Clin Infect Dis, 2005 Jan 15, 40(2), 246 - 50 Epub 2004 Dec 17. Comparing interferon- gamma release assay with tuberculin skin test readings at 48-72 hours and 144-168 hours with use of 2 commercial reagents; Tat D et al.; BACKGROUND: Despite widespread use, the tuberculin skin test (TST) has many limitations, including a requirement for a second visit between 48 and 72 hours . The goal of this study was to determine the reliability of a TST reading between 144 and 168 hours . METHODS: Tuberculin antigen was applied into both forearms (Aplisol in one arm and Tubersol in the other, from single lots of each product) by the Mantoux method . Blood samples were obtained for interferon- gamma release assay . Subjects were seen at 48-72 hours for the initial (day 2) TST reading and returned at 144-168 hours for a second (day 7) reading . RESULTS: A total of 116 subjects at increased risk for tuberculosis were studied; 25 (22%) had positive results at day 2 with Tubersol and 27 (23%) had positive results at day 2 with Aplisol . Overall agreement between Tubersol and Aplisol at day 2 was 93% (kappa = 0.80) and at day 7 was 94% (kappa = 0.76) . Overall agreement between day 2 and day 7 was 89% for Tubersol and 86% for Aplisol . Discordant results between day 2 and day 7 occurred mostly in persons with a history of bacille Calmette-Guerin vaccination . CONCLUSIONS: Subjects who fail to present at 48-72 hours for TST reading may still have a reliable TST reading at up to 168 hours . Aplisol and Tubersol reagents produce comparable results when compared with the interferon- gamma release assay. Clin Infect Dis, 2005 Jan 15, 40(2), 211 - 7 Epub 2004 Dec 20. Influence of bacille Calmette-Guérin vaccination on size of tuberculin skin test reaction: to what size? Tissot F, Zanetti G, Francioli P, Zellweger JP, Zysset F. BACKGROUND: Previous bacillus Calmette-Guerin (BCG) vaccination can confound the results of a tuberculin skin test (TST) . We sought to determine a cutoff diameter of TST induration beyond which the influence of BCG vaccination was negligible in evaluating potential Mycobacterium tuberculosis infection in a population of health care workers with a high vaccination rate and low incidence of tuberculosis . METHODS: From 1991 through 1998, all new employees at the University Hospital of Lausanne, Switzerland, underwent a 2-step TST at entry visit . We also gathered information on demographic characteristics, along with factors commonly associated with tuberculin positivity, including previous BCG vaccination, history of latent M . tuberculosis infection, and predictors for M . tuberculosis infection . RESULTS: Among the 5117 investigated subjects, we found that influence of BCG vaccination on TST results varied across categories of age (likelihood ratio test, 0.0001) . Prior BCG vaccination had a strong influence on skin test results of <or=18 mm in diameter among persons <40 years old, compared with the influence of factors predictive of M . tuberculosis infection . Prior latent M . tuberculosis infection and travel or employment in a country in which tuberculosis is endemic also had significant influences . CONCLUSIONS: Interpretation of TST reactions of <or=18 mm among BCG-vaccinated persons <40 years of age must be done with caution in areas with a low incidence of tuberculosis . In such a population, except for persons who have never been vaccinated, TST reactions of <or=18 mm are more likely to be the result of prior vaccination than infection and should not systematically lead to preventive treatment. Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 443 - 7 Bacillus patagoniensis sp . nov., a novel alkalitolerant bacterium from the rhizosphere of Atriplex lampa in Patagonia, Argentina; Olivera N et al.; A Gram-positive, rod-shaped, spore-forming bacterium (PAT 05(T)) was isolated from the rhizosphere of the perennial shrub Atriplex lampa in north-eastern Patagonia, Argentina . Its overall biochemical and physiological characteristics indicated that this strain should be placed in the alkaliphilic Bacillus group . Strain PAT 05(T) grew at pH 7-10 (optimum pH 8), but not at pH 6 . Its DNA G+C content was 39.7 mol% . Sequence analysis of the 16S rRNA gene of PAT 05(T) revealed the closest match (99.6 % similarity) with Bacillus sp . DSM 8714 . The highest level of DNA-DNA relatedness (88.6 %) was also found with this strain . On the basis of 16S rRNA gene sequence similarity and phylogenetic analysis, G+C content and DNA-DNA hybridization data, strain PAT 05(T) is related at the species level to Bacillus sp . DSM 8714, a member of a group referred as phenon 4a by Nielsen et al . {Nielsen, P., Fritze, D . & Priest, F . G . (1995) . Microbiology 141, 1745-1761}, which still lacks taxonomic standing . These results support the proposal of strain PAT 05(T) (=DSM 16117(T)=ATCC BAA-965(T)) as the type strain of Bacillus patagoniensis sp . nov. Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 159 - 63 Bacillus saliphilus sp . nov., isolated from a mineral pool in Campania, Italy; Romano I et al.; A haloalkaliphilic Gram-positive bacterium, strain 6AG(T), that grew aerobically at an optimum temperature of 37 degrees C and at pH 7-10 (optimum 9.0), was isolated from algal mat from a mineral pool located in Malvizza in the Campania region (southern Italy) . The isolate tolerated high concentrations of NaCl, up to 25 % . On the basis of 16S rRNA gene sequence similarity, the strain was shown to belong to the genus Bacillus . Analysis of the 16S rRNA gene sequence revealed high similarity between strain 6AG(T) and an unidentified isolate from Hailaer soda lake (China) (99.9 % identity) and two Kenyan isolates, 3E1 and WE4 (98.3 and 97.8 % identity, respectively) . The G+C content of the DNA was 48.4 mol% . The predominant respiratory quinones were MK-7(H(2)), MK-7(H(4)) and DMK-7(H(2)); phosphatidylglycerol and diphosphatidylglycerol were the predominant polar lipids . iC15 : 0 and aiC15 : 0 were the major fatty acids . Strain 6AG(T) accumulated osmolytes . The phylogenetic distance of strain 6AG(T) (=DSM 15402(T)=ATCC BAA-957(T)) from any recognized species within the genus Bacillus allowed it to be classified as the type strain of Bacillus saliphilus sp . nov. Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 119 - 31 Study of mural painting isolates, leading to the transfer of 'Bacillus maroccanus' and 'Bacillus carotarum' to Bacillus simplex, emended description of Bacillus simplex, re-examination of the strains previously attributed to 'Bacillus macroides' and description of Bacillus muralis sp . nov; Heyrman J et al.; A group of 24 strains was isolated from deteriorated mural paintings situated in Spain (necropolis of Carmona) and Germany (church of Greene-Kreiensen) . (GTG)(5)-PCR genomic fingerprinting was performed on these strains to assess their genomic variability and the strains were delineated into four groups . Representatives were studied by 16S rRNA gene sequencing and were found to be closely related to Bacillus simplex and the species 'Bacillus macroides' (strain NCIMB 8796) and 'Bacillus maroccanus' (names not validly published) according to a fasta search . The close similarity between B . simplex, 'B . macroides' NCIMB 8796, 'B . maroccanus' and the mural painting isolates was confirmed by additional (GTG)(5)-PCR, ARDRA, FAME and SDS-PAGE analyses . Furthermore, these techniques revealed that strains of 'Bacillus carotarum', another name that has not been validly published, also showed high similarity to this group of organisms . On the other hand, it was shown that the strains labelled 'B . macroides' in different collections do not all belong to the same species . Strain NCIMB 8796 can be allocated to B . simplex, while strain DSM 54 (=ATCC 12905) shares the highest 16S rRNA gene sequence similarity with Bacillus sphaericus and Bacillus fusiformis (both around 98.6 %) . On the basis of further DNA-DNA hybridization data and the study of phenotypic characteristics, one group of five mural painting strains was attributed to a novel species in the genus Bacillus, for which the name Bacillus muralis sp . nov . is proposed . Finally, the remaining mural painting strains, one (LMG 18508=NCIMB 8796) of two strains belonging to 'B . macroides' and strains belonging to 'B . maroccanus' and 'B . carotarum' are allocated to the species B . simplex and an emended description of B . simplex is given. Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 111 - 7 Bacillus arenosi sp . nov., Bacillus arvi sp . nov . and Bacillus humi sp . nov., isolated from soil; Heyrman J et al.; A group of nine Gram-positive endospore-forming bacteria was isolated from soil of the Drentse A agricultural research area in the Netherlands . Using (GTG)(5)-PCR genomic fingerprinting and fatty acid analysis, the nine isolates were divided into three consistent groups . On the basis of 16S rRNA gene sequence similarity of representative strains, the nine isolates were shown to belong to the genus Bacillus . The first group of four isolates was most closely related to Bacillus carboniphilus (95.5 %) and Bacillus sporothermodurans (95.5 %) . The second and third groups of three and two isolates, respectively, showed highest sequence similarity to Bacillus neidei (97.0 and 97.1 %, respectively) and Bacillus pycnus (both 96.7 %) . A DNA-DNA relatedness study confirmed the consistency of the three groups delineated by (GTG)(5)-PCR and fatty acid analysis . A small number of phenotypic characters allowed differentiation of the three groups of isolates . The three groups therefore represent novel species, for which the names Bacillus humi, Bacillus arenosi and Bacillus arvi are proposed, with LMG 22167(T) (=DSM 16318(T)), LMG 22166(T) (=DSM 16319(T)) and LMG 22165(T) (=DSM 16317(T)) as the respective type strains. Vaccine, 2005 Jan 26, 23(10), 1251 - 7 Acute lower respiratory tract infections and respiratory syncytial virus in infants in Guinea-Bissau: a beneficial effect of BCG vaccination for girls Community based case-control study; Stensballe LG et al.; Among measles unvaccinated infants in Guinea-Bissau, we tested whether case infants with acute lower respiratory tract infection (ALRI), especially ALRI caused by respiratory syncytial virus (RSV), were more likely to be Bacille Calmette Guerin (BCG)-unvaccinated and to have no scar after BCG vaccination than were control infants without symptoms of ALRI . Three hundred and eighty-six case infants with ALRI were identified at a paediatric clinic (N=84), a health centre (N=82), and in a community morbidity surveillance system (N=220) . Control infants were matched on sex, age, and district and were also measles unvaccinated . In ALRI case infants, the adjusted OR of being BCG unvaccinated was 2.87 (1.31-6.32), 1.72 (0.48-6.19) in boys and 4.45 (1.48-13.4) in girls . Among BCG vaccinated ALRI case infants, the adjusted OR of having no BCG scar was 1.54 (0.86-2.75), 0.93 (0.45-1.91) in boys and 2.70 (1.21-6.02) in girls . In ALRI case infants with RSV infection, similar trends were observed . BCG vaccination may have a non-targeted protective effect against ALRI, the effect being most marked in girls. Altern Lab Anim, 2004 Oct, 32(4), 407 - 16 Mitochondrial toxicity detected in a health product with a boar spermatozoan bioassay; Hoornstra D et al.; Seaweed and organic alfalfa capsules sold as "health promoting" products had repeatedly caused emesis in a consumer . Using the boar spermatozoan bioassay, the capsule contents were found to contain a toxic substance that inhibited boar sperm motility and depolarised mitochondria at low exposure concentrations of 10mug/ml . The capsule also contained high amounts (10(5)-10(7)cfu/g), of endospore-forming bacteria and Streptomyces-like bacteria . Bacteria from the capsule produced toxic substances when cultured in the laboratory . Three different toxic responses were provoked in the spermatozoa exposed to extracts from the Streptomyces-like isolates: a) hyperpolarisation of the plasma membrane and depolarisation of the mitochondria; b) depolarisation of mitochondria similar to that caused by the capsule content extract; and c) motility inhibition, with no observed change of any cytosolic transmembrane potential . Membrane potential changes in the sperm cells exposed to the bacterial extracts were similar to those provoked by exposure to valinomycin and bafilomycin A1, to nigericin, and to oligomycin and ionomycin, respectively . Extracts prepared from Bacillus isolated from the capsule non-specifically depolarised all the cellular transmembrane potentials . The results demonstrate the potential value of a cell toxicity assay with boar spermatozoa for detecting hazardous substances in products intended for human consumption, without whole-animal exposure or using fetal calf serum for cell cultures. Nippon Geka Gakkai Zasshi, 2004 Dec, 105(12), 751 - 6 {Pathogenesis and treatment of chronic empyema}; Iuchi K et al.; In chronic empyema (CE), thickened pleura, collapsed chest wall, and the accumulation of purulent fluid in the thoracic cavity are typical findings . Patients complaints of symptoms with bronchopleural fistula (BPF) . On the other hand, there is another type of CE in which the pleural space expands progressively to shift the neighboring lungs, mediastinum, and diaphragm . This type of CE is considered to be chronic expanding hematoma (Reid et al.) occurring in the thoracic cavity . In the empyemic cavity, mycobacterial infection is found approximately in 20-30% of cases, pyogenic bacillus or fungus in about 40%, but the cavity is aseptic in other 30-40% . Although the fundamental treatment procedures include decortication and pleuropneumonectomy, the method of muscle or omental plombage to manage dead space or BPF are far superior functionally in intractable CE . Recently, the methods of plastic and reconstructive surgery have been used to utilize the muscle or