus thuringiensis Cry1A toxins in Drosophila S2 cells; Hua G et al.; A fluorescence-based approach was developed to analyze in vivo the function of Manduca sexta cadherin (Bt-R(1)) as a Cry1 toxin receptor . We cloned a Bt-R(1a) cDNA that differs from Bt-R(1) by 37 nucleotides and two amino acids and expressed it transiently in Drosophila melanogaster Schneider 2 (S2) cells . Cells expressing Bt-R(1a) bound Cry1Aa, Cry1Ab, and Cry1Ac toxins on ligand blots, and in saturation binding assays . More Cry1Ab was bound relative to Cry1Aa and Cry1Ac, though each Cry1A toxin bound with high-affinity (Kd values from 1.7 to 3.3 nM) . Using fluorescent microscopy and flow cytometry assays, we show that Cry1Aa, Cry1Ab and Cry1Ac, but not Cry1Ba, killed S2 cells expressing Bt-R(1a) cadherin . These results demonstrate that M . sexta cadherin Bt-R(1a) functions as a receptor for the Cry1A toxins in vivo and validates our cytotoxicity assay for future receptor studies.

Oral Microbiol Immunol, 2004 Apr, 19(2), 88 - 94
The effect of oxygen on the growth and physiology of Porphyromonas gingivalis; Diaz PI et al.; Oxygen constitutes a constant challenge for the survival of strict anaerobes in the oral environment . The aim of this study was to investigate the effect of oxygen on the physiology and growth of Porphyromonas gingivalis in a continuous culture system when grown under conditions of hemin limitation and excess . Results showed that, when grown in the presence of hemin at 0.5 mg/l, P . gingivalis could tolerate low levels of oxygen, being able to reach steady-state when 6% oxygen was present in the incoming gas mixture . When the hemin concentration was increased to 5 mg/l, the culture tolerated 10% oxygen . Anaerobically-grown cells were coccoid in shape, whereas those grown in the presence of oxygen were bacillary . Acetate was the predominant end-product in cultures grown in the presence of oxygen or in cultures hemin-limited . Despite some changes in the activity of Arg- and Lys-gingipain, most of the proteolytic activity was retained in the presence of oxygen . Activity of each of the three anti-oxidant enzymes tested (NADH oxidase, NADH peroxidase and SOD) was detected under all conditions and usually increased under oxygenated environments . Higher activities were also seen in the hemin-limited cultures . These results show some of the changes that occur in the physiology of P . gingivalis as a result of oxidative stress and confirm that hemin has a protective effect on the growth of the microorganism in the presence of oxygen.

Biotechnol Appl Biochem, 2004 Oct, 40(Pt 2), 191 - 6
Optimization of medium composition for keratinase production on feather by Bacillus licheniformis RG1 using statistical methods involving response surface methodology; Ramnani P et al.; A 3.5-fold increase in keratinase production by Bacillus licheniformis RG1 was achieved by using statistical methods involving Plackett-Burman design and response surface methodology . Eight variables were screened using Plackett-Burman design . Of these, glucose, peptone and glutathione were found to affect the response signal positively, whereas CaCl(2) had a negative effect . Further interaction of these factors, along with phosphate and incubation time, was studied using response surface methodology . An optimum keratinase production of 1295 units/mg dry weight was obtained with the following medium composition: 1% glucose, 1% peptone, 1% phosphate, 0.05% glutathione, 0.5% feather and 2% inoculum under shaking at 250 rev./min with an incubation period of 72 h at 37 degrees C . Keratinase production was found to be a function of biomass and maximum production occurred during the stationary phase.

J Immunother, 2004 Mar-Apr, 27(2), 116 - 23
Immunotherapy of experimental bladder cancer with recombinant BCG expressing interferon-gamma; Arnold J et al.; One of the most potent immunotherapies presently used is the application of Bacillus Calmette Guerin (BCG) to prevent recurrences of superficial bladder cancer . Despite its successful use, nonresponders and certain side effects remain a major obstacle . Therefore, current studies aim at developing recombinant BCG (rBCG) strains to further improve the effectiveness of the therapy . In BCG-treated patients a strong local induction of Th1-like cytokines was observed . For this reason rBCG-strains secreting Th1-like cytokines might be potentially useful agents to improve this type of immunotherapy . Because we previously demonstrated the essential role of IFNgamma in BCG-induced antitumor responses, in this study a rBCG strain secreting murine IFNgamma (rBCG-IFNgamma) was generated and tested for its immunostimulatory capacity in several in vitro and in vivo test systems . In vitro rBCG-IFNgamma specifically up-regulated expression of MHC class I molecules on a murine bladder cancer cell line (MB49), compared to the rBCG control strain (transfected with an empty vector) . In a murine model of experimental bladder cancer, intravesical instillation of rBCG-IFNgamma resulted in an enhanced recruitment of CD4+ T-cells into the bladder and further induced the local expression of IL-2 and IL-4 cytokines (mRNA) compared to control rBCG . With a low-dose treatment regimen for murine orthotopic bladder cancer, rBCG-IFNgamma significantly prolonged survival, whereas the therapeutic effect of wild-type control BCG did not reach statistical significance . We conclude that this recombinant BCG strain has enhanced immunostimulatory potential and might offer new opportunities in the treatment of bladder cancer.

J Biochem (Tokyo), 2003 Dec, 134(6), 881 - 91
Effects of essential carbohydrate/aromatic stacking interaction with Tyr100 and Phe259 on substrate binding of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp . 1011; Haga K et al.; The stacking interaction between a tyrosine residue and the sugar ring at the catalytic subsite -1 is strictly conserved in the glycoside hydrolase family 13 enzymes . Replacing Tyr100 with leucine in cyclodextrin glycosyltransferase (CGTase) from Bacillus sp . 1011 to prevent stacking significantly decreased all CGTase activities . The adjacent stacking interaction with both Phe183 and Phe259 onto the sugar ring at subsite +2 is essentially conserved among CGTases . F183L/F259L mutant CGTase affects donor substrate binding and/or acceptor binding during transglycosylation {Nakamura et al . (1994) Biochemistry 33, 9929-9936} . To elucidate the precise role of carbohydrate/aromatic stacking interaction at subsites -1 and +2 on the substrate binding of CGTases, we analyzed the X-ray structures of wild-type (2.0 A resolution), and Y100L (2.2 A resolution) and F183L/F259L mutant (1.9 A resolution) CGTases complexed with the inhibitor, acarbose . The refined structures revealed that acarbose molecules bound to the Y100L mutant moved from the active center toward the side chain of Tyr195, and the hydrogen bonding and hydrophobic interaction between acarbose and subsites significantly diminished . The position of pseudo-tetrasaccharide binding in the F183L/F259L mutant was closer to the non-reducing end, and the torsion angles of glycosidic linkages at subsites -1 to +1 on molecule 1 and subsites -2 to -1 on molecule 2 significantly changed compared with that of each molecule of wild-type-acarbose complex to adopt the structural change of subsite +2 . These structural and biochemical data suggest that substrate binding in the active site of CGTase is critically affected by the carbohydrate/aromatic stacking interaction with Tyr100 at the catalytic subsite -1 and that this effect is likely a result of cooperation between Tyr100 and Phe259 through stacking interaction with substrate at subsite +2.

J Biol Chem, 2004 Apr 23, 279(17), 17466 - 72 Epub 2004 Feb 09.
Structural characterization of the nickel-binding properties of Bacillus pasteurii urease accessory protein (Ure)E in solution; Won HS et al.; Urease activation is critical to the virulence of many human and animal pathogens . Urease possesses multiple, nickel-containing active sites, and UreE, the only nickel-binding protein among the urease accessory proteins, activates urease by transporting nickel ions . We performed NMR experiments to investigate the solution structure and the nickel-binding properties of Bacillus pasteurii (Bp) UreE . The secondary structures and global folds of BpUreE were determined for its metal-free and nickel-bound forms . The results indicated that no major structural change of BpUreE arises from the nickel binding . In addition to the previously identified nickel-binding site (Gly(97)-Cys(103)), the C-terminal tail region (Lys(141)-His(147)) was confirmed for the first time to be involved in the nickel binding . The C-terminally conserved sequence ((144)GHQH(147)) was confirmed to have an inherent nickel-binding ability . Nickel addition to 1.6 mm subunit, a concentration where BpUreE predominantly forms a tetramer upon the nickel binding, induced a biphasic spectral change consistent with binding of up to at least three nickel ions per tetrameric unit . In contrast, nickel addition to 0.1 mm subunit, a concentration at which the protein is primarily a dimer, caused a monophasic spectral change consistent with more than 1 equivalent per dimeric unit . Combined with the equilibrium dialysis results, which indicated 2.5 nickel equivalents binding per dimer at a micromolar protein concentration, the nickel-binding stoichiometry of BpUreE at a physiological concentration could be three nickel ions per dimer . Altogether, the present results provide the first detailed structural data concerning the nickel-binding properties of intact, wild-type BpUreE in solution.

FEMS Microbiol Lett, 2004 Feb 9, 231(1), 91 - 8
Genotypic diversity among Bacillus licheniformis strains from various sources; De Clerck E et al.; Bacillus licheniformis is exploited industrially for the production of enzymes and has been shown to exhibit pathogenic properties . Because of these divergent characteristics, questions arise concerning intraspecies diversity . A comparative study by means of combined repetitive polymerase chain reaction, rpoB and gyrA sequencing, 16S rDNA targeted probe analysis, DNA-DNA hybridizations, gelatinase tests and antibiotic susceptibility tests was performed on a set of strains from diverse sources, including strains with pathogenic potential . B . licheniformis was found to consist of two lineages that are distinguished genotypically.

FEMS Microbiol Lett, 2004 Feb 9, 231(1), 45 - 52
Conjugative transfer, stability and expression of a plasmid encoding a cry1Ac gene in Bacillus cereus group strains; Hu X et al.; The plasmid pHT73 containing cry1Ac and tagged with an erythromycin resistance gene was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to several Bacillus cereus group strains by conjugation . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and phase contrast microscopy showed that the transconjugants containing plasmid pHT73 could express Cry1Ac toxin and produce bipyramidal crystalline inclusion bodies during sporulation . The study demonstrated that pHT73 could be transferred to B . thuringiensis subsp . kurstaki, several B . cereus strains and Bacillus mycoides . Under non-selective conditions, the stability of the pHT73 plasmid in the transconjugants was found to be 58.2-100% after 100 generations and 4-96% after 200 generations . The variations are mainly caused by the choice of receptor strain.

Rev Panam Salud Publica, 2003 Dec, 14(6), 402 - 8
{Educational strategy for improving patient compliance with the tuberculosis treatment regimen in Chiapas, Mexico}; Alvarez Gordillo Gdel C et al.; OBJECTIVE: To implement a training program for physicians and patients and assess its effectiveness in terms of patient compliance with the pulmonary tuberculosis treatment regimen in the border region of Chiapas, Mexico . METHODS: A controlled intervention study was performed with patients over 15 years of age who had pulmonary tuberculosis diagnosed by direct microscopy (bacilloscopy) between 1 February 2001 and 31 January 2002 in health units randomly selected in the border region of Chiapas, Mexico . The sample was made up of patients who sought consultation at 23 and 25 health units over that period (intervention and control group, respectively) . The intervention group took part in a training program for health personnel in which the following were discussed: the social, cultural, and economic aspects of tuberculosis; the theoretical and practical underpinnings of the diagnosis and treatment of the illness, and the establishment of self-help groups . Selfhelp groups were also created for all patients at the 23 units where the intervention group sought consultation . All patients were given a short-term treatment regimen with isoniazid, rifampin, pyrazinamide, and ethambutol for a total of 25 weeks, until completing a total of 105 doses . Patient follow-up was extended through December 2003 . The intervention and control groups were compared by means of the chi square test, and Student's t test was used to compare means . The relative risk of non-compliance (RR) was calculated along with 95% confidence intervals (95% CI) . RESULTS: Eighty-seven patients participated in the study; 44 were exposed to the intervention, and 43 made up the control group . Compliance with treatment was considerably greater in the intervention group than in the control group (97.7% vs . 81.4%, respectively; RR = 1.20; 95% CI: 1.03 to 1.39; P = 0.0015) . It was noted that physicians in the border region of Chiapas gear their activities toward curative medicine, rather than preventive medicine or understanding the social determinants of disease . CONCLUSIONS: As a result of the educational activities that were part of the intervention, there was an increase in the proportion of patients who complied with treatment . Health services can improve tuberculosis control in Chiapas with the resources that are available to them at present . Physicians should be taught to view health problems in Chiapas as part of an integral set of conditions, and efforts should be made to improve the doctor-patient relationship . Steps should also be taken to incorporate educational activities and community participation in health services in order to address public health problems in a comprehensive way.

Chirurgia (Bucur), 2000 Mar-Apr, 95(2), 179 - 91
{Intestinal tuberculosis in children . Differential diagnosis and treatment}; Sabetay C et al.; Lately, tuberculosis has became a top priority problem in health all over the world . The actual level of epidemiological indicates of tuberculosis Shaw that Romania is the country that has the most unfavorable situation in Europe except for Bosnia-Herzegovina . Tuberculosis incidence from 1996 to 1997 ranks Dolj county second in Romania from the point of view of the increasing rate (154.3% + 25.4% versus 107% + 4.3%) . The National Programme of Controlling Tuberculosis for 1997-2000 has as main object the neutralization of a least 85% of the discovered contamination sources . At the same time with the pulmonary tuberculosis were discovered 17.2% extrapulmonary localizations . The purpose of these works is that of presenting in the frame of extrapulmonary TBC the localization of intestinal TBC with children (peritonitic form with infants and pseudotumoral with child) . In class cooperation with the veterinary Medicine Service we have also studied the possibility of same sources of TBC infection of animal origin, knowing the fact that besides the human Koch bacillus were discovered bovine, ovine, poultry sources . Presenting a number of 16 cases of intestinal tuberculosis with children, rare cases of extra pulmonary tuberculosis, the authors also present 2 cases with children whose differential diagnosis had to be made ou a histopathological base between tuberculosis with an abdominal lymphoma or peritonitic carcinomatosis . The results of the surgical treatment that made the diagnosis with the help of the histopathological examination and the tuberculostatic treatment were very good, the evolution and the control in true of all the patients, proving that intestinal tuberculosis extrapulmonary form with children can be cured.

Cancer Immunol Immunother . 2004 Feb 7; {Epub ahead of print}
WT1 peptide vaccination combined with BCG-CWS is more efficient for tumor eradication than WT1 peptide vaccination alone; Nakajima H et al.; A Wilms' tumor gene WT1 is expressed at high levels not only in most types of leukemia but also in various types of solid tumors, including lung and breast cancer . WT1 protein has been reported to serve as a target antigen for tumor-specific immunotherapy both in vitro in human systems and in vivo in murine models . We have shown that mice immunized with WT1 peptide or WT1 cDNA could reject a challenge from WT1-expressing tumor cells (a "prophylactic" model) . However, it was not examined whether WT1 peptide vaccination had the potency to reject tumor cells in a "therapeutic" setting . In the present study, we demonstrated for the first time that WT1 peptide vaccination combined with Mycobacterium bovis bacillus Calmette-Guerin cell wall skeleton (BCG-CWS) was more effective for eradication of WT1-expressing tumor cells that had been implanted into mice before vaccination (a "therapeutic" model) compared with WT1 peptide vaccination alone . An intradermal injection of BCG-CWS into mice, followed by that of WT1 peptide at the same site on the next day, generated WT1-specific cytotoxic T lymphocytes (CTLs) and led to rejection of WT1-expressing leukemia or lung cancer cells . These results showed that BCG-CWS, which was well known to enhance innate immunity, could enhance WT1-specific immune responses (acquired immunity) in combination with WT1 peptide vaccination . Therefore, WT1 peptide vaccination combined with BCG-CWS may be applied to cancer immunotherapy in clinical settings.

J Urol, 2004 Mar, 171(3), 1343 - 7
Immunotherapy for bladder cancer using recombinant bacillus Calmette-Guerin DNA vaccines and interleukin-12 DNA vaccine; Lee CF et al.; PURPOSE: We investigated the efficacy of recombinant bacillus Calmette-Guerin (BCG) DNA (poly-rBCG) and murine interleukin (IL)-12 (mIL-12) vaccines in inducing T helper 1 polarized cytokines and suppressing bladder tumor growth in mice . MATERIALS AND METHODS: Four mycobacteria candidate genes (Ag85A, Ag85B, Mpt64 and PstS3) were cloned, fused with ESAT6 and ligated into eukaryotic expression vectors . Combined poly-rBCG and mIL-12 vaccines were transferred into a murine bladder tumor model . The efficiency of gene expression was detected using Western blotting, flow cytometry and semiquantitative reverse transcriptase-polymerase chain reaction . Systemic cytokine responses, tumor growth and cumulative survival rates were monitored . RESULTS: Transfected bladder cancer cells showed high in vitro and in vivo expression of the recombinant subcomponents . Mice with tumors injected with poly-rBCG plus mIL-12 produced serum interferon-gamma significantly within 21 days but no significant elevations in tumor necrosis factor-alpha, IL-2, IL-4 or IL-5 were found . On day 28 after electroporation the growth of MBT-2 implants treated with poly-rBCG, mIL-12 or poly-rBCG plus mIL-12 was significantly inhibited . The cumulative survival of mice treated with poly-rBCG plus mIL-12 was significantly higher than that of the other 3 groups . CONCLUSIONS: Highly immunopotent recombinant vaccines of bacillus Calmette-Guerin DNA were produced that elicited T helper 1 immune responses with a high serum interferon-gamma level, inhibited tumor growth and prolonged the survival of tumor bearing mice . Thus, electroporation immunogene therapy using poly-rBCG plus mIL-12 may be an attractive regimen for the treatment of bladder cancer.

J Pediatr Hematol Oncol, 2004 Feb, 26(2), 112 - 5
Reactivation of the bacille Calmette-Guérin scar following immune reconstitution during treatment of infant acute lymphoblastic leukemia; Swinson S et al.; The authors describe an infant presenting at 2 weeks of age with congenital acute lymphoblastic leukemia who had previously received routine bacille Calmette-Guerin (BCG) vaccination at birth . The risk of BCG dissemination in immunocompromised infants is discussed and the use of antimycobacterial prophylaxis in such cases considered.

Appl Environ Microbiol, 2004 Feb, 70(2), 656 - 63
Growth characteristics of Bartonella henselae in a novel liquid medium: primary isolation, growth-phase-dependent phage induction, and metabolic studies; Chenoweth MR et al.; Bartonella henselae is a zoonotic pathogen that usually causes a self-limiting infection in immunocompetent individuals but often causes potentially life-threatening infections, such as bacillary angiomatosis, in immunocompromised patients . Both diagnosis of infection and research into the molecular mechanisms of pathogenesis have been hindered by the absence of a suitable liquid growth medium . It has been difficult to isolate B . henselae directly from the blood of infected humans or animals or to grow the bacteria in liquid culture media under laboratory conditions . Therefore, we have developed a liquid growth medium that supports reproducible in vitro growth (3-h doubling time and a growth yield of approximately 5 x 10(8) CFU/ml) and permits the isolation of B . henselae from the blood of infected cats . During the development of this medium, we observed that B . henselae did not derive carbon and energy from the catabolism of glucose, which is consistent with genome nucleotide sequence data suggesting an incomplete glycolytic pathway . Of interest, B . henselae depleted amino acids from the culture medium and accumulated ammonia in the medium, an indicator of amino acid catabolism . Analysis of the culture medium throughout the growth cycle revealed that oxygen was consumed and carbon dioxide was generated, suggesting that amino acids were catabolized in a tricarboxylic acid (TCA) cycle-dependent mechanism . Additionally, phage particles were detected in the culture supernatants of stationary-phase B . henselae, but not in mid-logarithmic-phase culture supernatants . Enzymatic assays of whole-cell lysates revealed that B . henselae has a complete TCA cycle . Taken together, these data suggest B . henselae may catabolize amino acids but not glucose to derive carbon and energy from its host . Furthermore, the newly developed culture medium should improve isolation of B . henselae and basic research into the pathogenesis of the bacterium.

Protein Expr Purif, 2004 Mar, 34(1), 103 - 10
Expression, purification, and aggregation studies of His-tagged thermoalkalophilic lipase from Bacillus thermocatenulatus; Schlieben NH et al.; The His-tagged lipase BTL2 from Bacillus thermocatenulatus was expressed in Escherichia coli and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography . The success of protein separation and purification was pH-dependent and increased with decreasing pH . The purified BTL2 lipase showed a strong tendency to aggregate upon concentration, which prevented a reproducible crystallization . Aggregation studies using dynamic light-scattering (DLS) analysis were performed to improve the purification and concentration of BTL2 lipase . Different chemical classes of additives were tested to manipulate the aggregation behaviour of BTL2 lipase with the aim of obtaining a monodisperse sample to use for crystallization . For the process of concentration of BTL2 lipase in monomeric form, the alcohol 2-propanol and the ionic detergent dodecyl dimethylamine-N-oxide (LDAO) were found to be necessary . For the concentrated lipase, the availability of 5% 2-propanol was sufficient to hold the lipase in monomeric form and no additional detergent was needed.

J Med Entomol, 2003 Nov, 40(6), 841 - 8
Role of bacteria in mediating the oviposition responses of Aedes albopictus (Diptera: Culicidae); Trexler JD et al.; The responses of Aedes albopictus to sources of oviposition attractants and stimulants were evaluated with a behavioral bioassay in which females attracted to odorants emanating from water were trapped on screens coated with an adhesive . Gravid mosquitoes were attracted to volatiles from larval-rearing water and soil-contaminated cotton towels . Bacteria were isolated from these substrates and from an organic infusion made with oak leaves . Through fatty acid-methyl ester analyses, six bacterial isolates from larval-rearing water, two isolates from soil-contaminated cotton towels, and three isolates from oak leaf infusion were identified to species . The response of gravid mosquitoes to these isolates was also evaluated in behavioral bioassays . Water containing Psychrobacter immobilis (from larval-rearing water), Sphingobacterium multivorum (from soil-contaminated cotton towels), and an undetermined Bacillus species (from oak leaf infusion) elicited significantly higher oviposition than control water without bacteria . Only volatiles collected from larval rearing water elicited significant electroantennogram responses in females.

Int J Lepr Other Mycobact Dis, 2003 Dec, 71(4), 308 - 19
Long-term efficacy of 2 year WHO multiple drug therapy (MDT) in multibacillary (MB) leprosy patients; Cellona RV et al.; Relapse rate estimates after 2 year WHO multiple drug therapy (MDT) in multi-bacillary (MB) leprosy vary . Between 1987 and 1994, 500 MB leprosy patients completing 2 year MDT were enrolled in a prospective relapse study . The majority of patients (N = 316) were treated and followed at the physician-staffed Cebu Skin Clinic (CSC), whereas others (N = 184) received therapy from government clinics and were followed by CSC technicians in the field . Relapse definition was an increased bacteriologic index (BI) and new skin lesions, supplemented with mouse footpad inoculations . Through 2002, follow-up was 5368 person-years, with a mean of 10.8 years per patient . The absolute relapse rate was 3% (15/498; 0.28/100 person-years), with a cumulative risk estimate of 3.9% at 15 yrs . For a subset of 217 patients followed for >or=12 yrs or until relapse, relapses occurred in 9% (13/142) attending the CSC, versus 3% (2/75) assessed in the field (p = 0.09) . The rate for patients followed at CSC for >or=12 yrs and a pre-treatment BI >or=2.7+ was 13% (13/98) . All relapses were BL or LL, with pre-treatment BI's of >or=2.7+ . Relapses occurred long after completion of therapy, between 3 and 11 yrs from the midpoint of the examination without relapse to detection, or between 6 to 13 yrs to the actual year of detection, 7 occurring at >or=10 yrs . Lesion material from all relapses contained M . leprae that was rifampin and clofazimine sensitive, whereas 3 showed partial or full dapsone resistance . {Follow-up rigor and time}, medical expertise, and pre-treatment bacterial load influence relapse rates after 2 yr MDT.

Int J Lepr Other Mycobact Dis, 2003 Dec, 71(4), 328 - 40
Ultrastructural study of Schwann cells and endothelial cells in the pathogenesis of leprous neuropathy; Kumar V et al.; Peripheral nerve biopsies from 4 borderline tuberculoid (BT) and 4 lepromatous (LL) patients who were on multidrug therapy were investigated by light and electron microscopic studies . The variation of diameters and distribution of myelinated and unmyelinated fibers between BT and LL patients were not significant . This study has shown significant changes in peripheral nerves and endoneural blood vessels . It was revealed that besides Schwann cells (SC), the endothelial cells (EC) of endoneural blood vessels frequently harbor M . leprae . In BT, peripheral nerves in addition to the degenerative changes of SC and presence of perineural and perivascular cuffing by mononuclear cells, the endoneurial blood vessels showed thickening of basement membrane with hypertrophy of EC leading to narrowing or complete occlusion of lumen . On the other hand, peripheral nerves of LL patients were infiltrated with large number of M . leprae shown to be present in the electron transparent zone (ETZ) of the SC . The EC of endoneurial blood vessels were found to be loaded with M . leprae, and this bacillary loaded EC was found to release M . leprae into the lumen through its ruptured membrane.

Cad Saude Publica, 1994 Jun, 10(2), 222 - 30 Epub 2004 Feb 03.
{Mosquito control based on larvicides in the State of Rio Grande do Sul, Brazil: choice of the control agent}; Ruas-Neto AL et al.; A comparison between chemical and biological larvicides in routine operations against mosquitoes in Rio Grande do Sul State was carried out in this study . In laboratory bioassays against Culex quinquefasciatus, biological formulations Vectobac 12 AS and Teknar 3000 (Bacillus thuringiensis israelensis) as well as ABG 6262 (B . sphaericus 2362), both in liquid and powder form, were highly effective . Locally produced B . thuringiensis israelensis, formulations also yielded good results . Among chemical larvicides, pyrethroid compounds Pirisa and K-Othrine yielded better results than the organophosphates Lebaycid and Abate . These last formulations yielded responses ten weaker than predicted in other studies . Under field conditions, a dose of 1250 mg/m2 for biological formulations was considered adequate for routine application because at this level it is possible to overcome physical influences on results . Only B . sphaericus preparations caused important disruption of mosquito colonization in active breeding sites . Up to 39 weeks were tabulated without complete colonization in natural conditions and one month in artificially colonized tanks . This study suggests that biological alternatives should be considered in mosquito control programs . They may be a solution to such problems as resistance to larvicides, elimination of natural enemies, and short-lasting effects of applications.

J Ind Microbiol Biotechnol, 2004 Feb, 31(2), 51 - 6 Epub 2004 Jan 31.
Purification and characterisation of alkaline cellulase produced by a novel isolate, Bacillus sphaericus JS1; Singh J et al.; A novel strain of Bacillus sphaericus JS1 producing thermostable alkaline carboxymethyl cellulase (CMCase; endo-1,4-beta-glucanase, E.C . 3.2.1.4) was isolated from soil using Horikoshi medium at pH 9.5 . CMCase was purified 192-fold by (NH(4))(2)SO(4) precipitation, ion exchange and gel filtration chromatography, with an overall recovery of 23% . The CMCase is a multimeric protein with a molecular weight estimated by native-PAGE of 183 kDa . Using SDS-PAGE a single band is found at 42 kDa . This suggests presence of four homogeneous polypeptides, which would differentiate this enzyme from other known alkaline cellulases . The activity of the enzyme was significantly inhibited by bivalent cations (Fe(3+) and Hg(2+), 1.0 mM each) and activated by Co(2+), K(+) and Na(+) . The purified enzyme revealed the products of carboxymethyl cellulose (CMC) hydrolysis to be CM glucose, cellobiose and cellotriose . Thermostability, pH stability, good hydrolytic capability, and stability in the presence of detergents, surfactants, chelators and commercial proteases make this enzyme potentially useful in laundry detergents.