omentum more effectively . The classic thoracoplasty procedure should not be undertaken unnecessarily to avoid additional deterioration of respiratory function . Additionally, it should be remembered that malignant lymphoma occurs frequently in the empyemic chest wall. J Microbiol, 2004 Dec, 42(4), 340 - 5 Expression of a recombinant Cry1Ac crystal protein fused with a green fluorescent protein in Bacillus thuringiensis subsp . kurstaki Cry-B; Roh JY et al.; To investigate the co-expression and crystallization of a fusion gene between the Bacillus thuringiensis crystal protein and a foreign protein in B . thuringiensis, the expression of the Cry1Ac fused with green fluorescent protein (GFP) genes in a B . thuringiensis Cry(-)B strain was examined . The cry1Ac gene was cloned in the B . thuringiensis-E . coli shuttle vector, pHT3101, under the control of the native cry1Ac gene promoter, while the GFP gene was inserted into the XhoI site upstream of the proteolytic cleavage site, in the middle region of the cry1Ac gene (pProAc-GFP) . The B . thuringiensis Cry(-)B strain carrying pProAc-GFP (ProAc-GFP/CB) did not produce any inclusion bodies . However, the transformed strain expressed fusion protein forms although the expression level was relatively low . Furthermore, an immunoblot analysis using GFP and Cry1Ac antibodies showed that the fusion protein was not a single species, but rather multiple forms . In addition, the N-terminal fragment of Cry1Ac and a non-fused GFP were also found in the B . thuringiensis Cry(-)B strain after autolysis . The sporulated cells before autolysis and the spore-crystal mixture after autolysis of ProAc-GFP/CB exhibited insecticidal activities against Plutella xylostella larvae . Accordingly, the current results suggest that a fusion crystal protein produced by the transfomant, ProAc-GFP/CB, can be functionally expressed but easily degraded in B . thuringiensis. J Virol, 2005 Feb, 79(3), 1452 - 62 Vaccination of Rhesus Macaques with Recombinant Mycobacterium bovis Bacillus Calmette-Guerin Env V3 Elicits Neutralizing Antibody-Mediated Protection against Simian-Human Immunodeficiency Virus with a Homologous but Not a Heterologous V3 Motif; Someya K et al.; Although the correlates of vaccine-induced protection against human immunodeficiency virus type 1 (HIV-1) are not fully known, it is presumed that neutralizing antibodies (NAb) play a role in controlling virus infection . In this study, we examined immune responses elicited in rhesus macaques following vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guerin expressing an HIV-1 Env V3 antigen (rBCG Env V3) . We also determined the effect of vaccination on protection against challenge with either a simian-human immunodeficiency virus (SHIV-MN) or a highly pathogenic SHIV strain (SHIV-89.6PD) . Immunization with rBCG Env V3 elicited significant levels of NAb for the 24 weeks tested that were predominantly HIV-1 type specific . Sera from the immunized macaques neutralized primary HIV-1 isolates in vitro, including HIV-1(BZ167/X4), HIV-1(SF2/X4), HIV-1(CI2/X4), and, to a lesser extent, HIV-1(MNp/X4), all of which contain a V3 sequence homologous to that of rBCG Env V3 . In contrast, neutralization was not observed against HIV-1(SF33/X4), which has a heterologous V3 sequence, nor was it found against primary HIV-1 R5 isolates from either clade A or B . Furthermore, the viral load in the vaccinated macaques was significantly reduced following low-dose challenge with SHIV-MN, and early plasma viremia was markedly decreased after high-dose SHIV-MN challenge . In contrast, replication of pathogenic SHIV-89.6PD was not affected by vaccination in any of the macaques . Thus, we have shown that immunization with an rBCG Env V3 vaccine elicits a strong, type-specific V3 NAb response in rhesus macaques . While this response was not sufficient to provide protection against a pathogenic SHIV challenge, it was able to significantly reduce the viral load in macaques following challenge with a nonpathogenic SHIV . These observations suggest that rBCG vectors have the potential to deliver an appropriate virus immunogen for desirable immune elicitations. Anal Chem, 2005 Jan 15, 77(2), 631 - 8 Feasibility of detection and identification of individual bioaerosols using laser-induced breakdown spectroscopy; Dixon PB et al.; The detection and identification of individual bioaerosols using laser-induced breakdown spectroscopy (LIBS) is investigated using aerosolized Bacillus spores . Spores of Bacillus atrophaeous, Bacillus pumilus, and Bacillus stearothemophilus were introduced into an aerosol flow stream in a prescribed manner such that single-particle LIBS detection was realized . Bacillus spores were successfully detected based on the presence of the 393.4- and 396.9-nm calcium atomic emission lines . Statistical analyses based on the aerosol number density, the LIBS-based spore sampling frequency, and the distribution of the resulting calcium mass loadings support the conclusion of individual spore detection within single-shot laser-induced plasmas . The average mass loadings were in the range of 2-3 fg of calcium/Bacillus spore, which corresponds to a calcium mass percentage of approximately 0.5% . While individual spores were detected based on calcium emission, the resulting Bacillus spectra were free from CN emission bands, which has implications for the detection of elemental carbon, and LIBS-based detection of single spores based on the presence of magnesium or sodium atomic emission was unsuccessful . Based on the current instrumental setup and analyses, real-time LIBS-based detection and identification of single Bacillus spores in ambient (i.e., real life) conditions appears unfeasible. Appl Microbiol Biotechnol . 2005 Jan 13; {Epub ahead of print} The cry3Aa gene of Bacillus thuringiensis Bt886 encodes a toxin against long-horned beetles; Chen J et al.; This report describes the identification of a new toxigenic strain of Bacillus thuringiensis specific for long-horned beetles . B . thuringiensis Bt866 encodes a cry3Aa-like gene (Bt886cry3Aa) that is 1,956 bp in length and is predicted to encode an 85.78-kDa protein . The gene is highly similar to cry3Aa1, differing in only six nucleotides and four amino acids . The four disparate amino acids occur within the conserved domains of the Cry3Aa toxin . The expression of Bt866cry3A in Escherichia coli cells resulted in a high level of toxicity toward Apriona germari Hope larvae . More than 75% of the larvae were killed; and the remaining survivors exhibited slower growth . These results indicate that the toxigenic strain Bt886cry3Aa encodes a protein that is specific against long-horned beetles . Genetic engineering of the Bt866cry3Aa gene into poplar plantations may provide resistance to long-horned beetles. Microbiol Res, 2004, 159(4), 347 - 54 Aerobic chromate reduction by chromium-resistant bacteria isolated from serpentine soil; Pal A et al.; A group of 34 chromium-resistant bacteria were isolated from naturally occurring chromium percolated serpentine soil of Andaman (India) . These isolates displayed different degrees of chromate reduction under aerobic conditions . One of the 34 isolates identified as Bacillus sphaericus was tolerant to 800 mgl(-1) Cr(VI) and reduced > 80% Cr(VI) during growth . In Vogel Bonner broth, B . sphaericus cells (10(10) cells ml(-1)) reduced 62% of 20mg l(-1) of Cr(VI) in 48h with concomitant discoloring of yellow medium to white one . Reduction of chromate was pronounced by the addition of glucose and yeast extract as electron donors . In the presence of 4.0 g l(-1) of glucose, 20mg l(-1) of Cr(VI) was reduced to 2.45mg l(-1) after 96h of incubation . Optimum pH and temperature for reduction were 6.0 and 25 degrees C, respectively . Increase in cell density and initial Cr(VI) concentration increased chromate reduction but was inhibited by metal ions like, Ni2+, Co2+, Cd2+ and Pb2+ . Experiments with cell-free extracts indicated that the soluble fraction of the cell was responsible for aerobic reduction of Cr(VI) by this organism. Chang Gung Med J, 2004 Oct, 27(10), 766 - 9 Fatal aeromonas hydrophila infection of soft tissue after endoscopic injection sclerotherapy for gastric variceal bleeding; Liu BM et al.; Aeromonas hydrophila, an anaerobic gram-negative bacillus, can cause severe infections in immune-compromised patients . We present a 45-year-old cirrhotic man who suffered from hematemesis and received emergency endoscopic injection sclerotherapy (EIS) for gastric variceal bleeding . Twenty-one hours after EIS, painful swelling of the bilateral lower extremities and fever occurred . Severe soft-tissue infections with emergence of hemorrhagic bullae over the bilateral lower extremities followed . Even under aggressive treatment, the patient died of overwhelming sepsis 42 hours after EIS . Cultures of the blood and serosanguineous fluid from the hemorrhagic bullae revealed Aeromonas hydrophila . To the best of our knowledge, this is the first case of fatal Aeromonas hydrophila infection after emergancy EIS for gastric variceal bleeding reported in the English literature . It is worth emphasizing that physicians should consider Aeromonas hydrophila infection in cirrhotic patients who develop soft-tissue infections after variceal bleeding whether emergency EIS has been performed or not. New Microbiol, 2004 Oct, 27(4), 411 - 3 Detection of Bacillus species in selected meals from an apulian catering service; Fontanarosa M et al.; The authors have started a microbiological survey of the meals given in a factory canteen in the town of Bari with the evaluation of food contamination from the bacteria of the genus Bacillus . In the examined meals, all the detected Bacillus species have the potential to cause food-borne illnesses, even if the relatively low bacterial counts are not dangerous for healthy consumers . The study underlines the need of guidelines to enstablish which levels of food contamination are significant potential risks to all community catering service users. New Microbiol, 2004 Oct, 27(4), 353 - 60 Morphological changes and outer membrane protein patterns in Helicobacter pylori during conversion from bacillary to coccoid form; Citterio B et al.; Conversion from bacillary to fully coccoid form via an intermediate U-and V-shaped form has been described in prolonged cultures of H . pylori . This morphological transformation may be the expression of transitory adaptation to a particular environment and may play an important role in antibiotic resistance and the difficulty to eradicate the pathogen . The aim of this study was to evaluate morphological and outer membrane protein changes in H . pylori during ageing-induced conversion to coccoid morphology . We used two H . pylori strains (the reference NCTC 11639 and a fresh clinical isolate) cultivated in microaerophilic environment at 37 degrees C, monitoring their morphological and biochemical evolutions for 11 days . Microscopic examination revealed the passage from spiral to U- and V-shaped form after 5-8 days of incubation, the conversion to coccoid form and the entry into viable but non-culturable state (VBNC) between days 9 and 11 . Protein pattern difference appeared at 97.4 to 45 and 30 kDa molecular weight . Biochemical tests demonstrated not only a modification of outer membrane protein profiles, but also an intra-specific variability by comparison between the two analysed strains . Our findings suggest that structural and outer membrane changes associated with coccoid transformation represent a typical response in H . pylori and may constitute a survival strategy in adverse environmental conditions. Can J Microbiol, 2004 Nov, 50(11), 973 - 5 Fate of Bacillus thuringiensis strains in different insect larvae; Suzuki MT et al.; In favorable conditions Bacillus thuringiensis spores germinate and vegetative cells multiply, whereas in unfavorable conditions Bacillus thuringiensis sporulates and produces insecticidal crystal proteins . The development of B . thuringiensis strains was investigated in the larvae of insects belonging to the orders Lepidoptera and Diptera . Bacillus thuringiensis strains able to kill the insects did not always multiply in cadavers . Strains with no specificity to kill the insect sometimes multiplied when the insects were killed mechanically . These results indicate that some insect larvae represent an environment that favors the germination of B . thuringiensis spores and the multiplication of vegetative cells; however, there was no correlation between the toxin specificity and the specificity of the host. Can J Microbiol, 2004 Oct, 50(10), 771 - 8 Co-induction of methyltransferase Rv0560c by naphthoquinones and fibric acids suggests attenuation of isoprenoid quinone action in Mycobacterium tuberculosis; Garbe TR; The superoxide generator menadione was previously demonstrated as an inducer of growth stage dependent protein patterns in Mycobacterium tuberculosis . The present study refines this observation by characterizing a novel 27-kDa protein that had not been observed in previous studies relying on younger cultures . A very similar response, based on two-dimensional gel electrophoretic analyses, was induced by the closely related naphthoquinone plumbagin . The 27-kDa protein was also induced by the pro-oxidant peroxisome proliferator gemfibrozil and to a lesser extent by the structurally related compounds fenofibrate and clofibrate . N-terminal sequence data of proteolytic fragments from the 27-kDa protein demonstrated its identity with protein Rv0560c, previously demonstrated to be inducible by salicylate, which also possesses peroxisome proliferating properties . Protein Rv0560c bears three conserved motifs characteristic of S-adenosylmethionine-dependent methyltransferases . Further sequence similarities suggest a function in the bio syn thesis of isoprenoid compounds, e.g., tocopherol, ubiquinone, and sterols . Such involvement is supported by the recognized yet unexplained widespread interference of menadione, salicylate, and fibrates with the isoprenoid quinones ubiquinone, menaquinone, and vitamin K . Induction of Rv0560c by fibrates, salicylate, and naphthoquinones is thus suggested to be caused by action on the plasma membrane, reminiscent of cytochrome P450BM-3 induction by fibrates in Bacillus megaterium, which catalyzes the hydroxylation of fatty acids and thus modulates membrane properties. Environ Microbiol, 2005 Jan, 7(1), 34 - 9 The clonal structure of Bacillus thuringiensis isolates from north-east Poland does not correlate with their cry gene diversity; Rwiecicka I et al.; Summary The genetic relationship among 103 natural Bacillus thuringiensis isolates was investigated on the basis of polymerase chain reaction amplification of their specific crystal (cry) protein type genes and chromosomal DNA profiling by pulsed-field gel electrophoresis (PFGE) . The strains were recovered from the intestines of small wild rodents and insectivores from the Biebrza National Park and the Lomza Landscape Park of the Narew River Valley in north-east Poland . The percentage of B . thuringiensis strains harbouring genes coding for toxins active against Lepidoptera (cry1, cry2, cry9) was very high (64%) compared with that of Diptera-specific strains (cry4, 14%) . No strain with cry genes coding for proteins directed against coleopteran larvae and nematodes was found . After digestion with NotI and AscI, only nine PFGE pulsotypes were observed among all isolates, indicating a clonal structure for the B . thuringiensis population from NE Poland . Interestingly, no correlation was observed between the DNA pulsotype strains and their crystal gene content and diversity . These results therefore emphasize the importance of cry gene horizontal transfer occurring among natural isolates of B . thuringiensis. Colloids Surf B Biointerfaces, 2005 Feb 10, 40(2), 93 - 8 Inactivation of bacterial endospores by photocatalytic nanocomposites; Lee SH et al.; A novel biocidal photocatalytic nanocomposite, composed of TiO(2) and multi-walled carbon nanotubes (MWNTs), was synthesized via wet chemistry followed by a heat treatment . Uniform anatase coatings on MWNTs were successfully obtained with a thickness of a few nanometers . The nanostructure of the composite was determined by high resolution transmission electron microscopy (HRTEM) and X-ray diffraction (XRD) . The needle-like shape of the nanocomposite provided more than three times higher photocatalytic specific surface area than commercial TiO(2) nanoparticles (Degussa P25) when dispersed in water . Moreover, under ultraviolet (UV) radiation the excited electrons can be trapped at the interface between the TiO(2) layer and MWNTs and they can also be scavenged through the conductive graphitic layers . Thus, an intense photochemical reaction yielding a powerful biocide can be expected . Irradiating bacterial endospores (Bacillus cereus) with solar UV lamps in presence of the novel photocatalyst successfully inactivated the spores while solar UV lamps only or solar UV Lamps with Degussa P25 showed no significant inactivating behavior . Performance of photocatalytic nanocomposites was assessed based on time to achieve 90% inactivation of spores (LD(90)) and also in terms of time required to achieve a 1.0log(10) reduction of spores in the tail region of the inactivation curve. Biochemistry, 2005 Jan 18, 44(2), 589 - 597 A Detergent-like Mechanism of Action of the Cytolytic Toxin Cyt1A from Bacillus thuringiensis var . israelensis; Manceva SD et al.; The cytolytic delta-endotoxin Cyt1A from Bacillus thuringiensis var . israelensis is used in commercial preparations of environmentally safe insecticides . The current hypothesis on its mode of action is that the toxin self-assembles into well-defined cation-selective channels or pores, which results in colloid-osmotic lysis of the cell . Recently, a new hypothesis has been put forward suggesting that Cyt1A rather nonspecifically aggregates on the membrane surface and acts in a detergent-like manner . To distinguish between these two hypotheses, we investigated whether in the presence of lipid Cyt1A self-assembles into stoichiometric oligomers, which are characteristic of pores or channels, or aggregates into nonstoichiometric complexes, which would support the detergent-like model . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that in the presence of lipid Cyt1A forms protein aggregates with a broad range of molecular weights, some being too large to enter the gel . Cyt1A tryptophan (Trp) fluorescence in the presence of lipid exhibited a decrease in anisotropy and quantum yield, but an unchanged lifetime, which is consistent with the presence of toxin aggregates in the membrane . Electrostatic interactions between the charged amino acid residues and the lipid headgroups are responsible for bringing the protein to the membrane surface, while hydrophobic and/or van der Waals interactions make the membrane binding irreversible . Fluorescence photobleaching recovery, a technique that measures the diffusion coefficient of fluorescently labeled particles, and epifluorescence microscopy revealed that upon addition of Cyt1A lipid vesicles were broken into smaller, faster diffusing objects . Since no change in size or morphology of the vesicles is expected when pores are formed in the osmotically equilibrated membranes, our results support the detergent-like mode of action of Cyt1A. Crit Care Med, 2004 Nov, 32(11), 2183 - 90 De-escalation therapy in ventilator-associated pneumonia; Rello J et al.; OBJECTIVE: To evaluate de-escalation of antibiotic therapy in patients with ventilator-associated pneumonia . DESIGN: Prospective observational study during a 43-month period . SETTING: Medical-surgical intensive care unit . PATIENTS: One hundred and fifteen patients admitted to the intensive care unit with clinical diagnosis of ventilator-associated pneumonia . All the episodes of ventilator-associated pneumonia received initial broad-spectrum coverage followed by reevaluation according to clinical response and microbiology . Quantitative cultures obtained by bronchoscopic examination or tracheal aspirates were used to modify therapy . INTERVENTIONS:: None . MEASUREMENTS AND MAIN RESULTS: One hundred and twenty-one episodes of ventilator-associated pneumonia were diagnosed . Change of therapy was documented in 56.2%, including de-escalation (the most frequent cause) in 31.4% (increasing to 38% if isolates were sensitive) . Overall intensive care unit mortality rate was 32.2% . Inappropriate antibiotic therapy was identified in 9% of cases and was associated with 14.4% excess intensive care unit mortality . Quantitative tracheal aspirates and bronchoscopic samples (58 protected specimen brush and three bronchoalveolar lavage) were associated with 32.7% and 29.5% intensive care unit mortality and 29.3% and 34.4% de-escalation rate . De-escalation was lower (p < .05) in the presence of nonfermenting Gram-negative bacillus (2.7% vs . 49.3%) and in the presence of late-onset pneumonia (12.5% vs . 40.7%) . When the pathogen remained unknown, half of the patients died and de-escalation was not performed . CONCLUSION: De-escalation was the most important cause of antibiotic modification, being more feasible in early-onset pneumonia and less frequent in the presence of nonfermenting Gram-negative bacillus . The impact of quantitative tracheal aspirates or bronchoscopic techniques was comparable in terms of mortality. Appl Biochem Biotechnol, 2005 Jan, 120(1), 71 - 80 Characterization of a Novel Feather-Degrading Bacillus sp . Strain; Werlang PO et al.; Feather waste is generated in large amounts as a byproduct of commercial poultry processing . This residue is almost pure keratin, which is not easily degradable by common proteolytic enzymes . A feather-degrading bacterium was isolated from poultry feathers in decomposition . The strain identified as kr16 showed important feather-degrading activity when grown on basal medium containing 10 g/L of native feather as the source of energy, carbon, and nitrogen . The isolate was characterized according to the phenotypical characteristics and biochemical profiling that belong to the Bacillus genus . Keratinolytic activity of this isolate was monitored during cultivation of the bacterium on raw feathers at different temperatures . Maximum growth and feather-degrading activity were observed at 30-37 degrees C . The keratinolytic enzyme had a pH optimum ranging from 8.0 to 11.0 and a temperature optimum of 45-65 degrees C . The keratinase was strongly inhibited by EDTA and the metal ions Hg2+ and Sn2+. Appl Environ Microbiol, 2005 Jan, 71(1), 185 - 9 Cyt1A of Bacillus thuringiensis Delays Evolution of Resistance to Cry11A in the Mosquito Culex quinquefasciatus; Wirth MC et al.; Insecticides based on Bacillus thuringiensis subsp . israelensis have been used for mosquito and blackfly control for more than 20 years, yet no resistance to this bacterium has been reported . Moreover, in contrast to B . thuringiensis subspecies toxic to coleopteran or lepidopteran larvae, only low levels of resistance to B . thuringiensis subsp . israelensis have been obtained in laboratory experiments where mosquito larvae were placed under heavy selection pressure for more than 30 generations . Selection of Culex quinquefasciatus with mutants of B . thuringiensis subsp . israelensis that contained different combinations of its Cry proteins and Cyt1Aa suggested that the latter protein delayed resistance . This hypothesis, however, has not been tested experimentally . Here we report experiments in which separate C . quinquefasciatus populations were selected for 20 generations to recombinant strains of B . thuringiensis that produced either Cyt1Aa, Cry11Aa, or a 1:3 mixture of these strains . At the end of selection, the resistance ratio was 1,237 in the Cry11Aa-selected population and 242 in the Cyt1Aa-selected population . The resistance ratio, however, was only 8 in the population selected with the 1:3 ratio of Cyt1Aa and Cry11Aa strains . When the resistant mosquito strain developed by selection to the Cyt1Aa-Cry11Aa combination was assayed against Cry11Aa after 48 generations, resistance to this protein was 9.3-fold . This indicates that in the presence of Cyt1Aa, resistance to Cry11Aa evolved, but at a much lower rate than when Cyt1Aa was absent . These results indicate that Cyt1Aa is the principal factor responsible for delaying the evolution and expression of resistance to mosquitocidal Cry proteins. Appl Environ Microbiol, 2005 Jan, 71(1), 105 - 13 Identification and Partial Characterization of the Nonribosomal Peptide Synthetase Gene Responsible for Cereulide Production in Emetic Bacillus cereus; Ehling-Schulz M et al.; Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus . Due to its chemical structure, (d-O-Leu-d-Ala-l-O-Val-l-Val)(3), cereulide might be synthesized nonribosomally . Therefore, degenerate PCR primers targeted to conserved sequence motifs of known nonribosomal peptide synthetase (NRPS) genes were used to amplify gene fragments from a cereulide-producing B . cereus strain . Sequence analysis of one of the amplicons revealed a DNA fragment whose putative gene product showed significant homology to valine activation NRPS modules . The sequences of the flanking regions of this DNA fragment revealed a complete module that is predicted to activate valine, as well as a putative carboxyl-terminal thioesterase domain of the NRPS gene . Disruption of the peptide synthetase gene by insertion of a kanamycin cassette through homologous recombination produced cereulide-deficient mutants . The valine-activating module was highly conserved when sequences from nine emetic B . cereus strains isolated from diverse geographical locations were compared . Primers were designed based on the NRPS sequence, and the resulting PCR assay, targeting the ces gene, was tested by using a panel of 143 B . cereus group strains and 40 strains of other bacterial species showing PCR bands specific for only the cereulide-producing B . cereus strains. Yi Chuan . 2003 Jan;25(1):71. {Miniscale Preparation for Large Plasmid DNA from Bacillus thuringiensis.