Bull World Health Organ, 2003, 81(11), 821 - 6 Epub 2004 Jan 20.
Do childhood vaccines have non-specific effects on mortality?
Cooper WO, Boyce TG, Wright PF, Griffin MR.
A recent article by Kristensen et al . suggested that measles vaccine and bacille Calmette-Gu rin (BCG) vaccine might reduce mortality beyond what is expected simply from protection against measles and tuberculosis . Previous reviews of the potential effects of childhood vaccines on mortality have not considered methodological features of reviewed studies . Methodological considerations play an especially important role in observational assessments, in which selection factors for vaccination may be difficult to ascertain . We reviewed 782 English language articles on vaccines and childhood mortality and found only a few whose design met the criteria for methodological rigor . The data reviewed suggest that measles vaccine delivers its promised reduction in mortality, but there is insufficient evidence to suggest a mortality benefit above that caused by its effect on measles disease and its sequelae . Our review of the available data in the literature reinforces how difficult answering these considerations has been and how important study design will be in determining the effect of specific vaccines on all-cause mortality.

Biochim Biophys Acta, 2004 Jan 28, 1660(1-2), 99 - 105
Role of toxin activation on binding and pore formation activity of the Bacillus thuringiensis Cry3 toxins in membranes of Leptinotarsa decemlineata (Say); Rausell C et al.; Binding and pore formation constitute key steps in the mode of action of Bacillus thuringiensis delta-endotoxins . In this work, we present a comparative analysis of toxin-binding capacities of proteolytically processed Cry3A, Cry3B and Cry3C toxins to brush border membranes (BBMV) of the Colorado potato beetle Leptinotarsa decemlineata (CPB), a major potato coleopteran-insect pest . Competition experiments showed that the three Cry3 proteolytically activated toxins share a common binding site . Also heterologous competition experiments showed that Cry3Aa and Cry3Ca toxins have an extra binding site that is not shared with Cry3Ba toxin . The pore formation activity of the three different Cry3 toxins is analysed . High pore-formation activities were observed in Cry3 toxins obtained by proteolytical activation with CPB BBMV in contrast to toxins activated with either trypsin or chymotrypsin proteases . The pore-formation activity correlated with the formation of soluble oligomeric structures . Our data support that, similarly to the Cry1A toxins, the Cry3 oligomer is formed after receptor binding and before membrane insertion, forming a pre-pore structure that is insertion-competent.

J Mol Biol, 2004 Feb 13, 336(2), 395 - 408
DNA-binding orientation and domain conformation of the E . coli rep helicase monomer bound to a partial duplex junction: single-molecule studies of fluorescently labeled enzymes; Rasnik I et al.; The SF1 DNA helicases are multi-domain proteins that can unwind duplex DNA in reactions that are coupled to ATP binding and hydrolysis . Crystal structures of two such helicases, Escherichia coli Rep and Bacillus stearothermophilus PcrA, show that the 2B sub-domain of these proteins can be found in dramatically different orientations (closed versus open) with respect to the remainder of the protein, suggesting that the 2B domain is highly flexible . By systematically using fluorescence resonance energy transfer at the single-molecule level, we have determined both the orientation of an E.coli Rep monomer bound to a 3'-single-stranded-double-stranded (ss/ds) DNA junction in solution, as well as the relative orientation of its 2B sub-domain . To accomplish this, we developed a highly efficient procedure for site-specific fluorescence labeling of Rep and a bio-friendly immobilization scheme, which preserves its activities . Both ensemble and single-molecule experiments were carried out, although the single-molecule experiments proved to be essential here in providing quantitative distance information that could not be obtained by steady-state ensemble measurements . Using distance-constrained triangulation procedures we demonstrate that in solution the 2B sub-domain of a Rep monomer is primarily in the "closed" conformation when bound to a 3'-ss/ds DNA, similar to the orientation observed in the complex of PcrA bound to a 3'-ss/ds DNA . Previous biochemical studies have shown that a Rep monomer bound to such a 3'-ss/ds DNA substrate is unable to unwind the DNA and that a Rep oligomer is required for helicase activity . Therefore, the closed form of Rep bound to a partial duplex DNA appears to be an inhibited form of the enzyme.

J Mol Biol, 2004 Feb 13, 336(2), 381 - 93
The clamp-loader-helicase interaction in Bacillus . Atomic force microscopy reveals the structural organisation of the DnaB-tau complex in Bacillus; Haroniti A et al.; The clamp-loader-helicase interaction is an important feature of the replisome . Although significant biochemical and structural work has been carried out on the clamp-loader-clamp-DNA polymerase alpha interactions in Escherichia coli, the clamp-loader-helicase interaction is poorly understood by comparison . The tau subunit of the clamp-loader mediates the interaction with DnaB . We have recently characterised this interaction in the Bacillus system and established a tau(5)-DnaB(6) stoichiometry . Here, we have obtained atomic force microscopy images of the tau-DnaB complex that reveal the first structural insight into its architecture . We show that despite the reported absence of the shorter gamma version in Bacillus, tau has a domain organisation similar to its E.coli counterpart and possesses an equivalent C-terminal domain that interacts with DnaB . The interaction interface of DnaB is also localised in its C-terminal domain . The combined data contribute towards our understanding of the bacterial replisome.

Biochemistry, 2004 Feb 10, 43(5), 1393 - 400
Selective antagonism to the cadherin BT-R1 interferes with calcium-induced adhesion of epithelial membrane vesicles; Griko N et al.; BT-R(1) is a member of the cadherin superfamily of proteins and is expressed in the midgut epithelium of Manduca sexta during larval development . Previously, we showed that calcium ions influence the structure and stability of BT-R(1) on brush border membrane vesicles (BBMVs) prepared from M . sexta midgut epithelium . In the present study, the effects of calcium and Cry1Ab toxin, produced by Bacillus thuringiensis, on the adhesive properties of BBMVs were investigated . Addition of calcium to a suspension of BBMVs promoted adhesion and aggregation of the vesicles . Treatment of BBMVs with trypsin or lowering the pH (pH 4.0) of the BBMV suspension abolished calcium-induced vesicle aggregation, whereas treatment with deglycosylating enzymes did not affect the aggregation of vesicles, indicating that adhesion and clustering of BBMVs involves protein-protein interactions . Preincubation of BBMVs with Cry1Ab toxin, which specifically binds to BT-R(1) with high affinity and disrupts the midgut epithelium of M . sexta, caused a 50% decrease in calcium-induced vesicle aggregation . The inhibitory effects of the Cry1Ab toxin on BBMV aggregation was blocked completely when the toxin was preincubated with a peptide containing the toxin-binding site of BT-R(1) . Cry3A toxin, which is similar in molecular structure to Cry1Ab but does not bind to BT-R(1) and is not toxic to M . sexta larvae, did not affect BBMV aggregation . The results of this study demonstrate that the adhesive function of BT-R(1) is compromised by the Cry1Ab toxin, which acts as a selective antagonist, and supports the notion that BT-R(1) is critical in preserving the integrity of larval midgut epithelium in M . sexta.

Curr Microbiol, 2003 Dec, 47(6), 501 - 7
Occurrence and linkage between secreted insecticidal toxins in natural isolates of Bacillus thuringiensis; Espinasse S et al.; Little is known about the occurrence and linkage between secreted insecticidal virulence factors in natural populations of Bacillus thuringiensis (Bt) . We carried out a survey of 392 Bt strains isolated from various samples originating from 31 countries . The toxicity profile of the culture supernatants of these strains was determined individually against Anthonomus grandis (Coleoptera) and Spodoptera littoralis (Lepidoptera) . We analyzed beta-exotoxin I production and searched for the genes encoding Vip1-2, Vip3, and Cry1I toxins in 125 of these strains . Our results showed that these insecticidal toxins were widespread in Bt but that their distribution was nonrandom, with significant linkage observed between vip3 and cry1I and between vip1-2 and beta-exotoxin I . Strains producing significant amounts of beta-exotoxin I were more frequently isolated from invertebrate samples than from dust, water, soil, or plant samples.

Exp Appl Acarol, 2003, 30(1-3), 225 - 31
Passion fruit green spot virus vectored by Brevipalpus phoenicis (Acari: Tenuipalpidae) on passion fruit in Brazil; Kitajima EW et al.; Passion fruit green spot disease was first identified in 1997 after a severe outbreak at Vera Cruz County, state of Sao Paulo, Brazil . Mature yellow fruits of Passiflora edulis Simms f . flavicarpa Degener showed characteristic green spots, 2-5 mm in diameter and patches of green tissues were present on senescent leaves . The devastating effect to passion flower is caused by necrotic lesions that encircle the stems and kill the plant . In severe cases, entire orchards of a few hectares in size have been completely destroyed . The disease was always preceded by heavy infestations of Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae) . Transmission electron microscopy of affected tissues (fruits, leaves, and stems) consistently revealed the presence of short, bacilliform particles (50-70 nm x 100-120 nm) in the cisternae of the endoplasmic reticulum, as well as the presence of a dense viroplasm in the cytoplasm . This cytopathic effect has been found in several other Brevipalpus-transmitted or associated viruses and is classified as a cytoplasmic type of disease . Experimental reproduction of the leaf and stem symptoms was achieved by transferring B . phoenicis collected from affected field passion flower plants onto healthy plants . The evidence supports a viral etiology for the disease and the agent was named passion fruit green spot virus . Its relationship with other B . phoenicis related viruses continues to be studied . The disease was also found in the Brazilian states of Bahia, Sergipe, Rondonia, Minas Gerais, Rio de Janeiro, and in the Federal District . Use of one or more of the following acaricides (hexythiazox, fenbutatin-oxide, propargite, quinomethionate, or dicofol) has significantly reduced the incidence of the disease.

Exp Appl Acarol, 2003, 30(1-3), 215 - 23
Orchid fleck virus: Brevipalpus californicus mite transmission, biological properties and genome structure; Kondo H et al.; Orchid fleck virus (OFV) causes necrotic or chlorotic ring spots and fleck symptoms in many orchid species world-wide . The virus has non-enveloped, bacilliform particles of about 40 nm x 100-150 nm and is sap-transmissible to several plant species . OFV is transmitted by the mite Brevipalpus californicus (Banks) in a persistent manner and efficiently transmitted by both adults and nymphs, but not by larvae . Viruliferous mites retain their infectivity for 3 weeks on a virus-immune host . The genome of OFV consists of two molecules of 6431 (RNA1) and 6001 nucleotides (RNA2) . The RNAs have conserved and complementary terminal sequences . RNA1 contains five open reading frames (ORF), and RNA2 encodes a single ORF . Although some of the encoded proteins of OFV have sequences similar to those of proteins of plant rhabdoviruses, OFV differs from viruses in the family Rhabdoviridae in having a bipartite genome.

Exp Appl Acarol, 2003, 30(1-3), 203 - 13
Coffee ringspot virus vectored by Brevipalpus phoenicis (Acari: Tenuipalpidae) in coffee; Chagas CM et al.; Coffee ringspot is characterized by conspicuous ringspot symptoms on leaves, berries, and less frequently on twigs . It is caused by coffee ringspot virus (CoRSV), a short, bacilliform virus (40 nm x 100-110 nm) . The virus is not seed borne and is transmitted by Brevipalpus phoenicis (Geijskes) . Transovarial transmission within the mite does not occur . CoRSV has been mechanically transmitted to Chenopodium amaranticolor Coste and Reynaud, C . quinoa Wildenow, Beta vulgaris L., and Alternanthera tenella Colla resulting in local lesions . Systemic infection within both C . amaranticolor and C . quinoa occurs . Virions are found in the nucleus or cytoplasm of infected cells, commonly associated with membranes . Occasionally, membrane bounded particles are found within the cisternae of the endoplasmic reticulum . A characteristic electron lucent, nuclear inclusion is commonly found in many infected cells . These cytopathic effects place CoRSV among the nuclear type of Brevipalpus-borne viruses . The disease has been reported in several Brazilian states (Sao Paulo, Parana, Minas Gerais, and Federal District) and recently found in Costa Rica . A similar disease is known in the Philippines, but no information exists about its relationship to CoRSV . Coffee ringspot had no economical significance until recently when a large scale infection was reported in Minas Gerais that resulted in yield loss.

Exp Appl Acarol, 2003, 30(1-3), 135 - 60
Brevipalpus-transmitted plant virus and virus-like diseases: cytopathology and some recent cases; Kitajima EW et al.; An increasing number of diseases transmitted by Brevipalpus mite species (Acari: Tenuipalpidae) is being identified that affect economically important plants such as citrus, coffee, passion fruit, orchids, and several ornamentals . All of these diseases are characterized by localized lesions (chlorotic, green spots, or ringspots) on leaves, stems, and fruits . Virus or virus-like agents are considered to be the causal agents, possibly transmitted in a circulative-propagative manner by Brevipalpus mites . The virus or virus-like particles are short, rod-like, or bacilliform, that induce two characteristic types of cell alteration: (1) 'Nuclear type'--nuclei of parenchyma and epidermal cells in the lesions often contain a large electron lucent inclusion . Short, naked, rod-like (40-50 nm x 100-110 nm) particles may be seen in the viroplasm or nucleoplasm and in the cytoplasm . These particles are commonly arranged perpendicularly on the membranes of the nuclear envelope or endoplasmic reticulum (ER) . In a very few instances, they were found to be membrane-bound, within the ER cavities . (2) 'Cytoplasmic type'--short bacilliform particles (60-70 nm x 110-120 nm) are present within the cisternae of the ER and often have electron dense viroplasm of varied shapes present in the cytoplasm . Bacilliform particles may be seen budding into the ER lumen near the viroplasm . These particles resemble those of members of the Rhabdoviridae, but are shorter . The only sequenced virus of this group, orchid fleck virus (OFV), has a negative sense (bipartite) type ssRNA genome, but its organization is similar to known rhabdoviruses, which are monopartite . Both types of cytopathological effects have been found associated with citrus leprosis . In orchids, OFV has a 'nuclear type' of cytopathology, but in some species the 'cytoplasmic type' has been found associated with ringspot symptoms . In Hibiscus and Clerodendron, green spot symptoms have been associated with the cytoplasmic type of cell alteration, while chlorotic spots, in the same species, are associated with the nuclear type . In a few cases, both types of cytopathological effects have been found in the same tissue and cell.

Arch Biochem Biophys, 2004 Feb 15, 422(2), 211 - 20
Posttranslational processing of polysaccharide lyase: maturation route for gellan lyase in Bacillus sp . GL1; Miyake O et al.; Cells of Bacillus sp . GL1 extracellularly secrete a gellan lyase with a molecular mass of 130 kDa responsible for the depolymerization of a heteropolysaccharide (gellan), although the gene is capable of encoding a huge protein with a molecular mass of 263 kDa . A maturation route for gellan lyase in the bacterium was determined using anti-gellan lyase antibodies . The fluid of the bacterial exponentially growing cultures on gellan contained two proteins with molecular masses of 260 and 130 kDa, both of which reacted with the antibodies . The 260 kDa protein was purified from the cultured fluid and characterized . The protein exhibited gellan lyase activity and showed similar enzyme properties, such as optimal pH and temperature, thermal stability, and substrate specificity, to those of the 130 kDa gellan lyase . The N-terminal amino acid sequences of the 260 and 130 kDa enzymes were found to be identical . Determination of the C-terminal amino acid of the 130 kDa enzyme indicated that the 260 kDa enzyme is cleaved between the 1205Gly and 1206Leu residues to yield the mature form (130 kDa) of the gellan lyase . Therefore, the mature enzyme consists of 1170 amino acids (36Ala-1205Gly) with a molecular weight of 125,345, which is in good agreement with that calculated from SDS-PAGE analysis . Judging from these results, gellan lyase is first synthesized as a preproform (263 kDa) and then secreted as a precursor (260 kDa) into the medium through cleavage of the signal peptide . Finally, the precursor is post-translationally processed into the N-terminal half domain of 130 kDa as the mature form, the function of C-terminal half domain being unclear.

Phytother Res, 2004 Jan, 18(1), 30 - 5
Chemical and biological evaluation of the essential oils of different Melaleuca species; Farag RS et al.; The essential oils of the fresh leaves of M . ericifolia, M . leucadendron, M . armillaris and M . styphelioides were isolated by a hydrodistillation method and analysed by a gas chromatography/mass spectrometry (GC/MS) technique . The essential oil of M . ericifolia contained methyl eugenol (96.84%) as a major constituent, whereas M . leucadendron was rich in 1,8-cineole (64.30%) . The essential oil of M . armillaris was rich in 1,8-cineole (33.93%) followed by terpinen-4-ol (18.79%), whereas M . styphelioides was rich in caryophyllene oxide (43.78%) and (-) spathulenol (9.65%) . The essential oils of these species possessed antimicrobial and antifungal activities . M . ericifolia exhibited the highest inhibitory effects against Bacillus subtiles and Aspergillus niger . The antiviral activities of the essential oils against Herpes simplex virus type 1 (HSV-1) were studied in African green monkey kidney cells (Vero) by a plaque reduction assay . The volatile oil of M . armillaris was more effective as a virucidal (up to 99%) than that of M . leucadendron (92%) and M . ericifolia (91.5%) . The effects of the essential oils on the antioxidant system status in carbon tetrachloride treated animals were studied . The essential oil of M . armillaris exhibited a marked antioxidant effect, it improved vitamin E, vitamin C and superoxide dismutase parameters so it can be used as a free radical suppressor .

Microb Ecol, 2004 Feb, 47(2), 150 - 8 Epub 2004 Feb 02.
Microbial monitoring of spacecraft and associated environments; La Duc MT et al.; Rapid microbial monitoring technologies are invaluable in assessing contamination of spacecraft and associated environments . Universal and widespread elements of microbial structure and chemistry are logical targets for assessing microbial burden . Several biomarkers such as ATP, LPS, and DNA (ribosomal or spore-specific), were targeted to quantify either total bioburden or specific types of microbial contamination . The findings of these assays were compared with conventional, culture-dependent methods . This review evaluates the applicability and efficacy of some of these methods in monitoring the microbial burden of spacecraft and associated environments . Samples were collected from the surfaces of spacecraft, from surfaces of assembly facilities, and from drinking water reservoirs aboard the International Space Station (ISS) . Culture-dependent techniques found species of Bacillus to be dominant on these surfaces . In contrast, rapid, culture-independent techniques revealed the presence of many Gram-positive and Gram-negative microorganisms, as well as actinomycetes and fungi . These included both cultivable and noncultivable microbes, findings further confirmed by DNA-based microbial detection techniques . Although the ISS drinking water was devoid of cultivable microbes, molecular-based techniques retrieved DNA sequences of numerous opportunistic pathogens . Each of the methods tested in this study has its advantages, and by coupling two or more of these techniques even more reliable information as to microbial burden is rapidly obtained .

Org Lett, 2004 Feb 5, 6(3), 429 - 32
Asymmetric total synthesis of bacillariolide III, a marine oxylipin; Seo SY et al.; {reaction: see text} The asymmetric total synthesis of bacillariolide III has been achieved via 15 linear steps in 14.6% overall yield . The key feature of this synthetic route involves the highly stereoselective construction of the vinyl-substituted bicyclic lactone by an intramolecular Pd(0)-catalyzed allylic alkylation and its facile conversion to the hydroxy bicyclic lactone skeleton of bacillariolide III, induced by stereoselective vinylcerium addition to the aldehyde . In addition, the (Z)-pentenoic acid was efficiently introduced by the internal hydroxy group tethered ring-closing metathesis (RCM).

Proteins, 2004 Feb 15, 54(3), 412 - 23
Substrate binding to mononuclear metallo-beta-lactamase from Bacillus cereus; Dal Peraro M et al.; Structure and dynamics of substrate binding (cefotaxime) to the catalytic pocket of the mononuclear zinc-beta-lactamase from Bacillus cereus are investigated by molecular dynamics simulations . The calculations, which are based on the hydrogen-bond pattern recently proposed by Dal Peraro et al . (J Biol Inorg Chem 2002; 7:704-712), are carried out for both the free and the complexed enzyme . In the resting state, active site pattern and temperature B-factors are in agreement with crystallographic data . In the complexed form, cefotaxime is accommodated into a stable orientation in the catalytic pocket within the nanosecond timescale, interacting with the enzyme zinc-bound hydroxide and the surrounding loops . The beta-lactam ring remains stable and very close to the hydroxide nucleophile agent, giving a stable representation of the productive enzyme-substrate complex .

Rev Clin Esp, 2004 Jan, 204(1), 3 - 8
{Factors related to the tuberculous patient and the study of contacts.}; Alseda Graells Pere M et al.; CONTEXT: While the maximum priority of the programs for tuberculosis prevention and control is the identification and treatment of all the people with active tuberculosis, the second priority is the evaluation of their contacts . The goal of this study has been to evaluate the factors of the tuberculous patients associated with the study of their contacts . PATIENTS AND METHODS: The incident cases of tuberculosis occurred during the period 1992-1997 in the province of Lleida were studied . The variables of the study were: age, sex, place of residence, infection by HIV, parenteral drug use (PDU), excessive alcohol consumption, anatomical location of tuberculosis, existence of cavernous lesions in the chest X-ray, and microbiological result of the samples of respiratory secretions . The association of the dependent variable (study of the contacts in the incident cases of tuberculosis) with the rest of independent variables it was determined by the raw and adjusted odds ratio . RESULTS: The prevalence of tuberculosis patients whose contacts were studied was 60.4% (63.1% in the bacilliferous cases and 51.9% in the non-bacilliferous cases, p=0.003) . A higher number of patients whose contacts were studied was detected between those that were not HIV-infected (ORa: 4.53; 95% CI: 2.91-7.05), between those with lung as the anatomical localization of the tuberculous disease (ORa: 2.62; 95% CI: 1.70-4.05) and between those that did not showed excessive alcohol consumption (ORa: 1.69; 95 CI: 1.17-2.46) . CONCLUSIONS: The global prevalence of tuberculous whose contacts are studied should be increased, especially among the patients with sociosanitary risk (HIV, PDU and alcoholism), and among the patients with higher probability of transmission.

Biosci Biotechnol Biochem, 2004 Jan, 68(1), 235 - 7
High-level production of hyperthermophilic cellulase in the Bacillus brevis expression and secretion system; Kashima Y et al.; A hyperthermophilic cellulase derived from Pyrococcus horikoshii was successfully produced with the Bacillus brevis host-vector system . The production of the recombinant enzyme was increased about 20-fold (to a level of 100 mg per liter) by the insertion of certain amino acid such as alanine and peptides like AEEAADP between the carboxyl end of signal peptide and the N-terminus of the mature cellulase . These recombinant cellulases had the same characteristics as that of the cellulase expressed in Escherichia coli.

Biosci Biotechnol Biochem, 2004 Jan, 68(1), 36 - 43
Antioxidative effects of glycosyl-ascorbic acids synthesized by maltogenic amylase to reduce lipid oxidation and volatiles production in cooked chicken meat; Lee SB et al.; Glycosylated ascorbic acids were synthesized by using the transglycosylation activity of Bacillus stearothermophilus maltogenic amylase with maltotriose to show effective antioxidative activity with enhanced oxidative stability . The modified ascorbic acids comprised mono- and di-glycosyl transfer products with an alpha-(1,6)-glycosidic linkage . The antioxidative effects of the glycosyl derivatives of ascorbic acid on the lipid oxidation of cooked chicken breast meat patties were compared, and the synergistic effect when combined with alpha-tocopherol was determined in terms of thiobarbituric acid-reactive substances (TBARS) and volatiles production during storage . The results indicate that the glycosylated ascorbic acids had very effective antioxidative activity in preventing lipid oxidation, and were better in their synergistic effect in comparison to authentic ascorbic acid, with maltosyl-ascorbic acid being the most effective . Volatiles production was highly correlated with the TBARS values in the lipid oxidation of cooked meat . The antioxidative effect preventing the production of volatiles was particularly strong on pentanal, fairly strong on propanal and butanal, and not at all on ethanal . Propanal, pentanal, and the total volatiles thus provided a good representation of the lipid oxidation status of cooked chicken meat.

Cancer Res, 2004 Jan 15, 64(2), 757 - 64
Adjuvant-mediated tumor regression and tumor-specific cytotoxic response are impaired in MyD88-deficient mice; Akazawa T et al.; The Mycobacterium bovis bacillus Calmette-Guerin cell-wall skeleton (BCG-CWS) activates Toll-like receptor (TLR) 2 and TLR4, but unlike the typical TLR4 agonist bacterial lipopolysaccharide barely induces type 1 IFN . BCG-CWS has been used for adjuvant immunotherapy for patients with cancer . We investigated the adjuvant potential of BCG-CWS for induction of CTLs subsequent to TLR-mediated dendritic cell (DC) maturation, using a syngeneic mouse tumor model (B16 melanoma in C57BL/6) . We evaluated the retardation of tumor growth and cytotoxic response in wild-type and MyD88-/- mice immunized with tumor debris and/or BCG-CWS . Delays in tumor growth and cytotoxic response were induced by immunization with a mixture of BCG-CWS emulsion and the tumor . BCG-CWS was capable of activating DCs ex vivo by the criteria of CD80/CD86 up-regulation and cytokine (interleukin-12, tumor necrosis factor-alpha) induction . Efficient tumor suppression and ex vivo cytokine induction did not occur in MyD88-deficient mice and cells, suggesting that the MyD88 adapter is crucial for induction of tumor cytotoxicity . Because TLR4 is involved in both MyD88-dependent and -independent pathways and the latter affects DC maturation, our findings indicate that both pathways cooperate to induce CTL-based tumor immunity.

J Microbiol Methods, 2004 Feb, 56(2), 213 - 9
Colloidal chitin stained with Remazol Brilliant Blue R, a useful substrate to select chitinolytic microorganisms and to evaluate chitinases; Gomez Ramirez M et al.; A simple and sensitive method based on the use of colloidal chitin stained with Remazol Brilliant Blue R (RBB) is proposed to evaluate chitinase activity . If this colloidal-stained substrate is included as a carbon source in a liquid medium, this technique allows the selection or the comparison of chitinolytic microorganisms . The colloidal substrate is proportionally solubilized and the dye released is spectrophotometrically quantified at 595 nm . The procedures used for the staining and fixing of RBB in the colloidal chitin, and a comparison with the commercial substrate chitin-azure, are presented . The influence of several physicochemical and enzymatic parameters on the release of dyes is also shown . Both stained substrates were used for studying the effect of pH, substrate concentration, temperature and time on the chitinase reaction of Bacillus thuringiensis Bt-107.

Acta Microbiol Pol, 2003, 52(3), 293 - 300
Comparative delta-endotoxins of Bacillus thuringiensis against mosquito vectors (Aedes aegypti and Culex pipiens); Lonc E et al.; Pure crystals of seven Bacillus thuringiensis field isolates from the Lower Silesia region (Poland) were tested against larvae of Aedes aegypti L . and Culex pipiens L . (Culicidae, Diptera) . The crystals of OpQ3 phylloplane isolate (belonging to the first biochemical type of B . thuringiensis subsp . japonensis, yoso, jinghongiensis) killed from 68 +/- 7% to 84 +/- 7% of the fourth instar larvae of A . aegypti . The crystals of two other strains (KpF3 and KpC1) of this group caused mortality between 3 +/- 2% and 70 +/- 7% . The LC50 ranged from 3.2 +/- 0.4 to 34.1 +/- 4.8 microg/ml . The effect of B . thulringiensis wratislaviensis H-47 crystals was the lowest with larval mortality from 0% to 17 +/- 3% . No significant (0%-37 +/- 6%) effect of B . thuringiensis crystals on the larvae of C . pipiens was observed . Our results show that the delta-endotoxins of B . thuringiensis act very specifically.

J Pathol, 2004 Feb, 202(2), 164 - 71
Expression of IL-27 in human Th1-associated granulomatous diseases; Larousserie F et al.; Interleukin (IL)-27 is a newly described member of the IL-12 family . It is a heterodimeric cytokine composed of two subunits, p28 and Epstein-Barr virus-induced gene 3 (EBI3) . In vitro studies have shown that IL-27 is mainly produced by activated monocytes and dendritic cells . It induces the proliferation of naive CD4-positive T cells and synergizes with IL-12 for interferon-gamma (IFN-gamma) production . Knock-out mice for the IL-27 receptor (WSX-1/TCCR) have impaired Th1 responses and form abnormal granulomas when injected with bacillus Calmette-Guerin . However, the expression profile of IL-27 in vivo is currently unknown . To investigate the potential role of IL-27 in the development of a Th1 response in humans in vivo, this study has analysed the in situ expression of IL-27 subunits in three types of granulomatous disease (tuberculosis, sarcoidosis, and Crohn's disease), each characterized by a Th1 response . Tissue sections from patients with tuberculosis (n = 9), sarcoidosis (n = 8), or Crohn's disease (n = 7) were analysed by immunohistochemistry with anti-EBI3 and anti-p28 antibodies, in parallel with control tissues (control reactive lymph nodes, n = 14, and control intestinal tissues, n = 11) . In granulomatous tissues, EBI3 and p28 co-expression was detected in epithelioid and multinucleate giant cells in granulomas . In addition, sinus or tissue macrophages, endothelial cells, and plasma cells were found to co-express EBI3 and p28 . These data support a possible role for IL-27 in human Th1 responses .

J Leukoc Biol, 2004 Apr, 75(4), 689 - 97 Epub 2004 Jan 23.
Activation of phosphatidylinositol 3-kinase and c-Jun-N-terminal kinase cascades enhances NF-kappaB-dependent gene transcription in BCG-stimulated macrophages through promotion of p65/p300 binding; Darieva Z et al.; The proinflammatory response of infected macrophages is an important early host defense mechanism against mycobacterial infection . Mycobacteria have been demonstrated to induce proinflammatory gene transcription through the Toll-like receptors, (TLR)2 and TLR 4, which initiate signaling cascades leading to nuclear factor (NF)-kappaB activation . The main transduction pathway responsible for NF-kappaB activation has been established and involves the MyD88, interleukin-1 receptor-associated kinase, tumor necrosis factor receptor-associated factor-6, NF-kappaB-inducing kinase, and inhibitor of kappaB kinase complex . The role of other kinase cascades triggered by mycobacteria in the NF-kappaB activation is less clear . We herein examine the role of the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI-3K) cascades in the expression of the bacillus Calmette-Guerin (BCG) mycobacteria-induced NF-kappaB-dependent genes, macrophage-inflammatory protein-2 (MIP-2) and inducible nitric oxide (NO) synthase . Specific pharmacological inhibition of the PI-3K, c-jun-N-terminal kinase (JNK), and to a smaller extent, p38 MAPK but not extracellular-regulated kinase (ERK), suppressed NF-kappaB-dependent reporter gene transcription and MIP-2 and NO secretion in BCG-induced RAW264.7 macrophages . A similar effect was obtained following molecular inhibition of JNK via JNK-interacting protein-1 overexpression . In addition, a kinase-dead mutant of MEK kinase-1, the up-stream regulator of JNK, also proved to be a potent inhibitor of NF-kappaB-reporter activity . The effect of inhibitors was mediated by the down-regulation of NF-kappaB transcription activity and without effecting its nuclear translocation . These data suggest an indirect mechanism of the NF-kappaB regulation by these kinases, probably through p65 phosphorylation and improved binding to the p300 transcription coactivator . The data obtained demonstrate that PI-3K, JNK, and p38 MAPK activation by mycobacteria enhance NF-kappaB-driven gene expression contributing to the proinflammatory macrophage response.

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 47 - 57
Bacillus novalis sp . nov., Bacillus vireti sp . nov., Bacillus soli sp . nov., Bacillus bataviensis sp . nov . and Bacillus drentensis sp . nov., from the Drentse A grasslands; Heyrman J et al.; A group of 42 isolates were isolated from the soil of several disused hay fields, in the Drentse A agricultural research area (The Netherlands), that were taken out of production at different times . The group represents hitherto-uncultured Bacillus lineages that have previously been found, by a non-cultural method, to be predominant in soil . The strains were subjected to a polyphasic taxonomic study, including (GTG)5-PCR, 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base-ratio determination, fatty acid analysis and morphological and biochemical characterization . By comparing the groupings obtained by (GTG)5-PCR and 16S rDNA sequence analysis, six clusters of similar strains could be recognized . A DNA-DNA relatedness study showed that these clusters represented five novel genospecies . Further analysis supported the proposal of five novel species in the genus Bacillus, namely Bacillus novalis sp . nov . (type strain IDA3307T=R-15439T=LMG 21837T=DSM 15603T), Bacillus vireti sp . nov . (type strain IDA3632T=R-15447T=LMG 21834T=DSM 15602T), Bacillus soli sp . nov . (type strain IDA0086T=R-16300T=LMG 21838T=DSM 15604T), Bacillus bataviensis sp . nov . (type strain IDA1115T=R-16315T=LMG 21833T=DSM 15601T) and Bacillus drentensis sp . nov . (type strain IDA1967T=R-16337T=LMG 21831T=DSM 15600T).

FEBS Lett, 2004 Jan 16, 557(1-3), 248 - 52
Independent folding and conformational changes of the barnase module in the VL-barnase immunofusion: calorimetric evidence; Tsybovsky YI et al.; Although stability is critical for in vivo application of immunotoxins, a thermodynamic description of their folding/stability is still lacking . We applied differential scanning calorimetry (DSC) to RNase-based immunofusion comprising barnase, cytotoxic RNase from Bacillus amyloliquefaciens, fused to the light chain variable domain (VL) of anti-human ferritin antibody F11 . By analyzing DSC curves recorded with or without preheating and addition of the barnase-stabilizing ligand guanosine 3'-monophosphate, we (i) . assigned two well-resolved thermal transitions to the VL and barnase modules of VL-barnase, (ii) . demonstrated independent folding of these two modules, and (iii) . showed altered stability of the barnase module, which resulted from the dimeric state of VL-barnase.

Appl Microbiol Biotechnol, 2004 Jun, 64(6), 833 - 9 Epub 2004 Jan 23.
Enhanced secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae by translational fusion to cellulose-binding domain; Ahn JO et al.; The secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae was investigated by employing a fusion partner, a cellulose-binding domain (CBD) from Trichoderma harzianum endoglucanase II (THEG) . The CBD was connected to the N-terminal of L1 lipase through an endogenous linker peptide from THEG . The expression cassette for the fusion protein in S . cerevisiae was constructed using the alpha-amylase signal peptide and the galactose-inducible GAL10 promoter . Secretion of CBD-linker-L1 lipase by this fusion construct was dramatically 7-fold enhanced, compared with that of the mature L1 lipase without CBD-fusion . The fusion protein was secreted into the culture medium, reaching levels of approximately 1.3 g/l in high-cell-density fed-batch cultures . Insertion of a KEX2 cleavage site into the junction between CBD-linker and L1 lipase resulted in the same level of enhanced secretion, indicating that the CBD-linker fusion probably plays a critical role in secretion from endoplasmic reticulum to Golgi apparatus . Therefore, the CBD from THEG can be used both as an affinity tag and as a secretion enhancer for the secretory production of heterologous proteins in S . cerevisiae, since in vivo breakage at the linker was almost negligible.

Protein Sci, 2004 Feb, 13(2), 457 - 65
Role of Phe283 in enzymatic reaction of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp.1011: Substrate binding and arrangement of the catalytic site; Kanai R et al.; Cyclodextrin glycosyltransferase (CGTase) belonging to the alpha-amylase family mainly catalyzes transglycosylation and produces cyclodextrins from starch and related alpha-1,4-glucans . The catalytic site of CGTase specifically conserves four aromatic residues, Phe183, Tyr195, Phe259, and Phe283, which are not found in alpha-amylase . To elucidate the structural role of Phe283, we determined the crystal structures of native and acarbose-complexed mutant CGTases in which Phe283 was replaced with leucine (F283L) or tyrosine (F283Y) . The temperature factors of the region 259-269 in native F283L increased >10 A(2) compared with the wild type . The complex formation with acarbose not only increased the temperature factors (>10 A(2)) but also changed the structure of the region 257-267 . This region is stabilized by interactions of Phe283 with Phe259 and Leu260 and plays an important role in the cyclodextrin binding . The conformation of the side-chains of Glu257, Phe259, His327, and Asp328 in the catalytic site was altered by the mutation of Phe283 with leucine, and this indicates that Phe283 partly arranges the structure of the catalytic site through contacts with Glu257 and Phe259 . The replacement of Phe283 with tyrosine decreased the enzymatic activity in the basic pH range . The hydroxyl group of Tyr283 forms hydrogen bonds with the carboxyl group of Glu257, and the pK(a) of Glu257 in F283Y may be lower than that in the wild type.

Biochim Biophys Acta, 2004 Jan 22, 1670(2), 137 - 46
Low molecular weight chitosans: preparation with the aid of papain and characterization; Vishu Kumar AB et al.; Low molecular weight chitosans (LMWC) of different molecular weight (4.1-5.6 kDa) were obtained by the depolymerization of chitosan using papain (from Carica papaya latex, EC . 3.4.22.2) at optimum conditions of pH 3.5 and 37 degrees C for 1-5 h . Scanning electron microscopy (SEM) showed approximately 15-fold decrease in the particle size after depolymerization . Decrease in the molecular weight was associated with decrease in the degree of acetylation (DA) as evidenced by circular dichroism (CD), FT-IR and solid-state CP-MAS 13C-NMR data . X-ray diffraction pattern revealed slight decrease in the crystallinity index (CrI) whereas the 13C-NMR data showed molecular inhomogeneity . LMWC showed lytic effect towards Bacillus cereus and Escherichia coli more efficiently than native chitosan . The growth inhibitory effect was maximal towards B . cereus, with minimum inhibitory concentration (MIC) of 0.01% (w/v).

J Biol Chem, 2004 Apr 2, 279(14), 13645 - 51 Epub 2004 Jan 20.
Specificity and enzyme kinetics of the quorum-quenching N-Acyl homoserine lactone lactonase (AHL-lactonase); Wang LH et al.; N-Acyl homoserine lactone (AHL) quorum-sensing signals are the vital elements of bacterial quorum-sensing systems, which regulate diverse biological functions, including virulence . The AHL-lactonase, a quorumquenching enzyme encoded by aiiA from Bacillus sp., inactivates AHLs by hydrolyzing the lactone bond to produce corresponding N-acyl homoserines . To characterize the enzyme, the recombinant AHL-lactonase and its four variants were purified . Kinetic and substrate specificity analysis showed that AHL-lactonase had no or little residue activity to non-acyl lactones and noncyclic esters, but displayed strong enzyme activity toward all tested AHLs, varying in length and nature of the substitution at the C3 position of the acyl chain . The data also indicate that the amide group and the ketone at the C1 position of the acyl chain of AHLs could be important structural features in enzyme-substrate interaction . Surprisingly, although carrying a (104)HX- HXDH(109) short sequence identical to the zinc-binding motif of several groups of metallohydrolytic enzymes, AHL-lactonase does not contain or require zinc or other metal ions for enzyme activity . Except for the amino acid residue His-104, which was shown previously to not be required for catalysis, kinetic study and conformational analysis using circular dichroism spectrometry showed that substitution of the other key residues in the motif (His-106, Asp-108, and His-109), as well as His-169 with serine, respectively, caused conformational changes and significant loss of enzyme activity . We conclude that AHL-lactonase is a highly specific enzyme and that the (106)HXDH(109) approximately H(169) of AHL-lactonase represents a novel catalytic motif, which does not rely on zinc or other metal ions for activity.

Biochim Biophys Acta, 2004 Jan 5, 1676(1), 112 - 7
Cloning and molecular characterization of the atp operon encoding for the F1F0-ATP synthase from a thermoalkaliphilic Bacillus sp . strain TA2.A1; Keis S et al.; The genes encoding the subunits for the F(1)F(0)-ATP synthase from Bacillus sp . strain TA2.A1 were cloned as three overlapping fragments and sequenced . The nine genes were organized in an operon with the gene order atpIBEFHAGDC encoding the i, a, c, b, delta, alpha, gamma, beta, and epsilon subunits, respectively . Northern blot analysis showed a maximum transcript of approximately 7.2 kb, which corresponds to the size of the atp operon and demonstrated that the nine genes are transcribed as a single polycistronic message . The alkaliphilic-specific residues Lys(218) and Gly(245) were conserved in subunit a of strain TA2.A1 . Analysis of the C-terminal domain of the epsilon subunit showed several clusters of basic residues which are predicted to form a strong electrostatic interaction with the DELSDED motif in the beta subunit from strain TA2.A1, and may explain the blockage of this enzyme in the ATP hydrolysis direction.

J Gastroenterol Hepatol, 2004 Feb, 19(2), 200 - 4
Helicobacter pylori is a fragile bacteria when stored at low and ultra-low temperatures; Ohkusa T et al.; BACKGROUND AND AIM: Usually, bacteria are cryopreserved for short-term storage at low and ultra-low temperatures . There are no reports as to whether Helicobacter pylori is a fragile bacteria when stored at low and ultra-low temperatures as compared with other intestinal bacteria . A study was done on seven H . pylori strains and other intestinal bacteria to compare different temperatures for storage of organisms in saline solution . METHODS: Seven H . pylori strains, specifically American Type Culture Collection (ATCC) strains 43504 and TN2GF4, and five strains isolated from the present patients were grown on a modified Skirrow's agar for H . pylori . Escherichia coli and Bacteroides distasonis, both representing isolates from the present patients, were grown on trypticase soy blood agar for E . coli, and EG agar for B . distasonis . Culture was for 4-5 days under microaerobic, aerobic and anaerobic conditions at 37 degrees C . Cells were harvested by scraping growth from the solid medium and into sterile saline . The cells were adjusted to concentrations of 109 viable cells/mL in saline and preserved at 4 degrees C, -20 degrees C, or -80 degrees C for 3 weeks before reculture under microaerobic, aerobic and anaerobic conditions at 37 degrees C for 7 days . After incubation, morphologically distinct colonies were counted, isolated, and identified by standard bacteriologic techniques . The H . pylori were morphologically analyzed by electronic microscopy before and after preservation . Mongolian gerbils were inoculated with the cryopreserved H . pylori to evaluate the bacterial infectivity . RESULTS: Six of the seven H . pylori strains failed to culture after being preserved at 4 degrees C, -20 degrees C, or -80 degrees C . Only ATCC 43504 could be cultured after freezing at -80 degrees C . The number of H . pylori ATCC 43504 before preservation was 9.0 +/- 0.5 (log10 no . organisms/mL) and decreased to 5.7 +/- 0.6 after preservation . Morphologically, all H . pylori except ATCC 43504 strains transformed from a bacillary to a coccoid form after preservation . In addition, none of the H . pylori strains could infect Mongolian gerbils after preservation . Escherichia coli and B . distasonis were recovered . Titers before and after 4 degrees C, -20 degrees C, and -80 degrees C, respectively, were 9.1 +/- 0.2, 8.9 +/- 0.5, 8.6 +/- 0.3, and 8.7 +/- 0.3 for E . coli and 9.1 +/- 0.4, 8.7 +/- 0.6, 8.6 +/- 0.5, and 8.8 +/- 0.3 for B . distasonis . CONCLUSIONS: Helicobacter pylori is a fragile bacteria for storage at low and ultra-low temperatures in comparison with other intestinal bacteria.

Mar Biotechnol (NY), 2003 Sep-Oct, 5(5), 424 - 34
Antimicrobial activities and matrix-assisted laser desorption/ionization mass spectrometry of Bacillus isolates from the marine sponge Aplysina aerophoba; Pabel CT et al.; The aim of this study was to isolate bacteria that are resistant to the strong antimicrobial metabolites characteristic of Aplysina aerophoba . For this purpose, bacterial isolation was performed on agar plates to which sponge tissue extract had been added . Following screening for antifungal and antimicrobial activities, 5 strains were chosen for more detailed analyses . 16S ribosomal DNA sequencing revealed that all isolates belonged to the genus Bacillus, specifically B . subtilis and B . pumilus . Using a combination of matrix-assisted laser desorption/ ionization mass spectrometry typing of whole cells and antimicrobial bioassays against selected reference strains, the bioactive metabolites were identified as lipopeptides.

Eur J Biochem, 2004 Feb, 271(3), 601 - 6
Purification and functional characterization of insecticidal sphingomyelinase C produced by Bacillus cereus; Nishiwaki H et al.; Bacillus cereus isolated from the larvae of Myrmeleon bore was found to secrete proteins that paralyze and kill German cockroaches, Blattela germanica, when injected . One of these active proteins was purified from the culture broth of B . cereus using anion-exchange and gel-filtration chromatography . The purified toxin, with a molecular mass of 34 kDa, was identified as sphingomyelinase C (EC 3.1.4.12) on the basis of its N-terminal and internal amino-acid sequences . A recombinant sphingomyelinase C expressed in Escherichia coli was as potent as the native protein in killing the cockroaches . Site-directed mutagenesis (His151Ala) that inactivated the sphingomyelinase activity also abolished the insecticidal activity, suggesting that the rapid insect toxicity of sphingomyelinase C results from its phospholipid-degrading activity.

Am J Reprod Immunol, 2004 Jan, 51(1), 63 - 70
Anti-proliferative effect of mycobacteria, IFN-gamma and TNF-alpha on primary cultures derived from endometrial stroma: possible relevance to endometriosis?
Clayton RD, Duffy SR, Wilkinson N, Garry R, Jackson AM.
PROBLEM: To assess the effects of mycobacteria and inflammatory cytokines on proliferation of endometrial stromal cells . An effect on endometrial stromal cell proliferation in vitro may suggest a similar effect on endometriotic cells in vivo . METHOD OF STUDY: Primary cultures of endometrial stromal cells were grown from female volunteers . Proliferation of cells was assessed by cell counting and incorporation of tritiated thymidine after exposure to mycobacteria or inflammatory cytokines . RESULTS: When assessed by cell counting, stromal cell growth was reduced following treatment with Connaught Bacillus of Calmette and Guerin (BCG) and Pasteur BCG: Mycobacterium smegmatis demonstrated a cytotoxic effect . Addition of the cytokines interferon (IFN)-gamma or tumour necrosis factor (TNF)-alpha at high concentrations led to a reduction in cell growth by 24 hr in two of three cell lines . A reduction in proliferation was also found when assessed by tritiated thymidine incorporation, which was statistically significant for Connaught BCG and M . smegmatis . CONCLUSIONS: Endometrial stromal cells are susceptible to the anti-proliferative effects of mycobacteria . The BCG and other mycobacteria are known immunomodulators in other disease conditions . Further work is required to assess whether these in vitro effects might translate into a useful therapy for endometriosis.

Biochem J, 2004 Apr 15, 379(Pt 2), 441 - 8
A functional chimaeric S-layer-enhanced green fluorescent protein to follow the uptake of S-layer-coated liposomes into eukaryotic cells; Ilk N et al.; The chimaeric gene encoding a C-terminally truncated form of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 and the EGFP (enhanced green fluorescent protein) was ligated into plasmid pET28a and cloned and expressed in Escherichia coli . Just 1 h after induction of expression an intense EGFP fluorescence was detected in the cytoplasm of the host cells . Expression at 28 degrees C instead of 37 degrees C resulted in clearly increased fluorescence intensity, indicating that the folding process of the EGFP moiety was temperature sensitive . To maintain the EGFP fluorescence, isolation of the fusion protein from the host cells had to be performed in the presence of reducing agents . SDS/PAGE analysis, immunoblotting and N-terminal sequencing of the isolated and purified fusion protein confirmed the presence of both the S-layer protein and the EGFP moiety . The fusion protein had maintained the ability to self-assemble in suspension and to recrystallize on peptidoglycan-containing sacculi or on positively charged liposomes, as well as to fluoresce . Comparison of fluorescence excitation and emission spectra of recombinant EGFP and rSbpA(31-1068)/EGFP revealed identical maxima at 488 and 507 nm respectively . The uptake of liposomes coated with a fluorescent monomolecular protein lattice of rSbpA(31-1068)/EGFP into HeLa cells was studied by confocal laser-scanning microscopy . The major part of the liposomes was internalized within 2 h of incubation and entered the HeLa cells by endocytosis.

Shi Yan Sheng Wu Xue Bao, 2003 Dec, 36(6), 476 - 81
{Displaying polyhistidine peptide on the cell surface of Bacillus thuringiensis by S-layer protein}; Wang L et al.; Cell surface layer (S-layer) is the outmost structure of bacterium cell and covers the intact cell . S-layer protein of Bacillus thuringiensis strain CTC (CTC protein) was used as carrier protein to display polyhistidine (polyHis) peptides on the cell surface . Series fusion protein genes were constructed by inserting DNA fragments encoding (6His)1, (6His)2, (6His)3, (6His)9 and (6His)15 polyHis peptides into CTC protein gene at the site of downstream of slh domain . With the help of recombinant plasmid pBMB-CSA, which contains operon csaAB needed for S-layer protein anchoring on cell surface, fusion protein genes were expressed in crystal negative B . thuringiensis strain 4Q7 . SDS-PAGE profiles demonstrated that all the fusion proteins except CTC-(6His)15 were expressed . Ni-NTA-agarose beads binding test showed that all recombinant strains could attach to agarose beads except 4Q7 (pBMB-CSA, pBMB-SH15) . Cd2+ adsorption test indicated that the adsorption ability of all recombinant strains except 4Q7 (pBMB-CSA, pBMB-SH15) were higher than that of host strains . The Cd2+ adsorption quantity of the recombinant strain with strongest adsorption ability was twice higher than that of host strain.

J Membr Biol, 2003 Nov 1, 196(1), 51 - 9
Analysis of the properties of Bacillus thuringiensis insecticidal toxins using a potential-sensitive fluorescent probe; Kirouac M et al.; A potential-sensitive fluorescent probe, 3,3'-dipropylthiadicarbocyanine iodide, was used to analyze, at pH 7.5 and 10.5, the effects of Bacillus thuringiensis toxins on the membrane potential generated by the efflux of K(+) ions from brush border membrane vesicles purified from the midgut of the tobacco hornworm, Manduca sexta . Fluorescence levels were strongly influenced by the pH and ionic strength of the media . Therefore, characterization of the effects of the toxins was conducted at constant pH and ionic strength . Under these conditions, the toxins had little effect on the fluorescence levels measured in the presence or absence of ionic gradients, indicating that the ionic selectivity of their pores is similar to that of the intact membrane . Valinomycin greatly increased the potential generated by the diffusion of K(+) ions although membrane permeability to the other ions used to maintain the ionic strength constant also influenced fluorescence levels . In the presence of valinomycin, active toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C and Cry1E) efficiently depolarized the membrane at pH 7.5 and 10.5.

Insect Biochem Mol Biol, 2004 Jan, 34(1), 43 - 9
Identification of 1-lysophosphatidylethanolamine (C(16:1)) as an antimicrobial compound in the housefly, Musca domestica; Meylaers K et al.; We observed that a methanolic whole body extract of uninfected last instar larvae of the housefly, Musca domestica, displayed antifungal and antibacterial activity . We have further purified this extract to a single active fraction using reversed phase high performance liquid chromatography . The pure fraction inhibited growth of the Gram-positive bacteria Bacillus thuringiensis and the yeast Saccharomyces cerevisiae, but not the Gram-negative bacteria Escherichia coli . The active compound was determined to have a molecular mass of 451.2 Da . Further analysis by nuclear magnetic resonance identified the substance as mono-unsaturated 1-lysophosphatidylethanolamine (C(16:1)) (1-LPE) . The structurally different and more common 2-LPE have been described as mediators of the antimicrobial activity of rimenophenazine antibiotic agents (Van Rensburg et al., 1992) . Our results suggest that the isolated 1-LPE displays a higher activity in comparison, possibly based on structure-specific differences in activity.

J Appl Microbiol, 2004, 96(2), 320 - 7
Newly isolated Bacillus clausii GMBAE 42: an alkaline protease producer capable to grow under higly alkaline conditions; Denizci AA et al.; AIMS: The isolation and identification of new Bacillus sp . capable of growing under highly alkaline conditions as alkaline protease producers . METHODS AND RESULTS: A Bacillus strain capable of growing under highly alkaline conditions was isolated from compost . The strain is a Gram-positive, spore-forming, motile, aerobic, catalase- and oxidase-positive, alkaliphilic bacterium and designated as GMBAE 42 . Good growth of the strain was observed at pH 10 . The strain was identified as Bacillus clausii according to the physiological properties, cellular fatty acid composition, G + C content of genomic DNA and 16S rRNA gene sequence analyses . The result of 16S rRNA sequence analyses placed this bacterium in a cluster with B . clausii . The G + C content of the genomic DNA of the isolate GMBAE 42 was found to be 49 mol% . The crude extracellular alkaline protease produced by the isolate showed maximal activity at pH 11.0 and 60 degrees C . CONCLUSIONS: The results suggest that isolated strain GMBAE 42 is a new type of B . clausii capable of growing at pH 10.0 and produce extracellular alkaline protease very active at pH 11.0 . SIGNIFICANCE AND IMPACT OF THE STUDY: Isolated strain could be used in commercial alkaline protease production and its enzyme can be considered as a candidate as an additive for commercial detergents.

Scand J Immunol, 2004 Jan, 59(1), 66 - 70
Immunological crossreactivity of the Mycobacterium leprae CFP-10 with its homologue in Mycobacterium tuberculosis; Geluk A et al.; Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) (Rv3874) is considered a promising antigen for the immunodiagnosis of tuberculosis (TB) together with early secreted antigens of M . tuberculosis (ESAT-6) . Both ESAT-6 and CFP-10 are encoded by the RD1 region that is deleted from all tested M . bovis bacille Calmette-Guerin (BCG) strains but present in M . leprae, M . tuberculosis, M . bovis, M . kansasii, M . africanum and M . marinum . In this study, the homologue of CFP-10 in M . leprae (ML0050) is identified and characterized . Interferon-gamma production in response to this homologue by T cells from leprosy patients, TB patients and unexposed controls shows that CFP-10 of M . leprae is a potent antigen that crossreacts with CFP-10 of M . tuberculosis at the T-cell level . This crossreactivity has implications for the use of CFP-10 of these mycobacterial species as diagnostic tool in areas endemic for both the diseases.

Scand J Immunol, 2004 Jan, 59(1), 16 - 24
Computer-assisted prediction of HLA-DR binding and experimental analysis for human promiscuous Th1-cell peptides in the 24 kDa secreted lipoprotein (LppX) of Mycobacterium tuberculosis; Al-Attiyah R et al.; The secreted 24 kDa lipoprotein (LppX) is an antigen that is specific for Mycobacterium tuberculosis complex and M . leprae . The present study was carried out to identify the promiscuous T helper 1 (Th1)-cell epitopes of the M . tuberculosis LppX (MT24, Rv2945c) antigen by using 15 overlapping synthetic peptides (25 mers overlapping by 10 residues) covering the sequence of the complete protein . The analysis of Rv2945c sequence for binding to 51 alleles of nine serologically defined HLA-DR molecules, by using a virtual matrix-based prediction program (propred), showed that eight of the 15 peptides of Rv2945c were predicted to bind promiscuously to >/=10 alleles from more than or equal to three serologically defined HLA-DR molecules . The Th1-cell reactivity of all the peptides was assessed in antigen-induced proliferation and interferon-gamma (IFN-gamma)-secretion assays with peripheral blood mononuclear cells (PBMCs) from 37 bacille Calmette-Guerin (BCG)-vaccinated healthy subjects . The results showed that 17 of the 37 donors, which represented an HLA-DR-heterogeneous group, responded to one or more peptides of Rv2945c in the Th1-cell assays . Although each peptide stimulated PBMCs from one or more donors in the above assays, the best positive responses (12/17 (71%) responders) were observed with the peptide p14 (aa 196-220) . This suggested a highly promiscuous presentation of p14 to Th1 cells . In addition, the sequence of p14 is completely identical among the LppX of M . tuberculosis, M . bovis and M . leprae, which further supports the usefulness of Rv2945c and p14 in the subunit vaccine design against both tuberculosis and leprosy.