}; Zhong WF et al.; Miniprep of low copy number and large plasmid was done from three subspecies of Bacillus thuringiensis.The plasmid was purified using PEG 6000 instead of phenol and chloroform.Experiments demonstrate that consequences from this protocol were stable,the quality and yield of plasmid DNA produced from it have met the standard for many molecular biology experiments. Arch Biochem Biophys, 2005 Feb 15, 434(2), 289 - 98 Purification, characterization cloning, and sequencing of metalloendopeptidase from Streptomyces septatus TH-2; Hatanaka T et al.; Streptomyces septatus TH-2 secretes a large amount of a protease when cultured on a medium containing K(2)HPO(4) and glucose . The enzyme was purified to homogeneity by a three-step procedure . This enzyme had a molecular mass of approximately 35kDa, and was particularly inhibited by EDTA and phosphoramidon . Its substrate specificity was investigated using novel fluorescence energy transfer combinatorial libraries . The protease was found to prefer Phe and Tyr at the P(1) position, a hydrophobic or basic residue at the P(2) position, and a basic or small residue at the P(3) position . Its gene was cloned and sequenced, and its deduced amino acid sequence contained an HEXXH consensus sequence for zinc binding, confirming that it encodes metalloendopeptidase . The primary structure of the enzyme showed 40 and 69% identities with that of thermolysin from Bacillus thermoproteolyticus and that of a metalloendopeptidase from Streptomyces griseus, respectively. Nat Biotechnol, 2005 Jan, 23(1), 57 - 62 Insect resistance management in GM crops: past, present and future; Bates SL et al.; Transgenic plants expressing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) were first commercialized in 1996 amid concern from some scientists, regulators and environmentalists that the widespread use of Bt crops would inevitably lead to resistance and the loss of a 'public good,' specifically, the susceptibility of insect pests to Bt proteins . Eight years later, Bt corn and cotton have been grown on a cumulative area >80 million ha worldwide . Despite dire predictions to the contrary, resistance to a Bt crop has yet to be documented, suggesting that resistance management strategies have been effective thus far . However, current strategies to delay resistance remain far from ideal . Eight years without resistance provides a timely opportunity for researchers, regulators and industry to reassess the risk of resistance and the most effective strategies to preserve Bt and other novel insect-resistant crops in development. Int J Tuberc Lung Dis, 2004 Dec, 8(12), 1443 - 7 Physician attitudes regarding bacille Calmette-Guérin vaccination and treatment of latent tuberculosis infection; Salazar-Schicchi J et al.; OBJECTIVE: To investigate the attitudes of international medical graduates (IMGs) about treatment of latent tuberculosis infection (LTBI) . DESIGN: Anonymous survey among physicians in training, all IMGs from TB endemic countries . Attitudes were assessed through hypothetical scenarios regarding the protective effect of BCG and LTBI treatment . RESULTS: Of 77 participants, 72.4% were male, the mean age was 31.4 years, and mean years post-graduation 8 . Positive tuberculin skin tests (TSTs) were reported among 64.0%, and 89.6% had received BCG vaccine . Over a quarter of IMGs (27.4%) believed that BCG protects for many years . Only 59.2% believed that LTBI treatment was effective and 16% that the risks of treatment were greater than the benefits . Most would treat BCG-vaccinated patients with positive TST in various scenarios (63-94.7%), with less agreement about treating themselves (49.3%) or their family members (54.2%) . For recent converters, more than 80% would recommend LTBI treatment . Over half felt that LTBI treatment should be mandatory for new immigrants with positive TST . CONCLUSIONS: IMGs are cautious about LTBI treatment except for recent converters . They are less likely to treat themselves and family members than others . Educational efforts should address discrepancies between these physicians' attitudes and current guidelines for treating LTBI in the US. Int J Tuberc Lung Dis, 2004 Dec, 8(12), 1437 - 42 Can BCG-vaccinated children be included in tuberculin surveys to estimate the annual risk of tuberculous infection in India? Chadha VK, Jagannatha PS, Kumar P. SETTING: Selected villages in three defined zones of India . OBJECTIVES: To compare the estimated prevalence of tuberculous infection among children with and without bacille Calmette-Guerin (BCG) scar . STUDY DESIGN: During a nationwide tuberculin survey, 1-9-year-old children were tuberculin tested using 1TU-PPD RT23 with Tween 80 . RESULTS: In the 5-9 year age group, subgroups of tuberculous infected children could be seen as distinct humps among those with or without BCG scar, but not in those aged 1-4 years . In children aged 1-4 years, the estimated prevalence of infection was respectively 3.5%, 3.8% and 3.6% among children without BCG scar, and 4.8%, 4.7% and 4.5% among children with BCG scar in the western, northern and eastern zones . In those aged 5-9 years, the estimated prevalence was respectively 10.4%, 11.0% and 9.1% among children without BCG scar and 11%, 11.9% and 8.7% among children with BCG scar in the three zones . Thus, in children aged 1-4 years, the estimated prevalence among those with BCG scar was considerably higher than in those without BCG scar . This difference was small in those aged 5-9 years . CONCLUSION: Tuberculin surveys may be conducted irrespective of BCG scar status among children aged 5-9 years, when BCG vaccination is given using Danish 1331 strain during infancy under the Expanded Program of Immunization. Infect Control Hosp Epidemiol, 2004 Dec, 25(12), 1067 - 71 Risk of tuberculous infection among healthcare workers in a tertiary-care hospital in Ankara, Turkey; Keskiner R et al.; OBJECTIVE: To determine risk factors for tuberculin skin test (TST) positivity among healthcare workers (HCWs) . DESIGN: Two-step TST was performed in 2002 . SETTING: Tertiary-care hospital in Ankara, Turkey . PARTICIPANTS: A sample of 491 hospital HCWs were included . Information related to demographics, profession, work duration, department, and individual and family history of tuberculosis (TB) was obtained by a structured questionnaire . RESULTS: Four hundred eight (83%) had two-step TST positivity . On multivariate analysis, male physicians (relative risk {RR}, 1.5; 95% confidence interval {CI95}, 1.23-1.69; P = .001), nurses (RR, 1.5; CI95, 1.29-1.66; P = .005), radiology technicians (RR, 1.7; CI95, 1.35-1.73; P = .002), laboratory technicians (RR, 1.6; CI95, 1.3-1.74; P = .007), and male housekeepers (RR, 1.6; (HCWs) . CI95, 1.38-1.7; P < .001) had a higher risk than did female physicians . Among laboratory technicians, radiology technicians had the highest TST positivity (85%) . HCWs working for less than 1 year (RR, 0.8; CI95, 0.72-0.98; P = .027) had a lower risk of infection . The HCWs having bacille Calmette-Guerin vaccination (RR, 1.12; CI95, 1.08-1.45) had higher TST positivity . CONCLUSION: Male physicians, nurses, and laboratory technicians had increased risk of Mycobacterium tuberculosis infection in this setting, but community exposure likely accounted for most infections. J Membr Biol, 2004 Sep 1, 201(1), 9 - 24 Role of arginine residues on the S4 segment of the Bacillus halodurans Na+ channel in voltage-sensing; Chahine M et al.; The one-domain voltage-gated sodium channel of Bacillus halodurans (NaChBac) is composed of six transmembrane segments (S1-S6) comprising a pore-forming region flanked by segments S5 and S6 and a voltage-sensing element composed of segment S4 . To investigate the role of the S4 segment in NaChBac channel activation, we used the cysteine mutagenesis approach where the positive charges of single and multiple arginine (R) residues of the S4 segment were replaced by the neutrally charged amino acid cysteine (C) . To determine whether it was the arginine residue itself or its positive charge that was involved in channel activation, arginine to lysine (R to K) mutations were constructed . Wild-type (WT) and mutant NaChBac channels were expressed in tsA201 cells and Na+ currents were recorded using the whole-cell configuration of the patch-clamp technique . The current/voltage (I-V) and conductance/voltage (G-V) relationships steady-state inactivation (h(infinity)) and recovery from inactivation were evaluated to determine the effects of the S4 mutations on the biophysical properties of the NaChBac channel . R to C on the S4 segment resulted in a slowing of both activation and inactivation kinetics . Charge neutralization of arginine residues mostly resulted in a shift toward more positive potentials of G-V and h(infinity) curves . The G-V curve shifts were associated with a decrease in slope, which may reflect a decrease in the gating charge involved in channel activation . Single neutralization of R114, R117, or R120 by C resulted in a very slow recovery from inactivation . Double neutralization of R111 and R129 confirmed the role of R111 in activation and suggested that R129 is most probably not part of the voltage sensor . Most of the R to K mutants retained WT-like current kinetics but exhibited an intermediate G-V curve, a steady-state inactivation shifted to more hyperpolarized potentials, and intermediate time constants of recovery from inactivation . This indicates that R, at several positions, plays an important role in channel activation . The data are consistent with the notion that the S4 is most probably the voltage sensor of the NaChBac channel and that both positive charges and the nature of the arginine residues are essential for channel activation. J Clin Microbiol, 2005 Jan, 43(1), 523 - 5 Multiplex PCR-identified cutaneous tuberculosis evoked by Mycobacterium bovis BCG vaccination in a healthy baby; Okazaki T et al.; This is the first identified case of Mycobacterium bovis bacillus Calmette-Guerin (BCG)-derived cutaneous tuberculosis that localizes at a place different from the vaccination site in hosts without immune deficiency . A healthy baby with a developing abscess is described . A multiplex PCR identified the abscess as originating from M . bovis BCG Tokyo 172. J Clin Microbiol, 2005 Jan, 43(1), 41 - 8 Multispacer typing technique for sequence-based typing of Bartonella quintana; Foucault C et al.; Bartonella quintana is a worldwide fastidious bacterium of the Alphaproteobacteria responsible for bacillary angiomatosis, trench fever, chronic lymphadenopathy, and culture-negative endocarditis . The recent genome sequencing of a B . quintana isolate allowed us to propose a genome-wide sequence-based typing method . To ensure sequence discrimination based on highly polymorphic areas, we amplified and sequenced 34 spacers in a large collection of B . quintana isolates . Six of these exhibited polymorphisms and allowed the characterization of 4 genotypes . However, the strain variants suggested by the noncoding sequences did not correlate with the results of pulsed-field gel electrophoresis (PFGE), which suggested a higher degree of variability . Modification of the PFGE profile of one isolate after nine subcultures confirmed that rearrangement frequencies are high in this species, making PFGE unreliable for epidemiological purposes . The low extent of sequence heterogeneity in the species suggests a recent emergence of this bacterium as a human pathogen . Direct typing of natural samples allowed the identification of a fifth genotype in the DNA extracted from a human body louse collected in Burundi . We have named the typing technique herein described multispacer typing. FEBS J, 2005 Jan, 272(1), 259 - 68 Interaction of the E2 and E3 components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus . Use of a truncated protein domain in NMR spectroscopy; Allen MD et al.; A (15)N-labelled peripheral-subunit binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2p) and the dimer of a solubilized interface domain (E3int) derived from the dihydrolipoyl dehydrogenase (E3) were used to investigate the basis of the interaction of E2p with E3 in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus . Thirteen of the 55 amino acids in the PSBD show significant changes in either or both of the (15)N and (1)H amide chemical shifts when the PSBD forms a 1 : 1 complex with E3int . All of the 13 amino acids reside near the N-terminus of helix I of PSBD or in the loop region between helix II and helix III . (15)N backbone dynamics experiments on PSBD indicate that the structured region extends from Val129 to Ala168, with limited structure present in residues Asn126 to Arg128 . The presence of structure in the region before helix I was confirmed by a refinement of the NMR structure of uncomplexed PSBD . Comparison of the crystal structure of the PSBD bound to E3 with the solution structure of uncomplexed PSBD described here indicates that the PSBD undergoes almost no conformational change upon binding to E3 . These studies exemplify and validate the novel use of a solubilized, truncated protein domain in overcoming the limitations of high molecular mass on NMR spectroscopy. J Food Prot, 2004 Dec, 67(12), 2805 - 8 Incidence and characterization of Bacillus cereus isolated from traditional fermented meals in Nigeria; Oguntoyinbo FA et al.; The aim of this study was to examine the presence of Bacillus cereus in fermented meals used in food seasoning in Nigeria . The microbial profiles of iru and ogiri, two Nigerian fermented vegetable proteins, were examined for presence of B . cereus . In the 50 samples tested, B . cereus was detected in all the samples, with the level of detection ranging from log 6.3 to log 8.3 g(-1) sample . Phenotypic characteristics of the B . cereus isolates showed that all of them could not ferment many sugars, most especially mannitol, but they utilized propionate citrate as a source of carbon and grew anaerobically . The isolates do not produce gas from glucose but hydrolyzed starch, casein, and gelatin . API-50CHB combined with API-20E identified the isolates as B . cereus . The diarrheal enterotoxin was detected by a reversed passive latex agglutination test kit . Results showed no significant difference in toxin production between ogiri and iru B . cereus isolated from different sources; all the isolates also demonstrated positive hemolytic activity . The API-ZYM enzyme profile showed that the strains have poor hydrolytic enzyme potential; hence, their possible contributions to the fermentation of vegetable protein is doubtful . This study established the proliferation of B . cereus in fermented protein meal and determined the diarrheal toxin production potential of the organism. Eur J Pediatr . 