Mikrobiol Z, 2003 Sep-Oct, 65(5), 20 - 5
Biological activity of bacterial lectins and their molecular complexes with heterocyclic bis-adducts; Welchinskaya HV et al.; A new convenient method for the preparation of heterocyclic bis-adducts: of imidazole, benzimidazole, uracile with 1,1,1-trifluoro-2-bromo-2-chloroethane is described . The reactions are catalysed by the 18-crown-6-complex . The critical toxicity and antitumour activity of saprophytic strains Bacillus genus (B . subtilis 668 IMV and B . polymyxa 102 KSU) extracellular lectins were studies . It was discovered that these substances apply to a few toxic preparations and have a expression antitumour action on the tumours: Walker carcinosarcoma 256, Pliss' lymphosarcoma and Sarcoma 45 . The new molecular complexes were created with bacterial lectins and the same heterocyclic-bis-adducts of unsubstituted benzimidazole and 6-methyluracile . A strongly antitumour effect of these complexes has been discovered: of growth relaxation of Pliss' lymphosarcoma tumour mass was 62.5-82.01%.

J Vet Med B Infect Dis Vet Public Health, 2003 Dec, 50(10), 505 - 9
Use of a quantitative TaqMan-PCR for the fast quantification of mycobacteria in broth culture, eukaryotic cell culture and tissue; Lewin A et al.; The quantification of slow-growing mycobacteria such as Mycobacterium tuberculosis or M . bovis from in vitro and in vivo samples is complicated by their long generation time, their ability to form aggregates, and their capacity to persist in a state of dormancy . We compared different methods for the establishment of growth curves for broth cultures of M . bovis bacille Calmette-Guerin (BCG) . A quantitative TaqMan-PCR yielded results comparable with those obtained by protein quantification and measurement of the ATP content of the cultures . The quantitative TaqMan-PCR furthermore turned out to be particularly suitable for the measurement of multiplication of BCG within eukaryotic cells . Furthermore, it is a fast method allowing an estimation of the mycobacterial load in tissue long before colony counts can be obtained.

Anal Chem, 2004 Jan 15, 76(2), 373 - 8
Reagentless detection and classification of individual bioaerosol particles in seconds; Fergenson DP et al.; The rapid chemical analysis of individual cells is an analytical capability that will profoundly impact many fields including bioaerosol detection for biodefense and cellular diagnostics for clinical medicine . This article describes a mass spectrometry-based analytical technique for the real-time and reagentless characterization of individual airborne cells without sample preparation . We characterize the mass spectral signature of individual Bacillus spores and demonstrate the ability to distinguish two Bacillus spore species, B . thuringiensis and B.atrophaeus, from one another very accurately and from the other biological and nonbiological background materials tested with no false positives at a sensitivity of 92% . This example demonstrates that the chemical differences between these two Bacillus spore species are consistently and easily detected within single cells in seconds.

Biotechnol Lett, 2003 Dec, 25(23), 2029 - 33
Production of transglutaminase from Bacillus circulans on solid-state and submerged cultivations; Soares LH et al.; A Bacillus circulans strain, isolated in the Amazon basin, produced a transglutaminase (EC 2.3 x 2.13) in both submerged and solid-state cultivation . Enzyme activity in the former case reached 0.69 U ml(-1) after 160 h cultivation on starch based medium, and in the latter 0.61 U ml(-1) after 60 h cultivation on soybean industrial fibrous residue, a non-expensive agro-industrial residue.

Biochemistry, 2004 Jan 20, 43(2), 577 - 86
Disentangling the web of allosteric communication in a homotetramer: heterotropic inhibition of phosphofructokinase from Bacillus stearothermophilus; Ortigosa AD et al.; A strategy for isolating each of the four potentially unique heterotropic pairwise allosteric interactions that exist in the homotetramer phosphofructokinase from Bacillus stearothermophilus is described . The strategy involves the construction of hybrid tetramers containing one wild-type subunit and three mutant subunits that have been modified to block binding of both the substrate, fructose 6-phosphate (Fru-6-P), and the allosteric inhibitor, phospho(enol)pyruvate (PEP) . Each type of binding site occurs at a subunit interface, and mutations on either side of the interface have been identified that will greatly diminish binding at the respective site . Consequently, four different types of mutant subunits have been created, each containing a different active site and allosteric site modification . The corresponding 1:3 hybrids isolate a different pair of unmodified substrate and allosteric sites with a unique structural disposition located 22, 30, 32, and 45 A apart, respectively . The allosteric inhibition exhibited by the unmodified sites in each of these four hybrids has been quantitatively evaluated in terms of a coupling free energy . Each of the coupling free energies is unique in magnitude, and their relative magnitudes vary with pH . Importantly, the sum of these coupling free energies at each pH is equal to the total heterotropic coupling free energy associated with the tetrameric enzyme . The latter quantity was assessed from the overall inhibition of a control hybrid that removed the homotropic interactions in PEP binding . The results do not agree with either the concerted or sequential models that are often invoked to explain allosteric behavior in oligomeric enzymes.

Vet Pathol, 2004 Jan, 41(1), 44 - 9
Immunoglobulin-A nephropathy with crescentic glomerulonephritis in a pigtailed macaque (Macaca nemestrina); Borda JT et al.; A 4-year-old female pigtailed macaque (Macaca nemestrina), experimentally coinfected with simian immunodeficiency virus (SIVmac251) and Mycobacterium bovis(bacillus Calmette-Guerin), was euthanatized 1 year after infection because of weight loss and labored breathing . On gross examination, both kidneys were found to be markedly enlarged (right: 54.7 g and left: 51.7 g; normal < 20 g) . Renal lesions were evaluated by histopathologic, immunohistochemical, and ultrastructural methods . Light microscopy revealed that the glomeruli were diffusely hypercellular with expansion of the mesangial matrix, and crescent formation affected approximately 60% of the glomeruli . By immunohistochemical evaluation, it was found that the crescents were composed principally of macrophages, as seen by CD68 (KP1), MRP8, MAC387, and HAM56 expression . Electron microscopic examination of the glomeruli revealed extensive intramembranous, subendothelial, and mesangial electron-dense deposits and multifocal fusion of the visceral epithelial foot processes . Immunofluorescence, used to determine the composition of the electron-dense deposits, revealed diffuse granular mesangial and capillary staining for immunoglobulin A (IgA) . The renal changes described in this case report are most consistent with the findings of crescentic gloerulonephritis with IgA immune complex deposition in the glomerular basement membrane and mesangium as described in humans with IgA nephropathy.

J Biol Chem, 2004 Mar 26, 279(13), 12369 - 78 Epub 2004 Jan 10.
Revisiting the structure of the anti-neoplastic glucans of Mycobacterium bovis Bacille Calmette-Guerin . Structural analysis of the extracellular and boiling water extract-derived glucans of the vaccine substrains; Dinadayala P et al.; The attenuated strain of Mycobacterium bovis Bacille Calmette-Guerin (BCG), used worldwide to prevent tuberculosis and leprosy, is also clinically used as an immunotherapeutic agent against superficial bladder cancer . An anti-tumor polysaccharide has been isolated from the boiling water extract of the Tice substrain of BCG and tentatively characterized as consisting primarily of repeating units of 6-linked-glucosyl residues . Mycobacterium tuberculosis and other mycobacterial species produce a glycogen-like alpha-glucan composed of repeating units of 4-linked glucosyl residues substituted at some 6 positions by short oligoglucosyl units that also exhibits an anti-tumor activity . Therefore, the impression prevails that mycobacteria synthesize different types of anti-neoplastic glucans or, alternatively, the BCG substrains are singular in producing a unique type of glucan that may confer to them their immunotherapeutic property . The present study addresses this question through the comparative analysis of alpha-glucans purified from the extracellular materials and boiling water extracts of three vaccine substrains . The polysaccharides were purified, and their structural features were established by mono- and two-dimensional NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the enzymatic and chemical degradation products of the purified compounds . The glucans isolated by the two methods from the three substrains of BCG were shown to exhibit identical structural features shared with the glycogen-like alpha-glucan of M . tuberculosis and other mycobacteria . Incidentally, we observed an occasional release of dextrans from Sephadex columns that may explain the reported occurrence of 6-substituted alpha-glucans in mycobacteria.

Biomacromolecules, 2004 Jan-Feb, 5(1), 153 - 61
Isolation and characterization of gram-positive cyanophycin-degrading bacteria-kinetic studies on cyanophycin depolymerase activity in aerobic bacteria; Obst M et al.; This study is the first report on the extracellular degradation of cyanophycin (CGP) by Gram-positive bacteria . Three different Gram-positive bacteria were isolated from forest soil that were able to utilize CGP as the sole carbon source for growth . The isolates were assigned to species of the genera Bacillus and Micromonospora . From one of the isolates, which was taxonomically affiliated as Bacillus megaterium strain BAC19, the extracellular CGP depolymerase (extracellular CGPase; CphEBm) was purified to electrophoretic homogeneity by fast protein liquid chromatography and affinity binding to an arginine-agarose column . The purified enzyme was specific for hydrolytic cleavage of CGP, and inhibitor studies indicated that CphEBm is a serine-type peptidase . As CGP degradation products, (beta-Asp-Arg)2 tetrapeptides in addition to beta-Asp-Arg dipeptides occurred, which were identified by electrospray ionization mass spectrometry analysis . Furthermore, a novel quantitative enzyme assay was developed for kinetic studies on CGP depolymerases . For CphEBm, as well as for the extracellular CGPase of Pseudomonas anguilliseptica strain BI (CphEPa), KM values of 2.2 and 1.0 microM, respectively, for CGP were determined.

J Vector Ecol, 2003 Dec, 28(2), 184 - 9
Survey of ticks collected in Mississippi for Rickettsia, Ehrlichia, and Borrelia species; Goddard J et al.; From November 1999 through October 2000, we tested ticks collected from vegetation as well as from deer, dogs, and humans for spotted fever group (SFG) rickettsiae, Ehrlichia chaffeensis, and Borrelia spp . spirochetes . A total of 149 adult ticks representing four species was collected from 11 collection sites from southwestern to northern Mississippi . Amblyomma americanum was most commonly collected (n=68), followed by Ixodes scapularis (n=53) . The bird tick, Ixodes brunneus (usually rare), was the third most commonly collected tick (n=17) . Eleven Dermacentor variabilis were also collected . Ticks were cut longitudinally to make smears on three microscope slides . The remaining body parts were frozen at -65 degrees C for additional testing . Tick smears were stained by direct immunofluorescence assays (DFA) for Rickettsia spp . and Borrelia spp., while indirect immunofluorescence assays (IFA) were used for Ehrlichia spp . The corresponding tick for each positive smear was evaluated using PCR analysis . None of the 149 ticks tested was DFA positive for Borrelia spp . However, smears of 30 (20%) and 32 (22%) ticks reacted with anti-E . chaffeensis sera and anti-R . rickettsii conjugate (known to react with several members of the spotted fever group), respectively . None of the ticks staining with the IFA for Ehrlichia was positive for E . chaffeensis using PCR . However, 23 (72%) of 32 FA-positive ticks for SFG rickettsiae yielded amplicons of the appropriate size when tested using a PCR assay for SFG rickettsiae, corresponding to an overall infection rate with SFG rickettsiae among the collected ticks of 15% . Smears of 12 (71%) of 17 I . brunneus revealed abundant bacilliform bacteria . PCR amplification of DNA from a single I . brunneus containing these bacteria was performed using universal primers for the 16S rRNA gene as well as Borrelia-specific primers . The predominant sequence obtained using the universal primers did not match any sequence in GenBank, but it showed 91% identity with an endosymbiont of Acanthoamoeba . Other sequences represented in the top 50 Basic Local Alignment Search (BLAST) scores were primarily from soil bacteria, although some similarity to several Anaplasma species and Ehrlichia risticii was indicated . The significance of this finding remains undetermined.

Prikl Biokhim Mikrobiol, 2003 Nov-Dec, 39(6), 656 - 60
The action of Cu2+ on Bacillus thuringiensis growth investigated by microcalorimetry; Jun Y et al.; By using an LKB-2277 Bioactivity Monitor, ampoule mode, the heat output of Bacillus thuringiensis growth metabolism has been determined at 28 degrees C and effect of Cu2+ on B . thuringiensis growth was studied . Copper has been regarded as an essential trace element for life . Its deficiency may be the cause of diseases . Cu2+ of different concentration have different effects on B . thuringiensis growth metabolism, Cu2+ of low concentration (0-30 micrograms/ml) can promote the growth of B . thuringiensis, and Cu2+ of high concentration (40-120 micrograms/ml) is able to inhibit its growth and B . thuringiensis can't grow at all when the concentration of Cu2+ is up to 130 micrograms/ml.

Prikl Biokhim Mikrobiol, 2003 Nov-Dec, 39(6), 619 - 24
{Identification of catalytically active groups in inulinase from Bacillus polymyxa 722}; Zherebtsov NA et al.; Inulinase from Bacillus polymyxa 722 hydrolyzing a polyfructosan inulin was studied . The dependence of inulinase activity on pH, measurements of pK value, calculation of ionization heat, photoinactivation with methylene blue, and inhibition with p-chloromercuribenzoate suggest that the active center of this enzyme contains imidazole and sulfhydryl groups . A possible mechanism underlying cleavage of beta-2,1-fructoside bonds in the inulin molecule with inulinase is considered.

J Urol, 2004 Feb, 171(2 Pt 1), 870 - 6
Immunotherapy for urological malignancies; Krejci KG et al.; PURPOSE: For decades urologists have successfully used immunotherapy in the battle against cancer . Interleukin-2 in renal cell carcinoma and bacillus Calmette-Guerin in bladder cancer are standard primary and/or adjunctive therapies for these diseases . Recent advances in our understanding of mechanisms governing immune system activation have fostered a myriad of novel immunotherapeutic approaches that show great promise in vivo but have had limited success in human trials to date . This review highlights current immunotherapy strategies that may prove to be successful treatments for urological cancers . MATERIALS AND METHODS: We performed a MEDLINE literature search for articles relating to immunotherapy in bladder, prostate and renal cell carcinoma in animals and humans . We included the most promising developments in this review . RESULTS: In addition to combining existing therapies to improve their efficacy, novel approaches that attempt to exploit the immune system ability to identify, target and eradicate malignancies are now being developed . These therapies include the use of antitumoral monoclonal and bi-specific antibodies, manipulation of T-lymphocyte costimulatory molecules and the administration of newly discovered cytokines as well as the development of antitumor vaccines . CONCLUSIONS: To date the full potential of immunotherapy for the treatment of urological malignancies has not been recognized . As our knowledge of the immune system expands, so too may our ability to manipulate it to affect tumor regression . This review describes the most recent and most promising developments in immunotherapy for urological malignancies.

Clin Exp Nephrol, 2003 Dec, 7(4), 301 - 5
POEMS syndrome caused refractory ascites in a polycystic disease patient undergoing hemodialysis; Higuchi M et al.; A 60-year-old man with polycystic disease (PCD) undergoing hemodialysis was admitted to our hospital because of refractory ascites in September 2000 . He had been diagnosed with probable chronic inflammatory demyelinating polyradiculopathy 6 months before admission . Though the ascites was bloody and exudative, the cytology was normal and cultures of bacteria and acid-fast bacillus were both negative . Hepatic venous outflow obstruction was excluded by several radiological examinations . Because of the presence of polyneuropathy, organomegaly, endocrine abnormality, M-protein, plasma cell dyscrasia, and skin lesions, POEMS, syndrome was diagnosed; this had caused the refractory ascites . Initial prednisolone therapy was effective for the refractory ascites, but it was not effective in preventing recurrence . He died due to cachexia in December 2000 . This is a very rare case of the presence of both PCD and POEMS syndrome in a patient.

J Thorac Imaging, 2004 Jan, 19(1), 60 - 2
Granulomatous chest disease following intravesical bacillus Calmette-Guerin immunotherapy; Chang H et al.; Bacillus Calmette-Guerin (BCG) is well accepted as an intravesicular method of treatment of bladder cancer . Although well tolerated in the majority of patients, adverse local and systemic adverse effects of this therapy have been described . In particular, lung disease complicating intravesicular use of BCG has been reported, producing 5 recognized forms of chest disease: 1) allergic interstitial lung disease, 2) miliary infection, 3) empyema, 4) diffuse alveolar damage, and 5) localized consolidative or cavitary lung disease . We report an asymptomatic form of pulmonary disease complicating intravesical BCG therapy, that of pulmonary granulomas with mediastinal and hilar lymph node enlargement.

J Biol Chem, 2004 Mar 19, 279(12), 11156 - 62 Epub 2004 Jan 07.
Isolation and characterization of a HpyC1I restriction-modification system in Helicobacter pylori; Lin TL et al.; Using transposon shuttle mutagenesis, we identified six Helicobacter pylori mutants from the NTUH-C1 strain that exhibited decreased adherence and cell elongation . Inverse polymerase chain reaction and DNA sequencing revealed that the same locus was interrupted in these six mutants . Nucleotide and amino acid sequences showed no homologies with H . pylori 26695 and J99 strains . This novel open reading frame contained 1617 base pairs . The amino acid sequence shared 24% identity with a putative nicking enzyme in Bacillus halodurans and 23 and 20% identity with type IIS restriction endonucleases PleI and MlyI, respectively . The purified protein, HpyC1I, showed endonuclease activity with the recognition and cleavage site 5'-CCATC(4/5)-3' . Two open reading frames were located upstream of the gene encoding HpyC1I . Together, HpyC1I and these two putative methyltransferases (M1.HpyC1I and M2.HpyC1I) function as a restriction-modification (R-M) system . The HpyC1I R-M genes were found in 9 of the 15 H . pylori strains tested . When compared with the full genome, significantly lower G + C content of HpyC1I R-M genes implied that these genes might have been acquired by horizontal gene transfer . Plasmid DNA transformation efficiencies and chromosomal DNA digestion assays demonstrated protection from HpyC1I digestion by the R-M system . In conclusion, we have identified a novel R-M system present in approximately 60% of H . pylori strains . Disruption of this R-M system results in cell elongation and susceptibility to HpyC1I digestion.

Appl Environ Microbiol, 2004 Jan, 70(1), 631 - 4
Identification of bacilysin, chlorotetaine, and iturin a produced by Bacillus sp . strain CS93 isolated from pozol, a Mexican fermented maize dough; Phister TG et al.; Three antimicrobial compounds produced by Bacillus sp . strain CS93 isolated from pozol were identified by using high-performance liquid chromatography and mass spectrometry . The three compounds were iturin, bacilysin, and chlorotetaine . Production of these compounds by CS93 could account for the medicinal properties attributed to pozol.

Appl Environ Microbiol, 2004 Jan, 70(1), 104 - 13
Genetics of zwittermicin a production by Bacillus cereus; Emmert EA et al.; Zwittermicin A represents a new chemical class of antibiotic and has diverse biological activities, including suppression of oomycete diseases of plants and potentiation of the insecticidal activity of Bacillus thuringiensis . To identify genes involved in zwittermicin A production, we generated 4,800 transposon mutants of B . cereus UW101C and screened them for zwittermicin A accumulation . Nine mutants did not produce detectable zwittermicin A, and one mutant produced eightfold more than the parent strain . The DNA flanking the transposon insertions in six of the nine nonproducing mutants contains significant sequence similarity to genes involved in peptide and polyketide antibiotic biosynthesis . The mutant that overproduced zwittermicin A contained a transposon insertion immediately upstream from a gene that encodes a deduced protein that is a member of the MarR family of transcriptional regulators . Three genes identified by the mutant analysis mapped to a region that was previously shown to carry the zwittermicin A self-resistance gene, zmaR, and a biosynthetic gene (E . A . Stohl, J . L . Milner, and J . Handelsman, Gene 237:403-411, 1999) . Further sequencing of this region revealed genes proposed to encode zwittermicin A precursor biosynthetic enzymes, in particular, those involved in the formation of the aminomalonyl- and hydroxymalonyl-acyl carrier protein intermediates . Additionally, nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) homologs are present, suggesting that zwittermicin A is synthesized by a mixed NRPS/PKS pathway.

Appl Environ Microbiol, 2004 Jan, 70(1), 52 - 60
Structural and spectral features of selenium nanospheres produced by Se-respiring bacteria; Oremland RS et al.; Certain anaerobic bacteria respire toxic selenium oxyanions and in doing so produce extracellular accumulations of elemental selenium {Se(0)} . We examined three physiologically and phylogenetically diverse species of selenate- and selenite-respiring bacteria, Sulfurospirillum barnesii, Bacillus selenitireducens, and Selenihalanaerobacter shriftii, for the occurrence of this phenomenon . When grown with selenium oxyanions as the electron acceptor, all of these organisms formed extracellular granules consisting of stable, uniform nanospheres (diameter, approximately 300 nm) of Se(0) having monoclinic crystalline structures . Intracellular packets of Se(0) were also noted . The number of intracellular Se(0) packets could be reduced by first growing cells with nitrate as the electron acceptor and then adding selenite ions to washed suspensions of the nitrate-grown cells . This resulted in the formation of primarily extracellular Se nanospheres . After harvesting and cleansing of cellular debris, we observed large differences in the optical properties (UV-visible absorption and Raman spectra) of purified extracellular nanospheres produced in this manner by the three different bacterial species . The spectral properties in turn differed substantially from those of amorphous Se(0) formed by chemical oxidation of H(2)Se and of black, vitreous Se(0) formed chemically by reduction of selenite with ascorbate . The microbial synthesis of Se(0) nanospheres results in unique, complex, compacted nanostructural arrangements of Se atoms . These arrangements probably reflect a diversity of enzymes involved in the dissimilatory reduction that are subtly different in different microbes . Remarkably, these conditions cannot be achieved by current methods of chemical synthesis.

Clin Lab Med, 2003 Dec, 23(4), 823 - 41, v-vi
Molecular detection of resistance to antituberculous therapy; Marttila HJ et al.; Drug-resistant tuberculosis is becoming increasingly common and represents a worldwide threat . Therefore, new approaches for the rapid susceptibility testing of Mycobacterium tuberculosis are needed to replace traditional culture-based methods . This article presents the genetic background of drug resistance in tubercle bacillus, and the methods currently available for genotypic susceptibility testing.

J Am Mosq Control Assoc, 2003 Dec, 19(4), 424 - 9
A semifield evaluation of Vectobac DT (ABG-6499), a new formulation of Bacillus thuringiensis israelensis for control of Aedes albopictus; Toma L et al.; We evaluated the effectiveness and duration of effectiveness of a new formulation of Bacillus thuringiensis var . israelensis (Bti) for control of larval Aedes albopictus . The product tested was Vectobac DT (ABG-6499), a Bti tablet formulation containing 3.4% of active ingredient (3,400 ITU/mg) supplied by SCAE Valent BioSciences Italy S.r.l . The study was conducted at the Botanical Garden of the University of Rome "La Sapienza" between June and September 2002, the most favorable season for the development of Ae . albopictus in Italy . Black 20-liter plastic buckets containing water and an organic substrate were used as experimental breeding sites . The number of larvae in these buckets was estimated weekly, and positive buckets were treated with the recommended dose of the larvicide . The results showed that Vectobac DT induced 100% larval mortality after 24 h in all experimental breeding sites during the entire study period . Nonetheless, in most cases, the larvicidal activity only lasted about 48 h; thus, effective mosquito control would require that treatment be performed every 8-10 days in this habitat.

Atherosclerosis, 2004 Jan, 172(1), 13 - 20
Impairment of vascular function following BCG immunisation is associated with immune responses to HSP-60 in the cholesterol-fed rabbit; Lamb DJ et al.; An immune response to heat shock protein (HSP)-60/65 has recently been implicated in atherogenesis . The aim of this study was to determine whether this effect may be mediated by impairment of endothelial function . Rabbits were injected with bacillus Calmette-Guerin (BCG) vaccine (n=12) or saline (n=12) . A further injection of BCG or saline was administered after 2 weeks . After a further 2 weeks, animals were fed either a 0.25-1% cholesterol diet or a chow diet for 16 weeks . Blood cholesterol levels were maintained at 10-12mmol/l by altering the dietary cholesterol content . Plasma levels of anti-mycobacterial antibodies rose following BCG immunisation, but anti-HSP antibodies developed only in the BCG-immunised, cholesterol-fed rabbits . Aortic endothelium from cholesterol-fed, but not chow-fed, rabbits stained positively for HSP-60, independently of the immunisation protocol . Endothelial function was impaired in the BCG immunised, cholesterol-fed rabbits as measured by acetylcholine-mediated relaxation of isolated non-atherosclerotic carotid artery rings (P<0.05) . This impairment was positively associated with the level of plasma anti-HSP-60 antibodies (P<0.01) . These results suggest that BCG immunisation impairs endothelial responses, at least in part, by immune responses against mycobacterial and vascular HSP.

J Am Chem Soc, 2004 Jan 14, 126(1), 311 - 9
A comparative study of claisen and cope rearrangements catalyzed by chorismate mutase . An insight into enzymatic efficiency: transition state stabilization or substrate preorganization?
Marti S, Andres J, Moliner V, Silla E, Tunon I, Bertran J.
In this work we present a detailed analysis of the activation free energies and averaged interactions for the Claisen and Cope rearrangements of chorismate and carbachorismate catalyzed by Bacillus subtilischorismate mutase (BsCM) using quantum mechanics/molecular mechanics (QM/MM) simulation methods . In gas phase, both reactions are described as concerted processes, with the activation free energy for carbachorismate being about 10-15 kcal mol(-)(1) larger than for chorismate, at the AM1 and B3LYP/6-31G levels . Aqueous solution and BsCM active site environments reduce the free energy barriers for both reactions, due to the fact that in these media the two carboxylate groups can be approached more easily than in the gas phase . The enzyme specifically reduces the activation free energy of the Claisen rearrangement about 3 kcal mol(-)(1) more than that for the Cope reaction . This result is due to a larger transition state stabilization associated to the formation of a hydrogen bond between Arg90 and the ether oxygen . When this oxygen atom is changed by a methylene group, the interaction is lost and Arg90 moves inside the active site establishing stronger interactions with one of the carboxylate groups . This fact yields a more intense rearrangement of the substrate structure . Comparing two reactions in the same enzyme, we have been able to obtain conclusions about the relative magnitude of the substrate preorganization and transition state stabilization effects . Transition state stabilization seems to be the dominant effect in this case.

J Microbiol Methods, 2004 Jan, 56(1), 125 - 32
A facile analytical method for the identification of protease gene profiles from Bacillus thuringiensis strains; Chen FC et al.; Five pairs of degenerate universal primers have been designed to identify the general protease gene profiles from some distinct Bacillus thuringiensis strains . Based on the PCR amplification patterns and DNA sequences of the cloned fragments, it was noted that the protease gene profiles of the three distinct strains of B . thuringiensis subsp . kurstaki HD73, tenebrionis and israelensis T14001 are varied . Seven protease genes, neutral protease B (nprB), intracellular serine protease A (ispA), extracellular serine protease (vpr), envelope-associated protease (prtH), neutral protease F (nprF), thermostable alkaline serine protease and alkaline serine protease (aprS), with known functions were identified from three distinct B . thuringiensis strains . In addition, five DNA sequences with unknown functions were also identified by this facile analytical method . However, based on the alignment of the derived protein sequences with the protein domain database, it suggested that at least one of these unknown genes, yunA, might be highly protease-related . Thus, the proposed PCR-mediated amplification design could be a facile method for identifying the protease gene profiles as well as for detecting novel protease genes of the B . thuringiensis strains.

Biochemistry, 2004 Jan 13, 43(1), 166 - 74
Tryptophan spectroscopy studies and black lipid bilayer analysis indicate that the oligomeric structure of Cry1Ab toxin from Bacillus thuringiensis is the membrane-insertion intermediate; Rausell C et al.; During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins, which form ionic pores . Binding of Cry1A toxins to the cadherin receptor promotes the formation of a 250 kDa oligomer . In this work, we analyzed for the first time the structural changes presented by Cry1Ab toxin upon membrane insertion . Trp fluorescence of pure monomeric and oligomeric structures in solution and in a membrane-bound state was analyzed . Cry1Ab has nine Trp residues, seven of them in pore-forming domain I . Trp quenching analysis with iodide indicated that oligomerization caused a 27% reduction in the level of Trp exposed to the solvent . Most of the oligomeric structure (96%) inserts into the membrane as a function of the lipid:protein ratio, in contrast to the monomer (10%) . Additionally, the membrane-associated oligomer presented a blue shift of 5 nm in lambda(max) of the emission spectrum, indicating a more hydrophobic environment for some Trp residues . In agreement with this, iodide was unable to quench the Trp of the membrane-bound oligomer, suggesting that a significant part of the protein may be buried in the membrane . Quenching analysis using brominated and spin-labeled phospholipids in the vesicles indicates that most of the Trp residues are located close to the membrane-water interface . Finally, ionic currents in black lipid bilayers revealed that the oligomeric structure has kinetics different from those of the monomer, producing stable channels with a high probability of being open in contrast to the monomer that exhibited unstable opening patterns . These data show that the oligomer, in contrast to the monomer, is able to interact efficiently with phospholipid membranes forming stable pores.

Proteins, 2004 Jan 1, 54(1), 128 - 34
Improved thermostability of bacillus circulans cyclodextrin glycosyltransferase by the introduction of a salt bridge; Leemhuis H et al.; Cyclodextrin glycosyltransferase (CGTase) catalyzes the formation of cyclodextrins from starch . Among the CGTases with known three-dimensional structure, Thermoanaerobacterium thermosulfurigenes CGTase has the highest thermostability . By replacing amino acid residues in the B-domain of Bacillus circulans CGTase with those from T . thermosulfurigenes CGTase, we identified a B . circulans CGTase mutant (with N188D and K192R mutations), with a strongly increased activity half-life at 60 degrees C . Asp188 and Arg192 form a salt bridge in T . thermosulfurigenes CGTase . Structural analysis of the B . circulans CGTase mutant revealed that this salt bridge is also formed in the mutant . Thus, the activity half-life of this enzyme can be enhanced by rational protein engineering .

Proc Natl Acad Sci U S A, 2004 Jan 13, 101(2), 687 - 92 Epub 2004 Jan 02.
RF2b, a rice bZIP transcription activator, interacts with RF2a and is involved in symptom development of rice tungro disease; Dai S et al.; The phloem-specific promoter of rice tungro bacilliform virus (RTBV) is regulated in part by sequence-specific DNA-binding proteins that bind to Box II, an essential cis element . Previous studies demonstrated that the bZIP protein RF2a is involved in transcriptional regulation of the RTBV promoter . Here we report the identification and functional characterization of a second bZIP protein, RF2b . RF2b, identified by its interaction with RF2a, binds to Box II in in vitro assays as a homodimer and as RF2a/RF2b heterodimers . Like RF2a, RF2b activates the RTBV promoter in transient assays and in transgenic tobacco plants . Both RF2a and RF2b are predominantly expressed in vascular tissues . However, RF2a and RF2b have different DNA-binding affinities to Box II, show distinctive expression patterns in different rice organs, and exhibit different patterns of subcellular localization . Furthermore, transgenic rice plants with reduced levels of RF2b exhibit a disease-like phenotype . We propose that the regulation of phloem-specific expression of the RTBV promoter and potentially the control of RTBV replication are mainly achieved via interactions of the Box II cis element with multiple host factors, including RF2a and RF2b . We also propose that quenching/titration of these and perhaps other transcription factors by RTBV is involved in the development of the symptoms of rice tungro disease.

Pharmazie, 2003 Dec, 58(12), 857 - 9
Enzymatic synthesis of N-acetyllactosamine in aqueous-organic reaction media; Franke J et al.; Beta-galactosidase from Bacillus circulans was used for the biocatalytic transfer of D-galactose (D-Gal) from o-nitrophenyl-beta-D-galactoside (ONPG) to the O-4 position of 2-acetamido-2-deoxy-D-glucopyranose (D-GlcNAc) forming the disaccharide N-acetyllactosamine (LacNAc, beta-D-Gal-(1 --> 4)-D-GlcNAc) . In order to investigate the potential of this biocatalytic synthesis, first the optimal reactant ratio in an aqueous buffer system was determined . On the basis of these standard conditions we then performed reactions in aqueous-organic media applying organic cosolvents of different structure and polarity in various amounts . In this way we received in some cases appreciably better results than without organic cosolvent . The highest obtainable disaccharide yield was 50% in a mixture of 20% (v/v) cyclohexane/80% buffer versus 35% in buffer solution alone.

Zhonghua Yu Fang Yi Xue Za Zhi, 2003 Nov, 37(6), 408 - 11
{A case-control study on natural-resistance-associated macrophage protein 1 gene polymorphisms and susceptibility to pulmonary tuberculosis}; Liu W et al.; OBJECTIVE: To investigate association between the natural-resistance-associated macrophage protein 1 (NRAMP1) gene polymorphisms and susceptibility to pulmonary tuberculosis (TB) in Chinese Han population . METHODS: Hospital-based case-control study design was adopted . Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) technique were used to type three NRAMP1 polymorphisms (INT4, D543N and 3'UTR) . Information on related factors of tuberculosis was collected using a pre-tested standard questionnaire . Univariate and multivariate unconditional logistic analyses were conducted using SPSS for window software package . Totally, 110 cases of TB were selected during April 2001 to June 2002, with an average age of (27.7 +/- 12.7) years . Also, 180 cases of healthy control were selected, aged (27.3 +/- 9.2) years in average . Locus of NRAMP1 polymorphism was analysed with univariate method . RESULTS: Univariate analysis demonstrated that the D543N G/A and 3'UTR TGTG+/del genotype occurred more frequently in the cases than in the controls, with crude odds ratios (OR) (95% CI) of 2.22 (1.03 - 4.78) and 1.93 (1.14 - 3.26), respectively . No significant association was observed between TB and INT4 polymorphisms . In multivariate analysis, associations of TB and D543N G/A and 3'UTR TGTG+/del genotypes remained, adjusted for exposure history and bacille Camette-Guerin immunization . Adjusted OR (95% CI) was 3.04 (1.12 - 8.27) and 2.36 (1.20 - 4.64), respectively . Still, no significant association between INT4 polymorphisms and TB was found . CONCLUSION: Polymorphisms of D543N and 3'UTR locus in NRAMP1 gene might affect their susceptibility to TB in Chinese Han population.

Med Confl Surviv, 2003 Oct-Dec, 19(4), 285 - 302
Seascape with monkeys and guinea-pigs: Britain's biological weapons research programme, 1948-54; Willis EA; The British biological weapons (BW) research programme based at Porton Down continued after the Second World War . Five series of BW experiments with animals at sea were undertaken to supplement laboratory work . The causative organisms for plague, brucellosis, tularemia and later Venezuelan equine encephalomyelitis and Vaccinia viruses were tested in the Caribbean near Antigua in the late 1940s, in Hebridean waters (north-west Scotland) in the early 1950s and off Nassau in the Bahamas in 1953-54 . In September 1952, at the end of Operation 'Cauldron' off the Isle of Lewis, a trawler, the Carella, passed through the danger area when a toxic cloud had been released and was covertly watched until the incubation period had passed in case those on board had come into contact with the plague bacillus . Publicity about the trials was avoided, but a press statement was issued in March 1954 . The last series provoked sustained agitation in Cuba . More recently an outline of the sequence has emerged in the UK parliamentary record and in Porton's official history, and a fuller account of the Scottish trials has awakened some interest locally.

J Bacteriol, 2004 Jan, 186(2), 393 - 9
The BH1999 protein of Bacillus halodurans C-125 is gentisyl-coenzyme A thioesterase; Zhuang Z et al.; In this study, we have shown that recombinant BH1999 from Bacillus halodurans catalyzes the hydrolysis of gentisyl coenzyme A (CoA) (2,5-dihydroxybenzoyl-coenzyme A) at physiological pH with a k(cat)/K(m) of 1.6 x 10(6) M(-1) s(-1) and the hydrolysis of 3-hydroxybenzoyl-CoA with a k(cat)/K(m) of 3.0 x 10(5) M(-1) s(-1) . All other acyl-CoA thioesters tested had low or no substrate activity . The BH1999 gene is juxtaposed with a gene cluster that contains genes believed to function in gentisate oxidative degradation . It is hypothesized that BH1999 functions as a gentisyl-CoA thioesterase . Gentisyl-CoA thioesterase shares the backbone fold and the use of an active site aspartate residue to mediate catalysis with the 4-hydroxybenzoyl-CoA thioesterase of the hotdog fold enzyme superfamily . A comparative study of these two enzymes showed that they differ greatly in the rate contribution made by the catalytic aspartate, in the pH dependence of catalysis, and in substrate specificity.

J Bacteriol, 2004 Jan, 186(2), 316 - 25
The alternative sigma factor sigmaB of Bacillus cereus: response to stress and role in heat adaptation; van Schaik W et al.; A gene cluster encoding the alternative sigma factor sigma(B), three predicted regulators of sigma(B) (RsbV, RsbW, and RsbY), and one protein whose function is not known (Orf4) was identified in the genome sequence of the food pathogen Bacillus cereus ATCC 14579 . Western blotting with polyclonal antibodies raised against sigma(B) revealed that there was 20.1-fold activation of sigma(B) after a heat shock from 30 to 42 degrees C . Osmotic upshock and ethanol exposure also upregulated sigma(B), albeit less than a heat shock . When the intracellular ATP concentration was decreased by exposure to carbonyl cyanide m-chlorophenylhydrazone (CCCP), only limited increases in sigma(B) levels were observed, revealing that stress due to ATP depletion is not an important factor in sigma(B) activation in B . cereus . Analysis of transcription of the sigB operon by Northern blotting and primer extension revealed the presence of a sigma(B)-dependent promoter upstream of the first open reading frame (rsbV) of the sigB operon, indicating that transcription of sigB is autoregulated . A second sigma(B)-dependent promoter was identified upstream of the last open reading frame (orf4) of the sigB operon . Production of virulence factors and the nonhemolytic enterotoxin Nhe in a sigB null mutant was the same as in the parent strain . However, sigma(B) was found to play a role in the protective heat shock response of B . cereus . The sigB null mutant was less protected against the lethal temperature of 50 degrees C by a preadaptation to 42 degrees C than the parent strain was, resulting in a more-than-100-fold-reduced survival of the mutant after 40 min at 50 degrees C.

J Infect Dis, 2004 Jan 1, 189(1), 105 - 12 Epub 2003 Dec 22.
Mycobacterium tuberculosis defective in phthiocerol dimycocerosate translocation provides greater protective immunity against tuberculosis than the existing bacille Calmette-Guérin vaccine; Pinto R et al.; We demonstrate that Mycobacterium tuberculosis that is unable to export the complex lipid phthiocerol dimycocerosate has a decreased capacity to replicate in mice and affords sustained protective immunity against M . tuberculosis infection Protection was significantly better than that provided by the existing vaccine, Mycobacterium bovis bacille Calmette-Guerin (BCG), and this improved protective efficacy was maintained for at least 24 weeks after vaccination . Protection afforded by this attenuated strain coincided with a number of factors that were not associated with BCG vaccination: long-term persistence of the strain within the host, sustained and potent induction of antimycobacterial interferon-gamma-secreting cells equal to that induced by virulent M . tuberculosis, and elicitation of T cells recognizing dominant M . tuberculosis antigens absent from BCG . These results suggest that the BCG vaccine may be too attenuated to afford effective protective immunity against tuberculosis, and vaccine strains that can provide sustained delivery of mycobacterial antigens are promising antituberculosis vaccine candidates.

Indian J Med Res, 2003 Oct, 118, 147 - 51
Seroprevalence of human immunodeficiency virus (HIV) infection among tuberculosis patients in Tamil Nadu; Ramachandran R et al.; BACKGROUND & OBJECTIVES: The dual epidemic of HIV and tuberculosis is a cause for concern in those countries where these two infections are prevalent in epidemic proportions . We undertook a survey at two sites in North Arcot district of Tamil Nadu in 1992-1993, to know the seroprevalence of HIV infection among tuberculosis patients . The objective of this study was to re-examine the prevalence of HIV infection among tuberculosis patients in a repeat survey . METHODS: The study was undertaken in four centres: District Tuberculosis Centre (DTC), Vellore, Tuberculosis Sanatorium, Pennathur (Vellore), District TB Centre (DTC), Kancheepuram and the Government Thiruvotteswarar Tuberculosis Hospital (GTTH), Chennai in the northern part of Tamil Nadu during 1997-1998 . A total of 2361 newly diagnosed TB patients were registered in this study . HIV serology after pre-test counseling was done along with sputum examination for acid-fast bacillus by smear and culture for mycobacteria for all patients . RESULTS: The overall HIV seroprevalence among TB patients was 4.7 per cent . The highest HIV seropositivity rate was found among patients aged 30-39 yr (10.6%) . HIV seroprevalence showed a wide variation among the different centres ranging from 0.6 . per cent in DTC, Kancheepuram to 9.4 per cent in Pennathur Sanatorium, Vellore . Sputum smear positivity was 88 per cent among the HIV-negative and 83 per cent among HIV-positive tuberculosis patients . INTERPRETATION & CONCLUSION: HIV infection is on the rise among TB patients in Tamil Nadu . Acid-fast smear microscopy is adequate for the diagnosis of pulmonary tuberculosis, and drug resistance among HIV positive patients is not a major problem at this point of time; hence antituberculosis regimens recommended by the Revised National Tuberculosis Control Program (RNTCP) can be used to treat HIV positive patients with tuberculosis.

J Clin Oncol, 2004 Feb 1, 22(3), 403 - 15 Epub 2003 Dec 22.
Immunopharmacologic analysis of an autologous, hapten-modified human melanoma vaccine; Berd D et al.; PURPOSE: We have previously reported a clinical trial of a human cancer vaccine consisting of autologous tumor cells modified with the hapten, dinitrophenyl (DNP), in patients with clinical stage III melanoma . Here we present a follow-up report expanded to 214 patients with 5-year follow-up . PATIENTS AND METHODS: Two hundred fourteen patients with clinical stage III melanoma (117 patients with stage IIIC and 97 patients with stage IIIB) who were melanoma-free after standard lymphadenectomy were treated with multiple intradermal injections of autologous, DNP-modified vaccine mixed with bacille Calmette-Guerin . Four vaccine dosage schedules were tested sequentially, all of which included low-dose cyclophosphamide . Patients were tested for delayed-type hypersensitivity (DTH) to autologous melanoma cells, both DNP-modified and unmodified, and to control materials . RESULTS: The 5-year overall survival (OS) rate of the 214 patients was 44% . DTH responses to unmodified autologous melanoma were induced in 47% of patients . The OS of this DTH-positive group was double that of DTH-negative patients (59.3% v 29.3%; P <.001) . In contrast, positive DTH responses to DNP-modified autologous melanoma cells and to purified protein derivative developed in almost all patients but did not affect OS . Surprisingly, the OS after relapse was also significantly longer in patients who developed positive DTH to unmodified tumor cells (25.2% v 12.3%; P <.001) . Finally, the development of DTH was dependent on the schedule of administration of the vaccine, specifically, the timing of an induction dose administered at the beginning of the treatment program . CONCLUSION: This study underscores the importance of the immunopharmacology of the autologous, DNP-modified vaccine and may be relevant to other cancer vaccine technologies.

Infect Immun, 2004 Jan, 72(1), 219 - 28
Camelysin is a novel surface metalloproteinase from Bacillus cereus; Grass G et al.; Bacillus cereus frequently causes food poisoning or nosocomial diseases . Vegetative cells express the novel surface metalloproteinase camelysin (casein-cleaving metalloproteinase) during exponential growth on complex, peptide-rich media . Camelysin is strongly bound to the cell surface and can be solubilized only by detergents or butanol . Camelysin spontaneously migrates from the surface of intact bacterial cells to preformed liposomes . The complete sequence of the camelysin-encoding gene, calY, was determined by reverse PCR on the basis of the N-terminal sequence and some internal tryptic cleavage peptides . The calY gene codes for a polypeptide of 21.569 kDa with a putative signal peptide of 27 amino acids (2.513 kDa) preceding the mature protein (19.056 kDa) . Although the predicted amino acid sequence of CalY does not exhibit a typical metalloprotease consensus sequence, high-pressure liquid chromatography-purified camelysin contains one zinc ion per protein molecule . Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and tryptic peptide mass fingerprinting confirmed the identity of this zinc-binding protein as CalY . Disruption of the calY gene results in a strong decrease in the cell-bound proteolytic activity on various substrates.

J Mol Biol, 2004 Jan 16, 335(3), 679 - 84
The solution structure of ribosomal protein L18 from Bacillus stearothermophilus; Turner CF et al.; A medium resolution solution structure has been obtained for L18 from Bacillus stearothermophilus (BstL18), a ribosomal protein that stabilizes the tertiary structure of 5S rRNA and mediates its interaction with the rest of the large subunit . The N-terminal 22 amino acid residues of BstL18 are unstructured in solution . Its remaining 98 residues form a globular domain that has the same topology as the globular domains of other L18s, but the orientation of helices is different . This conformational peculiarity should not prevent BstL18 from functioning in the ribosome the same way as other L18s.

Clin Microbiol Infect, 2003 Dec, 9(12), 1234 - 7
Liver abscess due to Bacillus cereus: a case report; Latsios G et al.; Bacillus cereus is a food-borne pathogen that causes a self-limiting gastroenteritis . We describe the case of a 72-year-old woman admitted to our hospital because of acute abdominal colic pain . Over a 2-day period, her clinical condition deteriorated rapidly, with the appearance of acute abdomen . Computed tomography investigation of the abdomen showed a liver abscess (diameter approximately 3 cm) . At laparotomy, the abscess was found to be ruptured to the free peritoneal cavity . The final clinical diagnosis was acute peritonitis due to a ruptured liver abscess . Bacillus cereus was isolated from culture of the pus . Up to now, no case of liver abscess due to this organism has been reported.

Expert Rev Anticancer Ther, 2003 Dec, 3(6), 809 - 21
Combined bacillus Calmette-Guerin and interferon use in superficial bladder cancer; O'Donnell MA; Due to its high rate of local recurrence after transurethral surgery, superficial bladder cancer is often treated with adjuvant topical intravesical chemotherapy or biological agents, such as bacillus Calmette-Guerin or interferon-alpha . Recent laboratory testing has revealed that combination interferon-alpha with bacillus Calmette-Guerin results in remarkable synergy, affecting not only bladder tumors directly but also enhancing the immune response to bacillus Calmette-Guerin by orders of magnitude . Clinical studies are now demonstrating lower toxicity with combination low-dose bacillus Calmette-Guerin/interferon regimens, while providing a much needed salvage option for previous bacillus Calmette-Guerin failures.

Water Sci Technol, 2003, 48(8), 239 - 46
Production of biopesticides using wastewater sludge as a raw material--effect of process parameters; Tirado Montiel ML et al.; Production of Bacillus thuringiensis (Bt) based biopesticide was studied using hydrolysed or raw wastewater sludge as a raw material . The sludge hydrolysis was carried out at different pH (2, 4 and 6) . The sludge after hydrolysis was sterilised at 121 degrees C for 30 minutes . The effect of temperature, pH and agitation speed on growth and toxin production was also investigated . The pH in the shake flask was controlled by adding 0.1 M TRIS buffer . The progress of the biopesticide production process was monitored by measuring viable cell count (VC), spore count (SC) and entomotoxicity (Tx) . The entomotoxicity was measured against spruce budworm . Control of pH substantially increased the entomotoxicity of the final product . Increased agitation speed resulted in higher VC, SC and Tx values.

J Chem Ecol, 2003 Nov, 29(11), 2585 - 602
Leaf ontogeny influences leaf phenolics and the efficacy of genetically expressed Bacillus thuringiensis cry1A(a) d-endotoxin in hybrid poplar against gypsy moth; Kleiner KW et al.; We tested the hypothesis that ontogenetic variation in leaf chemistry could affect the efficacy of genetically expressed Bacillus thuringiensis cry1A(a) d-endotoxin, and thus provide spatial variation in (1) foliage protection and (2) selective pressures that could delay the resistance of folivores . Our model consisted of clonal hybrid Populus plants (NC5339) . Consumption of foliage and relative growth rates of gypsy moth, Lymantria dispar (L.) increased, and phenolic glycoside concentrations decreased, as leaves from transformed plants containing the cry1A(a) d-endotoxin and nontransformed plants matured from leaf plastochron index (LPI) 1-6 . Feeding and growth rates were negatively correlated with phenolic glycosides in both transformed and nontransformed foliage . The presence of the B . thuringiensis d-endotoxin was at most, additive to the effect of the phenolic glycosides . Feeding and growth rates were positively correlated with condensed tannins in transformed foliage, but there was no relationship with condensed tannins in nontransformed foliage . The results indicate that the presence of foliar allelochemicals of poplar can enhance the effectiveness of genetically expressed B . thuringiensis d-endotoxin against gypsy moth larvae . However, the spatial variation in gypsy moth performance in response to the combination of foliar allelochemicals and d-endotoxin was not greater than the effect of ontogenetic variation in foliar allelochemicals alone . These results suggest that for this important pest, foliage protection may be obtained without genetically engineered defenses, and instead, by relying on ontogenetic and clonal variation in allelochemicals . The benefits of combining novel resistance mechanisms with natural ones will depend upon the specific folivore's adaptation to natural resistance mechanisms, such as allelochemicals . Moreover, some of the greatest benefits from transgenic resistance may arise from the need to protect trees from multiple pests, some of which may not be deterred by, or may even prefer, allelochemicals that confer protection from a few species.

Yi Chuan Xue Bao, 2003 Aug, 30(8), 737 - 42
{Construction of insecticidal recombinant Bacillus thuringiensis using an integrative vector}; Yue CY et al.; Insecticidal crystal protein gene cry1C which is highly toxic to Spodoptera exigua was cloned into an integrative vector pBMB-F7E, which was derived from Bacillus thuringiensis transposon Tn4430 . The recombinant integrative plasmid pBMB-FLC harboring the cry1C gene was gained and transformed into a wild-type Bt strain YBT803-1 . A transformant BMB803-A was obtained, and grown at 46 degrees C for about 120 generations, From which three recombinants with cry1C gene integrating into the chromosome were achieved at a frequency of approximately 3.4 x 10(-5) . Southern blotting revealed that the integration occurred in different sites of the chromosome . The integrated cry1C were expressed effectively . The results of bioassays showed that the toxicity of recombinants BMB803-X and BMB803-Z against Plutella xylostella were similar to that of strain YBT803-1, and their toxicity to Spodoptera exigua were higher than that of strain YBT803-1.

RNA, 2004 Jan, 10(1), 66 - 74
Crystal structure of the BstDEAD N-terminal domain: a novel DEAD protein from Bacillus stearothermophilus; Carmel AB et al.; Most cellular processes requiring RNA structure rearrangement necessitate the action of Asp-Glu-Ala-Asp (DEAD) proteins . Members of the family, named originally for the conserved DEAD amino acid sequence, are thought to disrupt RNA structure and facilitate its rearrangement by unwinding short stretches of duplex RNA . BstDEAD is a novel 436 amino acid representative of the DEAD protein family from Bacillus stearothermophilus that contains all eight conserved motifs found in DEAD proteins and is homologous with other members of the family . Here, we describe the 1.85 A resolution structure of the N-terminal domain (residues 1-211) of BstDEAD (BstDEAD-NT) . Similar to the corresponding domains of related helicases, BstDEAD-NT adopts a parallel alpha/beta structure with RecA-like topology . In general, the conserved motifs superimpose on closely related DEAD proteins and on more distantly related helicases such as RecA . This affirms the current belief that the core helicase domains, responsible for mechanistic activity, are structurally similar in DEAD proteins . In contrast, however, the so-called Walker A P-loop, which binds the beta- and gamma-phosphates of ATP, adopts a rarely seen "closed" conformation that would sterically block ATP binding . The closed conformation may be indicative of a general regulatory feature among DEAD proteins (and RNA helicases) that differs from that used by DNA helicases . BstDEAD also contains a unique extension of approximately 60 residues at the C terminus that is highly basic, suggesting that it might bind nucleic acids and, in so doing, confer specificity to the helicase activity of the core region.

J Toxicol Environ Health A, 2004 Feb 13, 67(3), 221 - 31
Effect of diesel exhaust particulate (DEP) on immune responses: contributions of particulate versus organic soluble components; Siegel PD et al.; The effect of diesel exhaust particulate (DEP) exposure on innate, cellular and humoral pulmonary immunity was studied using high-dose, acute-exposure rat, mouse, and cell culture models . DEP consists of a complex mixture of petrochemical-derived organics adsorbed onto elemental carbon particles . DEP is a major component of particulate urban air pollution and a health concern in both urban and occupational environments . The alveolar macrophage is considered a key cellular component in pulmonary innate immunity . DEP and DEP organic extracts have been found to suppress alveolar macrophage function as demonstrated by reduced production of cytokines (interleukin-1 {IL-1}, tumor necrosis factor- alpha {TNF- alpha}) and reactive oxygen species (ROS) in response to a variety of agents, including lipopolysaccharide (LPS), interferon- gamma (IFN- gamma), and bacteria . Fractionation of DEP organic extract suggests that this activity was predominately in polyaromatic-containing and more polar (resin) fractions . Organic-stripped DEP did not alter these innate pulmonary immune responses . DEP also depressed pulmonary clearance of Listeria monocytogenes and Bacillus Calmette-Guerin (BCG) . The contribution of the organic component of DEP is less well defined with respect to acquired and humoral immunity . Indeed, both DEP and carbon black enhanced humoral immune responses (specific immunoglobulin {Ig} E and IgG) in an ovalbumin-sensitized rat model . It is concluded that both the particulate and adsorbed organics may contribute to DEP-mediated immune alterations.

Biochem Biophys Res Commun, 2003 Dec 19, 312(3), 708 - 15
The IspA protease's involvement in the regulation of the sporulation process of Bacillus thuringiensis is revealed by proteomic analysis; Chen FC et al.; We have observed that the process of sporulation of the ispA-deficient mutant was delayed under phase-contrast microscopy . The protein profiles of the ispA-deficient mutant have been analyzed using two-dimensional gel electrophoresis . The results of a proteomic analysis using MALDI-TOF MS indicated that a sporulation-associated protein, pro- {Formula: see text}, was upregulated, while two other sporulation-associated proteins, SpoVD and SpoVR, were downregulated in the ispA-deficient mutant . It has been known that pro- {Formula: see text} is a precursor of {Formula: see text} and is required for gene expression related to the late stage of sporulation . Moreover, SpoVD and SpoVR are known to be involved in the formation of the spore cortex . Based on these observations, we propose that the delay in the sporulation process observed in the ispA-deficient mutant may be due to a failure of {Formula: see text} to signal sporulation . This phenomenon may be further enhanced by insufficient amount of SpoVD and SpoVR for cortex formation . In this study, we have revealed for the first time a possible pathway for the regulation of sporulation-associated proteins via IspA.

Mikrobiologiia, 2003 Sep-Oct, 72(5), 707 - 13
{Microbial complexes from apogeotropic roots and from rhizosphere of cycad plants}; Lobakova ES et al.; The microbial complexes of soil, the rhizosphere, and the rhizoplane of the apogeotropic (coralloid) roots of cycad plants were comparatively studied . The aseptically prepared homogenates of the surface-sterilized coralloid roots did not contain bacterial microsymbiont, indicating that it was absent in the root tissues . At the same time, associated bacteria belonging to different taxonomic groups were detected in increasing amounts in the cycad rhizoplane, rhizosphere, and the surrounding soil . The bacterial communities found in the cycad rhizoplane and the surrounding soil were dominated by bacteria from the genus Bacillus . The saprotrophic bacteria and fungi colonizing the cycad rhizosphere and rhizoplane were dominated by microorganisms capable of degrading the plant cell walls . The local degradation of the cell wall was actually observed on the micrographs of the thin sections of cycad roots in the form of channels, through which symbiotic cyanobacterial filaments can penetrate into the cortical parenchyma.