2005 Jan 5; {Epub ahead of print} Variable outcome of experimental interferon-gamma therapy of disseminated Bacillus Calmette-Guerin infection in two unrelated interleukin-12Rbeta1-deficient Slovakian children; Ulrichs T et al.; Mycobacterium bovis Bacillus Calmette-Guerin (BCG) is an attenuated live vaccine that may cause life-threatening clinical disease in children with impaired immunity . In particular, patients with any of the nine known inherited disorders of the interleukin-12/23 interferon-gamma (IL-12/23-IFNgamma) axis are highly vulnerable to BCG . We describe two unrelated young Slovakian children suffering from disseminated BCG infection which developed shortly after routine BCG vaccination after birth . During treatment with selected anti-BCG antibiotics, resistance against several of these drugs developed . In both children, interleukin-12/23 receptor beta1 (IL-12/23Rbeta1) deficiency was diagnosed . Thus, in addition to chemotherapy, immunomodulatory treatment with recombinant IFN-gamma was performed as the pathogenesis of BCG disease in IL-12Rbeta1 deficiency involves impaired IL-12- and IL-23-dependent IFN-gamma production by lymphocytes . One child responded to treatment and is presently doing well whereas the second patient died . Conclusion:The marked variability of outcome of disseminated Bacillus Calmette-Guerin disease in interleukin-12/23 receptor beta1-deficient children sharing the same ethnic origin and exposed to a similar environment as presented in these case reports has to be taken into consideration for diagnosis and treatment of infections due to this genetic defect. Microbiology, 2005 Jan, 151(Pt 1), 183 - 97 Emetic toxin formation of Bacillus cereus is restricted to a single evolutionary lineage of closely related strains; Ehling-Schulz M et al.; An in-depth polyphasic approach was applied to study the population structure of the human pathogen Bacillus cereus . To assess the intraspecific biodiversity of this species, which is the causative agent of gastrointestinal diseases, a total of 90 isolates from diverse geographical origin were studied by genetic {M13-PCR, random amplification of polymorphic DNA (RAPD), multilocus sequence typing (MLST)} and phenetic {Fourier transform Infrared (FTIR), protein profiling, biochemical assays} methods . The strain set included clinical strains, isolates from food remnants connected to outbreaks, as well as isolates from diverse food environments with a well documented strain history . The phenotypic and genotypic analysis of the compiled panel of strains illustrated a considerable diversity among B . cereus connected to diarrhoeal syndrome and other non-emetic food strains, but a very low diversity among emetic isolates . Using all typing methods, cluster analysis revealed a single, distinct cluster of emetic B . cereus strains . The isolates belonging to this cluster were neither able to degrade starch nor could they ferment salicin; they did not possess the genes encoding haemolysin BL (Hbl) and showed only weak or no haemolysis . In contrast, haemolytic-enterotoxin-producing B . cereus strains showed a high degree of heterogeneity and were scattered over different clusters when different typing methods were applied . These data provide evidence for a clonal population structure of cereulide-producing emetic B . cereus and indicate that emetic strains represent a highly clonal complex within a potentially panmictic or weakly clonal background population structure of the species . It may have originated only recently through acquisition of specific virulence factors such as the cereulide synthetase gene. J Biochem (Tokyo), 2004 Nov, 136(5), 635 - 41 Nickel-Binding Properties of the C-Terminal Tail Peptide of Bacillus pasteurii UreE; Won HS et al.; Urease activation, which is critical to the virulence of many human and animal pathogens, is mediated by several accessory proteins . UreE, the only nickel-binding protein among the urease accessory proteins, catalyzes the activation of urease by transporting nickel ions into the urease active sites . The nickel-binding properties of UreE are still not clear, particularly for the protein from Bacillus pasteurii (Bp) . Since the flexible C-terminal tail of BpUreE possesses two conserved histidines, the nickel-binding properties of the tail peptide were examined by circular dichroism spectroscopy, size-exclusion chromatography, and nuclear magnetic resonance spectroscopy . Specific nickel binding leading to alteration of the peptide backbone geometry was clearly observed . Side-chains of the two conserved histidines were identified as the main ligands for nickel coordination . The peptide became dimerized upon nickel binding and the binding stoichiometry was estimated as 1 equivalent of nickel per peptide dimer . Altogether, it is postulated that the C-terminal tail of BpUreE contributes to the nickel binding of the protein in different ways between the dimeric and tetrameric protein folds. Acta Med Port, 2003 Nov-Dec, 16(6), 389 - 394 Epub 2003 Dec 1. {Monitoring serum levels of gentamicin in neonates.}; Rocha MJ et al.; Newborn infants in intensive care units demonstrated a higher incidence of gestational age below 31 week . Obstetrical and neonatal diseases procedures required for the management of critically ill neonates are associated with an increased risk of infections . Gentamicin is an aminoglycoside antibiotic often used to treat gramnegative bacillary infections and suspected sepsis in neonates . The risk of toxicity or poor efficacy is further increased due to the recognised wide intra and interpatient variability of the newborn . The present work involved 49 infants of 31.3 +/- 4.1 (mean +/- SD) weeks of gestational age, weighing 1.7 +/- 0.8 kg and were treated with standard doses of gentamicin (3.8 +/- 0.9 mg/kg/day) . Routine clinical care data were retrospectively collected from the medical records in the neonatal intensivecare unit at Coimbra University Hospital . Data analysis demonstrated that potentially toxic serum levels were observed in 49% of newborn infants (trough > 2 mg/L) . Additionally, the obtained results also showed that 7.5% of peak concentrations were found to be higher than 10 mg/L . Potentially sub-therapeutic concentrations were observed in 15% of the patients (peak > 6 mg/L). J Biomol NMR, 2004 Dec, 30(4), 431 - 42 Determination of protein rotational correlation time from NMR relaxation data at various solvent viscosities; Korchuganov DS et al.; An accurate determination of the overall rotation of a protein plays a crucial role in the investigation of its internal motions by NMR . In the present work, an innovative approach to the determination of the protein rotational correlation time tau(R) from the heteronuclear relaxation data is proposed . The approach is based on a joint fit of relaxation data acquired at several viscosities of a protein solution . The method has been tested on computer simulated relaxation data as compared to the traditional tau(R) determination method from T(1)/T(2) ratio . The approach has been applied to ribonuclease barnase from Bacillus amyloliquefaciens dissolved in an aqueous solution and deuterated glycerol as a viscous component . The resulting rotational correlation time of 5.56 +/- 0.01 ns and other rotational diffusion tensor parameters are in good agreement with those determined from T(1)/T(2) ratio. Hinyokika Kiyo, 2004 Nov, 50(11), 767 - 71 {Results of adjuvant intravesical Bacillus Calmette-Guérin therapy for grade 3 superficial bladder cancer}; Yumura Y et al.; To examine the incidence of recurrence, progression and survival in patients with grade 3 superficial bladder cancer after transurethral resection (TUR) and adjuvant intravesical instillation of Bacillus Calmette-Guerin (BCG), we retrospectively studied 39 patients with grade 3 superficial bladder cancer . Nineteen patients with high-grade superficial bladder cancer (pTa, pT1) and 5 patients with grade 3 carcinoma in situ (CIS) received intravesical instillation of BCG after transurethral resection of the bladder tumor (BCG group and CIS-BCG group) . The Tokyo 172 strain BCG was given for 8 weeks, as a rule, in a dose of 80 mg in 40 ml of saline instilled into the bladder . As a control, 15 patients with grade 3 superficial bladder cancer who did not receive BCG therapy after TUR were compared (non-BCG group) . Of the BCG group (n=19), 4 patients (21.1%) had recurrent tumor and 3 had invasive progression after BCG therapy and died as a result of tumor progression, while in the non-BCG group (n=15), 8 cases (53.3%) developed recurrence, only one case had progression and died of cancer . In the CIS-BCG group (n=5), 3 patients (60.0%) had recurrent tumor and 2 had invasive progression . Univariate analysis (Logrank test) demonstrated that tumor size and adjuvant instillation of BCG were associated with tumor recurrence except for carcinoma in situ, but tumor progression and survival did not differ significantly . Our results suggest that BCG therapy prevents grade 3 superficial bladder cancer (pT1, pTa) recurrence. J Biochem (Tokyo), 2004 Oct, 136(4), 549 - 56 The Production of Recombinant APRP, an Alkaline Protease Derived from Bacillus pumilus TYO-67, by In Vitro Refolding of Pro-enzyme Fixed on a Solid Surface; Takahashi M et al.; Bacillus pumilus TYO-67 has been isolated from tofuyo, a traditional fermented food made from soybean milk in Okinawa, Japan . This bacterium secretes a soybean-milk-coagulating enzyme (SMCE), which can be applied for the production of processed foods from soybean milk . Thus, an easy method of producing the recombinant enzyme was developed in this study . SMCE is an alkaline serine protease belonging to the subtilisin family; its candidate gene, aprP, which encodes a prepro-enzyme, was isolated in a previous study . Recombinant APRP was then produced by in vitro refolding of pro-APRP-His, i.e., N-terminally His-tagged pro-APRP . A large amount of pro-APRP-His was produced in Esherichia coli BL21(DE3) (ca . 8 mg from a 20-ml culture), collected as insoluble protein, dissolved in 6 M guanidine-HCl (pH 8.0), bound to Ni-NTA, and refolded on the resin at pH 10.0 to become mature APRP by autocleavage . Then, 0.16 mg of purified mature APRP was obtained through single-step chromatography from the refolded sample using 10 mg of pro-APRP-His . The N-terminal sequence and the enzymatic properties of refolded APRP were identical to those of SMCE . In addition, the pro-sequence was found to be essential for the production of mature APRP, suggesting that it could function as an intramolecular chaperone. J Biochem (Tokyo), 2004 Oct, 136(4), 471 - 6 Possible Structure and Function of the Extra C-Terminal Sequence of Pyruvate Kinase from Bacillus stearothermophilus; Sakai H; The pyruvate kinases from Genus Bacillus and a few other bacteria have an extra C-terminal sequence with a phosphoenolpyruvate binding motif composed of about 110 amino acids . To elucidate the possible structure and function of this sequence, the enzyme lacking the sequence was prepared and characterized . The N-terminal sequences of the peptides, which were found only in the lysylendopeptidase digest of the wild enzyme and not in that of the truncated enzyme, were determined . All the determined sequences were found in the extra C-terminal sequence deduced from the DNA sequence . The truncated enzyme showed decreased affinity for phosphoenolpyruvate and the allosteric effector ribose 5-phosphate, and had a reduced thermostability . Other properties, such as tetrameric structure, specific activity, and allosteric characteristics were unchanged . A comparison of the CD spectra of the truncated enzyme and the recombinant enzyme indicated that the structure of the C-terminal sequence should be rich in beta-sheet . These findings suggest that the sequence actually exists and that it may form a steady domain interacting with the A-domain and C-domain, which are the catalytic domain and allosteric effector binding domain, respectively. J Clin Pathol, 2005 Jan, 58(1), 22 - 5 Antimicrobial activity of UMFix tissue fixative; Cleary TJ et al.; AIMS: The aim of this study was to determine the antimicrobial effects of UMFix, an alcohol based tissue fixative, on various microorganisms . The UMFix solution was compared with 10% neutral buffered formalin . METHODS: Standard methods to determine microorganism colony counts were performed after exposure of the microorganisms to UMFix and 10% neutral buffered formalin . RESULTS: After a short exposure, UMFix rapidly killed vegetative bacteria, yeasts, moulds, and viruses . Bacterial spores were resistant to killing by UMFix . All organisms were killed by the 10% neutral buffered formalin preparation . CONCLUSIONS: UMFix was microbicidal for vegetative bacteria, yeasts, and aspergillus species after a short exposure, although it was not active against spore forming bacillus species . The methanol content of the fixative was responsible for the killing effect of this fixative . No killing was seen when polyethylene glycol was used alone. Huan Jing Ke Xue, 2004 Sep, 25(5), 109 - 11 {Control of chironomids in water by immobilized Bacillus thuringiensis var . israelensis de Barjac}; Zhang JS et al.; A strain from Bacillus thuringiensis var . israelensis de Barjac (B . t . i), having a fatal toxicity to chironomids, was immobilized in the PVA-H3BO3 gel beads confected with a bit of sodium alginate and activated carbon . The result showed that optimal weight ratios were 10% PVA, 1% sodium alginate and 20% activated carbon, respectively . When 4% calcium chloride saturated boric acid solution (pH=6.7) was used as gelatin agent for gelating 24 h, the produced gel beads had good performance in mechanical strength . This method is proved to be simple and cheap . The toxicity of the beads to chironomids is high and the release is slow. FEMS Microbiol Lett, 2005 Jan 15, 242(2), 325 - 32 Targeted mutagenesis of loop residues in the receptor-binding domain of the Bacillus thuringiensis Cry4Ba toxin affects larvicidal activity; Tuntitippawan T et al.; Loop residues in domain II of Bacillus thuringiensis Cry delta-endotoxins have been demonstrated to be involved in insecticidal specificity . In this study, selected residues in loops beta6-beta7 (S(387)SPS(390)), beta8-beta9 (S(410), N(411), T(413), T(415), E(417) and G(418)) and beta10-beta11 (D(454)YNS(457)) in domain II of the Cry4Ba mosquito-larvicidal protein were changed individually to alanine by PCR-based directed mutagenesis . All mutant toxins were expressed in Escherichia coli JM109 cells as 130-kDa protoxins at levels comparable to the wild type . Only E . coli cells that express the P389A, S410A, E417A, Y455A or N456A mutants exhibited a loss in toxicity against Aedes aegypti mosquito larvae of approximately 30% when compared to the wild type . In addition, E . coli cells expressing double mutants, S410A/E417A or Y455A/N456A, at wild-type levels revealed a significantly higher loss in larvicidal activity of approximately 70% . Similar to the wild-type protoxin, both double mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0 . These results indicate that S(410) and E(417) in the beta8-beta9 loop, and Y(455) and N(456) in the beta10-beta11 loop are involved in larvicidal activity of the Cry4Ba toxin. FEMS Microbiol Lett, 2005 Jan 15, 242(2), 313 - 7 Heat-stable toxin production by strains of Bacillus cereus, Bacillus firmus, Bacillus megaterium, Bacillus simplex and Bacillus licheniformis; Taylor JM et al.; Strains of Bacillus cereus can produce a heat-stable toxin (cereulide) . In this study, 101 Bacillus strains representing 7 Bacillus species were tested for production of heat-stable toxins . Strains of B . megaterium, B . firmus and B . simplex were found to produce novel heat-stable toxins, which showed varying levels of toxicity . B . cereus strains (18 out of 54) were positive for toxin production . Thirteen were of serovar H1, and it was of interest that some were of clinical origin . Two were of serovars 17B and 20, which are not usually implicated in the emetic syndrome . Partial purification of the novel B . megaterium, B . simplex and B . firmus toxins showed they had similar physical characteristics to the B . cereus emetic toxin, cereulide. FEMS Microbiol Lett, 2005 Jan 1, 242(1), 127 - 36 Discrimination of pathogenic clinical isolates and laboratory strains of Bacillus cereus by NMR-based metabolomic profiling; Bundy JG et al.; Six different Bacillus cereus strains were selected from two different ecotypes: (1) three commonly used laboratory strains that are considered avirulent, and (2) three clinical isolates from meningitis patients . Screening of genomic DNA for the presence of genes encoding known toxins gave no candidate genes that were unambiguously able to distinguish between the two groups . However, the application of multivariate pattern-recognition methods to metabolite profiles derived from the different strains using (1)H nuclear magnetic resonance spectroscopy (metabolomics) was able to classify the different profiles . The two different ecotypes were clearly separated on the basis of their metabolite profiles, showing that it is possible to use metabolomic methods to classify pathogens on the basis of their expressed physiology, even when it is not possible to infer a direct mechanistic link to specific virulence factors . This metabolomic approach could also have a wide range of possible applications in both general microbiology and microbial ecology for distinguishing and identifying different functional/physiological ecotypes of bacterial strains or species. Biosci Biotechnol Biochem, 2004 Dec, 68(12), 2529 - 40 Cyclic Tetrasaccharide-Synthesizing Enzymes from Arthrobacter globiformis A19; Mukai K et al.; A bacterial strain Arthrobacter globiformis A19 producing cyclic tetrasaccharide (CTS) was isolated from soil . The enzymes, 6-alpha-glucosyltransferase (6GT) and 3-alpha-isomaltosyltransferase (IMT), involved in the synthesis of CTS were purified to homogeneity . The molecular and enzymatic properties of IMT from A . globiformis were similar to those of enzymes from Bacillus globisporus C11 and N75 . Arthrobacter 6GT had a smaller molecular mass of 108 kDa and a higher optimum pH of 8.4 than the enzymes from strains of B . globisporus . The genes for IMT (ctsY) and 6GT (ctsZ) were cloned from the genome of A . globiformis A19 . The two genes linked together in tandem and formed a gene cluster, ctsYZ . Both of the gene products showed similarities to alpha-glucosidases belonging to glycoside hydrolase family 31, and conserved two aspartic acids corresponding to the putative catalytic residues of the family enzymes . The enzymatic system for the production of CTS consisting of 6GT and IMT might be widespread among bacteria. Eur J Cancer, 2005 Jan, 41(1), 118 - 25 Prior immunisation of patients with malignant melanoma with vaccinia or BCG is associated with better survival . An European Organization for Research and Treatment of Cancer cohort study on 542 patients; Kolmel KF et al.; There is increasing evidence that infections and vaccinations play an important role in the normal maturation of the immune system . It was therefore of interest to determine whether these immune events also affect the prognosis of melanoma patients . A cohort study of 542 melanoma patients in six European countries and Israel was conducted . Patients were followed up for a mean of 5 years and overall survival was recorded . Biometric evaluations included Kaplan-Meier estimates of survival over time and Hazard Ratios (HRs), taking into account all known prognostic factors . During the follow-up between 1993 and 2002, 182 of the 542 patients (34%) died . Survival curves, related to Breslow's thickness as the most important prognostic marker, were in accordance with those observed in previous studies where the cause of death was known to be due to disseminated melanoma . In a separate analysis of patients, vaccinated with vaccinia or Bacille Calmette-Guerin (BCG), HRs and the corresponding 95% Confidence Intervals (CIs) were 0.52 (0.34-0.79) and 0.69 (0.49-0.98), respectively . Joint analyses yielded HRs (and 95% CIs) of 0.55 (0.34-0.89) for patients vaccinated with vaccinia, 0.75 (0.30-1.86) with BCG, and 0.41 (0.25-0.69) with both vaccines . In contrast, infectious diseases occurring before the excision of the tumour had little, or, at the most, a minor influence on the outcome of the melanoma patients . These data reveal, for the first time, that vaccination with vaccinia in early life significantly prolongs the survival of patients with a malignant tumour after initial surgical management . BCG vaccination seems to have a similar, although weaker, effect . The underlying immune mechanisms involved remain to be determined. Eur J Cancer, 2005 Jan, 41(1), 104 - 17 Protection against melanoma by vaccination with Bacille Calmette-Guerin (BCG) and/or vaccinia: an epidemiology-based hypothesis on the nature of a melanoma risk factor and its immunological control; Krone B et al.; A multicentre case-control study conducted by the FEBrile Infections and Melanoma (FEBIM) group has demonstrated a reduced risk of melanoma associated with Bacille Calmette-Guerin (BCG) and/or vaccinia vaccination in early childhood and/or with infectious diseases later in life . This has led to the recognition of a new risk indicator of melanoma; namely 'not being vaccinated with either with BCG or vaccinia' . On the basis of these findings, we propose a hypothesis of immune surveillance for melanoma induced or enhanced by prior contacts with pathogens unexpectedly cross-reactive to a cellular 'marker of melanoma risk' . The reduced risk of melanoma due to BCG and vaccinia, as well as certain common causes of infectious disease, is shown to be associated with antigenic determinants exhibiting sequence homologies with the HERV-K-MEL-antigen . The latter is a product of a pseudo-gene that is closely associated with the env-gene of the endogenous human retrovirus K (HERV-K) . A suppressive immune reaction appears to inhibit the expression of endogenous retroviral genes, such as the HERV-K env-gene, that could otherwise result in malignant transformation years or even decades later . The HERV-K env-protein has homologous amino acid sequences with the human nuclear factor Oxygen Responsive Element Binding Protein (OREBP) that controls the expression of glutathione peroxidase . The formation of this and other redox-enzymes, needed to maintain appropriate levels of the normal intracellular redox potential, seems to be suppressed by the OREBP-homologous protein . The present hypothesis is in accordance with the concept that immune dysregulation due to adverse environmental impacts is a risk factor not only for some autoimmune disorders, as previously described, but also for certain malignancies such as melanoma. J Cataract Refract Surg, 2004 Dec, 30(12), 2641 - 3 Chronic postoperative endophthalmitis caused by Actinomyces neuii; Raman VS et al.; Uneventful phacoemulsification with implantation of a foldable, acrylic posterior chamber intraocular lens was performed in the right eye of a 73-year-old white man . Postoperatively, the patient developed a chronic, low-grade intraocular inflammation . Cultures from the aqueous specimen grew Actinomyces neuii, an unusual gram-positive bacillus . The low-grade intraocular inflammation persisted with intensive topical steroid-antibiotic medication and systemic antibiotics . A diagnostic, 3-port, pars plana vitrectomy was performed, and aqueous aspirate specimens were sent for culture and sensitivity and to look for abnormal cells . Intracameral antibiotics were not injected . The specimens were sterile to culture, and chronic inflammatory cells were reported on the vitreous specimen . On examination 6 months later, inflammation had not recurred and the best corrected visual acuity was 6/18. Int J Food Microbiol, 2005 Jan 15, 98(1), 23 - 34 Phenotypic and genotypic characterisation of Bacillus cereus isolates from Bangladeshi rice; Haque A et al.; We report the first phenotypic and genotypic characterisation of Bacillus cereus from Bangladeshi rice . Seven strains of B . cereus were isolated from two high-yielding rice varieties (BR5 and BRRI Dhan28) which are cultivated during different rice-growing seasons in Bangladesh . The strains were identified as B . cereus based on colonial and cellular (light microscopic) morphology and were confirmed using the API 50 CHB test system and 16S rDNA analysis . They could also be distinguished from each other based on their substrate-utilisation patterns using the API system . No psychrotrophic (cold-tolerant) isolates were obtained and all were mesophilic with a minimum growth temperature of 10 degrees C, but with an ability to grow at a higher rate than normal temperature (45 degrees C) for B . cereus . All strains had temperature optima between 35 and 45 degrees C, but two groups could be distinguished based on having either a narrow or a broad optimum range . The different strains could not be distinguished based on their phospholipid compositions including major fatty acyl chains which were typical for B . cereus . However, capillary-GC/MS analysis of trace fatty acyl components of the lipids has been used for the first time to group the isolates . Genotypic analysis of the rDNA region has also been used to distinguish the strains . Restriction fragment length polymorphism (RFLP) analysis of the spacer region between the 16S and 23S rRNA genes yielded two groups based on different base sequences, whereas sequence analysis of variable regions in the 16S rRNA gene gave four different groups by base differences at two positions . Despite their association with rice, none of the strains produced emetic toxin . The spore germination response of heat-activated spores of the Bangladeshi B . cereus strains was typical of the bacterium, but three different groups could be identified based on differences in germination rates . The use of this polyphasic approach to taxonomy has enabled all the B . cereus strains to be distinguished from each other. Cell Microbiol, 2005 Jan, 7(1), 91 - 104 Bartonella henselae inhibits apoptosis in Mono Mac 6 cells; Kempf VA et al.; Summary Bartonella henselae causes the vasculoproliferative disorders bacillary angiomatosis and peliosis probably resulting from the release of vasculoendothelial growth factor (VEGF) from infected epithelial or monocytic host cells . Here we demonstrate that B . henselae in addition to VEGF induction was also capable of inhibiting the endogenous sucide programme of monocytic host cells . Our results show that B . henselae inhibits pyrrolidine dithiocarbamate (PDTC)-induced apoptosis in Mono Mac 6 cells . B . henselae was observed to be present in a vacuolic compartment of Mono Mac 6 cells . Direct contact of B . henselae with Mono Mac 6 cells was crucial for inhibition of apoptosis as shown by the use of a two-chamber model . Inhibition of apoptosis was paralleled by diminished caspase-3 activity which was significantly reduced in PDTC-stimulated and B . henselae-infected cells . The anti-apoptotic effect of B . henselae was accompanied by (i) the activation of the transcription factor NF-kappaB and (ii) the induction of cellular inhibitor of apoptosis proteins-1 and -2 (cIAP-1, -2) . Our results suggest a new synergistic mechanism in B . henselae pathogenicity by (i) inhibition of host cell apoptosis via activation of NF-kappaB and (ii) induction of host cell VEGF secretion. Gastric Cancer, 2004, 7(4), 240 - 5 Efficiency of adjuvant immunochemotherapy following curative resection in patients with locally advanced gastric cancer; Popiela T et al.; BACKGROUND: Despite curative resection, 50%-90% of gastric cancer patients die of disease relapse . Although some clinical trials have indicated that chemotherapy and immunochemotherapy may be effective modalities, more recent studies have not been able to define the standard treatment for advanced gastric cancer . The present study evaluated the effect of adjuvant immunochemotherapy with the use of BCG (bacille Calmette-Guerin) and FAM (5-fluorouracil, adriamycin, mitomycin C) chemotherapy on the survival of patients with locally advanced resectable gastric cancer . METHODS: A total of 156 patients with stage III or IV gastric cancer who had undergone curative resection were randomly assigned to three treatment groups: BCG + FAM (immunochemotherapy), FAM (chemotherapy), and control (surgery only) . Treatment was continued for 2 years or until death . Further postsurgical follow up was carried on for up to 10 years . RESULTS: Overall 10-year survival was 47.1% for the immunochemotherapy group (P < 0.037 vs FAM and P < 0.0006 vs control), 30% for the chemotherapy group (vs control, NS), and 15.2% for the control group . In patients with pT2/T3 primary tumors, 10-year survival was 55.3% for BCG + FAM vs 28.2% for FAM (P < 0.01) and 14.6% for the control group (P < 0.00018) . BCG + FAM significantly improved the survival of patients with intestinal-type but not diffuse-type cancer . Immunochemotherapy was well tolerated . CONCLUSION: This study, based on a limited number of patients, indicates that adjuvant immunochemotherapy (BCG + FAM) may prolong the survival of gastric cancer patients after curative gastrectomy; in particular, in patients with pT2/T3 tumors and intestinal-type primary tumors . There was no survival benefit from FAM adjuvant chemotherapy. Appl Microbiol Biotechnol . 