Mikrobiologiia, 2003 Sep-Oct, 72(5), 689 - 94
{On the adaptive nature of the dissociation process in Bacillus thuringiensis}; Sekerina OA et al.; The process of dissociation into variants differing in colony morphology occurring in batch cloned cultures of two Bacillus thuringiensis strains belonging to different subspecies was studied at optimal and elevated temperatures . An increase in the cultivation temperature to 40 degrees C resulted in an increase in the fraction of R variants to 100% after 72 h of cultivation of either of the strains . This increase was not due to the selection of forms with greater resistance to elevated temperature . The level of resistance to elevated temperature was determined by the strain genotype and did not correlate with morphological characteristics of the colonies.

Mikrobiologiia, 2003 Sep-Oct, 72(5), 639 - 44
{Membrane-bound forms of serine proteases of Bacillus intermedius}; Sharipova MR et al.; Proteolytic proteins solubilized from the membrane of Bacillus intermedius were studied by electrophoresis . The content of membrane-bound proteinases was lower in cells grown in the presence of glucose . Proteinase enzymograms revealed four molecular forms of subtilisin and four molecular forms of glutamyl endopeptidase . The electrophoretic mobility of one of the molecular forms was similar to those of the mature extracellular proteinases . Chromatography of membrane proteins on a MonoS column yielded four protein fractions that caused hydrolysis of Z-Glu-pNA and four fractions that caused hydrolysis of Z-Ala-Ala-Leu-pNA, which is in agreement with the results of electrophoresis . The molecular forms of proteinases identified in the membrane may reflect various stages of biogenesis of the corresponding extracellular enzymes.

Semin Respir Infect, 2003 Dec, 18(4), 320 - 38
TB vaccines at the turn of the century: insights into immunity to M . tuberculosis and modern approaches for prevention of an ancient disease; Lewinsohn DM et al.; Tuberculosis (TB) remains a leading cause of infectious disease mortality worldwide, accounting for nearly 2 million deaths annually . Despite the availability of effective anti-TB therapy, the world's case burden of TB continues to climb, in part owing to the concurrent acquired immune deficiency syndrome pandemic . The widespread use of the current TB vaccine, the bacille Calmette-Guerin strain of M . bovis (BCG), has failed to curtail the TB epidemic and therefore strategies for the eradication of TB have centered on aggressive case-finding and managed treatment, such as directly observed treatment short course (DOTS) . Although DOTS continues to have a significant impact, the ability of DOTS to eradicate TB is limited . Therefore, TB eradication will require the development of an improved vaccine, which, in turn, will require application of state-of-the-art vaccine technology and of our knowledge of TB immunology and TB genomics to this problem . In this article, knowledge about the requisite components of protective immunity, modern vaccine strategies, and elements of clinical trials required to evaluate vaccine efficacy, as well as the status of current TB vaccine candidates, are reviewed.

J Bacteriol, 2004 Jan, 186(1), 104 - 9
Genomic interrogation of the dassie bacillus reveals it as a unique RD1 mutant within the Mycobacterium tuberculosis complex; Mostowy S et al.; Despite their remarkable genetic homology, members of the Mycobacterium tuberculosis complex express very different phenotypes, most notably in their spectra of clinical presentation . For example, M . tuberculosis is regarded as pathogenic to humans, whereas members having deleted RD1, such as Mycobacterium microti and Mycobacterium bovis BCG, are not . The dassie bacillus, an infrequent variant of the M . tuberculosis complex characterized as being most similar to M . microti, is the causative agent of tuberculosis (TB) in the dassie (Procavia capensis) . Intriguingly, the dassie bacillus is not pathogenic to rabbits or guinea pigs and has never been documented to infect humans . Although it was identified more than a half-century ago, the reasons behind its attenuation are unknown . Because large sequence polymorphisms have presented themselves as the most obvious genomic distinction among members of the M . tuberculosis complex, the DNA content of the dassie bacillus was interrogated by Affymetrix GeneChip to identify regions that are absent from it but present in M . tuberculosis H37Rv . Comparison has led to the identification of nine regions of difference (RD), five of which are shared with M . microti (RDs 3, 7, 8, 9, and 10) . Although the dassie bacillus does not share the other documented deletions in M . microti (RD1(mic), RD5(mic), MID1, MID2, and MID3), it has endured unique deletions in the regions of RD1, RD5, N-RD25, and Rv3081-Rv3082c (virS) . RD1(das), affecting only Rv3874-Rv3877, is the smallest natural deletion of the RD1 region uncovered and points to genes within this region that are likely implicated in virulence . Newfound deletions from the dassie bacillus are discussed in relation to their evolutionary and biological significance.

J Eur Acad Dermatol Venereol, 2004 Jan, 18(1), 83 - 5
First case of Mycobacterium haemophilum infection in an AIDS patient in Italy; Martinelli C et al.; Mycobacterium haemophilum, a strongly acid- and alcohol-fast bacillus belonging to the group of non-tuberculous mycobacteria was first described in 1978 as the cause of cutaneous ulcerating lesions in a woman with Hodgkin's disease . Infection due to M . haemophilum is rare but increasing in prevalence in immnunosuppressed subjects, particularly in patients with acquired immunodeficiency syndrome (AIDS) patients . The skin is the most common site of infection with erythematous or violaceous papules and/or nodules that are usually painless at first, but some elements develop into abscesses or ulcers that can become very painful . The incidence of M . haemophilum is unknown, but cases of infection have been reported in Australia, Canada, the United States, France, Israel, the United Kingdom and Taiwan; to date no cases have been reported in Italy, thus the case reported here is apparently the first one observed in our country.

BJU Int, 2004 Jan, 93(1), 60 - 3
T1G3 transitional cell carcinoma of the bladder: recurrence, progression and survival; Peyromaure M et al.; OBJECTIVES: To report our experience with T1G3 bladder tumours over the last 10 years . PATIENTS AND METHODS: We analysed the outcome of 74 consecutive patients treated for a T1G3 bladder cancer between 1991 and 2001 . Fifty-seven patients (77%) were treated with transurethral resection (TUR) plus six weekly instillations of bacillus Calmette-Guerin (BCG) therapy . Ten patients (13.5%) with contraindications to BCG or with a small T1a tumour were treated with TUR plus mitomycin-C, and seven (9.5%) were treated with TUR alone because of their age . Patients treated with BCG had systematic biopsies taken at the end of the first course . Patients with residual tumour received a second course of six weekly instillations . Patients with negative biopsies received maintenance BCG therapy consisting of intravesical instillations each week for 3 weeks given 3, 6, 12, 18, 24, 30 and 36 months after the first course . RESULTS: The median follow-up was 53 months . The overall recurrence rate was 46% and the overall progression rate 19% . The rate of delayed cystectomy was 8% and that of disease-specific survival 91% . In patients who received BCG therapy, the recurrence and progression rates were 42% and 23%, respectively . In this group the rate of disease-specific survival was 88% . CONCLUSION: This study confirms that maintenance BCG therapy is an effective treatment for T1G3 bladder tumours, with an acceptable rate of bladder preservation.

J Appl Microbiol, 2004, 96(1), 194 - 200
Inorganic compounds have dual effect on recombinant protein production: influence of anions and cations on serine alkaline protease production; Calik P et al.; AIMS: Investigation of concerted effects of cations, i.e . Mg2+ and Mn2+, in combination with their anions, i.e . sulphate, chloride and acetate (Ac), on the physiology of Bacillus licheniformis carrying pHV1431::subC to improve the fermentation medium for serine alkaline protease (SAP) production, whereupon, determination of the acid that can be used in pH control . METHODS AND RESULTS: The cell concentrations increased with the increase in MnSO4 and Mn(Ac)2 concentrations, and the highest values were obtained at Co(MnSO4) = 0.20 mmol l-1 and Co(Mn(CH3COO)2) = 4.0 mmol l-1, as 2.3 and 2.2 g l-1, respectively . However, Co(MnCl2) did not influence biomass concentration . SAP production was inhibited with MnCl2 after Co(MnCl2) = 0.60 mmol l-1, but with MnSO4 SAP production was inhibited drastically . Whereas, at high concentrations of Mn(Ac)2 SAP production increased and the highest activity was obtained as ASAP = 1285 U ml-1 at t = 65 h . With the Mg compounds, cell concentrations increased with the increase in the concentrations of MgSO4, MgCl2 and Mg(Ac)2; and the anions did not show any influence on the cell growth . Similar to the results of Mn compounds, the glucose consumption rate increased with the increase in MgSO4 and MgCl2 concentrations; contrariwise, decreased with the increase in Mg(Ac)2 concentrations, due to the use of acetate as the second carbon source . Co(MgSO4) = 0.40 mmol l-1, Co(MgCl2) = 1.60 mmol l-1 and Co(Mg(Ac)2) = 0.40 mmol l-1 were the optimum concentrations separately, and the highest SAP activity was obtained with Mg(Ac)2 as ASAP = 1338 U ml-1 at t = 47 h . Consequently, ion acetate and its acid HAc appear, respectively, as the superior anion for the essential cations and the control agent for pH control in the bioreactor . Finally, optimum initial concentrations and the concerted effects of Mg(Ac)2 and Mn(Ac)2 were investigated, and the optimum concentrations were found respectively as 0.40 and 0.80 mmol l-1, while the maximum activity was obtained as ASAP = 1010 U ml-1 at a shortened cultivation time of t = 39 h . CONCLUSIONS: Mn(Ac)2 and Mg(Ac)2 together enhanced the cell formation and SAP synthesis rates, moreover, SAP synthesis started at an earlier cultivation time . SIGNIFICANCE AND IMPACT OF THE STUDY: Each inorganic compound with its cation and anion has dual effect on the metabolism . Mg2+ and Mn2+ at their specific concentrations influence the regulation of the pathways that might cause better coupling of supply and demand for the amino acids on the basis of the amino acid composition of the enzyme molecule.

Int J Tuberc Lung Dis, 2003 Dec, 7(12 Suppl 3), S494 - 500
Tuberculosis infection and disease in children living in households of Filipino patients with tuberculosis: a preliminary report; Salazar-Vergara RM et al.; SETTING: DOTS Clinic with a DOTS-Plus pilot project for the management of multidrug-resistant tuberculosis (MDR-TB) in a high burden country . OBJECTIVE: To determine the prevalence of tuberculosis (TB) infection and disease among pediatric household contacts of patients with pulmonary TB (PTB) . DESIGN: Cross-sectional study . METHODOLOGY: One hundred and fifty-three children aged 0-15 years in the households of 62 bacteriologically confirmed PTB patients, including 44 with MDR-TB, were studied . BCG scars were noted, and tuberculin skin test (TST), screening chest radiography, and sputum or gastric aspirate smear and culture for Mycobacterium tuberculosis in those with radiographic findings suggestive of PTB were done . RESULTS: For children in this study, the prevalences of latent TB infection (LTBI), radiographically diagnosed pulmonary TB, and bacillary pulmonary TB were 69.2%, 3.3%, and 0.65%, respectively . Only age > or = 5 years was found to be a significant predictor of LTBI (OR 3.17, 95%CI 1.43-7.01) . CONCLUSION: Contact investigation for active case-finding and early treatment of TB in children from households of patients with active PTB is essential for TB control . Further study on a more precise definition of TB infection and strategies for control in this population will be pursued.

Biochemistry, 2003 Dec 23, 42(50), 14994 - 5002
Fluorescence based structural analysis of tryptophan analogue-AMP formation in single tryptophan mutants of Bacillus stearothermophilus tryptophanyl-tRNA synthetase; Acchione M et al.; The symmetrical dimer structure of tryptophanyl-tRNA synthetase is similar to that of tyrosyl-tRNA synthetase whose binding behavior and structural details have been elucidated in detail . The structure of both subunits after forming the intermediate tryptophanyl-AMP has important implications for the binding of the cognate tRNA(Trp) . Single tryptophan mutants of Bacillus stearothermophilus tryptophanyl-tRNA synthetase have been constructed and expressed and used to probe structural changes in different domains of the enzyme in both subunits . Substrate titrations using the Trp analogues 4-fluorotryptophan and 7-azatryptophan in the presence of ATP to form the corresponding aminoacyl-adenylate reveal significant structural changes occurring throughout the active subunit in regions not confined to the active site . Changes in environment around the specific Trp residues were monitored using UV absorbance and steady-state fluorescence measurements . When titrated with 4-fluorotryptophan, both Trp 91 and Trp 290 fluorescence is quenched (49 and 22%, respectively) when one subunit has formed Trp-AMP . The fluorescence of Trp 48 is enhanced 19% . No further change in signal was observed after a 1:1 dimer/L-4FW-AMP complex ratio had been established . Using an anion-exchange filter binding assay with radiolabeled l-Trp as a substrate, binding to only one subunit was observed under nonsaturating conditions . This agrees with the results of the assay using 7-azatryptophan as a substrate . The observed changes extend to the unfilled subunit where a similar structure is believed to form after one subunit has formed tryptophan-AMP . Movement in the regions of the enzyme containing Trp 290 and Trp 91 suggests a mechanism for cross-subunit communication involving the helical backbone and dimer interface containing these two residues.

Acta Cytol, 2003 Nov-Dec, 47(6), 960 - 4
Exploring the links between quality assurance and laboratory resources . An audit-based study; Singh N et al.; OBJECTIVE: To investigate and rectify the problems related to Ziehl-Neelsen (Z-N) staining in a cytology laboratory in the context of quality assurance . STUDY DESIGN: An audit based quality assurance study of 1,421 patients with clinical diagnoses of tubercular lymphadenopathy who underwent fine needle aspiration cytology . Data from 8 months were audited (group 1) . Laboratory practices related to selection of smears for Z-N staining were studied . A 2-step corrective measure based on results of the audit was introduced for 2 months (group 2) . Results were subjected to statistical analysis using the chi 2 test . RESULTS: Of 1,172 patients in group 1,368 had diagnoses other than tuberculosis . Overall acid-fast bacillus (AFB) positivity was 42% . AFB positivity in 249 patients in group 2 was 89% (P < .0001) . CONCLUSION: Several issues in the laboratory are linked to quality assurance . Solving everyday problems can have far-reaching benefits for the performance of laboratory personnel, resources and work flow.

J Zhejiang Univ Sci, 2004 Feb, 5(2), 149 - 56
Improved elastase production by Bacillus sp . EL31410--further optimization and kinetics studies of culture medium for batch fermentation; He GQ et al.; An efficient culture medium producing a bacterial elastase with high yields was developed further following preliminary studies by means of response surface method . Central composite design (CCD) and response surface methodology were applied to optimize the medium constituents . A central composite design was used to explain the combined effect of three medium constituents, viz, glucose, K(2)HPO(4), MgSO(4).7H(2)O . The strain produced more elastase in the completely optimized medium, as compared with the partially optimized medium . The fitted model of the second model, as per RSM, showed that glucose was 7.4 g/100 ml, casein 1.13 g/100 ml, corn steep flour 0.616 g/100 ml, K(2)HPO(4) 0.206 g/100 ml and MgSO(4).7H(2)O 0.034 g/100 ml . The fermentation kinetics of these two culture media in the flask experiments were analyzed . It was found that the highest elastase productivity occurred at 54 hours . Higher glucose concentration had inhibitory effect on elastase production . At the same time, we observed that the glucose consumption rate was slow in the completely optimized medium, which can explain the lag period of the highest elastase production . Some metal ions and surfactant additives also affected elastase production and cell growth.

Urol Oncol, 2003 Sep-Oct, 21(5), 354 - 60
Bacillus Calmete-Guérin plus interferon-alpha2B intravesical therapy maintains an extended treatment plan for superficial bladder cancer with minimal toxicity; Lam JS et al.; Bacillus Calmette-Guerin (BCG) and interferon-alpha2B (IFN-alpha2B) have both been individually used for the intravesical treatment of superficial bladder cancer . We report our experience on the therapeutic efficacy and toxicity of combined intravesical BCG plus IFN-alpha2B for treating superficial bladder cancer, including patients failing previous BCG therapy . Thirty-two patients with superficial bladder cancer underwent 6 weekly treatments with full-, one-third, or one-tenth-dose of BCG plus 50 or 100 MU of IFN-alpha2B based on prior BCG exposure and tolerance . Patients with no evidence of disease proceeded onto maintenance therapy of 3 weekly treatments at 3 months followed by 2 additional maintenance cycles given 6 months apart . Response was assessed by cystoscopy/biopsy every 3 months after treatment . Before BCG plus IFN-alpha2B treatment, 20 patients (63%) had previously failed intravesical BCG therapy, 27 (84%) had aggressive disease (stage T1, grade 3, or carcinoma in situ), 27 (84%) had recurrent disease, 14 (44%) had multifocal disease, and 6 (19%) had disease of over 4 years duration . At median follow-up of 22 months, 21 patients (66%) remain disease-free and 11 patients (34%) had disease-recurrence . Nineteen of 32 patients (59%) were disease-free after the initial induction cycle . Six of 11 patients 55% ultimately failing combination therapy did so at the first 3 to 4 month evaluation . Four of 7 patients (57%) benefited from salvage re-induction therapy . Of the 20 patients previously treated with BCG, 12 patients (60%) remain disease-free . Combination BCG plus IFN-alpha2B intravesical therapy was well tolerated . Combination intravesical BCG plus IFN-alpha2B is an effective and tolerable alternative for patients with superficial bladder cancer, including those patients in whom intravesical BCG therapy had previously failed . Benefits of this combination therapy may include potentially less morbidity, improved clinical efficacy, and in the long term, fewer patients undergoing radical therapy . However, radical treatment options should be pursued for early failures of this combination regimen in those patients with risk factors for recurrence and progression.

Tuberculosis (Edinb), 2004, 84(1-2), 93 - 101
Immunology of tuberculosis and implications in vaccine development; Flynn JL; Mycobacterium tuberculosis is a very successful pathogen that can survive and persist in the human host in the face of a robust immune response . This immune response is sufficient to prevent disease in the majority of infected persons, providing compelling evidence that immunity to tuberculosis is possible . However, it is more striking that the strong immune response is not generally effective at eliminating the organisms, during either initial infection or the persistent or latent phase of infection . Studies in animal models and in humans have demonstrated the wide range of immune components involved in the effective response against M . tuberculosis . These components include T cells (both CD4+ and CD8+), cytokines, including IFN-gamma, IL-12, TNF-alpha, and IL-6, and macrophages . The precise roles and functions of these cells and molecules (and others) are still being defined and may differ in acute and chronic infection . These immune responses are directed towards containing or eliminating the tubercle bacillus within the tissues of the host . The estimated eight million new cases of tuberculosis each year clearly demonstrate that these responses are not always effective . M . tuberculosis has obviously evolved a variety of mechanisms to evade destruction by the immune response . Studying both the host and the pathogen will elucidate potential vaccine candidates . In this review, the known functions of immune components in the response to M . tuberculosis and implications for vaccine development will be discussed.

Curr Microbiol, 2003 Nov, 47(5), 417 - 24
Distribution analysis of IS231-like sequences among Bacillus thuringiensis serovars inferred from restriction fragment length polymorphisms; Joung KB et al.; The distribution of IS231 has been analyzed in Bacillus thuringiensis serovars . A 723-bp HaeII conserved fragment from IS231M has been used as a probe against EcoRI-digested B . thuringiensis total DNA to yield serovar-specific hybridization profiles . The approach was useful at revealing the extent of distribution of IS231-like sequences between and within strains . Of the 88 B . thuringiensis strains tested, 70 showed hybridization banding patterns that comprised between one and 20 distinct bands . These 70 B . thuringiensis strains were grouped based on banding pattern similarities . Interestingly, intraserovar strains did not necessarily cluster together.

Curr Microbiol, 2003 Nov, 47(5), 383 - 7
Efficient expression of the mosquito larvicidal binary toxin gene from Bacillus sphaericus in Escherichia coli; Promdonkoy B et al.; The binary toxin gene encoding BinA (42 kDa) and BinB (51 kDa) from Bacillus sphaericus strain 2297 was cloned and expressed in E . coli . Low expression level was found when both proteins were expressed from a single operon . High expression was observed when the gene encoding an individual protein was placed downstream of the T7 promoter . The expression level of BinB was not different when expressed alone (non-fusion) or as a fusion form with T7 peptide (T7-BinB) . Both forms of BinB were equally stable . Unlike BinB, the non-fusion form of BinA was less stable than T7-BinA . The mosquito larvicidal test showed that BinA or BinB alone was not toxic to mosquito larvae, but high toxicity was found when both BinA and BinB were applied . The results suggest that a short peptide of T7 linked to the N-terminus of either BinA or BinB does not affect their toxicity, but may make the toxin, especially BinA, more stable.

Curr Microbiol, 2003 Nov, 47(5), 372 - 5
Expression of binary toxin genes in the mosquito-colonizable bacteria, Bacillus cereus, leads to high toxicity against Culex quinquefasciatus larvae; Luxananil P et al.; Two B . cereus strains, Ae10 and Cx5, isolated from mosquito larval guts, were transformed with a recombinant plasmid, pBS373, harboring binary toxin genes from Bacillus sphaericus 2297 . Immunoblotting analysis clearly revealed the production and presence of the 51-kDa toxin protein in both strains . Two recombinant B . cereus strains Ae10 and Cx5 showed very high toxicity against C . quinquefasciatus larvae . Since both strains have a close relationship with the mosquito larvae in the native environment and are capable of recolonizing in the guts of mosquito larvae, these strains can be considered promising new hosts for an effective delivery of mosquito-larvicidal toxins.

Mol Membr Biol, 2004 Jan-Feb, 21(1), 67 - 74
Ion channels formed in planar lipid bilayers by the dipteran-specific Cry4B Bacillus thuringiensis toxin and its alpha1-alpha5 fragment; Puntheeranurak T et al.; Trypsin activation of Cry4B, a 130-kDa Bacillus thuringiensis (Bt) protein, produces a 65-kDa toxin active against mosquito larvae . The active toxin is made of two protease resistant-products of ca . 45 kDa and ca . 20 kDa . The cloned 21-kDa fragment consisting of the N-terminal region of the toxin was previously shown to be capable of permeabilizing liposomes . The present study was designed to test the following hypotheses: (1) Cry4B, like several other Bt toxins, is a channel-forming toxin in plannar lipid bilayers; and (2) the 21-kDa N-terminal region, which maps for the first five helices (alpha1-alpha5) of domain 1 in other Cry toxins, and which putatively shares a similar tri-dimensional structure, is sufficient to account for the ion channel activity of the whole toxin . Using circular dichroism spectroscopy and planar lipid bilayers, we showed that the 21-kDa polypeptide existed as an alpha-helical structure and that both Cry4B and its alpha1-alpha5 fragment formed ion channels of 248 +/- 44 pS and 207 +/- 23 pS, respectively . The channels were cation-selective with a potassium-to-chloride permeability ratio of 6.7 for Cry4B and 4.5 for its fragment . However, contrary to the full-length toxin, the alpha1-alpha5 region formed channels at low dose; they tended to remain locked in their open state and displayed flickering activity bouts . Thus, like the full-length toxin, the alpha1-alpha5 region is a functional channel former . A pH-dependent, yet undefined region of the toxin may be involved in regulating the channel properties.

Syst Appl Microbiol, 2003 Nov, 26(4), 502 - 4
Cry29A and Cry30A: two novel delta-endotoxins isolated from Bacillus thuringiensis serovar medellin; Juarez-Perez V et al.; Two novel crystal protein genes from a highly mosquitocidal Bacillus thuringiensis serovar medellin strain were cloned and sequenced . The corresponding proteins, Cry29A and Cry30A, were nontoxic when tested individually against the mosquito species bioassayed (Aedes aegypti, Culex pipiens and Anopheles stephensi) . However, Cry29A synergized the toxicity of Cry11Bb against Aedes aegypti by a four-fold factor.

Rev Saude Publica, 2003 Dec, 37(6), 813 - 6 Epub 2003 Nov 27.
{Potential of Bacillus thuringiensis israelensis Berliner for controlling Aedes aegypti}; Polanczyk RA et al.; The importance of the entomopathogenic bacterium Bacillus thuringiensis israelensis in the control of Aedes aegypti is presented . The use and potential of B . thuringiensis israelensis against the mosquito vector of dengue fever is described . Other aspects such as insect's resistance development against chemicals and advantages and constraints of using microbial control are discussed . Emphasis is given to the importance of the use of this bacterium in Brazil, which could contribute significantly to solving the mosquito problem without affecting the environment, humans and others invertebrate organisms in critical regions.

Appl Biochem Biotechnol, 2003 Dec, 111(3), 185 - 90
Asymmetric reduction of benzil to (S)-benzoin with whole cells of Bacillus cereus; Saito T et al.; Benzil (1) was selectively reduced to (S)-benzoin (2) in the presence of a wild-type Bacillus cereus Tim-r01 . A 92% yield of 2 with 94% enantiomeric excess ratio was attained in phosphate-buffered saline (PBS) (pH 7.5) by using glucose as a nutrient at 37 degrees C for 12 h . Compound 2 was not reduced further to hydrobenzoin (3) at all . The reduction activity differed greatly depending on the strain of B . cereus . Under these conditions the B . cereus strains IFO3001, IFO15305, IAM1110, IAM1229, IAM1656, and IAM1729 gave 2 in yields ranging from 23 to 46% and the configuration of 2 was (S)-form (7 to 86% ee).

Biol Trace Elem Res, 2003 Dec, 95(3), 269 - 78
Effect of Sm(3+) ion on growth of Bacillus thuringiensis by microcalorimetry; Ruming Z et al.; By using an LKB-2277 Bioactivity Monitor, cycle-flow method, the thermogenic curves of aerobic growth for Bacillus thuringiensis cry II strain at 28 degrees C have been obtained . The metabolic thermogenic curves of B . thuringiensis cry II contained two distinct patterns: the first reflects the changes during the bacterial growth phase and the second corresponds to the sporulation phase . From these thermogenic curves in the absence and presence of Sm(3+) ions, the thermokinetic parameters such as the growth rate constants k, the interval time tau(I), the maximum power P (max 1) and heat-output Q(log) for log phase, the maximum power P (max 2) and heatoutput Q(stat) for stationary phase, the heat-output Q(spor) for sporulation phase and total heat effects QT are calculated . Sm(3+) ion has promoting action on the growth of B . thuringiensis cry II in its lower concentration range; on the other hand, this ion has inhibitory action on the sporulation of B . thuringiensis in its higher concentration range . We also found that the effects of Sm(3+) ion on B . thuringiensis during the sporulation phase were far greater than that during the bacterial phase . It is concluded that the application of B . thuringiensis for controlling insecticides is not affected by the presence of the rare-earth elements in the environmental ecosystem.

Environ Int, 2004 Mar, 30(1), 117 - 22
Development of a rapid method for direct detection of tet(M) genes in soil from Danish farmland; Agerso Y et al.; A method for direct detection of antibiotic resistance genes in soil samples has been developed . The tetracycline resistance gene, tet(M), was used as a model . The method was validated on Danish farmland soil that had repeatedly been treated with pig manure slurry containing resistant bacteria . The tet(M) gene was directly detected in 10-80% of the samples from the various farmland soils and could be detected in all samples tested after selective enrichment . To validate the obtained results, the method was applied to garden soil samples where lower prevalence of resistance was found . RESULT: A detection limit of 10(2)-10(3) copies of the tet(M) gene per gram of soil (in a Bacillus cereus group bacterium) was achieved . tet(M) gene was detected in soil samples with the highest prevalence on farmland treated with pig manure slurry.

J Agric Food Chem, 2003 Dec 17, 51(26), 7664 - 70
Purification and characterization of proteases from Bacillus amyloliquefaciens isolated from traditional soybean fermentation starter; Cho SJ et al.; Bacillus amyloliquefaciens FSE-68 isolated from meju, a Korean soybean fermentation starter, was identified on the basis of biophysical tests and 16S rRNA gene sequence . A neutral metalloprotease (NPR68) and an alkaline serine-protease (APR68) were purified by ammonium sulfate precipitation and cation exchange chromatography and identified on the basis of their activities at different pH values and the selective protease inhibitors . The molecular weights of NPR68 and APR68 measured with ESI-MS were 32743 (+/- 0.8) and 27443 (+/- 0.5) Da, respectively . Against oxidized insulin chains, the NPR68 has a cleavage preference at the site where leucine is located as a P1' residue followed by phenylalanine, and the APR68 has broad specificity and favors leucine at the P1 site . These results indicate that the proteases are natural variants of subtilisin and bacillolysin.