2004 Dec 22; {Epub ahead of print} Bacterial community profiles on feathers during composting as determined by terminal restriction fragment length polymorphism analysis of 16S rDNA genes; Tiquia SM et al.; Composting is one of the more economical and environmentally safe methods of recycling feather waste generated by the poultry industry, since 90% of the feather weight consists of crude keratin protein, and feathers contain 15% N . However, the keratin in waste feathers is resistant to biodegradation and may require the addition of bacterial inocula to enhance the degradation process during composting . Two keratin-degrading bacteria isolated from plumage of wild songbirds and identified as Bacillus licheneformis (OWU 1411T) and Streptomyces sp . (OWU 1441) were inoculated into poultry feather composts (1.13x10(8) cfu g(-1) feathers) and co-composted with poultry litter and straw in 200-l compost vessels . Composting temperatures, as well as CO(2) and NH(3) evolution, were measured in these vessels to determine the effects of inoculation on the rate and extent of poultry feather decomposition during composting . Terminal restriction fragment length polymorphisms of 16S rRNA genes were used to follow changes in microbial community structure during composting . The results indicated that extensive carbon conversion occurred in both treatments (55.5 and 56.1%) . The addition of the bacterial inocula did not enhance the rate of waste feather composting . The microbial community structure over time was very similar in inoculated and uninoculated waste feather composts. J Immunother, 2005 Jan-Feb, 28(1), 20 - 7 CpG oligonucleotide therapy cures subcutaneous and orthotopic tumors and evokes protective immunity in murine bladder cancer; Ninalga C et al.; Bacillus Calmette-Guerin (BCG) instillation is standard immunotherapy for superficial bladder carcinoma . However, many patients become refractory to BCG, giving impetus to the development of alternative therapies . CpG oligodeoxynucleotide (ODN) therapy has been shown to promote T(H)1-oriented antitumor responses in various tumor models . To investigate its therapeutic effect in bladder cancer, we used different CpG ODNs to treat C57BL/6 mice bearing the subcutaneous murine bladder tumor MB49 . CpG type B ODN 1668 was superior at inhibiting tumor growth, leading to complete regression of large tumors . More importantly, CpG ODN 1668 also regressed orthotopically growing MB49 tumors for the first time . Rechallenge of CpG ODN-cured mice with MB49 showed that a majority of the mice were protected long term, demonstrating that CpG ODN therapy evokes a memory response . Adenoviral vectors (Ad) encoding CD40L, tumor necrosis factor-related activation-induced cytokine, lymphotactin, interleukin (IL) 2, and IL-15 were also investigated . AdCD40L and AdIL-15 transduction could abolish MB49 tumorigenicity, and these vectors were combined with CpG ODN 1668 to investigate any enhanced effects . No such effects were seen . All groups of mice treated with CpG ODNs, alone or in combination with adenoviral vector, exhibited increased serum concentrations of IL-12, indicative of a T(H)1 response . Our results show that CpG ODN therapy cures established subcutaneous and orthotopic bladder cancer via a T(H)1-mediated response and provides long-lasting protective immunity. J Agric Food Chem, 2004 Dec 29, 52(26), 7862 - 6 Antibacterial activity of Coriandrum sativum L . and Foeniculum vulgare Miller Var . vulgare (Miller) essential oils; Lo Cantore P et al.; Essential oils were extracted from the fruits of Coriandrum sativum L . and Foeniculum vulgare Miller var . vulgare (Miller) and assayed in vitro for antibacterial activity to Escherichia coli and Bacillus megaterium, bacteria routinely used for comparison in the antimicrobial assays, and 27 phytopathogenic bacterial species and two mycopathogenic ones responsible for cultivated mushroom diseases . A significant antibacterial activity, as determined with the agar diffusion method, was shown by C . sativum essential oil whereas a much reduced effect was observed for F . vulgare var . vulgare oil . C . sativum and F . vulgare var . vulgare essential oils may be useful natural bactericides for the control of bacterial diseases of plants and for seed treatment, in particular, in organic agriculture . The significant antibacterial activity of essential oils to the bacterial pathogens of mushrooms appears promising. Environ Biosafety Res, 2004 Apr-Jun, 3(2), 73 - 81 Hybridization and backcrossing between transgenic oilseed rape and two related weed species under field conditions; Halfhill MD et al.; Determining the frequency of crop-wild transgene flow under field conditions is a necessity for the development of regulatory strategies to manage transgenic hybrids . Gene flow of green fluorescent protein (GFP) and Bacillus thuringiensis (Bt) transgenes was quantified in three field experiments using eleven independent transformed Brassica napus L . lines and the wild relatives, B . rapa L . and Raphanus raphanistrum L . Under a high crop to wild relative ratio (600:1), hybridization frequency with B . rapa differed among the individual transformed B . napus lines (ranging from ca . 4% to 22%), however, this difference could be caused by the insertion events or other factors, e.g., differences in the hybridization frequencies among the B . rapa plants . The average hybridization frequency over all transformed lines was close to 10% . No hybridization with R . raphanistrum was detected . Under a lower crop to wild relative ratio (180:1), hybridization frequency with B . rapa was consistent among the transformed B . napus lines at ca . 2% . Interspecific hybridization was higher when B . rapa occurred within the B . napus plot (ca . 37.2%) compared with plot margins (ca . 5.2%) . No significant differences were detected among marginal plants grown at 1, 2, and 3 m from the field plot . Transgene backcrossing frequency between B . rapa and transgenic hybrids was determined in two field experiments in which the wild relative to transgenic hybrid ratio was 5-15 plants of B . rapa to 1 transgenic hybrid . As expected, ca . 50% of the seeds produced were transgenic backcrosses when the transgenic hybrid plants served as the maternal parent . When B . rapa plants served as the maternal parent, transgene backcrossing frequencies were 0.088% and 0.060% . Results show that transgene flow from many independent transformed lines of B . napus to B . rapa can occur under a range of field conditions, and that transgenic hybrids have a high potential to produce transgenic seeds in backcrosses. Environ Biosafety Res, 2003 Jul-Sep, 2(3), 181 - 206 Effects of Bacillus thuringiensis on non-target herbivore and natural enemy assemblages in tropical irrigated rice; Schoenly KG et al.; Endotoxins from Bacillus thuringiensis (Bt) produced in transgenic pest-resistant Bt crops are generally not toxic to predatory and parasitic arthropods . However, elimination of Bt-susceptible prey and hosts in Bt crops could reduce predator and parasitoid abundance and thereby disrupt biological control of other herbivorous pests . Here we report results of a field study evaluating the effects of Bt sprays on non-target terrestrial herbivore and natural enemy assemblages from three rice (Oryza sativa L.) fields on Luzon Island, Philippines . Because of restrictions on field-testing of transgenic rice, Bt sprays were used to remove foliage-feeding lepidopteran larvae that would be targeted by Bt rice . Data from a 546-taxa Philippines-wide food web, matched abundance plots, species accumulation curves, time-series analysis, and ecostatistical tests for species richness and ranked abundance were used to compare different subsets of non-target herbivores, predators, and parasitoids in Bt sprayed and water-sprayed (control) plots . For whole communities of terrestrial predators and parasitoids, Bt sprays altered parasitoid richness in 3 of 3 sites and predator richness in 1 of 3 sites, as measured by rarefaction (in half of these cases, richness was greater in Bt plots), while Spearman tests on ranked abundances showed that correlations, although significantly positive between all treatment pairs, were stronger for predators than for parasitoids, suggesting that parasitoid complexes may have been more sensitive than predators to the effects of Bt sprays . Species accumulation curves and time-series analyses of population trends revealed no evidence that Bt sprays altered the overall buildup of predator or parasitoid communities or population trajectories of non-target herbivores (planthoppers and leafhoppers) nor was evidence found for bottom-up effects in total abundances of non-target species identified in the food web from the addition of spores in the Bt spray formulation . When the same methods were applied to natural enemies (predators and parasitoids) of foliage-feeding lepidopteran and non-lepidopteran (homopteran, hemipteran and dipteran) herbivores, significant differences between treatments were detected in 7 of 12 cases . However, no treatment differences were found in mean abundances of these natural enemies, either in time-series plots or in total (seasonal) abundance . Analysis of guild-level trajectories revealed population behavior and treatment differences that could not be predicted in whole-community studies of predators and parasitoids . A more conclusive test of the impact of Bt rice will require field experiments with transgenic plants, conducted in a range of Asian environments, and over multiple cropping seasons. Environ Biosafety Res, 2004 Jan-Mar, 3(1), 45 - 54 Inheritance of GFP-Bt transgenes from Brassica napus in backcrosses with three wild B . rapa accessions; Zhu B et al.; Transgenes from transgenic oilseed rape, Brassica napus (AACC genome), can introgress into populations of wild B . rapa (AA genome), but little is known about the long-term persistence of transgenes from different transformation events . For example, transgenes that are located on the crop's C chromosomes may be lost during the process of introgression . We investigated the genetic behavior of transgenes in backcross generations of wild B . rapa after nine GFP (green fluorescent protein)-Bt (Bacillus thuringiensis) B . napus lines, named GT lines, were hybridized with three wild B . rapa accessions, respectively . Each backcross generation involved crosses between hemizygous GT plants and non-GT B . rapa pollen recipients . In some cases, sample sizes were too small to allow the detection of major deviations from Mendelian segregation ratios, but the segregation of GT:non-GT was consistent with an expected ratio of 1:1 in all crosses in the BC1 generation . Starting with the BC2 generation, significantly different genetic behavior of the transgenes was observed among the nine GT B . napus lines . In some lines, the segregation of GT:non-GT showed a ratio of 1:1 in the BC2, BC3, and BC4 generations . However, in other GT B . napus lines the segregation ratio of GT:non-GT significantly deviated from 1:1 in the BC2 and BC3 generations, which had fewer transgenic progeny than expected, but not in the BC4 generation . Most importantly, in two GT B . napus lines the segregation of GT:non-GT did not fit into a ratio of 1:1 in the BC2, BC3 or BC4 generations due to a deficiency of transgenic progeny . For these lines, a strong reduction of transgene introgression was observed in all three B . rapa accessions . These findings imply that the genomic location of transgenes in B . napus may affect the long-term persistence of transgenes in B . rapa after hybridization has occurred. Environ Biosafety Res, 2003 Apr-Jun, 2(2), 117 - 32 Selection of relevant non-target herbivores for monitoring the environmental effects of Bt maize pollen; Schmitz G et al.; Genes of Bacillus thuringiensis var . kurstaki (Berliner) that encode lepidopteran-specific toxins were engineered into maize for protection against the European Corn Borer, Ostrinia nubilalis (Hbn.) . Recent data suggest that Lepidoptera may be negatively affected, if maize pollen contains high amounts of Bt toxin and is diposited on host plants near maize fields . Monitoring the environmental effects of commercial Bt maize fields requires effective use of limited financial and logistical resources . The aim of this study was to develop and apply tools for selecting relevant herbivore species for the field monitoring of environmental Bt toxin effects via pollen deposition . We first present a theoretical selection tree based on "risk index of Bt pollen for herbivores" (I(Btp)) . Our index consists of five classes from zero (not relevant) to four (highly relevant) derived from data on potential temporal and spatial coincidence of pollen exposure (A), feeding mode (B), susceptibility to lepidopteran-specific Bt toxins (C) and hazard to rare and/or endangered species ("Red List") (D) . We then screened the Macrolepidoptera database LEPIDAT to identify relevant species in Germany . Finally, we also applied the index to species found in a local biocoenotic field study (Bonn, Western Rhineland, Germany) . Approximately 7% of the German Macrolepidoptera species mainly occur in farmland areas and were selected as being potentially affected by Bt pollen exposure . Of these species, 14% (= 1% of total) were found to be potentially exposed on a regional scale . The combination of I(Btp) and database screening enables us to pre-select species for monitoring purposes. Environ Biosafety Res, 2002 Oct, 1(1), 49 - 60 Ostrinia nubilalis parasitism and the field abundance of non-target insects in transgenic Bacillus thuringiensis corn (Zea mays); Bourguet D et al.; In this study, we evaluated in field trials the effects on non-target species, of transgenic corn producing the Cry1Ab toxin of Bacillus thuringiensis (Bt) . In 1998, we collected Ostrinia nubilalis (Hubner) larvae from transgenic Bt corn (Novartis Hybrid 176) and non-Bt corn at four geographical sites . We found a significant variation in parasitism by the tachinids Lydella thompsoni (Herting) and Pseudoperichaeta nigrolineata (Walker) among sites, and more parasitism in non-Bt than in Bt fields . The Bt effect did not vary significantly among fields . In 1999, we performed a field experiment at two sites, comparing the temporal abundance of non-target arthropods in Bt corn (Monsanto Hybrid MON810) and non-Bt corn . The non-target insects studied included the aphids Metopolophium dirhodum (Walker), Rhopalosiphum padi (L.) and Sitobion avenae (F.), the bug Orius insidiosus (Say), the syrphid Syrphus corollae (Meigen), the ladybird Coccinella septempunctata (L.), the lacewing Chrysoperla carnea (Stephens), thrips and hymenopteran parasitoids . For all species but one, the number of individuals varied greatly over the season but did not differ between the types of corn . The only exception was thrips which, at one site, was significantly more abundant in Bt corn than in non-Bt corn . However this difference did not remain significant when we took the multiple tests into account . Implications for pest resistance management, population dynamics and risk assessment are discussed. Environ Biosafety Res, 2002 Oct, 1(1), 19 - 28 Bt-transgenic oilseed rape hybridization with its weedy relative, Brassica rapa; Halfhill MD et al.; The movement of transgenes from crops to weeds and the resulting consequences are concerns of modern agriculture . The possible generation of "superweeds" from the escape of fitness-enhancing transgenes into wild populations is a risk that is often discussed, but rarely studied . Oilseed rape, Brassica napus (L.), is a crop with sexually compatible weedy relatives, such as birdseed rape (Brassica rapa (L.)) . Hybridization of this crop with weedy relatives is an extant risk and an excellent interspecific gene flow model system . In laboratory crosses, T3 lines of seven independent transformation events of Bacillus thuringiensis (Bt) oilseed rape were hybridized with two weedy accessions of B . rapa . Transgenic hybrids were generated from six of these oilseed rape lines, and the hybrids exhibited an intermediate morphology between the parental species . The Bt transgene was present in the hybrids, and the protein was synthesized at similar levels to the corresponding independent oilseed rape lines . Insect bioassays were performed and confirmed that the hybrid material was insecticidal . The hybrids were backcrossed with the weedy parent, and only half the oilseed rape lines were able to produce transgenic backcrosses . After two backcrosses, the ploidy level and morphology of the resultant plants were indistinguishable from B . rapa . Hybridization was monitored under field conditions (Tifton, GA, USA) with four independent lines of Bt oilseed rape with a crop to wild relative ratio of 1200:1 . When B . rapa was used as the female parent, hybridization frequency varied among oilseed rape lines and ranged from 16.9% to 0.7%. J Exp Med, 2004 Dec 20, 200(12), 1559 - 69 Mycobacterium tuberculosis pks12 Produces a Novel Polyketide Presented by CD1c to T Cells; Matsunaga I et al.; CD1c-mediated T cells are activated by a mycobacterial phospholipid antigen whose carbohydrate structure precisely corresponds to mammalian mannosyl beta-1-phosphodolichol (MPD), but contains an unusual lipid moiety . Here, we show that this T cell antigen is a member of a family of branched, alkane lipids that vary in length (C(30-34)) and are produced by medically important mycobacteria such as M . tuberculosis and M . bovis Bacille-Calmette-Guerin . The alkane moiety distinguished these mycobacterial lipid antigens from mammalian MPDs and was necessary for activation of CD1c-restricted T cells, but could not be accounted for by any known lipid biosynthetic pathway . Metabolic labeling and mass spectrometric analyses suggested a mechanism for elongating lipids using alternating C(2) and C(3) units, rather than C(5) isopentenyl pyrophosphate . Inspection of the M . tuberculosis genome identified one candidate gene, pks12, which was predicted to encode the largest protein in M . tuberculosis, consisting of 12 catalytic domains that correspond to key steps in the proposed pathway . Genetic deletion and complementation showed that Pks12 was necessary for antigen production, but did not affect synthesis of true isoprenols . These studies establish the genetic and enzymatic basis for a previously unknown type of polyketide, designated mycoketide, which contains a lipidic pathogen-associated molecular pattern. Lancet, 2004 Dec 18, 364(9452), 2204 - 11 Effect of infant immunisation on childhood mortality in rural Bangladesh: analysis of health and demographic surveillance data; Breiman RF et al.; BACKGROUND: In developing countries, immunisation programmes must compete with other strategies to improve public health and quality of life . Studies of long-term effects of immunisation programmes are rare . We assessed associations between vaccinations and mortality over 15 years after the introduction of routine infant immunisation programmes in Matlab, Bangladesh . METHODS: We analysed data recorded in a comprehensive health and demographic surveillance system from 1986 to 2001 . We did univariate analyses and assessed vaccinations as independent factors with other variables in Cox models with time dependent covariates . FINDINGS: Diphtheria-tetanus-pertussis (DTP) and oral polio vaccination were independently associated with decreased risk of death before age 9 months, as were amount of maternal education, maternal age, and birth order of the child . DTP vaccination was associated with increased survival (hazard ratio=0.76, 95% CI 0.67-0.88; p=0.001) in a model evaluating mortality between 6 weeks and 9 months of age . Measles vaccination was also associated with increased survival when data after late immunisation with DTP and Bacille Calmette-Guerin (BCG) were excluded . BCG vaccination was associated with reduced survival; however, children vaccinated with BCG during the first 6 months of life had significantly lower risk of death than those vaccinated later (hazard ratio=0.59; 95% CI 0.47-0.73; p=0.0001) . INTERPRETATION: By contrast with previous findings, we noted substantially reduced mortality among children who received DTP vaccine . This effect could be due to actual protection against pertussis disease and secondary illnesses or to a non-specific benefit, although we cannot rule out epidemiological artifact . Our findings show the value of population-based health surveillance systems. Int Braz J Urol, 2004 Sep-Oct, 30(5), 400 - 2 Interstitial pneumonitis secondary to intravesical bacillus calmette-guerin for carcinoma in-situ of the bladder; Diner EK et al.; We report an 81-year-old male who developed severe interstitial pneumonitis on maintenance intravesical Bacillus Calmette-Guerin (BCG) for in-situ carcinoma of the bladder . The patient was treated with steroids and anti-tuberculin therapy with complete response . While there is no established standard of care for the treatment of interstitial pneumonitis, recent reports describe success with combination of corticosteroids and anti-tuberculin medications . We elected to follow this precedent and treated our patient with corticosteroids and antituberculin therapy with good outcome. J Appl Microbiol, 2005, 98(1), 210 - 5 GroEL-GroES solubilizes abundantly expressed xylulokinase in Escherichia coli; Bu S et al.; Abstract s . bu, p.w.k . tsang and r.z . fu . 2004.Aims: The aims of the present work were to solubilize the abundantly expressed recombinant xylulokinase in Escherichia coli and to develop a reliable xylulokinase assay . Methods and Results: Three mutants of xylulokinase of Bacillus megaterium that were expressed at high level but formed insoluble protein in E . coli BL21(DE3)pLysS were selected for solubility study . The solubility of xylulokinase increased eight to 77-fold after introduction of molecular chaperones GroEL-GroES into the host . Conclusion: This investigation reports that GroEL-GroES minimizes the formation of insoluble protein in three highly expressed recombinant xylulokinases and an improved xylulokinase assay . Significance and Impact of the Study: Commercial production of bioethanol is critically dependent on the development of an efficient and low-cost process of enzymatic conversion of xylan, a major component in lignocellulose biomass, to xylulose-5-phosphate, which can then be channelled into pentose phosphate pathway and metabolized to ethanol . The improved intracellular xylulokinase activity is expected to facilitate the xylose degradation. J Appl Microbiol, 2005, 98(1), 24 - 32 A natural variant of Bacillus licheniformis alpha-amylase isolated from flour mill wastewaters sheds light on the origin of high thermostability; Shahhoseini M et al.; Abstract m . shahhoseini, a.-a . ziaee, a.-a . pourbabai, n . ghaemi and n . declerck . 2004.Aims: Understanding the origin of high thermostability exhibited by the alpha-amylase produced by a natural strain of Bacillus licheniformis . Methods and Results: The MSH320 alpha-amylase gene has been cloned from a native strain of B . licheniformis isolated from flour mill wastewaters in Kashan, central Iran, and its nucleotide sequence was determined (GenBank Accession Number AF438149) . Whereas previously cloned B . licheniformisalpha-amylase (BLA) genes are nearly identical, the MSH320 gene coding sequence presents only 93% identity with the reference 'wild-type' BLA gene, most of the nucleotide changes leading to silent mutations . Amino acid substitutions occurred at 19 of the 483 residues of the matured protein, distributed all along the protein sequence . Nevertheless, the natural BLA variant presents thermoinactivation kinetics similar to that of the reference BLA . Protein modelling and structural predictions at the substitution sites suggest that half of the mutations may have a significant stabilizing or destabilizing effect on the protein structure . Compensatory mutations thus occurred in the natural variant in order to maintain thermostability to the level of the reference enzyme . Conclusions: The exceptional high thermostability of BLA, although produced by a nonthermophilic organism, is not fortuitous but subject to a selective pressure still at work in natural environments . Significance and Impact of the Study: BLA thermal performances are not naturally maximized and can be substantially improved by protein engineering. Biochemistry, 2004 Dec 28, 43(51), 16416 - 31 Interaction of nitric oxide with cytochrome P450 BM3; Quaroni LG et al.; The interaction of nitric oxide with cytochrome P450 BM3 from Bacillus megaterium has been analyzed by spectroscopic techniques and enzyme assays . Nitric oxide ligates tightly to the ferric heme iron, inducing large changes in each of the main visible bands of the heme and inhibiting the fatty acid hydroxylase function of the protein . However, the ferrous adduct is unstable under aerobic conditions, and activity recovers rapidly after addition of NADPH to the flavocytochrome due to reduction of the heme via the reductase domain and displacement of the ligand . The visible spectral properties revert to that of the oxidized resting form . Aerobic reduction of the nitrosyl complex of the BM3 holoenzyme or heme domain by sodium dithionite also displaces the ligand . A single electron reduction destabilizes the ferric-nitrosyl complex such that nitric oxide is released directly, as shown by the trapping of released nitric oxide . Aerobically and in the absence of exogenous reductant, nitric oxide dissociates completely from the P450 over periods of several minutes . However, recovery of the nativelike visible spectrum is accompanied by alterations in the catalytic activity of the enzyme and changes in the resonance Raman spectrum . Specifically, resonance Raman spectroscopy identifies the presence of internally located nitrated tyrosine residue(s) following treatment with nitric oxide . Analysis of a Y51F mutant indicates that this is the major nitration target under these conditions . While wild-type P450 BM3 does not form an aerobically stable ferrous-nitrosyl complex, a site-directed mutant of P450 BM3 (F393H) does form an isolatable ferrous-nitrosyl complex, providing strong evidence for the role of this residue in controlling the electronic properties of the heme iron . We report here the spectroscopic characterization of the ferric- and ferrous-nitrosyl complexes of P450 BM3 and describe the use of resonance Raman spectroscopy to identify nitrated tyrosine residue(s) in the enzyme . Nitration of tyrosine in P450 BM3 may exemplify a typical mechanism by which the ubiquitous messenger molecule nitric oxide exerts a regulatory function over the cytochromes P450. Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D344 - 7 The Diatom EST Database; Maheswari U et al.; The Diatom EST database provides integrat