Microbiology, 2003 Dec, 149(Pt 12), 3565 - 73
Gradual evolution in bacteria: evidence from Bacillus systematics; Feldgarden M et al.; The bacterial genome projects have suggested a central role for horizontal transfer in bacterial adaptation, but it is difficult to rule out an adaptive role for ordinary genetic change in existing genes . The bacterial systematics literature can readily address the importance of gene acquisition in adaptive evolution, since phenotypic characterization typically assesses presence versus absence of metabolic capabilities, and metabolic gains and losses are most likely due to horizontal transfer and/or gene loss . Bacterial systematists have not geared their studies toward quantitative differences in metabolic capabilities, which are more likely to involve adjustments of existing genes . Here, quantitative variation in metabolism within and between three closely related Bacillus taxa has been assayed . While these taxa show no qualitative (i.e . presence versus absence) differences in resource utilization, they are quantitatively different in utilization of 8 % of 95 resources tested . Moreover, 93 % of the resources tested showed significant quantitative variation among strains within a single taxon . These results suggest that ordinary genetic changes in existing genes may play an important role in adaptation . If these results are typical, future genomically based assays of quantitative variation in phenotype (e.g . microarray analysis of mRNA concentrations) may identify hundreds of genes whose expression has been modified . A protocol is presented for identifying those modifications of gene expression and those gene acquisitions that are most likely to have played a role in adaptive evolution.

Microbiology, 2003 Dec, 149(Pt 12), 3553 - 64
Compaction of the Escherichia coli nucleoid caused by Cyt1Aa; Manasherob R et al.; Compaction of the Escherichia coli nucleoid in the cell's centre was associated with the loss of colony-forming ability; these effects were caused by induction of Cyt1Aa, the cytotoxic 27 kDa protein from Bacillus thuringiensis subsp . israelensis . Cyt1Aa-affected compaction of the nucleoids was delayed but eventually more intense than compaction caused by chloramphenicol . The possibility that small, compact nucleoids in Cyt1Aa-expressing cells resulted in DNA replication run-out and segregation following cell division was ruled out by measuring relative nucleoid length . Treatments with membrane-perforating substances other than Cyt1Aa did not cause such compaction of the nucleoids, but rather the nucleoids overexpanded to occupy nearly all of the cell volume . These findings support the suggestion that, in addition to its perforating ability, Cyt1Aa causes specific disruption of nucleoid associations with the cytoplasmic membrane . In situ immunofluorescence labelling with Alexa did not demonstrate a great amount of Cyt1Aa associated with the membrane . Clear separation between Alexa-labelled Cyt1Aa and 4',6-diamidino-2-phenylindole (DAPI)-stained DNA indicates that the nucleoid does not bind Cyt1Aa . Around 2 h after induction, nucleoids in Cyt1Aa-expressing cells started to decompact and expanded to fill the whole cell volume, most likely due to partial cell lysis without massive peptidoglycan destruction.

J Immunol, 2003 Dec 15, 171(12), 6788 - 94
Role of monocyte chemotactic protein-1/CC chemokine ligand 2 on gamma delta T lymphocyte trafficking during inflammation induced by lipopolysaccharide or Mycobacterium bovis bacille Calmette-Guérin; Penido C et al.; Gammadelta T lymphocytes are involved in a great variety of inflammatory and infectious responses . However, the mechanisms by which gammadelta T lymphocytes migrate to inflamed sites are poorly understood . In this study we investigate the role of monocyte chemotactic protein (MCP)-1 in regulating gammadelta T cell migration after LPS or Mycobacterium bovis bacille Calmette-Guerin (BCG) challenge . LPS-induced gammadelta T cell influx was significantly inhibited by either pretreatment with dexamethasone or vaccinia virus Lister 35-kDa chemokine binding protein, vCKBP, a CC chemokine neutralizing protein, suggesting a role for CC chemokines in this phenomenon . LPS stimulation increased the expression of MCP-1 mRNA and protein at the inflammation site within 6 h . It is noteworthy that LPS was unable to increase MCP-1 production or gammadelta T cell recruitment in C3H/HeJ, indicative of the involvement of Toll-like receptor 4 . Gammadelta T cells express MCP-1 receptor CCR2 . Pretreatment with anti-MCP-1 mAb drastically inhibited LPS-induced in vivo gammadelta T cell mobilization . Indeed, MCP-1 knockout mice were unable to recruit gammadelta T cells to the pleural cavity after LPS stimulation, effect that could be restored by coadministration of MCP-1 . In addition, BCG-induced gammadelta lymphocyte accumulation was significantly reduced in MCP-1 knockout mice when compared with wild-type mice . In conclusion, our results indicate that LPS-induced gammadelta T lymphocyte migration is dependent on Toll-like receptor 4 and sensitive to both dexamethasone and CC chemokine-binding protein inhibition . Moreover, by using MCP-1 neutralizing Abs and genetically deficient mice we show that LPS- and BCG-induced gammadelta T lymphocyte influx to the pleural cavity of mice is mainly orchestrated by the CC chemokine MCP-1.

Curr Opin Microbiol, 2003 Dec, 6(6), 550 - 6
Spore germination; Setlow P; The germination of dormant spores of Bacillus species is the first crucial step in the return of spores to vegetative growth, and is induced by nutrients and a variety of non-nutrient agents . Nutrient germinants bind to receptors in the spore's inner membrane and this interaction triggers the release of the spore core's huge depot of dipicolinic acid and cations, and replacement of these components by water . These latter events trigger the hydrolysis of the spore's peptidoglycan cortex by either of two redundant enzymes in B . subtilis, and completion of cortex hydrolysis and subsequent germ cell wall expansion allows full spore core hydration and resumption of spore metabolism and macromolecular synthesis.

J Biol Chem, 2004 Feb 27, 279(9), 8378 - 88 Epub 2003 Dec 05.
Crystal structure of human tryptophanyl-tRNA synthetase catalytic fragment: insights into substrate recognition, tRNA binding, and angiogenesis activity; Yu Y et al.; Human tryptophanyl-tRNA synthetase (hTrpRS) produces a full-length and three N terminus-truncated forms through alternative splicing and proteolysis . The shortest fragment that contains the aminoacylation catalytic fragment (T2-hTrpRS) exhibits the most potent angiostatic activity . We report here the crystal structure of T2-hTrpRS at 2.5 A resolution, which was solved using the multi-wavelength anomalous diffraction method . T2-hTrpRS shares a very low sequence homology of 22% with Bacillus stearothermophilus TrpRS (bTrpRS); however, their overall structures are strikingly similar . Structural comparison of T2-hTrpRS with bTrpRS reveals substantial structural differences in the substrate-binding pocket and at the entrance to the pocket that play important roles in substrate binding and tRNA binding . T2-hTrpRS has a wide opening to the active site and adopts a compact conformation similar to the closed conformation of bTrpRS . These results suggest that mammalian and bacterial TrpRSs might use different mechanisms to recognize the substrate . Modeling studies indicate that tRNA binds with the dimeric enzyme and interacts primarily with the connective polypeptide 1 of hTrpRS via its acceptor arm and the alpha-helical domain of hTrpRS via its anticodon loop . Our results also suggest that the angiostatic activity is likely located at the alpha-helical domain, which resembles the short chain cytokines.

FEMS Microbiol Lett, 2003 Dec 5, 229(1), 111 - 7
Expression of green fluorescent protein in Bacillus brevis under the control of a novel constitutive promoter F1 and insertion mutagenesis of F1 in Escherichia coli DH5alpha; Chen Y et al.; The constitutive expression vector pHY300-F1gfp was constructed to test the function of a promoter, F1, cloned from the rice epiphyte Bacillus brevis strain DX01 . The DX01 cells harboring the plasmid pHY300-F1gfp were shown to produce bright green fluorescence . The results were confirmed by Western blot analysis and fluorescence-activated cell sorting . Expression of the F1 promoter was constitutive . To improve the activity of F1, insertion mutagenesis of F1 based on in vitro transposition reaction was performed . Seven mutants with enhanced transcription activity in Escherichia coli DH5alpha were obtained . The enhanced promoters showed similar high activities in B . brevis strain DX01.

J Econ Entomol, 2000 Feb, 93(1), 149 - 56
Colorado potato beetle (Coleoptera: Chrysomelidae) feeding, development, and survival to adulthood after continuous exposure to Bacillus thuringiensis subsp . tenebrionis-treated potato foliage from the field; Nault BA et al.; Colorado potato beetle, Leptinotarsa decemlineata (Say), feeding, development, and survival to adulthood were examined after continuously exposing large larvae to Bacillus thuringiensis subsp . tenebrionis-treated potato foliage from the field . In laboratory assays, the overall consumption and the length of period to become prepupae were determined for larvae, which began as 3rd and 4th instars, that were offered potato leaf disks with naturally declining levels of B . thuringiensis residue . In small-cage field experiments, survival to adulthood and the period to adult emergence for beetles confined to potato plants treated with B . thuringiensis beginning as 3rd and 4th instars also were examined . Third instars remaining on plants after a B . thuringiensis application were unlikely to feed and 4th instars consumed only approximately 50% as much foliage as those fed untreated foliage . Many late instars subjected to B . thuringiensis-treated foliage failed to survive to adulthood; 58-83% of these beetles died during the larval stage . Reduced feeding and poor survival of late instars suggest that counts of large larvae after application do not provide a complete picture of the efficacy of the B . thuringiensis treatment . Late instar Colorado potato beetles that were exposed continually to naturally declining levels of B . thuringiensis-treated potato foliage took an average of 1.8-4.5 d longer to become prepupae and 4-8 d longer to emerge as adults compared with those provided with untreated foliage . Delayed emergence of adults that fed on B . thuringiensis-treated potatoes as late instars indicated that development was prolonged in these insects because of ingestion of a sublethal dose of B . thuringiensis.

J Econ Entomol, 2000 Feb, 93(1), 26 - 30
Frequency of resistance to Bacillus thuringiensis toxin Cry1Ab in an Iowa population of European corn borer (Lepidoptera: Crambidae); Andow DA et al.; The refuge plus high-dose strategy for resistance management assumes that the frequency of resistance alleles is low . We used an F2 screen to estimate the frequency of resistance to transgenic corn that produces Bacillus thuringiensis Berliner Cry1Ab toxin (Bt corn) in an Iowa population of European corn borer, Ostrinia nubilalis (Hubner) . We also proposed a modification to the statistical analysis of the F2 screen that extends its application for nonuniform prior distributions and for repeated sampling of a single population . Based on a sample of 188 isofemale lines derived from females caught at light traps during the 2nd flight of 1997, we show with 95% confidence that the frequency of resistance to Bt corn was <3.9 x 10(-3) in this Iowa population . These results provide weak evidence that the refuge plus high-dose strategy may be effective for managing resistance in O . nubilalis to Bt corn . Partial resistance to Cry1Ab toxin was found commonly . The 95% CI for the frequency of partial resistance were {8.2 x 10(-4), 9.4 x 10(-3)} for the Iowa population . Variable costs of the method were 14.90 dollars per isofemale line, which was a reduction of 25% compared with our initial estimate.

J Econ Entomol, 2000 Feb, 93(1), 14 - 7
Heritability of tolerance to the Cry1Ab toxin of Bacillus thuringiensis in Chilo suppressalis (Lepidoptera: Crambidae); Alinia F et al.; Heritability of Chilo suppressalis (Walker) tolerance to the Cry1Ab toxin of Bacillus thuringiensis Berliner was estimated using a half-sibling design . Artificial diet with and without Cry1Ab was infested with progenies of 20 males, each mated with 2 females, and mortality was scored 5 d after infestation . The progeny of each female was reared and scored separately . Mean mortality of the 20 families on the Cry1Ab diet was 46.5% . The effects of both male parent and of female parent within male parent were significant . Heritability was estimated to be 0.52, suggesting that a high proportion of phenotypic variation was because of genetic differences . Mortality on the Cry1Ab diet was not correlated with mortality on control diet, indicating that differences among families in tolerance to Cry1Ab were not attributable to differences in general fitness . Our results indicate that "high dose" Bt rice plants may be particularly important for Cry1Ab resistance management in C . suppressalis populations.

J Econ Entomol, 2000 Feb, 93(1), 1 - 6
Binding and toxicity of Bacillus thuringiensis protein Cry1C to susceptible and resistant diamondback moth (Lepidoptera: Plutellidae); Liu YB et al.; We studied mechanisms of resistance to Bacillus thuringiensis insecticidal crystal protein Cry1C in the diamondback moth, Plutella xylostella (L.) . Binding assays with midgut brush border membrane vesicles prepared from whole larvae showed no significant difference between resistant and susceptible strains in binding of radioactively-labeled Cry1C . These results indicate that reduced binding of Cry1C to midgut membrane target sites did not cause resistance to Cry1C . Thus, the mechanism of resistance to Cry1C differs from that observed in several previously reported cases of resistance to Cry1A toxins in diamondback moth . We tested Cry1C toxin and Cry1C crystalline protoxin against resistant and susceptible larvae using leaf disk bioassays . After adjusting for the size difference between Cry1C toxin and protoxin, we found that with resistant larvae, toxin was significantly more toxic than protoxin . In contrast, with susceptible larvae, no significant difference in toxicity occurred between Cry1C toxin and protoxin . The resistance ratios for Cry1C were 19 for toxin and 48 for protoxin . These results suggest that reduced conversion of Cry1C protoxin to toxin is a minor mechanism of resistance to Cry1C . Because neither reduced binding nor reduced conversion of protoxin to toxin appear to be major mechanisms, one or more other mechanisms are important in diamondback moth resistance to Cry1C.

Appl Spectrosc, 2003 Nov, 57(11), 1340 - 5
Near-infrared surface-enhanced-Raman-scattering-mediated detection of single optically trapped bacterial spores; Alexander TA et al.; A novel methodology has been developed for the investigation of bacterial spores . Specifically, this method has been used to probe the spore coat composition of two different Bacillus stearothermophilus variants . This technique may be useful in many applications; most notably, development of novel detection schemes toward potentially harmful bacteria . This method would also be useful as an ancillary environmental monitoring system where sterility is of importance (i.e., food preparation areas as well as invasive and minimally invasive medical applications) . This unique detection scheme is based on the near-infrared (NIR) surface-enhanced Raman scattering (SERS) from single, optically trapped, bacterial spores . The SERS spectra of bacterial spores in aqueous media have been measured using SERS substrates based on approximately 60-nm-diameter gold colloids bound to 3-aminopropyltriethoxysilane derivatized glass . The light from a 787-nm laser diode was used to trap and manipulate as well as simultaneously excite the SERS of an individual bacterial spore . The collected SERS spectra were examined for uniqueness and the applicability of this technique for the strain discrimination of Bacillus stearothermophilus spores . Comparison of normal Raman and SERS spectra reveals not only an enhancement of the normal Raman spectral features but also the appearance of spectral features absent in the normal Raman spectrum.

Transplantation, 2003 Nov 27, 76(10), 1514 - 6
Intravesical bacillus Calmette-Guèrin for the treatment of superficial bladder cancer in renal transplant patients; Palou J et al.; BACKGROUND: Intravesical instillations with bacillus Calmette-Guerin (BCG) is considered the treatment of choice in the prophylaxis of high-grade superficial bladder carcinoma and in the treatment of carcinoma in situ (CIS) of the bladder . METHODS: There is no previous experience with BCG treatment in patients with renal transplantation . Theoretically, immunosuppression is a contraindication because of the risk of severe morbidity and sepsis . We present our experience with endovesical BCG in three renal transplant patients, under immunosuppressive treatment, with high-grade superficial bladder cancer and CIS . RESULTS: Two patients are free of disease at 17 and 60 months . One patient developed disease recurrence and underwent a radical cystectomy . There was neither change in renal function nor any clinical evidence of tuberculous infection . CONCLUSIONS: Intravesical BCG in superficial bladder cancer and/or CIS is a valid option, with no added morbidity to renal transplant patients.

Cell Mol Biol (Noisy-le-grand), 2003 Sep, 49(6), 985 - 90
Conformational changes induced by cloxacillin in class a beta-lactamase from Bacillus cereus; Salas-Burgos A et al.; Class A beta-lactamases are enzymes that hydrolyse beta-lactam antibiotics such as penicillins and cephalosporins . They also hydrolyse substrate analogues such as oxacillin and cloxacillin, with a biphasic kinetic as it has been reported for Bacillus cereus beta-lactamase . A molecular model of Bacillus cereus beta-lactamase was built and the conformational changes that the substrates benzylpenicillin and cloxacilline produced in the conformation of selected regions of the protein were analyzed . This study was performed using the docking of the substrates, their tetrahedral intermediates and the corresponding acids on the active site, followed by molecular dynamic and subsequent optimisation procedures . The most important changes were produced on Tyr105 and Tyr273, when the tetrahedral intermediate of cloxacillin was docked at the active site, these amino acids are partially responsible for the stabilisation of the substrates at the active site . These changes may explain the kinetic differences observed during the hydrolysis of substrates type S and type A by beta-lactamases class A.

Hinyokika Kiyo, 2003 Oct, 49(10), 599 - 601
{Reiter syndrome after intravesical Bacillus Calmette-Guerin (BCG) immunotherapy: a case report}; Shiba M et al.; A 56-year-old man who had previously undergone transurethral resection and intra-arterial chemotherapy for bladder cancer developed irritable bladder, bilateral conjunctivitis and arthritis including the knees, ankles and sacroiliac joints after starting intravesical Calmette-Guerin bacillus (BCG) immunotherapy . These symptoms were in agreement with the features of Reiter syndrome . One month after cessation of the intravesical BCG immunotherapy and initiation of the treatment with a non-steroidal anti-inflammatory drug (NSAID), he was not complaining of symptoms . Reiter syndrome is an uncommon complication after intravesical BCG immunotherapy . Nevertheless, since the prolonged arthritis has a possibility to cause joint deformity, we must pay serious attention to this side effect.

Rev Panam Salud Publica, 2003 Sep, 14(3), 209 - 14
{Tuberculin reactivity among ninth-grade schoolchildren in the city of Havana, Cuba}; Borroto Gutierrez SM et al.; OBJECTIVE: To determine the proportion of 14-year-old schoolchildren in the city of Havana, Cuba, with a positive tuberculin skin test, as an indicator of the prevalence of tuberculosis infection among them . METHODS: Using single-stage cluster sampling, 1 936 Mantoux (tuberculin) tests were carried out with ninth-grade students (cohort born in 1985) during the 1999- 2000 school year in 20 basic high schools randomly selected in Havana . The tests were performed according to the standard technique recommended by the World Health Organization, and they were read after 72 hours . The percentage of skin tests that were positive and the average diameter of the indurations were calculated for the cohort overall and for the two genders . The means and the percentages were compared using the chi-square test, with 95% confidence intervals . The computer software used was Epi Info version 6.0 . RESULTS: Of the tests read, 96% of them were negative (0-4 mm), 2.5% were doubtful (5-9 mm), and 1.5% were positive (>/=10 mm) . The percentage of reactivity was 0.1% when a cutoff value of 15 mm was used . The mean diameter of the indurations was 0.41 mm . No statistically significant difference was found between the genders . CONCLUSIONS: In this study the proportion of schoolchildren with tuberculin reactivity, using an induration-diameter cutoff point of 10 mm, was very low (1.5%), and it was much lower (0.1%) when a cutoff point of 15 mm was used . The skin reactions with an induration diameter of >/=10 mm could be the expression of a natural infection if one takes into account the low frequency of bacillary tuberculosis in Cuba and that there is an inverse relationship between the time elapsed from the BCG vaccination and the intensity of the response to tuberculin . Therefore, that would mean that in this case (l)the point (.)prevalence of tuberculosis infection in this group of schoolchildren wou d be 1.5% ResumenObjetivos . Determinar la proporcion de personas que reaccionan a la tuberculina como indicador de la prevalencia de infeccion tuberculosa en escolares de 14 anos de Ciudad de La Habana, Cuba . Metodos . Mediante un muestreo monoetapico por conglomerados se aplicaron 1 936 pruebas de Mantoux (tuberculina) a estudiantes de noveno grado del curso escolar 1999-2000 (cohorte de los nacidos en 1985) de 20 escuelas secundarias basicas seleccionadas aleatoriamente en Ciudad de La Habana, Cuba . Las pruebas se realizaron segun la tecnica estandar recomendada por la Organizacion Mundial de la Salud y se evaluaron a las 72 horas . Se calcularon los porcentajes de reactividad y el diametro medio de las induraciones cutaneas en la cohorte y segun el sexo . Las comparaciones de medias y porcentajes se realizaron mediante la prueba de ji al cuadrado con 95% de confiabilidad . Se uso el programa Epi Info v . 6.0 . Resultados . Noventa y seis por ciento de las pruebas leidas fueron negativas (0-4 mm), 2,5% fueron dudosas (5-9 mm) y 1,5% resultaron positivas ( >/= 10 mm) . El porcentaje de reactividad disminuyo a 0,1% cuando se utilizo un valor de corte de 15 mm . El diametro medio de las induraciones fue de 0,41 mm y no se detecto ninguna diferencia estadisticamente significativa en funcion del sexo . Conclusiones . La proporcion de personas que reaccionaron a la tuberculina, usando como punto de corte un diametro de induracion cutanea de 10 mm, fue muy baja (1,5%) en este estudio y mucho mas baja (0,1%) cuando se utilizo 15 mm como punto de corte . Si se toma en cuenta la baja frecuencia de tuberculosis bacilifera en Cuba y que hay una relacion inversa entre el tiempo transcurrido desde la aplicacion de la vacuna a base del bacilo de Calmette-Guerin (BCG) y la intensidad de la respuesta a la tuberculina, las reacciones cutaneas con un diametro de induracion >/= 10 mm podrian ser la expresion de una infeccion natural, por lo que, en ese caso, la prevalencia puntual de infeccion tuberculosa en este grupo de escolares seria de 1,5%.

Mol Ecol, 2004 Jan, 13(1), 237 - 41
Stable Bacillus thuringiensis (Bt) toxin content in interspecific F1 and backcross populations of wild Brassica rapa after Bt gene transfer; Zhu B et al.; Stable expression of a transgene may lead to increased fitness for wild plants after acquiring the transgene via crop-weed hybridization . Here, we investigate the stability of Bt toxin content in wild Brassica rapa acquiring the Bt gene from Bt Brassica napus . The Bt toxin content in nine Bt-expressing B . napus lines was 0.80-1.70 micro g/g leaf tissue throughout the growing season . These nine lines were crossed with three accessions of wild B . rapa and the Bt gene was successfully transferred to interspecific hybrids (F1) and successive backcross generations (BC1 to BC4) . The Bt toxin level in F1 and BC progenies containing the Bt gene remained at 0.90-3.10 micro g/g leaf tissue . This study indicates that the Bt gene can persist and be stably expressed in wild B . rapa.

Plant Cell Rep, 2003 Dec, 22(5), 338 - 43 Epub 2003 Sep 04.
Spatial and temporal patterns of green fluorescent protein (GFP) fluorescence during leaf canopy development in transgenic oilseed rape, Brassica napus L; Halfhill MD et al.; The green fluorescent protein (GFP) holds promise as a field-level transgene marker . One obstacle to the use of GFP is fluorescence variability observed within leaf canopies . In growth chamber and field experiments, GFP fluorescence in transgenic oilseed rape ( Brassica napus) was shown to be variable at each leaf position over time and among different leaves on the same plant . A leaf had its highest GFP fluorescence after emergence and, subsequently, its fluorescence intensity decreased . GFP fluorescence intensity was directly correlated with the concentration of soluble protein . The concentration of the genetically linked recombinant Bacillus thuringiensis (Bt) cry1Ac endotoxin protein also was examined, and GFP fluorescence was positively correlated with Bt throughout development . The results show that GFP can be used as an accurate transgene marker but that aspects of plant developmental should be taken into account when interpreting fluorescence measurements.

Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2319 - 21 Epub 2003 Nov 27.
Expression, purification, crystallization and preliminary crystallographic analysis of phosphoserine aminotransferase from Bacillus alcalophilus; Dubnovitsky AP et al.; Phosphoserine aminotransferase (PSAT; EC 2.6.1.52) from Bacillus alcalophilus, an obligatory alkalophile with optimum growth at pH 10.6, was overexpressed in Escherichia coli, purified and crystallized under two different conditions using the hanging-drop vapour-diffusion method . Crystals were obtained using trisodium citrate dihydrate or PEG 400 as a precipitating agent . Crystals grown in the presence of trisodium citrate belong to the orthorhombic space group C222(1), with unit-cell parameters a = 105.6, b = 136.6, c = 152.0 A, and those grown in the presence of PEG 400 belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 143.7, b = 84.3, c = 67.4 A . Complete data sets were collected to 1.7 and 1.6 A resolution, respectively, at 100 K using synchrotron radiation . Analysis of the structure of B . alcalophilus PSAT may reveal structural features that contribute to enzyme adaptability at high pH values.

Bioresour Technol, 2004 Mar, 92(1), 49 - 54
Optimization of cyclodextrin production from sago starch; Charoenlap N et al.; Cyclodextrin (CD) is synthesized by bacterial cyclodextrin glycosyltransferase (CGTase) and is widely used in food, pharmaceutical, cosmetic, and agricultural industries . In this study, Bacillus circulans CGTase was partially purified by ammonium sulfate precipitation at 50-70% saturation . The optimum pH and temperature for CD production from sago starch were found to be in the ranges of 4.5-5.0 and 55-60 degrees C, respectively . beta-CD was the predominant product, constituting 65% of all CD products . The beta-CD produced using partially purified and crude CGTase were compared and found to have no significant difference in yield and productivity . The appropriate proportion of CGTase to sago starch for beta-CD production was determined by response surface methodology . The most appropriate enzyme:substrate ratio was 50 U g sago starch(-1) CGTase and 60 g l(-1) sago starch.

Microb Pathog, 2004 Jan, 36(1), 11 - 7
Mycobacterium bovis infection of vitamin D-deficient NOS2-/- mice; Waters WR et al.; Vitamin D deficiency is associated with an increased risk for tuberculosis infection . Studies using in vitro systems indicate that 1,25-dihydroxyvitamin D(3) {i.e . 1,25(OH)(2)D(3)}, the most active form of the vitamin, enhances mycobacterial killing by increasing nitric oxide (NO) production . To evaluate concurrently the role of 1,25(OH)(2)D(3) and NO on the host response to tuberculosis infection, mice deficient in NO synthase 2 (NOS2(-/-)) and/or vitamin D were aerosol-challenged with Mycobacterium bovis and subsequently evaluated for mycobacterial colonization and lesion formation . Infected NOS2(-/-) mice developed severe necrotizing pyogranulomatous inflammation of the lungs with heavy M . bovis colonization and systemic dissemination of the bacillus . Colonization and lung lesion area of NOS2(-/-) mice exceeded that of NOS2(+/+) mice . Additionally, disease progression was more rapid in NOS2(-/-) mice than in NOS2(+/+) mice . Lung colonization and lesion area of vitamin D deficient mice exceeded that of vitamin D replete mice, regardless of NOS2 phenotype . However, effects of vitamin D on colonization, but not lesion area, were more pronounced in NOS2(+/+) mice than in NOS2(-/-) mice . These findings are consistent with the current hypothesis that 1,25(OH)(2)D(3) enhances mycobacterial killing through a NO-dependent mechanism . As responses of NOS2(-/-) mice were affected by 1,25(OH)(2)D(3) deficiency, albeit to a lesser extent than were those of NOS2(+/+) mice, NO-independent actions of 1,25(OH)(2)D(3) also likely exist.

Biochemistry (Mosc), 2003 Nov, 68(11), 1217 - 24
Isolation and characterization of glutamyl endopeptidase 2 from Bacillus intermedius 3-19; Balaban NP et al.; The culture filtrate of Bacillus intermedius 3-19 was used for isolation by chromatography on CM-cellulose and Mono S columns of a proteinase that is secreted during the late stages of growth . The enzyme is irreversibly inhibited by the inhibitor of serine proteinases diisopropyl fluorophosphate, has two pH optima (7.2 and 9.5) for casein hydrolysis and one at pH 8.5 for Z-Glu-pNA hydrolysis . The molecular weight of the enzyme is 26.5 kD . The K(m) for Z-Glu-pNA hydrolysis is 0.5 mM . The temperature and pH dependences of the stability of the proteinase were studied . The enzyme was identified as glutamyl endopeptidase 2 . The N-terminal sequence (10 residues) and amino acid composition of the enzyme were determined . The enzyme hydrolyzes Glu4-Gln5, Glu17-Asp18, and Cys11-Ser12 bonds in the oxidized A-chain of insulin and Glu13-Ala14, Glu21-Arg22, Cys7-Gly8, and Cys19-Gly20 bonds in the oxidized B-chain of insulin.

J Econ Entomol, 2003 Oct, 96(5), 1433 - 47
Overwintering of Heliothis virescens (F.) and Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in cotton fields of northeast Mississippi; Schneider JC; Following the 1995-2001 crop seasons, population densities of pupae of tobacco budworm, Heliothis virescens (F.), and corn earworm, Helicoverpa zea (Boddie), overwintering in cotton fields, Gossypium hirsutum L., in Monroe County, MS, were estimated by digging a total of 43,542 m-row . Densities of pupae varied greatly among years with the highest densities occurring in years with low rainfall in the late summer and early fall . The sex ratio of pupae was variable and significantly male-biased in some years . The total area-wide, tobacco budworm population emerging from all overwintering sites was estimated from catches in pheromone traps whose absolute efficiency is known . After the introduction of Bacillus thuringiensis Berliner (Bt)-transgenic cotton in 1996 but before a several-fold increase in use of herbicide resistant (HR)-transgenic cotton cultivars in 2001, local cotton fields accounted for <2% of the total overwintered tobacco budworm population . In the pretransgenic cotton production era, it is estimated cotton fields typically accounted for <10% of the overwintered tobacco budworm population . Driving surveys of the tillage status of cotton fields and pheromone trapping to determine the timing of adult emergence in the spring indicate that--until the 2000 crop season--postseason tillage destroyed most tobacco budworm and corn earworm pupae overwintering in cotton fields . Adoption of HR-transgenic cultivars has quintupled cotton field area planted no-till, but the contribution of cotton fields to overwintered tobacco budworm populations has remained low . Preemptive tillage of cotton fields to kill overwintering tobacco budworm pupae would not appreciably suppress in-season population densities of this pest . However, the absence of such tillage might increase the rate at which the tobacco budworm develops resistance to insecticides and counteradaptation to antibiotic host plant resistance traits including the Bt-transgenic trait.

Hist Cienc Saude Manguinhos, 2003, 10(Suppl 1), 13 - 40
Leprosy and the elusive M . leprae: colonial and Imperial medical exchanges in the nineteenth century; Robertson J; In the 1800s, humoral understandings of leprosy successively give way to disease models based on morbid anatomy, physiopathology, and bacteriology . Linkages between these disease models were reinforced by the ubiquitous seed/soil metaphor deployed both before and after the identification of M.leprae . While this metaphor provided a continuous link between medical descriptions, Henry Vandyke Carter's On leprosy (1874) marks a convergence of different models of disease . Simultaneously, this metaphor can be traced in popular medical debates in the late nineteenth century, accompanying fears of a resurgence of leprosy in Europe . Later the mapping of the genome ushers in a new model of disease but, ironically, while leprosy research draws its logic from a view of the world in which a seed and soil metaphor expresses many different aspects of the activity of the disease, the bacillus itself continues to be unreceptive to cultivation.

Infect Control Hosp Epidemiol, 2003 Nov, 24(11), 801 - 6
A survey of latent tuberculosis infection among laboratory healthcare workers in New York City; Garber E et al.; OBJECTIVE: To determine the prevalence of positive tuberculin skin tests (TSTs), incidence of TST conversion, risk factors for positive TSTs, and history of active TB among HCWs in microbiology laboratories in New York City . DESIGN: Two-year survey from May 1999 to June 2001 . SETTING: Nineteen microbiology laboratories . RESULTS: During the first year, interviews were conducted with 345 laboratory HCWs (mean, 18 HCWs per site; range, 2 to 51) to assess the prevalence of positive TSTs, but 3 (1%) could not recall their result and were excluded from further analyses . The mean age of the remaining 342 HCWs was 48 years; 68% (n = 233) were female, 54% (n = 183) received bacille Calmette-Guerin (BCG) vaccination, and 71% (n = 244) were foreign born . The prevalence of a positive TST was 57% (n = 196), but only 20% (n = 39) of the HCWs received isoniazid . The incidence of TST conversion in the second year of the study was 1% (1 of 108) . Multivariate analysis identified age (odds ratio {OR} per year, 1.05; 95% confidence interval {CI95}, 1.02-1.08), foreign birth (OR, 3.80; CI95, 1.98-7.28), BCG immunization (OR, 4.89; CI95, 2.72-8.80), and employment in a mycobacteriology laboratory (OR, 2.14; CI95, 1.25-3.68) as risk factors for a positive TST . Only one HCW had been treated for active TB . CONCLUSIONS: The prevalence of positive TSTs was high among laboratory HCWs, but the TST conversion rate was low . Higher rates of treatment for latent TB infection are desirable.

J Ind Microbiol Biotechnol, 2003 Dec, 30(12), 677 - 81 Epub 2003 Nov 29.
Fed-batch culture of Bacillus thuringiensis based on motile intensity; Chen S et al.; The operation of a fed-batch culture is more complicated than that of batch or continuous culture . Thus, an appropriate feeding strategy for fed-batch cultures should be carefully designed . In this study, a simple feeding strategy for fed-batch culture of Bacillus thuringiensis based on "motile intensity" is described . The feeding strategy consisted of two steps: (1) initiating feeding at the peak of motile intensity; (2) terminating feeding at low motile intensity (or non-motility) of the cells . In addition, the motile intensity of B . thuringiensis was used to determine the optimum environmental conditions (pH, temperature, and dissolved oxygen) and optimum medium composition . Using this fed-batch strategy, the production of thuringiensin increased 34% compared with batch culture using the same environmental conditions and medium composition . The proposed strategy for fed-batch culture helps to avoid overfeeding of substrate and facilitates on-line control . A comparison of several alternative strategies for fed-batch culture demonstrated that strategies such as glucose-stat and DO-stat result in a lower productivity than that obtained using the motility intensity method.

J Infect Dis, 2003 Dec 1, 188(11), 1782 - 9 Epub 2003 Nov 18.
Enzymatic degradation of prion protein in brain stem from infected cattle and sheep; Langeveld JP et al.; Prions-infectious agents involved in transmissible spongiform encephalopathies-normally survive proteolytic and mild protein-destructive processes . Using bacterial keratinase produced by Bacillus licheniformis strain PWD-1, we tested conditions to accomplish the full degradation of prion protein (PrP) in brain-stem tissue from animals with bovine spongiform encephalopathy and scrapie . The detection of PrPSc, the disease-associated isoform of PrP, in homogenates was done by Western blotting and various antibodies . The results indicated that only in the presence of detergents did heat pretreatment at >100 degrees C allow the extensive enzymatic breakdown of PrPSc to a state where it is immunochemically undetectable . Proteinase K and 2 other subtilisin proteases, but not trypsin and pepsin, were also effective . This enzymatic process could lead to the development of a method for the decontamination of medical and laboratory equipment . The ultimate effectiveness of this method of prion inactivation has to be tested in mouse bioassays.

Infect Immun, 2003 Dec, 71(12), 6933 - 42
Mitogenic effect of Bartonella bacilliformis on human vascular endothelial cells and involvement of GroEL; Minnick MF et al.; Bartonellae are bacterial pathogens for a wide variety of mammals . In humans, bartonellosis can result in angioproliferative lesions that are potentially life threatening to the patient, including bacillary angiomatosis, bacillary peliosis, and verruga peruana . The results of this study show that Bartonella bacilliformis, the agent of Oroya fever and verruga peruana, produces a proteinaceous mitogen for human vascular endothelial cells (HUVECs) that acts in a dose-dependent fashion in vitro with maximal activity at >or=72 h of exposure and results in a 6- to 20-fold increase in cell numbers relative to controls . The mitogen increases bromodeoxyuridine (BrdU) incorporation into HUVECs by almost twofold relative to controls . The mitogen is sensitive to heat and trypsin but is not affected by the lipopolysaccharide inhibitor polymyxin B . The mitogen does not affect caspase 3 activity in HUVECs undergoing serum starvation-induced apoptosis . The Bartonella mitogen was found in bacterial culture supernatants, the soluble cell lysate fraction, and, to a lesser degree, in insoluble cell fractions of the bacterium . In contrast, soluble cell lysate fractions from closely related B . henselae, although possessing significant mitogenicity for HUVECs, resulted in only about a twofold increase in cell numbers . Biochemical and immunological analyses identified GroEL as a participant in the observed HUVEC mitogenicity . A B . bacilliformis strain containing the intact groES-groEL operon on a multicopy plasmid was generated and used to demonstrate a correlation between HUVEC mitogenicity and GroEL levels in the lysate (r(2) = 0.85) . Antiserum to GroEL significantly inhibited mitogenicity of the lysate . Data also show that GroEL is located in the soluble and insoluble fractions (including inner and outer membranes) of the cell and is actively secreted by B . bacilliformis.

J Biol Chem, 2004 Feb 20, 279(8), 7287 - 95 Epub 2003 Nov 24.
Structural and enzymatic analysis of soybean beta-amylase mutants with increased pH optimum; Hirata A et al.; Comparison of the architecture around the active site of soybean beta-amylase and Bacillus cereus beta-amylase showed that the hydrogen bond networks (Glu380-(Lys295-Met51) and Glu380-Asn340-Glu178) in soybean beta-amylase around the base catalytic residue, Glu380, seem to contribute to the lower pH optimum of soybean beta-amylase . To convert the pH optimum of soybean beta-amylase (pH 5.4) to that of the bacterial type enzyme (pH 6.7), three mutants of soybean beta-amylase, M51T, E178Y, and N340T, were constructed such that the hydrogen bond networks were removed by site-directed mutagenesis . The kinetic analysis showed that the pH optimum of all mutants shifted dramatically to a neutral pH (range, from 5.4 to 6.0-6.6) . The Km values of the mutants were almost the same as that of soybean beta-amylase except in the case of M51T, while the Vmax values of all mutants were low compared with that of soybean beta-amylase . The crystal structure analysis of the wild type-maltose and mutant-maltose complexes showed that the direct hydrogen bond between Glu380 and Asn340 was completely disrupted in the mutants M51T, E178Y, and N340T . In the case of M51T, the hydrogen bond between Glu380 and Lys295 was also disrupted . These results indicated that the reduced pKa value of Glu380 is stabilized by the hydrogen bond network and is responsible for the lower pH optimum of soybean beta-amylase compared with that of the bacterial beta-amylase.

FEMS Microbiol Lett, 2003 Nov 21, 228(2), 259 - 63
A plasmid encoding a combination of mosquito-larvicidal genes from Bacillus thuringiensis subsp . israelensis and Bacillus sphaericus confers toxicity against a broad range of mosquito larvae when expressed in Gram-negative bacteria; Tanapongpipat S et al.; A recombinant plasmid harboring cry4A, cry4B and cry11A from Bacillus thuringiensis subsp . israelensis and binary toxin genes from Bacillus sphaericus has been constructed . The three cry genes were placed under the control of the cry4B promoter whereas the binary toxin gene was controlled by its native promoter . The expression of toxins in Escherichia coli harboring the resulting plasmid, p4BDA-5142, was investigated . Cry4B expression was highest compared to other toxins . Although the level of toxin expression was low compared with E . coli expressing single toxins, the recombinant E . coli strain harboring p4BDA-5142 exhibited broad range mosquito-larvicidal activity against all Aedes, Culex and Anopheles larvae . This work has shown that the development of the recombinant plasmid can be used to broaden the host range spectrum of the appropriate bacterial host for mosquito control.

Biochem Biophys Res Commun, 2003 Dec 12, 312(2), 405 - 13
On the structural significance of the linkage region constituents of N-glycoproteins: an X-ray crystallographic investigation using models and analogs; Lakshmanan T et al.; The linkage region constituents, namely, 2-acetamido-2-deoxy-beta-D-glucopyranose and asparagine are conserved in the N-glycoproteins of all the eukaryotes . The present work is aimed at understanding the reasons for the occurrence of GlcNAc and Asn as the linkage region constituents . A total of six sugar amides have been designed as models and analogs of the linkage region and their crystal structures have been solved . This is the first report on the X-ray crystallographic investigation of the effect of systematic changes in the linkage sugar as well as its aglycon moiety on the N-glycosidic torsion, psi(N) (O5-C1-N1-C1(')) . This also forms the first report on the crystal structure of a model of L-RhabetaAsn, a variant linkage found in the surface layer glycoprotein of Bacillus stearothermophillus . Among the models and analogs examined, the acetamido derivatives of Man and Xyl, the linkage sugars of O-glycoproteins, show a psi(N) value of -114.5 degrees and -121.2 degrees, respectively, deviating maximum from the value of -89.8 degrees reported for the model compound GlcNAcbetaNHAc . The L-Rha and Gal derivatives also show noticeable deviations . The psi(N) values, -89.5 degrees and -91.0 degrees, of the propionamide derivatives of Glc and GlcNAc (analogs of GlcbetaGln and GlcNAcbetaGln, respectively) agree well with those (-93.8 degrees and -89.8 degrees ) reported for their corresponding acetamide derivatives suggesting Gln could serve as well as Asn as the linkage region amino acid . However, the rotational freedom about the additional C-C bond would lead to altered rigidity of the linkage region . An analysis of packing reveals that the molecular assembly of these compounds is driven by different infinite and finite chains of hydrogen bonds . The double pillaring of hydrogen bonds involving the amide groups at C1 and C2 is seen as a unique packing feature characteristic of beta-1-N-acyl derivatives of GlcNAc . Based on the findings of the present study, it is speculated that the linkage region constituents of the eukaryotic N-glycoproteins appear to fulfill three essential structural requirements: rigidity, planarity, and linearity and these are met by the trisaccharide core and Asn at the linkage region.

Mol Cell, 2003 Nov, 12(5), 1165 - 72
Crystal structures of an archaeal class I CCA-adding enzyme and its nucleotide complexes; Xiong Y et al.; CCA-adding enzymes catalyze the addition of CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template and have been divided into two classes based on their amino acid sequences . We have determined the crystal structures of a class I CCA-adding enzyme from Archeoglobus fulgidus (AfCCA) and its complexes with ATP, CTP, or UTP . Although it and the class II bacterial Bacillus stearothermophilus CCA enzyme (BstCCA) have similar dimensions and domain architectures (head, neck, body, and tail), only the polymerase domain is structurally homologous . Moreover, the relative orientation of the head domain with respect to the body and tail domains, which appear likely to bind tRNA, differs significantly between the two enzyme classes . Unlike the class II BstCCA, this enzyme binds nucleotides nonspecifically in the absence of bound tRNA . The shape and electrostatic charge distribution of the AfCCA enzyme suggests a model for tRNA binding that accounts for the phosphates that are protected from chemical modification by tRNA binding to AfCCA . The structures of the AfCCA enzyme and the eukaryotic poly(A) polymerase are very similar, implying a close evolutionary relationship between them.

Crit Rev Microbiol, 2003, 29(4), 297 - 312
How far have we reached in tuberculosis vaccine development?
Kumar H, Malhotra D, Goswami S, Bamezai RN.
Tuberculosis, a bacterial disease prevalent since ancient times, continues to cause the most deaths globally compared with all other diseases . The causative agent Mycobacterium tuberculosis is responsible for different types of tuberculosis in humans; however, pulmonary tuberculosis is the most common and causes the most deaths . Mycobacterium tuberculosis is an intracellular pathogenic bacterium, which has developed sophisticated mechanisms to survive inside host mononuclear phagocytes and thus evade the host immune system . This is attributed primarily to an inadequate immune response toward infecting bacteria, which results in temporary growth inhibition rather than death and subsequently allows the bacteria to multiply immensely, leading to full-blown disease in an individual . This disease has become a challenge due to poor diagnosis, a low-efficiency tuberculosis vaccine (Mycobacterium bovis Bacillus Calmette-Guerin {BCG}), a long-term antibacterial chemotherapy regimen (approximately 6 months), and an emergence of multiple drug resistant strains of Mycobacterium tuberculosis especially in people with human immune deficiency virus (HIV) infection, for whom researchers worldwide must develop effective short-term chemotherapy and an effective vaccine . In this review different aspects of vaccines in tuberculosis are discussed, and these include the traditional BCG vaccine, the modern auxotrophic vaccine, the subunit or acellular vaccine; and a DNA vaccine . We discuss also the potential of mycobacterial lipids as a vaccine or as an adjuvant in the future . Since complete genome information of Mycobacterium tuberculosis H37Rv and bioinformatics tools are available, it is possible to develop new strategies for a better and effective tuberculosis vaccine, which can replace the traditional BCG vaccine.

J Evol Biol, 2003 May, 16(3), 378 - 87
Modelling the spatial configuration of refuges for a sustainable control of pests: a case study of Bt cotton; Vacher C et al.; The 'high-dose-refuge' (HDR) strategy is widely recommended by the biotechnology industry and regulatory authorities to delay pest adaptation to transgenic crops that produce Bacillus thuringiensis (Bt) toxins . This involves cultivating nontoxic plants (refuges) in close proximity to crops producing a high dose of Bt toxin . The principal cost associated with this strategy is due to yield losses suffered by farmers growing unprotected, refuge plants . Using a population genetic model of selection in a spatially heterogeneous environment, we show the existence of an optimal spatial configuration of refuges that could prevent the evolution of resistance whilst reducing the use of costly refuges . In particular, the sustainable control of pests is achievable with the use of more aggregated distributions of nontransgenic plants and transgenic plants producing lower doses of toxin . The HDR strategy is thus suboptimal within the context of sustainable agricultural development.

Nihon Rinsho Meneki Gakkai Kaishi, 2003 Oct, 26(5), 299 - 303
{A case of Williams syndrome with p47-phox-deficient chronic granulomatous disease}; Kabuki T et al.; A 2-month-old boy with a characteristic elfin face was diagnosed as having Williams syndrome by means of specific fluorescence in situ hybridization (FISH) analysis for a chromosomal microdeletion located in 7q11.23 . He was suspected to have immunodeficiency because of a persistent enlargement of axillary lymphnodes after immunization with Bacille Calmette-Guerin (BCG) vaccine since 7 month-old of age . The nitroblue tetrazolium test (NBT) and the chemiluminescence test revealed an absence of superoxide production . Western blotting and DNA sequence analysis confirmed the diagnosis of p47-phox-deficient autosomal recessive chronic granulomatous disease (CGD) (A47 degrees CGD) . The predominant genetic defect in A47 degrees CGD was a GT deletion at the beginning of exon 2 in neutrophil cytosol factor 1 gene (NCF1) located in 7q11.23 . It suggests that CGD in this patient resulted from the hemizygosity of recessive genetic mutation in NCF1 located at 7q11.23 associated with Williams syndrome . In such a disease with the chromosomal microdeletion like Williams syndrome, we should consider a combination with autosomal recessive diseases, the genes of which are located in the hemizygous region.

Arch Insect Biochem Physiol, 2003 Dec, 54(4), 200 - 11
Bt: mode of action and use; Whalon ME et al.; The insecticidal toxins from Bacillus thuringiensis (Bt) represent a class of biopesticides that are attractive alternatives to broad-spectrum "hard" chemistries . The U.S . Food Quality Protection Act and the European Economic Council directives aimed at reducing the use of carbamate and organophosphate insecticides were expected to increase the use of narrowly targeted, "soft" compounds like Bt . Here we summarize the unique mode of action of Bt, which contributes to pest selectivity . We also review the patterns of Bt use in general agriculture and in specific niche markets . Despite continued predictions of dramatic growth for biopesticides due to US Food Quality Protection Act-induced cancellations of older insecticides, Bt use has remained relatively constant, even in niche markets where Bt has traditionally been relatively high .

Int J Urol, 2003 Nov, 10(11), 622 - 4
Acute eosinophilic pneumonia associated with intravesical bacillus Calmette-Guérin therapy of carcinoma in situ of the bladder; Orikasa K et al.; A 71-year-old man with a history of rheumatoid arthritis was treated with intravesical bacillus Calmette-Guerin (BCG) instillation of 80 mg once-a-week for carcinoma in situ . He developed low-grade fever followed by dyspnea and severe hypoxemia . Radiological and laboratory studies revealed bilateral diffuse reticulonodular infiltrates and hypereosinophilia . A lymphocyte stimulation test for BCG was strongly positive . From these findings, a pulmonary hypersensitivity reaction to immunotherapy was suspected, and therefore, methylprednisolone (500 mg per day) was started . After that, the fever and dyspnea disappeared, the hypereosinophilia was normalised and chest radiography results were clear . The present case is the first reported case of eosinophilic pneumonia following intravesical BCG therapy.

J Appl Microbiol, 2003, 95(5), 982 - 9
Characterization of a mosquitocidal Bacillus thuringiensis serovar sotto strain isolated from Okinawa, Japan; Ohgushi A et al.; AIMS: To characterize the mosquitocidal activity of parasporal inclusions of the Bacillus thuringiensis serovar sotto strain 96-OK-85-24, for comparison with two well-characterized mosquitocidal strains . METHODS AND RESULTS: The strain 96-OK-85-24 significantly differed from the existing mosquitocidal B . thuringiensis strains in: (1) lacking the larvicidal activity against Culex pipiens molestus and haemolytic activity, and (2) SDS-PAGE profiles, immunological properties and N-terminal amino acid sequences of parasporal inclusion proteins . CONCLUSIONS: It is clear from the results that the strain 96-OK-85-24 synthesizes a novel mosquitocidal Cry protein with a unique toxicity spectrum . SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the occurrence of a mosquitocidal B . thuringiensis strain with an unusual toxicity spectrum, lacking the activity against the culicine mosquito.

J Appl Microbiol, 2003, 95(6), 1361 - 6
A molecular method to detect Bacillus cereus from a coffee concentrate sample used in industrial preparations; Manzano M et al.; AIMS: The aim of this work was to develop specific primers which are able to detect Bacillus cereus in a coffee concentrate sample . METHODS AND RESULTS: A pre-PCR step to clean the DNA, used for PCR, was developed to avoid PCR inhibition by Maillard products . The combination of centrifugation and washing the pellet, employing EDTA and water, before DNA extraction improved the detection of low numbers of B . cereus cells (10 cells ml-1) . The development of specific primers enabled to detect low numbers of B . cereus without the need of a pre-enrichment step . CONCLUSIONS: The data obtained demonstrated the specificity and the sensitivity of the primers that could be used to check the presence of B . cereus in different food products, avoiding the need for labourious and time-consuming culture-based techniques . SIGNIFICANCE AND IMPACT OF THE STUDY: The method could help food microbiologists to check food samples quickly for the presence of B . cereus.

J Appl Microbiol, 2003, 95(6), 1250 - 4
Expression and secretion of an alpha-amylase gene from a native strain of Bacillus licheniformis in Escherichia coli by T7 promoter and putative signal peptide of the gene; Shahhoseini M et al.; The gene encoding a hyperthermostable alpha-amylase from a Bacillus licheniformis native strain was cloned in pET24d transcription vector containing T7 promoter, and expressed in Escherichia coli BL21(DE3) cells . Having confirmed the alpha-amylase activity through activity staining method on SDS-PAGE gel, the yields of production were determined in two separated intra and inter-cellular phases and compared using enzymatic assay methods . Extracellular production of the active recombinant enzyme implies the recognition of the putative signal peptide of this Bacillus sp . by E . coli secretory system . This may be because of the amino acid sequence of this signal peptide which covers all the structural parameters of a standard signal peptide processed by Lep B, the major signal peptidase in E . coli secretory system . This study recommends the use of this signal peptide for extracellular production of other foreign proteins in E . coli.

Immunology, 2003 Dec, 110(4), 507 - 12
Human T-cell responses to the RD1-encoded protein TB27.4 (Rv3878) from Mycobacterium tuberculosis; Agger EM et al.; In recent years, there has been considerable focus on the discovery and characterization of proteins derived from Mycobacterium tuberculosis leading to the identification of a number of candidate antigens for use in vaccine development or for diagnostic purposes . Previous experiments have demonstrated an important immunological role for proteins encoded by the RD1 region, which is absent from all strains of bacillus Calmette-Guerin (BCG) but present in the genomes of virulent M . bovis and M . tuberculosis . Herein, we have studied human T-cell responses to the antigen encoded by the putative open reading frame (rv3878) of the RD1 region . Immunoblot analysis revealed that rv3878 was expressed and the native protein was designated TB27.4 . Immunological evaluations demonstrate that TB27.4 elicits a prominent immune response in human tuberculosis patients with a dominant region in the C-terminal part of the molecule . In contrast, very limited responses were seen in M . bovis BCG-vaccinated donors . This study therefore emphasizes the diagnostic potential of proteins encoded by the RD1 region.

J Microsc, 2003 Dec, 212(Pt 3), 300 - 6
Self-assembly and recrystallization of bacterial S-layer proteins at silicon supports imaged in real time by atomic force microscopy; Gyorvary ES et al.; The self-assembly of bacterial surface-layer (S-layer) proteins (SbpA of Bacillus sphaericus CCM 2177) at silicon supports (hydrophobic, non-plasma-treated and hydrophilic, O2 plasma-treated silicon supports) was imaged in real time by atomic force microscopy (AFM) . A closed mosaic layer consisting of small crystals (less than 200 nm in diameter) was formed at a hydrophobic silicon support, whereas a coherent crystalline lattice consisting of large domains (2-10 micro m in size) was generated at O2 plasma-treated, hydrophilic silicon wafers . The structure of the formed layers was a monolayer (9 nm in height) at the hydrophobic silicon and a bilayer (15 nm in height) at the hydrophilic silicon . In situ AFM measurements confirmed the importance of ionic bonds in the formation of crystalline SbpA layers at silicon supports . Rupture of the protein subunits with a metal chelator from the crystalline lattice of SbpA was visualized in situ by AFM . The stability of solid-supported SbpA layers could be enhanced by cross-linking the S-layers with amino-amino or amino-carboxyl group directed cross-linkers.

Curr Microbiol, 2003 Oct, 47(4), 355 - 7
A Bacillus thuringiensis isolate possessing a spore-associated filament; Rampersad J et al.; We report on a novel bacterium, isolated during a screen for environmental isolates of Bacillus thuringiensis, that possesses a novel filamentous structure . Nucleotide sequence from the isolate's 16S rRNA gene places the bacterium unambiguously within the Bacillus thuringiensis/Bacillus cereus group . Phase-contrast and electron microscopy indicate the presence of both a parasporal body and a long filament which are retained after sporulation . The filament is shown to consistently arise from the end of the exosporium and next to the parasporal body . Upon spore germination, the parasporal body/filament complex is retained on the cell wall of the resulting bacterium.

Curr Microbiol, 2003 Oct, 47(4), 337 - 40
Nucleotide and deduced amino acid sequences of a new subtilisin from an alkaliphilic Bacillus isolate; Saeki K et al.; The gene for a new subtilisin from the alkaliphilic Bacillus sp . KSM-LD1 was cloned and sequenced . The open reading frame of the gene encoded a 97 amino-acid prepro-peptide plus a 307 amino-acid mature enzyme that contained a possible catalytic triad of residues, Asp32, His66, and Ser224 . The deduced amino acid sequence of the mature enzyme (LD1) showed approximately 65% identity to those of subtilisins SprC and SprD from alkaliphilic Bacillus sp . LG12 . The amino acid sequence identities of LD1 to those of previously reported true subtilisins and high-alkaline proteases were below 60% . LD1 was characteristically stable during incubation with surfactants and chemical oxidants . Interestingly, an oxidizable Met residue is located next to the catalytic Ser224 of the enzyme as in the cases of the oxidation-susceptible subtilisins reported to date.

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